A method of making a chimeric antigen receptor-expressing cell

Abstract

Provided is a method for preparing a chimeric antigen receptor-expressing cell, comprising: (1) contacting a cell with an activator for activation; (2) contacting the cell with a nucleic acid molecule encoding a CAR, the nucleic acid molecule encoding the CAR being on a non-viral vector, to obtain a cell comprising the nucleic acid molecule; and (3) harvesting the cell. Step (3) is performed no later than 72 hours after the start of step (1). Compared with cells prepared by other similar methods, the cells of step (3) show a higher CAR positivity rate, a higher proportion of undifferentiated cell phenotype, a lower proportion of exhausted cells, and stronger expansion capacity.

Description

A method for preparing cells expressing chimeric antigen receptor Technical Field

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

[0002] Chimeric antigen receptors (CARs) are genetically engineered receptors that typically consist of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. These receptors can be transduced into various immune cells, such as T lymphocytes and NK cells. Several CAR-T cell products are currently on the market for the treatment of hematologic malignancies.

[0003] The main technical problems currently facing CAR-T products are: CAR-T is prepared using autologous cells, and the preparation process is cumbersome and time-consuming, resulting in high costs; CAR-T products mostly use viral vectors, and the products are limited by the output and quality of the 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, there will be obvious T cell exhaustion.

[0004] To address these issues, rapid CAR-T production is currently being explored. By shortening the production cycle from 14 days to 2 days or less, this approach reduces T cell proliferation and differentiation, thereby increasing the potential for CAR-T cells to expand in the human body. Rapid CAR-T can significantly shorten the production cycle, reduce costs, and prolong the duration of CAR-T cell activity in the body. Novartis and Gracell already have rapid CAR-T production technologies based on viral vectors, but the use of non-viral vectors for rapid CAR-T cell production has not yet been reported.

[0005] Summary of the Invention

[0006] The object of the present invention is to provide a method for rapidly preparing cells expressing chimeric antigen receptors based on non-viral delivery technology.

[0007] A 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;

[0008] The method further satisfies at least any one of the following conditions (a) to (c):

[0009] (a) step (2) is performed together with step (1), or not later than 48, 36, 24, 20, 16, 12, 8, 5, 4, 3, 2 or 1 hour after the start of step (1);

[0010] (b) step (3) is performed no later than 48, 36, 30, 24, 18, 12, 6, 3, 2, or 1 hour after the start of step (2)

[0011] (c) step (3) is performed no later than 72, 60, 48, 36, 30, 24, 20, 18, 12, 6, 5, 4, 3 or 2 hours after the start of step (1);

[0012] In some embodiments, the cells from step (3) do not expand or expand by no more than 5%, 10%, 20%, 30%, 40%, 50%, or 100% compared to the cells at the beginning of step (1), as assessed by viable cell number.

[0013] In some embodiments, the nucleic acid molecule encoding CAR is DNA, and the non-viral vector is a plasmid vector.

[0014] In some embodiments, the nucleic acid molecule encoding CAR is RNA, such as mRNA, saRNA, and the non-viral vector is LNP, LPX, VLP, inorganic nanoparticles or exosomes.

[0015] In some embodiments, the non-viral vector is a plasmid vector containing a transposon, and the transposon contains a nucleic acid molecule encoding CAR, and the cell in step (2) is also contacted with a transposase or a nucleic acid molecule encoding a transposase.

[0016] The transposon and 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, Helaizer 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.

[0017] In one or more embodiments, the transposon system is a PB transposon system, a BZ transposon system, or a JL transposon system.

[0018] In some embodiments, the nucleic acid molecule encoding the transposase is DNA or RNA.

[0019] In some embodiments, cells are contacted with a transposase or a nucleic acid molecule encoding a transposase and the cells are transduced by electroporation.

[0020] In some embodiments, the introducing is by electroporation.

[0021] In some embodiments, step (2) comprises: contacting the cell 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 repeat sequences located on both sides of the CAR gene expression cassette.

[0022] In some embodiments, the amino acid sequence of the JL transposase is shown in SEQ ID NO:5.

[0023] In some embodiments, the inverted terminal repeat sequence is set forth in SEQ ID NO: 6 (3' ITR) and SEQ ID NO: 7 (5' ITR).

[0024] In some embodiments, the DNA vector is an antimicrobial plasmid vector, which comprises a nucleotide sequence encoding an antitoxin protein and a replicon; the amino acid sequence of the antitoxin protein comprises the following sequence: (1) the amino acid sequence as shown in SEQ ID NO: 14, or an amino acid sequence having one or more mutations of E24D, 136V, or V43I compared with SEQ ID NO: 14; or (2) the 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, or N54K compared with SEQ ID NO: 17; the length of the replicon is ≤800 bp, preferably ≤600 bp or ≤300 bp.

