Preparation method of cell for expressing chimeric antigen receptor

Abstract

The invention provides a preparation method of a cell for expressing a chimeric antigen receptor. The preparation method comprises the following steps: (1) enabling the cell to be in contact with an activating agent for activation; (2) contacting the cell with a nucleic acid molecule for coding the CAR, the nucleic acid molecule for coding the CAR being on a non-viral vector, and obtaining the cell containing the nucleic acid molecule; and (3) harvesting cells. Wherein the step (3) is not later than 72 hours after the step (1) is started. Compared with cells prepared by other similar methods, the cells in the step (3) show a higher CAR positive rate, a higher proportion of undifferentiated cell phenotypes, a lower proportion of depleted cells and a stronger amplification ability.

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 a chimeric antigen receptor. Background Art

[0002] A chimeric antigen receptor (CAR) is a genetically recombinant receptor. A chimeric antigen receptor generally 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] The main technical problems currently faced by CAR-T products are as follows: CAR-T is prepared using autologous cells, and the preparation process is cumbersome and time-consuming, resulting in high costs; most CAR-T products use viral vectors, and the products are limited by the yield and quality of viral vectors, which also leads to a sharp increase in costs; when CAR-T is injected into the human body, due to the long culture time and high degree of differentiation, obvious T cell exhaustion will occur.

[0004] To solve the above problems, rapid CAR-T preparation is a current direction of exploration. By shortening the preparation cycle from 14 days to 2 days or less, reducing T cell expansion and differentiation, and enhancing the expansion potential of CAR-T cells in the human body. Rapid CAR-T can significantly shorten the cycle, reduce costs, and make CAR-T cells act more persistently in the human body. Novartis and GenScript have rapid CAR-T preparation technologies based on viral vectors, and there has been no report on the preparation of rapid CAR-T cells using non-viral vectors. Summary of the Invention

[0005] The object of the present invention is to provide a rapid method for preparing cells expressing a chimeric antigen receptor based on non-viral delivery technology.

[0006] The first aspect of the present invention provides a method for preparing cells expressing a chimeric antigen receptor, the method comprising: (1) contacting the cells with an activator for activation; (2) 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; (3) harvesting the cells;

[0007] wherein the method further satisfies at least any one of the following conditions (a)-(c):

[0008] (a) Step (2) is carried out together with step (1), 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);

[0009] (b) Step (3) is no later than 48, 36, 30, 24, 18, 12, 6, 3, 2 or 1 hour after the start of step (2).

[0010] (c) Step (3) is no later than 72, 60, 48, 36, 30, 24, 20, 18 or 12 hours after the start of step (1).

[0011] In some embodiments, the evaluation is carried out by the number of live cells. Compared with the cells at the start of step (1), the cells from step (3) do not expand or expand by no more than 5%, 10%, 20%, 30%, 40%, 50% or 100%.

[0012] In some embodiments, the nucleic acid molecule encoding the CAR is DNA, and the non-viral vector is a plasmid vector.

[0013] In some embodiments, the nucleic acid molecule encoding the CAR is RNA, such as mRNA, saRNA, and the non-viral vector is LNP, LPX, VLP, inorganic nanoparticle or exosome.

[0014] 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 cells in step (2) are also contacted with a transposase or a nucleic acid molecule encoding a transposase.

[0015] 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.

[0016] In one or more embodiments, the transposon system is the PB transposon system, the BZ transposon system or the JL transposon system.

[0017] In some embodiments, the nucleic acid molecule encoding the transposase is DNA or RNA.

[0018] In some embodiments, the cells are contacted with a transposase or a nucleic acid molecule encoding a transposase, and cell transduction is carried out by electroporation.

[0019] In some embodiments, the introduction is carried out by electroporation.

[0020] In some embodiments, step (2) includes: contacting the cells with a DNA vector comprising a JL transposon and mRNA encoding a JL transposase, wherein the JL transposon comprises a CAR gene expression cassette and terminal inverted repeats located on both sides of the CAR gene expression cassette.

[0021] In some embodiments, the amino acid sequence of the JL transposase is as shown in SEQ ID NO:5.

[0022] In some embodiments, the terminal inverted repeats are as shown in SEQ ID NO:6 (3' ITR) and SEQ ID NO:7 (5' ITR).

[0023] In some embodiments, the DNA vector is an antibiotic-free minicircle vector, and the antibiotic-free minicircle vector comprises a nucleotide sequence encoding an antitoxin protein and a replicon; the amino acid sequence of the antitoxin protein contains the following sequences: (1) an amino acid sequence as 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) an amino acid sequence as 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.

[0024] In a preferred embodiment, the amino acid sequence of the antitoxin protein is as shown in any one of SEQ ID NO:14-20.

[0025] In some embodiments, the replicon is R6K.

[0026] In some embodiments, the activator is an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a co-stimulatory molecule on the cell surface.

[0027] In some embodiments, the agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3; the agent that stimulates the CD3 / TCR complex is selected from antibodies (such as single-domain antibodies, peptibodies, Fab fragments, or scFvs), small molecules, or ligands (such as naturally occurring ligands, recombinant ligands, or chimeric ligands).

[0028] In some embodiments, the agent that stimulates the CD3 / TCR complex is an anti-CD3 antibody.

[0029] In some embodiments, the agent that stimulates a co-stimulatory molecule is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 41BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof; the agent that stimulates a co-stimulatory molecule is selected from an antibody (such as a single-domain antibody, peptibody, Fab fragment, or scFv), a small molecule, or a ligand (such as a naturally occurring ligand, a recombinant ligand, or a chimeric ligand).

[0030] In some embodiments, the agent that stimulates a co-stimulatory molecule is an agent that stimulates CD28, preferably an anti-CD28 antibody.

[0031] In some embodiments, the agent that stimulates the CD3 / TCR complex and the agent that stimulates a co-stimulatory molecule are CD3 / 28 magnetic beads, such as TransAct TM .

[0032] In some embodiments, compared to cells prepared by other similar methods, the cells of step (3) show a higher percentage (such as at least 0.1%, 1%, 5%, 10%, 15%, 20% or higher) of CAR-expressing naive cells (such as CAR-expressing naive T cells, such as CD3+CD45RO-CCR7+ T cells expressing CAR), in the other similar methods, step (3) is carried out more than 72 hours after the start of step (i) (such as more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (1)).

[0033] In some embodiments, compared to the percentage of stem cell memory T cells (such as CD45RO+CCR7+CD95+ T cells) in the cells at the start of step (1), the percentage of stem cell memory T cells (such as CD45RO+CCR7+CD95+ T cells) in the cells of step (3) is increased.

[0034] In some embodiments, compared to cells prepared by other similar methods, the percentage of CAR-expressing stem cell memory T cells (such as CD3+CD45RO+CCR7+CD95+ T cells expressing CAR) in the cells of step (3) is higher (such as at least 1%, 5%, 10%, 15%, 20%, 30% or higher), in the other similar methods, step (3) is carried out more than 72 hours after the start of step (1) (such as more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i)).