[0025] In a preferred embodiment, the amino acid sequence of the antitoxin protein is shown in any one of SEQ ID NOs: 14-20.

[0026] In some embodiments, the replicon is R6K.

[0027] In some embodiments, the activating agent is an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a co-stimulatory molecule on the surface of a cell.

[0028] 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 an antibody (such as a single domain antibody, a peptibody, a Fab fragment, or a scFv), a small molecule, or a ligand (such as a naturally occurring ligand, a recombinant ligand, or a chimeric ligand).

[0029] In some embodiments, the agent that stimulates the CD3 / TCR complex is an anti-CD3 antibody.

[0030] 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, a peptibody, a Fab fragment, or a scFv), a small molecule, or a ligand (such as a naturally occurring ligand, a recombinant ligand, or a chimeric ligand).

[0031] In some embodiments, the agent that stimulates a co-stimulatory molecule is an agent that stimulates CD28, preferably an anti-CD28 antibody.

[0032] In some embodiments, the agent that stimulates the CD3 / TCR complex and the agent that stimulates the co-stimulatory molecule are CD3 / 28 magnetic beads, such as TransAct TM .

[0033] In some embodiments, the cells of step (3) exhibit a higher percentage (e.g., at least 0.1%, 1%, 5%, 10%, 15%, 20% or more) of CAR-expressing naive cells (e.g., CAR-expressing naive T cells, such as CAR-expressing CD3+CD45RO-CCR7+ T cells) compared to cells prepared by other similar methods, in which 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)).

[0034] 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 beginning of step (1).

[0035] In some embodiments, the percentage of CAR 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 more) than cells prepared by other similar methods, in which 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)).

[0036] 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 more) compared to cells prepared by other similar methods, in which 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)).

[0037] In some embodiments, the cells of step (3) have a greater expansion capacity (e.g., can expand 3-fold, 5-fold, 10-fold, or more at day 5, 10, or 15 in an organoid) than cells prepared by other similar methods, in which 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)).

[0038] In some embodiments, in step (2), after introducing the nucleic acid molecule into the cells, there is no step of culturing the cells.

[0039] In some embodiments, in step (2), the step of introducing the nucleic acid molecule into the cells further includes culturing the cells, and the time for culturing the cells is no longer than 24, 18, 13, 10, 6, 3, 2 or 1 hour.

[0040] In some embodiments, steps (1) and (2) are performed in a cell culture medium (e.g., serum-free medium) comprising IL-2, IL-15, IL-6, an LSD1 inhibitor, or a MALT1 inhibitor. In some embodiments, steps (1) and (2) are performed in a cell culture medium (e.g., serum-free medium) comprising IL-7, IL-21, or a combination thereof. In some embodiments, steps (1) and (2) are performed in a cell culture medium (e.g., serum-free medium) comprising 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 comprising serum replacement (SR).

[0041] In some embodiments, the method further comprises, before step (1), step (4): obtaining fresh or cryopreserved blood, leukapheresis product, or PBMC from the entity. In some embodiments, step (4) further comprises: isolating T cells from the fresh or cryopreserved blood, leukapheresis product, or PBMC. In some embodiments, step (4) further comprises: isolating, for example, CD3+, CD4+, and / or CD8+ T cells from the fresh or cryopreserved blood, leukapheresis product, or PBMC. The leukocytes include lymphocytes, basophils, neutrophils, eosinophils, and monocytes.

[0042] In some embodiments, step (3) is initiated no later than 72 hours after initiation of step (4) (e.g., no later than 6, 12, 24, 26, 28, 30, 36, 40, 48, or 72 hours after initiation of step (4).

[0043] In some embodiments, the method is performed in a closed system.

[0044] 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.

[0045] In some embodiments, the signal peptide is selected from the group consisting of a CD8 signal peptide, a CD28 signal peptide, a CD4 signal peptide, and a light chain signal peptide.

[0046] 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, leguman, 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, legumin, 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.

[0047] In some embodiments, the hinge region is selected from the extracellular hinge region of CD8, IgG1 Fc CH2CH3 hinge region, IgD hinge region, the extracellular hinge region of CD28, IgG4 Fc CH2CH3 hinge region and the extracellular hinge region of CD4.

[0048] In some embodiments, the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of 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.

[0049] In some embodiments, the intracellular costimulatory signaling domain comprises an intracellular domain derived from CD28, CD134 / OX40, CD137 / 4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T cell costimulator, and DNAX activating protein 10.

[0050] In some embodiments, the intracellular signaling domain is a CD3ζ intracellular signaling domain or an FcεRIγ intracellular signaling domain.

[0051] The present invention also provides cells expressing a chimeric antigen receptor prepared by the preparation method of any embodiment.

[0052] The present invention also provides the use of the CAR-expressing cells in the preparation of drugs for treating and / or preventing malignant tumors.

[0053] In some embodiments, the tumor is a solid cancer, for example, 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, 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 metastases thereof. In some embodiments, the cancer is a liquid cancer, for example, selected from the group consisting of chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphocytic 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 neoplasms, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), marginal Marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's 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, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, marginal lymph node Primary mediastinal (thymic) large B-cell lymphoma, pediatric 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 unclassifiable lymphoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 is a plasmid map of the P19V21 plasmid;

[0055] Figure 2 is a plasmid map of the MSLN CAR plasmid;

[0056] Figure 3 shows the cell viability of T cells transfected with different transposon systems after culturing for 24 h, 30 h, or 9 days;

[0057] Figure 4 shows the MSLN CAR+T positive rate after T cells were transfected with different transposon systems and cultured for 24 h, 30 h, or 9 days;

[0058] Figure 5 shows the cell differentiation subpopulations after T cells were transfected with different transposon systems and cultured for 24 hours, 30 hours or 9 days;

[0059] Figure 6 shows the ratio of CD4+CAR+ / CD8+CAR+ T cells after being transfected with different transposon systems and cultured for 24 h, 30 h, or 9 days;

[0060] Figure 7 shows T cell depletion after T cells were transfected with different transposon systems and cultured for 24 h, 30 h, or 9 days;

[0061] Figure 8 shows the expansion folds of CAR-T cells in organoid co-culture after T cells were transfected with different transposon systems and cultured for 24 hours, 30 hours, or 9 days;

[0062] Figure 9 is a map of the pCpGfree MCS-0637 empty vector miniplasmid, including the nucleotide sequence of antitoxin 0637 and the R6K replicon;

[0063] Figure 10 is a map of the pCpGfree MCS-43009 empty vector miniplasmid, including the nucleotide sequence of antitoxin 43009 and the R6K replicon.

[0064] Figure 11 shows the in vitro killing rate of 30h CAR-T and 6h CAR-T cells co-cultured with Raji cells. DETAILED DESCRIPTION

[0065] definition

[0066] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0067] The term "chimeric antigen receptor" (CAR) is an artificially modified receptor that can anchor specific molecules (such as antibodies) that recognize tumor cell surface antigens on immune cells (such as T cells), allowing immune cells to recognize tumor antigens or viral antigens and kill tumor cells or virus-infected cells. CAR usually contains 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 in sequence. Generally, polypeptides that bind to tumor cell membrane antigens can bind to membrane antigens widely expressed by tumor cells with moderate affinity. The polypeptide that binds to a tumor cell membrane antigen can be a natural polypeptide or an artificially synthesized polypeptide; preferably, the artificially synthesized polypeptide is a single-chain antibody, a single-domain antibody, a Fab fragment, a F(ab')2 fragment, and an Fv fragment.

[0068] The term "single-chain antibody" (scFv) refers to an antibody fragment that is composed of the amino acid sequence of the variable region of the antibody light chain (VL region) and the amino acid sequence of the variable region of the heavy chain (VH region) connected by a hinge and has the ability to bind to 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.

[0069] The terms "single-domain antibody," "heavy chain variable region domain of a heavy chain antibody," "VHH," "nanobody," and "single variable domain" are used interchangeably to 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. Typically, a single-domain antibody contains three CDRs and four FRs. A single-domain antibody is the smallest functional antigen-binding fragment. Typically, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only a single heavy chain variable region.

[0070] The term "co-stimulatory molecule" refers to a molecule present on the surface of antigen-presenting cells that can bind to the co-stimulatory molecule receptors on Th cells to generate a co-stimulatory signal. The proliferation of lymphocytes requires not only the binding of antigens but also the reception of co-stimulatory molecule signals. Co-stimulatory signals are transmitted to T cells mainly through the binding of co-stimulatory molecules CD80 and CD86 expressed on the surface of antigen-presenting cells to CD28 molecules on the surface of T cells. B cells can receive co-stimulatory signals through common pathogen components such as LPS, or through complement components, or through CD40L on the surface of activated antigen-specific Th cells.

[0071] The term "naive T cells" refers to T cells that have not experienced antigenic stimulation. In some embodiments, T cells that have not experienced antigenic stimulation are in the thymus and do not encounter their cognate antigens 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 isoforms. 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 assess the surface expression levels of markers.

[0072] The term "central memory T cells" refers to a subpopulation 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 beta, CCR7, and CD62L. In some embodiments, flow cytometry is used to assess the surface expression level of markers.