[0035] In some embodiments, the percentage of CD4+ T cells expressing CAR in the cells of step (3) is higher (e.g., at least 10%, 15%, 20%, 30%, 40%, 50% or higher) compared to cells prepared by other similar methods, in which step (3) is carried out more than 72 hours after the start of step (1) (e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i)).

[0036] In some embodiments, the cells of step (3) have a higher expansion capacity (e.g., can expand 3-fold, 5-fold, 10-fold or more at day 5, 10 or 15 in organoids) compared to cells prepared by other similar methods, in which step (3) is carried out more than 72 hours after the start of step (1) (e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i)).

[0037] In some embodiments, in step (2), after introducing the nucleic acid molecule into the cells, there is no step of culturing the cells.

[0038] In some embodiments, in step (2), after introducing the nucleic acid molecule into the cells, it further includes a step of culturing the cells, and the time for culturing the cells is not longer than 24, 18, 13, 10, 6, 3, 2 or 1 hour.

[0039] In some embodiments, steps (1) and (2) are carried out in a cell culture medium (e.g., serum-free medium) containing IL-2, IL-15, IL-6, LSD1 inhibitor, or MALT1 inhibitor. In some embodiments, steps (1) and (2) are carried out in a cell culture medium (e.g., serum-free medium) containing IL-7, IL-21, or a combination thereof. In some embodiments, steps (1) and (2) are carried out in a cell culture medium (e.g., serum-free medium) containing IL-2, IL-15, IL-21, IL-7, IL-6, LSD1 inhibitor, MALT1 inhibitor, or a combination thereof. In some embodiments, the cell culture medium is a serum-free medium containing a serum replacement (SR).

[0040] In some embodiments, the method further comprises step (4) before step (1): obtaining a fresh or cryopreserved leukapheresis product from an entity. In some embodiments, step (4) further comprises: isolating T cells from the fresh or cryopreserved leukapheresis product. In some embodiments, step (4) further comprises: isolating CD3+, CD4+ and / or CD8+ T cells from the fresh or cryopreserved leukapheresis product. The leukocytes include lymphocytes, basophils, neutrophils, eosinophils and monocytes, and the monocytes can be PBMC (peripheral blood mononuclear cells).

[0041] In some embodiments, step (3) is initiated no later than 72 hours after the start of step (4) (e.g., no later than 6, 12, 24, 26, 28, 30, 36, 40, 48 or 72 hours after the start of step (4).

[0042] In some embodiments, the method is performed in a closed system.

[0043] 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.

[0044] 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.

[0045] 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 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.

[0046] 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.

[0047] In some embodiments, the transmembrane domain comprises the transmembrane domain of a protein selected from the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

[0048] 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.

[0049] In some embodiments, the intracellular signaling domain is a CD3ζ intracellular signaling domain or an FcεRIγ intracellular signaling domain.

[0050] The present invention also provides a cell expressing a chimeric antigen receptor prepared by the preparation method of any one of the embodiments.

[0051] The present invention also provides the use of the cell expressing a CAR in the preparation of a medicament for treating and / or preventing a malignant tumor.

[0052] 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 its 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 It is the plasmid map of plasmid P19V21;

[0054] Figure 2 It is the plasmid map of MSLN CAR plasmid;

[0055] Figure 3 It is the cell viability after culturing for 24 h, 30 h or 9 days after transfection of T cells with different transposon systems;

[0056] Figure 4 It is the positive rate of MSLN CAR+T after culturing for 24 h, 30 h or 9 days after transfection of T cells with different transposon systems;

[0057] Figure 5 It is the cell differentiation subsets after culturing for 24 h, 30 h or 9 days after transfection of T cells with different transposon systems;

[0058] Figure 6 It is the ratio of CD4+CAR+ / CD8+CAR+T after culturing for 24 h, 30 h or 9 days after transfection of T cells with different transposon systems;

[0059] Figure 7 It is the T cell exhaustion after culturing for 24 h, 30 h or 9 days after transfection of T cells with different transposon systems;

[0060] Figure 8 It is the amplification fold of CAR-T cells in organoid co-culture after culturing for 24 h, 30 h or 9 days after transfection of T cells with different transposon systems;

[0061] Figure 9 It is the plasmid map of pCpGfree MCS-0637 empty microplasmid, including the nucleotide sequence of antitoxin 0637 and R6K replicon;

[0062] Figure 10 It is the plasmid map of pCpGfree MCS-43009 empty microplasmid, including the nucleotide sequence of antitoxin 43009 and R6K replicon. Detailed implementation mode

[0063] Definition

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs.

[0065] The term "chimeric antigen receptor" (CAR) refers to an artificially engineered receptor that can anchor a specific molecule (such as an antibody) that recognizes an antigen on the surface of tumor cells to an immune cell (such as a T cell), enabling the immune cell to recognize tumor antigens or viral antigens and kill tumor cells or virus-infected cells. CAR typically sequentially includes 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 on 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.

[0066] 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.

[0067] The terms "single-domain antibody", "variable domain of the 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 cloned to construct a single-domain antibody consisting of only one variable region of the heavy chain.

[0068] The term "costimulatory molecule" refers to a molecule that exists on the surface of antigen-presenting cells and can bind to a costimulatory molecule receptor on Th cells to generate a co-stimulatory signal. The proliferation of lymphocytes requires not only the binding of an antigen but also the receipt of a signal from a costimulatory molecule. The co-stimulatory signal is transmitted to T cells mainly through the costimulatory molecules CD80 and CD86 expressed on the surface of antigen-presenting cells binding to the CD28 molecule on the surface of T cells. B cells can receive co-stimulatory signals through general pathogen components such as LPS, or through complement components, or through CD40L on the surface of activated antigen-specific Th cells.

[0069] The term "naive T cell" refers to a T cell that has not experienced antigen stimulation. In some embodiments, a T cell that has not experienced antigen stimulation encounters its cognate antigen in the thymus but not in the periphery. In some embodiments, naive T cells are precursors of memory cells. In some embodiments, naive T cells express CD45RA and CCR7, but do not express CD45RO. In some embodiments, naive T cells can be characterized by the expression of CD62L, CD27, CCR7, CD45RA, CD28, and CD127 and the absence of CD95 or CD45RO isotype. In some embodiments, naive T cells express CD62L, IL-7 receptor-α, IL-6 receptor, and CD132, but do not express CD25, CD44, CD69, or CD45RO. In some embodiments, naive T cells express CD45RA, CCR7, and CD62L, but do not express CD95 or IL-2 receptor β. In some embodiments, flow cytometry is used to evaluate the surface expression levels of the markers.

[0070] The term "central memory T cell" refers to a subset of T cells in humans that are CD45RO positive and express CCR7. In some embodiments, central memory T cells express CD95. In some embodiments, central memory T cells express IL-2R, IL-7R, and / or IL-15R. In some embodiments, central memory T cells express CD45RO, CD95, IL-2 receptor β, CCR7, and CD62L. In some embodiments, flow cytometry is used to evaluate the surface expression levels of the markers.

[0071] The term "stem memory T cell", "stemcellmemory T cell", "stem-like memory T cell", "memory stem T cell", "T memory stem cell", "T stem cell memory cell", or "TSCM cell" refers to a subset of memory T cells with stem cell-like capabilities, e.g., the ability to self-renew and / or the pluripotent ability to reconstitute memory and / or effector T cell subsets. In some embodiments, stem memory T cells express CD45RA, CD95, IL-2 receptor β, CCR7, and CD62L. In some embodiments, flow cytometry is used to evaluate the surface expression levels of the markers. In some embodiments, exemplary stem memory T cells are disclosed in Gattinoni et al., Nat Med. [Nature Medicine] January 06, 2017; 23(1):18-27, which is incorporated herein by reference in its entirety.

[0072] The term "transduction" refers to the process of transferring or introducing exogenous nucleic acid into a host cell.

[0073] 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.

[0074] The term "viral vector" refers to a vector that uses the molecular mechanism by which a virus transfers its genome into other cells for infection to mediate gene transfer. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, etc.

[0075] The term "non-viral vector" refers to a non-viral vector used to mediate gene transfer, including plasmid vectors, non-viral materials (such as LNP, LPX, VLP, inorganic nanoparticles, exosomes, etc.).

[0076] The first aspect of the present invention provides a method for preparing cells expressing a chimeric antigen receptor, and the cells are, for example, T cells. The method includes: (1) contacting the cells with an activator for activation; (2) contacting the cells with a nucleic acid molecule encoding a CAR, and the nucleic acid molecule encoding a CAR is on a non-viral vector to introduce the nucleic acid molecule into the cells; (3) harvesting the cells;

[0077] Wherein, the method further satisfies at least any one or more of the following conditions (a)-(d):

[0078] (a) Step (2) is carried out together with step (1) or no later than 48, 36, 24, 20, 16, 12, 8, 5, 4, 3, 2 or 1 hour after the start of step (1), and

[0079] (b) Step (3) is no later than 48, 36, 30, 24, 18, 12, 6, 3, 2 or 1 hour after the start of step (2);

[0080] (c) Step (3) is no later than 72, 60, 48, 36, 30, 24, 20, 18, 12 hours after the start of step (1);

[0081] (d) Evaluated by the number of cells, compared with the cells at the start of step (2), the cells from step (3) do not expand or expand by no more than 5%, 10%, 20%, 30%, 40%, 50% or 100%.

[0082] The following is an exemplary description of the method herein.

[0083] Cell collection

[0084] The cells can be fresh or cryopreserved leukapheresis products obtained from a subject. The subject can be a healthy individual or a cancer patient. In some embodiments, T cells are isolated (cell sorting) from the fresh or cryopreserved leukapheresis products, such as CD3+, CD4+ and / or CD8+ T cells. The leukocytes include lymphocytes, basophils, neutrophils, eosinophils, and monocytes, wherein the monocytes can be PBMCs (peripheral blood mononuclear cells).

[0085] Cell activation

[0086] Step (1) The cells are activated by contacting with an activator. In some embodiments, the activator is an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates co-stimulatory molecules on the cell surface.

[0087] In some embodiments, the agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3; the agent that stimulates the CD3 / TCR complex is selected from antibodies (such as single-domain antibodies, peptibodies, Fab fragments, or scFvs), small molecules, or ligands (such as naturally occurring ligands, recombinant ligands, or chimeric ligands).

[0088] In some embodiments, the agent that stimulates the CD3 / TCR complex is an anti-CD3 antibody.

[0089] In some embodiments, the agent that stimulates co-stimulatory molecules is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 41BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof; the agent that stimulates co-stimulatory molecules is selected from antibodies (such as single-domain antibodies, peptibodies, Fab fragments, or scFvs), small molecules, or ligands (such as naturally occurring ligands, recombinant ligands, or chimeric ligands).

[0090] In some embodiments, the agent that stimulates co-stimulatory molecules is an agent that stimulates CD28, preferably an anti-CD28 antibody.

[0091] The stimulating agent may be present in the incubation mixture in the form of a solute, or it may be immobilized on a solid-phase carrier. Solid-phase carriers suitable for immobilizing activators (such as antibodies) are well known in the art, such as magnetic beads or the walls of containers. 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, a CD3 antibody and a CD28 antibody, a CD3 antibody and a 4-1BB antibody, or a CD3 antibody and a 4-1BBL antigen immobilized on the wall of a container; preferably, the container is a T75 flask.

[0092] In some embodiments, the activation time is 1 to 48 hours, for example, the activation time can be 48, 36, 24, 20, 16, 12, 8, 5, 4, 3, 2, or 1 hour. Preferably, the activation time is 2 to 36, 3 to 36, 4 to 24, or 5 to 24 hours.

[0093] Plasmid vector

[0094] In some embodiments, the nucleic acid molecule encoding the CAR is DNA, and the non-viral vector is a plasmid vector.

[0095] 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) typically 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 antibodies to be expressed, and optional marker elements. See, for example, WO 01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193.

[0096] When the nucleic acid molecule encoding the CAR is DNA, the nucleic acid molecule is typically 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.

[0097] Transposons and transposases

[0098] In some embodiments, the non-viral vector is a plasmid vector containing a transposon, and the transposon contains a nucleic acid molecule encoding a CAR.

[0099] DNA transposons can transpose through a non-replicative "cut and paste" mechanism. This requires the recognition of two terminal inverted repeats (ITRs) by a transposase, which can cleave its target, thereby releasing the DNA transposon from its donor template. After excision, the DNA transposon can then integrate into the recipient DNA that has been cleaved by the same transposase.

[0100] The transposon and the corresponding transposase form a transposon system. Depending on the type of transposon system, a transposase and a transposon containing the corresponding ITR sequence are selected. The nucleic acid molecule encoding the CAR contained in the transposon is located between the ITR sequences. 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).

[0101] 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) involves contacting the cells with a plasmid vector containing a nucleic acid molecule encoding a transposase and a transposon. In some embodiments, step (2) involves contacting the cells with a plasmid vector containing a nucleic acid molecule encoding a transposase and a plasmid vector containing a transposon.

[0102] 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.

[0103] The ZB transposon system is the ZB transposon system described in any embodiment of Patent CN201510429987.3, the entire content of which 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, the entire content of which is incorporated herein by reference. The BZ transposon system includes a BZ transposase and a BZ transposon containing ITR sequences recognizable by the BZ transposase.