[0073] The terms "stem memory T cells," "stem cell memory T cells," "stem cell-like memory T cells," "memory stem cell T cells," "T memory stem cells," "T stem cell memory cells," or "TSCM cells" refer to a subpopulation of memory T cells with stem cell-like capabilities, e.g., the ability to self-renew and / or reconstitute memory and / or the multipotency of effector T cell subsets. In some embodiments, stem cell memory T cells express CD45RA, CD95, IL-2 receptor beta, CCR7, and CD62L. In some embodiments, flow cytometry is used to assess the surface expression levels of markers. In some embodiments, exemplary stem cell memory T cells are disclosed in Gattinoni et al., Nat Med. 2017 Jan 06; 23(1): 18-27, which is incorporated herein by reference in its entirety.

[0074] The term "transduction" refers to the process of transferring or introducing exogenous nucleic acid into a host cell.

[0075] The term "vector" is intended to include any element capable of transferring and / or transporting a nucleic acid composition to a host cell, into a host cell and / or to a specific location and / or compartment in a host cell, such as a plasmid, a phage, a transposon, a cosmid, a chromosome, an artificial chromosome (YAC or BAC), a virus, a viral capsid, a virion, etc.

[0076] The term "viral vector" refers to the use of the molecular mechanism of viruses to transmit their genomes into other cells for infection, mediating gene transfer. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, etc.

[0077] The term "non-viral vector" refers to the use of non-viral vectors to mediate gene transfer, including plasmid vectors, non-viral materials (such as LNP, LPX, VLP, inorganic nanoparticles, exosomes, etc.).

[0078] The first aspect of the present invention provides a method for preparing a cell expressing a chimeric antigen receptor, wherein the cell is, for example, a T cell. The method comprises: (1) contacting the cell with an activator for activation; (2) contacting the cell with a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule encoding the CAR is on a non-viral vector, so as to introduce the nucleic acid molecule into the cell; (3) harvesting the cell;

[0079] The method further satisfies at least one or more of the following conditions (a) to (d):

[0080] (a) step (2) is performed together with step (1) or not later than 48, 36, 24, 20, 16, 12, 8, 5, 4, 3, 2 or 1 hour after the start of step (1), and

[0081] (b) step (3) is performed no later than 48, 36, 30, 24, 18, 12, 6, 3, 2, or 1 hour after the start of step (2);

[0082] (c) step (3) is performed no later than 72, 60, 48, 36, 30, 24, 20, 18, 12, 6 or 2 hours after the start of step (1);

[0083] (d) The cells from step (3) do not expand or expand by no more than 5%, 10%, 20%, 30%, 40%, 50% or 100% compared to the cells at the beginning of step (2), as assessed by cell number.

[0084] The following is an exemplary description of the method herein.

[0085] Cell collection

[0086] The cells can be fresh or cryopreserved blood, leukapheresis products, or PBMCs obtained from an entity. The entity can be a healthy person or a tumor patient. In some embodiments, T cells are isolated (cell sorting) from fresh or cryopreserved blood, leukapheresis products, or PBMCs, for example, CD3+, CD4+, and / or CD8+ T cells are isolated. The white blood cells include lymphocytes, basophils, neutrophils, eosinophils, and monocytes.

[0087] Cell activation

[0088] Step (1) activates the cells by contacting them 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.

[0089] 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 an antibody (such as a single domain antibody, a peptibody, a Fab fragment, or a scFv), a small molecule, or a ligand (such as a naturally occurring ligand, a recombinant ligand, or a chimeric ligand).

[0090] In some embodiments, the agent that stimulates the CD3 / TCR complex is an anti-CD3 antibody.

[0091] 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, a peptibody, a Fab fragment, or a scFv), a small molecule, or a ligand (such as a naturally occurring ligand, a recombinant ligand, or a chimeric ligand).

[0092] In some embodiments, the agent that stimulates a co-stimulatory molecule is an agent that stimulates CD28, preferably an anti-CD28 antibody.

[0093] The stimulating agent may be present in the incubation mixture in the form of a solute, or it may be immobilized on a solid support. Solid supports that can be used to immobilize activators (e.g., antibodies) are well known in the art, such as magnetic beads or container walls. In some embodiments, the activators are CD3 antibodies and CD28 antibodies immobilized on magnetic beads; preferably, the activators are Miltenyi MACS GMP TransAct CD3 / 28 magnetic beads and / or CTS Dynabeads CD3 / 28. In some embodiments, the activators are CD3 antibodies, CD3 antibodies and CD28 antibodies, CD3 antibodies and 4-1BB antibodies, or CD3 antibodies and 4-1BBL antigens immobilized on the container wall; preferably, the container is a T75 flask.