[0104] The BZ transposase is a transposase having any one or more sets of the following mutations compared with SEQ ID NO: 1:

[0105] Q71R\H110R,

[0106] Q71R\Q79R\H110R,

[0107] G216A\Q71R\Q79R\H110R,

[0108] H208V\Q71R\Q79R\H110R,

[0109] H208V\G216A\Q71R\Q79R\H110R, F21K\D22A\Q71R\H110R,

[0110] 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,

[0111] H208V\Q71R\H110R,

[0112] H208V\G216A\Q71R\H110R,

[0113] 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, G216A\H208V\K251T\G189A\Q138R\Q71R\H110R, G216A\H208V\K251T\G189A\K134A\Q71R\H110R,

[0114] G216A\H208V\K251T\G189A\Q138K\K134A\Q71R\H110R,

[0115] G216A\H208V\K251T\G189A\Q138R\K134A\Q71R\H110R,

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

[0117] G216A\H208V\K251T\G189A\Q138K\K137T\Q71R\H110R, or

[0118] N005S\F21K / D22A\Q71R\H110R.

[0119] G216A\H208V,

[0120] G216A\H208V\G189A,

[0121] G216A\H208V\K251T,

[0122] G216A\H208V\K251T\G189A,

[0123] G216A\H208V\K251T\G189A\Q138K,

[0124] G216A\H208V\K251T\G189A\Q138R,

[0125] G216A\H208V\K251T\G189A\K134A,

[0126] G216A\H208V\K251T\G189A\Q138K\K134A,

[0127] G216A\H208V\K251T\G189A\Q138R\K134A,

[0128] G216A\H208V\K251T\G189A\Q138K\V144E, or

[0129] G216A\H208V\K251T\G189A\Q138K\K137T.

[0130] 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.

[0131] The BZ transposon contains a nucleic acid molecule encoding a CAR and ITR sequences recognizable by the BZ transposase located 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 motifs therein are mutated to TpG or CpA.

[0132] 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.

[0133] In some embodiments, the JL transposon system includes a JL transposase and a JL transposon containing ITR sequences recognizable by the JL transposase.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] In some embodiments, the transposon system is a PB transposon system, a BZ transposon system, or a JL transposon system.

[0138] 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 without 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 within 600 bp in length, 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 (minicircle plasmid without an antibiotic expression cassette), also known as a tiny or tiniplasmid. The antibiotic-free minicircle plasmid applicable to the present invention can refer to patent application 202310072956., the entire content of which is incorporated herein by reference.

[0139] 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.

[0140] In some embodiments, the amino acid sequence of the antitoxin protein is any one of SEQ ID NO: 14-20.

[0141] In some embodiments, the replicon is selected from ColE1, ColE2, pMB1, pSC101, RSF, R6K, pUC57, RK2 and p15A; preferably R6K or pUC57.

[0142] 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.

[0143] 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.

[0144] In some embodiments, the nucleotide sequence of the replicon does not contain CpG motifs.

[0145] 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.

[0146] 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 9 shown in or 10.

[0147] Cell transduction

[0148] Step (2) contacting the cell with a nucleic acid molecule encoding a CAR to introduce the nucleic acid molecule into the cell.

[0149] 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 nanoparticle or exosome. The RNA molecule encoding a CAR is transfected into the cell 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 the cell directly by electroporation without a vector.

[0150] In some embodiments, in step (2), the cell is contacted with a plasmid vector of a transposon and a transposase or an 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.

[0151] In some embodiments, in step (2), the cell is 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.

[0152] In some embodiments, in step (2), the cell is 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 respectively.

[0153] In some embodiments, step (2) includes: contacting the cells with a DNA vector comprising a JL transposon and mRNA encoding a JL transposase, wherein the JL transposon comprises a CAR gene expression cassette and terminal inverted repeats located on both sides of the CAR gene expression cassette.

[0154] In some embodiments, the amino acid sequence of the JL transposase is as shown in SEQ ID NO:5.

[0155] In some embodiments, the terminal inverted repeats are as shown in SEQ ID NO:6 (3' ITR) and SEQ ID NO:7 (5' ITR).

[0156] In some embodiments, the plasmid map of the DNA vector comprising the JL transposon is as Figure 2 shown.

[0157] In some embodiments, in step (2), 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 (LNPs), particle bombardment, fugene, direct acoustic loading, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, nucleofection or any combination thereof.

[0158] In some embodiments, the introduction includes contacting the cells with 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 1 - 5 μg per 1×10 7 cells, and the dosage of the plasmid is at a concentration of 0.1 - 2 μg per 1×10 7 cells.

[0159] 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 the activator.

[0160] In some embodiments, the contact is adding a transposon plasmid containing a nucleic acid molecule encoding a CAR and mRNA encoding a transposase to the culture medium of the cells and the activator after the activation in step (1) is completed, and then introducing the transposon plasmid containing a nucleic acid molecule encoding a CAR and mRNA encoding a transposase into the cells by electroporation.

[0161] In some embodiments, after the activation in step (1) 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.

[0162] Different from viral vectors, the electroporation can be completed within 1 hour or less, and thus the time for introducing the nucleic acid molecule into the cell in the present invention is much lower than that of viral vectors.

[0163] In some embodiments, the cell can express a therapeutic agent and / or contain a coding sequence of a therapeutic agent, and in step (2), the cell further contacts with a nucleic acid molecule of the therapeutic agent to introduce the nucleic acid molecule of the therapeutic agent into the cell.

[0164] In some embodiments, the nucleic acid molecule of the therapeutic agent is also located on the plasmid vector of the transposon.

[0165] 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 connected 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.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] In some embodiments, the therapeutic agent is an immune checkpoint inhibitor.

[0171] In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof targeting 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.

[0172] In some embodiments, the therapeutic agent is an antibody targeting PD-1, preferably a single-domain antibody targeting PD-1. The sequence of the single-domain antibody targeting PD-1 is the single-domain antibody targeting PD-1 described in any embodiment of Patent CN202011582908.X, the entire content of which is incorporated herein by reference.

[0173] In some embodiments, the sequence of the single-domain antibody targeting PD-1 is as shown in any of SEQ ID NOs: 26-29.

[0174] In some embodiments, the therapeutic agent is an antibody targeting CTLA-4, preferably a single-domain antibody targeting CTLA-4. The sequence of the single-domain antibody targeting CTLA-4 is the single-domain antibody targeting CTLA-4 described in any embodiment of Patent CN202111152925.4, the entire content of which is incorporated herein by reference.

[0175] In some embodiments, the sequence of the single-domain antibody targeting CTLA-4 is as shown in SEQ ID NO: 30.

[0176] In some embodiments, the therapeutic agent is a bispecific antibody containing a first functional region targeting PD-1 and a second functional region targeting 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.

[0177] 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.

[0178] 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.

[0179] In some embodiments, the sequence of the bispecific antibody is as shown in any of SEQ ID NOs: 31-34.

[0180] Cytokine

[0181] In some embodiments, step (1) and / or (2) are 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 (1) and (2) 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 (1) and / or (2) are 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).

[0182] Other steps

[0183] After introducing the nucleic acid molecule encoding the CAR into the cell, the cell can be cultured for a period of time or the cell can be directly harvested. In some embodiments, after introducing the nucleic acid molecule encoding the CAR into the cell, the cell suspension is transferred to a new medium, which can be a serum-free medium, such as AIM-V medium + 5% SR.

[0184] In some embodiments, the culture time can be less than 24 hours, such as no more than 24, 18, 13, 10, 6, 3, 2, or 1 hour. The harvested cells can be formulated for storage or administration.

[0185] In some embodiments, after cell collection, a sorting step (separating T cells) is carried out, and the sorting time is 1 - 5 hours, such as 1, 2, 3, 4, or 5 hours.

[0186] In some embodiments, the total duration from sorting to harvesting CAR-T cells is 72, 60, 48, 36, 30, 28, 24, 22, 20, 18, or 12 hours or less. In an exemplary protocol, the total duration is 22 hours or 28 hours.

[0187] In some embodiments, the total duration from the start of contact between the cell and the activator to harvesting CAR-T cells is 72, 60, 48, 36, 30, 25, 24, 20, 19, 18, or 12 hours or less. In an exemplary protocol, the total duration is 19 hours or 25 hours.

[0188] 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 all carried out in a closed system.

[0189] Cell phenotype

[0190] In some embodiments, the percentage of naïve cells (e.g., naïve T cells such as CD45RO-CCR7+ T cells) in the cells from step (3) (i.e., the harvested cells) differs by no more than 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, or 12% from the percentage of naïve cells (e.g., naïve T cells such as CD45RO-CCR7+ cells) in the cells at the start of step (1).

[0191] In some embodiments, the cells of step (3) show a higher percentage (e.g., at least 0.1%, 1%, 5%, 10%, 15%, 20% or higher) of CAR-expressing naïve cells (e.g., CAR-expressing naïve T cells such as CAR-expressing CD3+CD45RO-CCR7+ T cells) compared to cells prepared by other similar methods, where step (3) is performed more than 72 hours after the start of step (i) (e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (1)).

[0192] In some embodiments, the percentage of stem cell memory T cells (e.g., CD45RO+CCR7+CD95+ T cells) in the cells of step (3) is increased compared to the percentage of stem cell memory T cells (e.g., CD45RO+CCR7+CD95+ T cells) in the cells at the start of step (1).

[0193] In some embodiments, the percentage of CAR-expressing stem cell memory T cells (e.g., CAR-expressing CD3+CD45RO+CCR7+CD95+ T cells) in the cells of step (3) is higher (e.g., at least 1%, 5%, 10%, 15%, 20%, 30% or higher) compared to cells prepared by other similar methods, where step (3) is performed more than 72 hours after the start of step (1) (e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i)).

[0194] In some embodiments, the percentage of CAR-expressing CD4+ T cells in the cells of step (3) is higher (e.g., at least 10%, 15%, 20%, 30%, 40%, 50% or higher) compared to cells prepared by other similar methods, where step (3) is performed more than 72 hours after the start of step (1) (e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i)).

[0195] In some embodiments, the cells of step (3) have a higher amplification capacity (e.g., can be amplified 3-fold, 5-fold, 10-fold or more at day 5, 10 or 15 in the organoids) compared to cells prepared by other similar methods, where step (3) is carried out more than 72 hours after the start of step (1) (e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i)).

[0196] In some embodiments, step (3) is carried out no later than 72 hours after the start of step (4) (e.g., no later than 6, 12, 24, 26, 28, 30, 36, 40, 48 or 72 hours after the start of step (4)). In some embodiments, for example, as evaluated by the number of live cells, compared to the cells at the end of step (4), the cells of step (3) do not expand, or expand by no more than 5%, 10%, 20%, 30%, 40%, 50% or 100%.

[0197] Chimeric antigen receptor

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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.

[0204] In some embodiments, the intracellular signaling domain is the CD3ζ intracellular signaling domain or the FcεRIγ intracellular signaling domain.

[0205] In some embodiments, the immune cell is a mesothelin-targeted 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.

[0206] Nucleic acid construct encoding the CAR

[0207] The present invention includes polynucleotide sequences encoding the CAR of the present invention. The polynucleotide sequences of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or synthetic DNA. The DNA can be single-stranded or double-stranded.

[0208] The polynucleotide sequences described herein can generally be obtained by PCR amplification. Specifically, primers can be designed according to the nucleotide sequences disclosed herein, especially the open reading frame sequences, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art can be used as a template for amplification to obtain the relevant sequences. When the sequence is long, it is often necessary to perform PCR amplification two or more times, and then splice the fragments amplified each time together in the correct order.

[0209] The present invention also relates to nucleic acid constructs. The term "nucleic acid construct" or "polynucleotide construct" refers to one or more single-stranded or double-stranded nucleic acid molecules, which are isolated from naturally occurring genes or modified to contain nucleic acid fragments in a manner not found in nature. The term "nucleic acid molecule" mainly refers to physical nucleic acid molecules, and the term "nucleic acid sequence" mainly refers to the nucleotide sequence on the nucleic acid molecule, but the two terms can be used interchangeably, especially with respect to nucleic acid molecules or nucleic acid sequences that can encode proteins or protein domains. The nucleic acid construct contains the polynucleotide sequences described herein, as well as one or more regulatory sequences operably linked to these sequences. The polynucleotide sequences of the present invention can be manipulated in various ways to ensure the expression of the CAR. The nucleic acid construct can be manipulated according to the different requirements of the expression vector before being inserted into the vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0210] The regulatory sequence can be a suitable promoter sequence. The promoter sequence is usually operably linked to the coding sequence of the protein to be expressed. The promoter can be any nucleotide sequence that shows transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in the present invention. The regulatory sequence can also be a suitable leader sequence, the untranslated region of the mRNA that is important for translation in the host cell. The leader sequence is operably linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in the present invention.

[0211] In certain embodiments, the nucleic acid construct is a vector. The term "vector" is capable of transferring a gene sequence into a target cell. Generally, the terms "vector construct", "expression vector", and "gene transfer vector" mean any nucleic acid construct that is capable of directing the expression of a gene of interest and can transfer the gene sequence into a target cell, which can be achieved by genomic integration of the whole or part of the vector, or by transient or heritable maintenance of the vector as an extrachromosomal element. Thus, the term includes cloning vectors, expression vectors, and integration vectors. Generally, expression of the polynucleotide sequence of the present invention is achieved by operably linking the polynucleotide sequence of the present invention to a promoter and incorporating the construct into an expression vector. The vector can be suitable for replication and integration in eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence. The nucleic acid construct can be one or more vectors, each vector containing one or two or three expression cassettes described in any of the embodiments herein.