[0094] In some embodiments, the activation time is 1-48 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 2-36, 3-36, 4-24 or 5-24 hours.

[0095] Plasmid vector

[0096] In some embodiments, the nucleic acid molecule encoding CAR is DNA, and the non-viral vector is a plasmid vector.

[0097] Vectors typically contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The 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 multilinker region for inserting a nucleic acid encoding an antibody to be expressed, and a selectable marker element. See, for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193.

[0098] When the nucleic acid molecule encoding CAR is DNA, the nucleic acid molecule is generally integrated into the cell genome by gene editing technology to stably express the CAR gene. Gene editing technology includes but is not limited to homologous recombination; gene editing technology based on zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR, such as those using Cas9 or cpf1), large-range nucleases, integrases, recombinases and transposases.

[0099] Transposons and transposases

[0100] In some embodiments, the non-viral vector is a plasmid vector containing a transposon comprising a nucleic acid molecule encoding a CAR.

[0101] DNA transposons can transpose via a non-replicative "cut and paste" mechanism. This requires recognition of two inverted terminal repeats (ITRs) by a transposase, which cleaves its target, releasing the DNA transposon from its donor template. After excision, the DNA transposon can then integrate into a recipient DNA cleaved by the same transposase.

[0102] The transposon and the corresponding transposase constitute a transposon system. According to the type of transposon system, a transposase and a transposon comprising a 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, the ITR sequences at both ends of the transposon DNA sequence have a cleavage site sequence for the transposase, and the cleavage site sequence is TA (nucleotide sequence).

[0103] In some embodiments, the cell is further contacted with a transposase or a nucleic acid molecule encoding a transposase in step (2). In some embodiments, the nucleic acid molecule encoding a transposase is DNA or RNA. In some embodiments, the cell is contacted with a plasmid vector containing a nucleic acid molecule encoding a transposase and a transposon in step (2). In some embodiments, the cell is contacted with a plasmid vector containing a nucleic acid molecule encoding a transposase and a plasmid vector containing a transposon in step (2).

[0104] The transposon system is selected from the group consisting of: Tol1 transposon system, Tol2 transposon system, Frog Prince transposon system, Minos transposon system, Hsmar1 transposon system, Helaizer 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.

[0105] The ZB transposon system is the ZB transposon system described in any embodiment of patent CN201510429987.3, and this application incorporates its entire contents herein by reference. A specific embodiment of a variant of the ZB transposon system is the BZ transposon system, which is the BZ transposon system described in any embodiment of patent CN202211150935.9, and this application incorporates its entire contents herein by reference. The BZ transposon system includes a BZ transposase and a BZ transposon comprising an ITR sequence recognizable by the BZ transposase.

[0106] The BZ transposase is a transposase having any one or more of the following mutations compared to SEQ ID NO: 1:

[0107] Q71R\H110R,

[0108] Q71R\Q79R\H110R,

[0109] G216A\Q71R\Q79R\H110R,

[0110] H208V\Q71R\Q79R\H110R、

[0111] H208V\G216A\Q71R\Q79R\H110R、

[0112] F21K\D22A\Q71R\H110R,

[0113] N005S\F21K / D22A\Q71R\Q79R\H110R、

[0114] K120S\N125L\Q71R\Q79R\H110R、

[0115] G216A\H208V\G189A\Q71R\Q79R\H110R、

[0116] G216A\H208V\K251T\Q71R\Q79R\H110R、

[0117] G216A\H208V\K251T\G189A\Q71R\Q79R\H110R、

[0118] G216A\H208V\K251T\G189A\Q138K\Q71R\Q79R\H110R,

[0119] G216A\H208V\K251T\G189A\Q138R\Q71R\Q79R\H110R,

[0120] G216A\H208V\K251T\G189A\K134A\Q71R\Q79R\H110R、

[0121] G216A\H208V\K251T\G189A\Q138K\K134A\Q71R\Q79R\H110R、

[0122] G216A\H208V\K251T\G189A\Q138R\K134A\Q71R\Q79R\H110R、

[0123] G216A\H208V\K251T\G189A\Q138K\V144E\Q71R\Q79R\H110R、

[0124] G216A\H208V\K251T\G189A\Q138K\K137T\Q71R\Q79R\H110R、

[0125] G216A\Q71R\H110R、

[0126] H208V\Q71R\H110R、

[0127] H208V\G216A\Q71R\H110R、

[0128] G216A\H208V\G189A\Q71R\H110R、

[0129] G216A\H208V\K251T\Q71R\H110R、

[0130] G216A\H208V\K251T\G189A\Q71R\H110R、

[0131] G216A\H208V\K251T\G189A\Q138K\Q71R\H110R、

[0132] G216A\H208V\K251T\G189A\Q138R\Q71R\H110R、

[0133] G216A\H208V\K251T\G189A\K134A\Q71R\H110R、

[0134] G216A\H208V\K251T\G189A\Q138K\K134A\Q71R\H110R、

[0135] G216A\H208V\K251T\G189A\Q138R\K134A\Q71R\H110R,

[0136] G216A\H208V\K251T\G189A\Q138K\V144E\Q71R\H110R,

[0137] G216A\H208V\K251T\G189A\Q138K\Q71R\H110R,

[0138] G216A\H208V\K251T\G189A\Q138R\Q71R\H110R、

[0139] G216A\H208V\K251T\G189A\K134A\Q71R\H110R,

[0140] G216A\H208V\K251T\G189A\Q138K\K134A\Q71R\H110R,

[0141] G216A\H208V\K251T\G189A\Q138R\K134A\Q71R\H110R,

[0142] G216A\H208V\K251T\G189A\Q138K\V144E\Q71R\H110R,

[0143] G216A\H208V\K251T\G189A\Q138K\K137T\Q71R\H110R, or

[0144] N005S\F21K / D22A\Q71R\H110R.

[0145] G216A\H208V,

[0146] G216A\H208V\G189A、

[0147] G216A\H208V\K251T,

[0148] G216A\H208V\K251T\G189A、

[0149] G216A\H208V\K251T\G189A\Q138K、

[0150] G216A\H208V\K251T\G189A\Q138R、

[0151] G216A\H208V\K251T\G189A\K134A、

[0152] G216A\H208V\K251T\G189A\Q138K\K134A,

[0153] G216A\H208V\K251T\G189A\Q138R\K134A、

[0154] G216A\H208V\K251T\G189A\Q138K\V144E, or

[0155] G216A\H208V\K251T\G189A\Q138K\K137T.

[0156] Among them, the first group of mutations Q71R\H110R refers to the BZ transposase containing mutation sites Q71R and H110R compared with SEQ ID NO: 1, and the other groups of mutations are similar.

[0157] The BZ transposon comprises a nucleic acid molecule encoding a CAR and an ITR sequence recognizable by a BZ transposase located at both ends of the nucleic acid molecule encoding the CAR. The ITR sequence is as shown in SEQ ID NO: 2 or 3, or compared with SEQ ID NO: 2 or 3, wherein the CpG motif is mutated to TpG or CpA.

[0158] The Passer (PS) transposon system is the PS transposon system described in any embodiment of patent CN201910366530.0, the entire contents of which are incorporated herein by reference. A specific example of a variant of the PS transposon system is the JL transposon system, which is the JL transposon system described in any embodiment of CN202310081106.8, the entire contents of which are incorporated herein by reference.

[0159] In some embodiments, the JL transposon system includes a JL transposase and a JL transposon comprising an ITR sequence recognizable by the JL transposase.

[0160] In some embodiments, the JL transposase is a mutant transposase of the PS transposase shown in SEQ ID NO: 4, which has one or more of the following mutations compared to the PS transposase 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, E4 7K, TQ57-58RR, E32K\T57R\Q58R, T57R, T57K, Q58K, Q58R, S59A, M95L, Y46Q, A8S, T187K, I35V, N199H, N193S, T350S, Q22K, T368E, N21 3D, H24R, T150A, H165D, K55R, K73R, L228M, E335S, K159H, V359L, T129Q, H215K, R51K, A84L, Q69E, I284L, K45R, H215E, H215Q, I237V.

[0161] In some embodiments, the JL transposase is a transposase fused to a wild-type PS transposase or a mutant transposase containing the above-mentioned mutation, wherein 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 comprises 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 comprises an SV40 NLS, a C-myc NLS, a TAF1 NLS, a TP53 NLS, a STAT3 NLS, or any combination thereof.

[0162] The JL transposon comprises a nucleic acid molecule encoding CAR and ITR sequences recognizable by JL transposase located at both ends of the nucleic acid molecule encoding CAR. The ITR sequence is shown in any one of SEQ ID NOs: 6-13.

[0163] In some embodiments, the transposon system is a PB transposon system, a BZ transposon system, or a JL transposon system.