[0212] The polynucleotide sequence of the present invention can be cloned into many types of vectors. For example, it can be cloned into plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Further, the vector is an expression vector. The expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.

[0213] Typically, suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (e.g., WO 01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0214] The nucleic acid construct can be a cloning vector or an expression vector. The expression vector is preferably a constitutive expression vector, such as a transposon vector (or "transposon vector").

[0215] Thus, in some embodiments, the nucleic acid construct contains the coding sequences of a CAR and a transposase. In some embodiments, the nucleic acid construct contains the expression cassette of the chimeric antigen receptor and the expression cassette of the transposase. The two expression cassettes are contained in one or two vectors. Alternatively, the nucleic acid construct is an expression cassette in which the coding sequence of the chimeric antigen receptor and the coding sequence of the transposase are within the expression cassette.

[0216] Pharmaceutical composition

[0217] The present invention also provides cells expressing a chimeric antigen receptor prepared by the preparation method of any of the embodiments.

[0218] 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.

[0219] 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 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-related multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, acute myeloid leukemia (AML), or lymphoma of undetermined classification.

[0220] The cells expressing CAR 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 cells expressing CAR 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.

[0221] 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.

[0222] When referring to an "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive 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, e.g., 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.

[0223] 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 compositions of cells expressing CAR of the present invention are preferably administered by intravenous injection. The compositions of cells expressing CAR can be directly injected into tumors, lymph nodes or sites of infection.

[0224] The present invention will be illustrated below in the form of specific examples. 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.

[0225] Examples

[0226] Example 1, Preparation of CAR-T cells

[0227] 1. Obtain a leukapheresis sample from the patient, and then perform T cell sorting using Miltenyi CD4 / CD8 magnetic beads. Add 200 μL of CD4 magnetic beads and 200 μL of CD8 magnetic beads to 1×10 9 WBC, incubate for 30 min, and then screen for CD4+T and CD8+T cells through an XS sorting column.

[0228] 2. After sorting, take 1.05×10 8 cells cell suspension into a coated bottle containing Miltenyi MACS GMP TransAct CD3 / 28 magnetic beads, and supplement the culture medium (AIM-V + 5% SR) to 30 mL / bottle. Add IL-7 & IL-15 with a final concentration of 25 ng / mL & 25 ng / mL, and place it at 37°C, 5% CO 2 for culture for 1 - 48 h.

[0229] 3. For Group 1 PB D9, take the T cells after activation for 48 h, 1*10 7 cells / group, add piggybac enzyme mRNA at 320 μg / mL and plasmid P19V21 expressing the MSLN CAR sequence at 84 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, and place it at 37°C, 5% CO 2 for culture for 9 days, and then observe the cell growth. Among them, the amino acid sequence of the piggybac enzyme is as shown in SEQ ID NO:39. The plasmid map of plasmid P19V21 is as Figure 1 shown, and the sequence is as shown in SEQ ID NO:35. The structure of MSLN CAR is: from the N-terminus to the C-terminus, it sequentially contains a CD8α signal peptide, mesothelin VHH at position 1444, a CD8α hinge region, a CD28 transmembrane region and an intracellular co-stimulatory signal region, and a CD3ζ intracellular signal domain; the amino acid sequence of mesothelin VHH at position 1444 is as shown in SEQ ID NO:36, and the amino acid sequence of MSLN CAR is as shown in SEQ ID NO:37.

[0230] 4. For Group 2 PB 24 h and Group 4 PB 30 h, take the T cells after activation for 5 and 24 h respectively, 1*10 7Cells / group, add 320 μg / mL of piggybac mRNA and 84 μg / mL of plasmid P19V21 expressing the MSLN CAR sequence. Transfer the mixture 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; transfer the electroporated cell suspension into a T75 culture flask (the culture medium is AIM-V medium containing 5% SR), mix well, and place it at 37°C, 5% CO 2 Culture for no more than 24 h and harvest the CAR-T cells.

[0231] Among them, the total duration of the product preparation cycle for Group 2 is 24 h, including 3 h of sorting, 5 h of activation, 1 h of electroporation, 13 h of culture, and 2 h of formulation; the total duration of the product preparation cycle for Group 4 is 30 h, including 3 h of sorting, 24 h of activation, 1 h of electroporation, and 2 h of formulation.

[0232] 5. For Group 3 JL 24 h and Group 5 JL 30 h, take the T cells after 5 and 24 h of activation, 1×10 7 Cells / group, add 5 μg / mL of the plasmid expressing MSLN CAR and 2.5 μg / mL of JL enzyme mRNA. Transfer the mixture 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; transfer the electroporated cell suspension into a T75 culture flask (the culture medium is AIM-V medium containing 5% SR), mix well, and place it at 37°C, 5% CO 2 Culture for no more than 24 h and harvest the CAR-T cells. From cell sorting to harvesting CAR-T cells, the total duration of the product preparation cycle for Group 3 and Group 5 is 24 h and 30 h respectively, and the duration of each step is the same as that of Group 2 and Group 4.

[0233] Among them, the plasmid map of the plasmid expressing MSLN CAR is as Figure 2 shown, and the sequence is SEQ ID NO: 38; except for the transposon, the plasmid backbone includes the nucleotide sequences of the R6K replicon and the antitoxin protein 0637. The nucleotide sequence of the R6K replicon is as shown in SEQ ID NO: 23, and the nucleotide sequence of the antitoxin protein 0637 is as shown in SEQ ID NO: 21. The MSLN CAR structure is the same as in Step 3, and the amino acid sequence of the JL enzyme is SEQ ID NO: 5.

[0234] 6. Use an NC-200 cell counter to detect the cell viability and cell density of the cells in the above five groups after culture. The results of cell viability are as Figure 3As shown, the viability of the cells prepared by the five groups of processes is greater than 70%, meeting the quality standards of the product; the viability of the CAR-T cells prepared by the 30h process is the highest (97.4%, 95.1%).

[0235] Example 2, Detection of CAR-T Positive Rate

[0236] The harvested cells of the above five groups were subjected to CAR+T positive rate detection, and the specific steps are as follows:

[0237] 1. Take 1444-Fc-biotin (which is a fusion protein of biotin-labeled VHH No. 1444 and IgG4 Fc, and the preparation method is shown in Example 1 of CN111381020A) and PE-streptavidin (purchased from Shanghai Genechem Co., Ltd.) and dissolve them in PBS to prepare a 100× stock solution with a concentration of 10.0 mg / mL. Dilute it 100 times with PBS before use to obtain a PE-fluorescein-labeled 1444-Fc dilution;

[0238] 2. Take 1×10 6 cells of each CAR-T cell prepared in Example 1 respectively, centrifuge at 400g for 5 min, discard the upper layer of the culture medium, resuspend with 1 mL of fresh culture medium, and add 1, 2, and 5 μL of the PE-fluorescein-labeled 1444-Fc dilution prepared in step 1 to all the cells respectively, and incubate at 37°C for 1 h;

[0239] 3. Wash each CAR-T cell incubated in step 2 three times with cold PBS. Each time, resuspend the cells with 1 mL of cold PBS and centrifuge at 1000 rpm for 3 min. After three times, detect the fluorescence intensity of the cells with a flow cytometer, analyze the positive rate, and make a comparison.