[0164] In some embodiments, the plasmid vector of the transposon includes but is not limited to conventional circular DNA plasmids, linear DNA plasmids, minicircular plasmids, nanoplasmids, Doggybone and other DNA forms that do not contain antibiotics or / and replicon DNA sequences. In some embodiments, the DNA vector is a DNA microcarrier, the DNA backbone sequence of the microcarrier does not contain an antibiotic expression cassette and is preferably limited to a length of 600bp or less, and / or does not contain a CpG DNA motif. In some embodiments, the DNA vector is an anti-microplasmid, that is, a microplasmid without an antibiotic resistance gene (microplasmid without an antibiotic expression cassette), also known as a tiny or tiniplasmid. The anti-microplasmid suitable for the present invention can be referred to patent application 202310072956., the entire contents of which are incorporated herein by reference.

[0165] In some embodiments, the antitoxin-free plasmid comprises a nucleotide sequence encoding an antitoxin protein and a replicon; the amino acid sequence of the antitoxin protein comprises the following sequence: (1) the amino acid sequence as shown in SEQ ID NO: 14, or an amino acid sequence having one or more mutations of E24D, I35V, V43I compared to SEQ ID NO: 14; or (2) the 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, N54K compared to SEQ ID NO: 17; the length of the replicon is ≤800 bp, preferably ≤600 bp or ≤300 bp.

[0166] In some embodiments, the amino acid sequence of the antitoxin protein is as shown in any one of SEQ ID NOs: 14-20.

[0167] In some embodiments, the replicon is selected from ColE1, ColE2, pMB1, pSC101, RSF, R6K, pUC57, RK2, and p15A; preferably R6K or pUC57.

[0168] In some embodiments, the length of the plasmid backbone of the microplasmid-free plasmid is ≤1000 bp, preferably ≤900 bp, ≤800 bp or ≤600 bp.

[0169] In some embodiments, the nucleotide sequence encoding the antitoxin protein does not contain a CpG motif. Preferably, the nucleotide sequence encoding the antitoxin protein is as shown in SEQ ID NO: 21 or 22.

[0170] In some embodiments, the nucleotide sequence of the replicon does not contain a CpG motif.

[0171] In a preferred embodiment, the backbone sequence of the microplasmid without antimicrobial activity is ≤600 bp in length, and the replicon is an R6K replicon without a CpG motif. The nucleotide sequence of the R6K replicon without a CpG motif is shown in SEQ ID NO: 23.

[0172] In some embodiments, the nucleotide sequence without the anti-microplasmid (empty vector) is shown in SEQ ID NO: 24 or 25; the map structure is shown in FIG9 or 10 .

[0173] Cell transduction

[0174] Step (2) contacting the cell with a nucleic acid molecule encoding CAR to introduce the nucleic acid molecule into the cell.

[0175] In some embodiments, the nucleic acid molecule encoding CAR is RNA, such as mRNA, saRNA, and the non-viral vector is LNP, LPX, VLP, inorganic nanoparticles or exosomes. The RNA molecule encoding CAR is transduced into cells via a non-viral vector and can be used for transient expression of CAR. In some embodiments, the RNA molecule encoding CAR can also be introduced into cells directly by electroporation without passing through a vector.

[0176] In some embodiments, the step (2) contacts the cell with a transposon plasmid vector, and a transposase or mRNA encoding a transposase, wherein the transposon plasmid vector comprises a CAR gene expression cassette and transposase-recognizable ITR sequences located at both ends of the CAR gene expression cassette. The CAR gene expression cassette may comprise gene functional elements such as a promoter, a nucleic acid molecule encoding CAR, and a polyA signal sequence.

[0177] In some embodiments, the step (2) contacts the cell with a plasmid vector of a transposon, wherein the plasmid vector of the transposon comprises a CAR gene expression cassette, an ITR sequence recognizable by a transposase at both ends of the CAR gene expression cassette, and a nucleic acid molecule encoding a transposase. In this case, the transposon and the nucleic acid molecule encoding the transposase are located on the same plasmid vector.

[0178] In some embodiments, step (2) contacts the cell with a plasmid vector of a transposon and a plasmid vector of a transposase, wherein the plasmid vector of the transposon comprises a CAR gene expression cassette and an ITR sequence recognizable by the transposase at both ends of the CAR gene expression cassette. The plasmid vector of the transposase comprises a transposase gene expression cassette. At this point, the transposon and the nucleic acid molecule encoding the transposase are respectively located on different plasmid vectors.

[0179] In some embodiments, step (2) comprises: contacting the cell 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 repeat sequences located on both sides of the CAR gene expression cassette.

[0180] In some embodiments, the amino acid sequence of the JL transposase is shown in SEQ ID NO:5.

[0181] In some embodiments, the inverted terminal repeat sequences are set forth in SEQ ID NO: 6 (3' ITR) and SEQ ID NO: 7 (5' ITR).

[0182] In some embodiments, the plasmid map of the DNA vector comprising the JL transposon is shown in FIG2 .