[0240] The results are as Figure 4 shown, indicating that the CAR-T positive rate prepared by the JL transposon system is the highest (76.11%, 78.34%).

[0241] Example 3, Detection of CAR-T Cell Differentiation Phenotype

[0242] The cells of the above five groups after culturing were subjected to cell differentiation phenotype detection, and the specific steps are as follows:

[0243] 1. Prepare a 1X working solution by mixing lysis storage solution and PBS phosphate buffer. Take 400g of each of the five groups of CAR-T cells prepared in Example 1 and centrifuge for 5 min, discard the upper layer of the culture medium, and take 1×10 6Separate tubes were added with 1 μL of BrilliantViolet 421TM anti-human CD45RO (purchased from Shanghai GenScript Biotech Corporation), 2 μL of PE-labeled CCR7 (purchased from Shanghai GenScript Biotech Corporation), 1 μL of Anti-human CD95 (purchased from Shanghai GenScript Biotech Corporation), and 1 μL of APC, and incubated at 2-8 °C in the dark for 15 min. 1 μL of PE-streptavidin was added to the CAR-T cell sample tubes and incubated at 2-8 °C in the dark for 15 min.

[0244] 2. The CAR-T cells after the incubation in Step 2 were washed three times with cold PBS. Each time, the cells were resuspended with 1 mL of cold PBS and centrifuged at 400 g for 5 min. After three washes, the fluorescence intensity of the cells was detected by flow cytometry, the cell differentiation phenotypes were analyzed, and comparisons were made.

[0245] The results are as Figure 5 shown, indicating that compared with the CAR-T cells conventionally prepared by PB D9, the subpopulations of less-differentiated cells in the rapidly prepared CAR-T cells (Tnaive: CD45RO-CCR7+ and Tscm: CD45RO+CCR7+CD95+ subpopulations) were significantly increased. Among them, the proportion of CD3+CAR+Tnaive cells in the PB 24h group was 11.34%, and the proportion of CD3+CAR+Tscm cells was 46.10%. The proportion of CD3+CAR+Tnaive cells in the JL 24h group was 19.23%, and the proportion of CD3+CAR+Tscm cells was 25.41%. The proportion of CD3+CAR+Tnaive cells in the PB 30h group was 0.16%, and the proportion of CD3+CAR+Tscm cells was 46.34%. The proportion of CD3+CAR+Tnaive cells in the JL 30h group was 0.40%, and the proportion of CD3+CAR+Tscm cells was 43.60%. The proportion of CD3+CAR+Tnaive cells in the PB D9 group was 0, and the proportion of CD3+CAR+Tscm cells was 17.58%.

[0246] Example 4, Proportions of CD4+ and CD8+ in CAR-T Positive Cells

[0247] The cells in the above five groups after the culture was ended were subjected to the detection of the CD4+CAR+ / CD8+CAR+T ratio. The specific steps were as follows:

[0248] 1. The lysis storage solution and PBS phosphate buffer were prepared into a 1X working solution. The CAR-T cells prepared in Example 1 were centrifuged at 400 g for 5 min respectively, the upper-layer culture medium was discarded, and 1×10 6Take each CAR-T cell sample, add 1 μl of FITC anti-human CD4 (purchased from BD Biosciences Co., Ltd.) and 1 μl of APC anti-human CD8 (purchased from BD Biosciences Co., Ltd.) respectively, and incubate in the dark at 2-8 °C for 15 min. Add 1 μl of PE-streptavidin to the CAR-T cell sample tube and incubate in the dark at 2-8 °C for 15 min.

[0249] 2. Wash each CAR-T cell after incubation in step 2 three times with cold PBS. Resuspend the cells with 1 mL of cold PBS each time and centrifuge at 400 g for 5 min. After three washes, detect the fluorescence intensity of the cells with a flow cytometer, analyze the cell differentiation phenotype, and make comparisons.

[0250] The results are as Figure 6 shown, indicating that compared with the CAR-T cells conventionally prepared by PB D9, the CD4+ T cell subset of the rapidly prepared CAR-T cells is significantly increased. Among them, the proportion of CD4+ T cells in the PB D9 group is 8.92%. The proportion of CD4+ CAR+ T cells in the PB 24h group is 58.47%, the proportion of CD4+ CAR+ T cells in the JL 24h group is 43.56%, the proportion of CD4+ CAR+ T cells in the PB 30h group is 50.45%, and the proportion of CD4+ CAR+ T cells in the JL 30h group is 41.09%.

[0251] Example 5, proportion of exhausted cells in CAR-T cells

[0252] 1. Prepare a 1X working solution by mixing the red blood cell lysis storage solution and PBS phosphate buffer. Centrifuge each CAR-T cell prepared in Example 1 at 400 g for 5 min, discard the upper layer of the culture medium, and take 1×10 6 cells, add 1 μl of APC anti-human CD8 (purchased from BD Biosciences Co., Ltd.), 2 μl of PE-labeled PD1 (purchased from BD Biosciences Co., Ltd.), 1 μl of Anti-human TOX1 (purchased from BD Biosciences Co., Ltd.), and 1 μl of Anti-human TIGIH (purchased from BD Biosciences Co., Ltd.) respectively, and incubate in the dark at 2-8 °C for 15 min. Add 1 μl of PE-streptavidin (purchased from BD Biosciences Co., Ltd.) to the CAR-T cell sample tube and incubate in the dark at 2-8 °C for 15 min.

[0253] 2. Wash each CAR-T cell after incubation in Step 2 three times with cold PBS. Resuspend the cells with 1 mL of cold PBS each time and centrifuge at 400 g for 5 min. After three washes, detect the fluorescence intensity of the cells with a flow cytometer, analyze the cell differentiation phenotype, and make a comparison. The results are as Figure 7 shown. Compared with the conventional preparation of CAR-T by PB D9, the proportion of the exhausted cell subset CD8+CAR+PD1+TOX+TIGIT+ in the rapidly prepared CAR-T cells is significantly reduced. The proportion of CD8+CAR+PD1+TOX+TIGIT+ cells in the PB D9 group is 46.68%, the proportion of CD8+CAR+PD1+TOX+TIGIT+ cells in the PB 24h group is 25.35%, and the

[0254] proportion of CD8+CAR+PD1+TOX+TIGIT+ cells in the JL 24h group

[0255] is 30.3%, and the proportion of CD8+CAR+PD1+TOX+TIGIT+ cells in the PB 30h group

[0256] is 18.66%, and the proportion of CD8+CAR+PD1+TOX+TIGIT+ cells in the JL 30h group

[0256] is 20.66%.

[0257] Example 6. Co-culture of CAR-T cells with organoids

[0258] Take the respective CAR-T cells prepared in Example 1 and co-culture them with thymic mesothelioma organoids, and detect the amplification ability of the CAR-T cells. The preparation of thymic mesothelioma organoids refers to the literature Shi, Huaikai, et al. "3-Dimensional mesothelioma spheroids provide closer to natural pathophysiological tumor microenvironment for drug response studies." Frontiers in Oncology 12 (2022): 973576.

[0259] The specific steps are as follows:

[0260] 1. Tumor organoid preparation: One week in advance, resuscitate and culture the organoids until they are in good condition. Take an appropriate number of organoids, wash them several times with pre-cooled PBS to remove most of the Matrigel, and resuspend the organoids with the culture medium.

[0261] 2. Co-culture of CAR-T cells and tumor organoids: Using a culture medium (RPMI1640, 10% FBS, 1% penicillin-streptomycin, 2 mM glutamine, 5 ng / mL IL-4 and GM-CSF), suspend and adjust to an appropriate concentration. Add 5×10 5 100 μL of each CAR-T cell prepared in Example 1 to a 96-well plate, resuspend the organoid mixture, and add an equal amount of the sample to the 96-well plate to a total volume of 200 μL. Incubate at 37°C under 5% CO 2 2 conditions and observe continuously every day.

[0262] 3. Sample and count the cell numbers on DAY0 and DAY5 respectively. The results are as Figure 8 shown, indicating that compared with the conventional preparation of CAR-T cells from PB D9, the rapidly prepared CAR-T cells have stronger amplification ability. Among them, the amplification fold of CAR-T cells in the PB D9 group co-cultured with organoids on DAY5 / DAY0 is 1.97. The amplification fold of CAR-T cells in the JL 30h group co-cultured with organoids on DAY5 / DAY0 is 10.81.

Claims

1. A method for preparing cells expressing a chimeric antigen receptor, characterized in that, the preparation method comprises: (1) contacting cells with an activator for activation; (2) contacting the cells with a nucleic acid molecule encoding a CAR, the nucleic acid molecule encoding a CAR being on a non-viral vector, to introduce the nucleic acid molecule into the cells; (3) harvesting the cells; wherein, the method further satisfies at least any one or more of the following conditions (a)-(c): (a) Step (2) is carried out together with step (1) 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 (1), and (b) Step (3) is carried out no later than 48, 36, 30, 24, 18, 12, 6, 3, 2 or 1 hour after the start of step (2); (c) Step (3) is carried out no later than 72, 60, 48, 36, 30, 24, 20, 18 or 12 hours after the start of step (1).

2. The preparation method according to claim 1, 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, saRNA, and the non-viral vector is LNP, LPX, VLP, inorganic nanoparticles or exosomes.

3. The preparation method according to claim 1, 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 in step (2), the cells 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 the PB transposon system, the BZ transposon system or the JL transposon system.

4. The preparation method according to claim 1, characterized in that, the introduction is carried out by electroporation.

5. The preparation method according to any one of claims 1-4, characterized in that, the activator is an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates co-stimulatory molecules on the cell surface; Preferably, the agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3, more preferably a CD3 antibody. Preferably, the agent that stimulates costimulatory molecules is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof, more preferably a CD28 or 4-1BB antibody.

6. The preparation method according to any one of claims 1-5, wherein, the cells in step (3) further satisfy at least one of the following conditions ①-⑤: ① Compared with cells prepared by other similar methods, the cells in step (3) show a higher percentage (e.g., at least 0.1%, 1%, 5%, 10%, 15%, 20% or higher) of CAR-expressing naive cells (e.g., CAR-expressing naive T cells, such as CD3+CD45RO-CCR7+ T cells expressing CAR), in the other similar methods, step (3) is carried out more than 72 hours after the start of step (i) (e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (1)); ② Compared with the percentage of stem cell memory T cells (e.g., CD45RO+CCR7+CD95+ T cells) in the cells at the start of step (1), the percentage of stem cell memory T cells (e.g., CD45RO+CCR7+CD95+ T cells) in the cells in step (3) increases; ③ Compared with cells prepared by other similar methods, the percentage of CAR-expressing stem cell memory T cells (e.g., CD3+CD45RO+CCR7+CD95+ T cells expressing CAR) in the cells in step (3) is higher (e.g., at least 1%, 5%, 10%, 15%, 20%, 30% or higher), in the other similar methods, step (3) is carried out more than 72 hours after the start of step (1) (e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i)); ④ Compared with cells prepared by other similar methods, the percentage of CAR-expressing CD4+ T cells in the cells in step (3) is higher (e.g., at least 10%, 15%, 20%, 30%, 40%, 50% or higher), in the other similar methods, step (3) is carried out more than 72 hours after the start of step (1) (e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i)); ⑤ Compared with cells prepared by other similar methods, the cells in step (3) have higher expansion ability (e.g., can expand 3-fold, 5-fold, 10-fold or higher at day 5, 10 or 15 in organoids), in the other similar methods, step (3) is carried out more than 72 hours after the start of step (1) (e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i)).

7. The preparation method according to any one of claims 1-6, wherein, in step (2), after introducing the nucleic acid molecule into the cells, there is no step of culturing the cells; Or in step (2), after introducing the nucleic acid molecule into the cell, it further includes a step of culturing the cell, and the time for culturing the cell is not longer than 24, 18, 13, 10, 6, 3, 2 or 1 hour.

8. The preparation method according to any one of claims 1-7, characterized in that steps (1) and (2) are carried out in a cell culture medium containing IL-2, IL-15, IL-21, IL-7, IL-6, LSD1 inhibitor, MALT1 inhibitor or a combination thereof; preferably, the cell culture medium is a serum-free medium.

9. The preparation method according to any one of claims 1-8, characterized in that before step (1), it further includes step (4): obtaining a fresh or cryopreserved leukapheresis product from an entity; preferably, step (4) further includes separating T cells from the fresh or cryopreserved leukapheresis product; more preferably, step (4) further includes separating CD3+, CD4+ and / or CD8+ T cells from the fresh or cryopreserved leukapheresis product.

10. The preparation method according to claim 9, characterized in that step (3) is not later than 72 hours after the start of step (4) (for example, not later than 6, 12, 24, 26, 28, 30, 36, 40, 48 or 72 hours after the start of step (4)).

11. The preparation method according to any one of claims 1-10, characterized in that the preparation method is carried out in a closed system.

12. The preparation method according to any one of claims 1-11, characterized in that 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.

13. A cell expressing a chimeric antigen receptor prepared by the preparation method according to any one of claims 1-12.

14. Use of the cell expressing a chimeric antigen receptor according to claim 13 in the preparation of a medicament for treating and / or preventing malignant tumors.

Images