[0183] In some embodiments, in step (2), when the cell is contacted with the nucleic acid molecule encoding CAR, the nucleic acid molecule encoding CAR is introduced into the cell, and the introduction includes transfecting the cell by means of electroporation, microinjection, calcium phosphate precipitation, cationic polymers, dendrimers, liposomes, lipid nanoparticles (LNP), microparticle bombardment, fugene, direct acoustic loading, cell extrusion, optical transfection, protoplast fusion, impalefection, magnetic transfection, nuclear transfection or any combination thereof.

[0184] In some embodiments, the introduction comprises contacting the cell with mRNA encoding the transposase and a plasmid containing the transposon. Preferably, the mRNA is used at a dosage of 1×10 7 1-30 μg of cells, the dosage of the plasmid is per 1×10 7 The most preferred dosage of the mRNA is 0.1-5 μg per 1×10 7 The concentration of the plasmid used is 1-5 μg per 1×10 cells. 7 0.1-2μg cells.

[0185] In some embodiments, the cell is contacted with the nucleic acid molecule encoding the CAR no later than 48, 36, 24, 20, 16, 12, 8, 5, 4, 3, 2, or 1 hour after initial contact of the cell with the activator.

[0186] In some embodiments, the contacting is to add the transposon plasmid containing the nucleic acid molecule encoding CAR and the mRNA encoding the transposase to the culture medium of the cells and the activator after the activation in step (1) is completed, and then use electroporation to introduce the transposon plasmid containing the nucleic acid molecule encoding CAR and the mRNA encoding the transposase into the cells.

[0187] In some embodiments, after activation in step (1) is completed, the activator is removed from the culture medium, and then the transposon plasmid containing the nucleic acid molecule encoding CAR and the mRNA encoding the transposase are added to the culture medium containing the activated cells.

[0188] Unlike viral vectors, the electroporation can be completed in 1 hour or less, so the time for introducing the nucleic acid molecule into cells in the present invention is much shorter than that of viral vectors.

[0189] In some embodiments, the cells may express a therapeutic agent and / or contain a coding sequence for a therapeutic agent, and in step (2), the cells are further contacted with a nucleic acid molecule of the therapeutic agent to introduce the nucleic acid molecule of the therapeutic agent into the cells.

[0190] In some embodiments, the nucleic acid molecule of the therapeutic agent is also located on a plasmid vector of the transposon.

[0191] In some embodiments, the nucleic acid molecule of the therapeutic agent and the nucleic acid molecule encoding CAR are located in the same transposon plasmid vector. The gene expression cassette of the therapeutic agent and the gene expression cassette of CAR can be connected by a cleavable linker (e.g., 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 CAR are respectively located between 2 groups of ITRs.

[0192] In some embodiments, the nucleic acid molecule of the therapeutic agent and the nucleic acid molecule encoding CAR are located in different transposon plasmid vectors. The transposon plasmid vector containing the nucleic acid molecule of the therapeutic agent is similar to the transposon plasmid vector structure containing the nucleic acid molecule encoding CAR above, except that the gene expression cassette of CAR is replaced with the gene expression cassette of the therapeutic agent.

[0193] In some embodiments, the nucleic acid molecule of the therapeutic agent and the nucleic acid molecule encoding the CAR are located in different transposon plasmid vectors.

[0194] In some embodiments, the step (2) contacts the cell with a plasmid vector comprising a nucleic acid molecule of a therapeutic agent, a plasmid vector comprising a nucleic acid molecule encoding CAR, and a transposase or mRNA encoding a transposase to simultaneously introduce the nucleic acid molecule encoding CAR and the nucleic acid molecule of the therapeutic agent into the cell.

[0195] In some embodiments, the therapeutic agent is an antibody (eg, a single chain antibody, a single domain antibody, a bispecific antibody) or a cytokine.

[0196] In some embodiments, the therapeutic agent is an immune checkpoint inhibitor.

[0197] In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that targets any one or more of PD-1, LAG-3, TIM3, B7-H1, CD160, P1H, 2B4, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), TIGIT, CTLA-4, BTLA and LAIR1.

[0198] 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 contents of which are incorporated herein by reference.

[0199] In some embodiments, the sequence of the single-domain antibody targeting PD-1 is shown in any one of SEQ ID NOs: 26-29.

[0200] 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 contents of which are incorporated herein by reference.

[0201] In some embodiments, the sequence of the single-domain antibody targeting CTLA-4 is shown in SEQ ID NO: 30.

[0202] In some embodiments, the therapeutic agent is a bispecific antibody comprising a first domain targeting PD-1 and a second domain targeting CTLA4. In some embodiments, the bispecific antibody is a bispecific antibody as described in any embodiment of patent CN CN202310338674.1, the entire contents of which are incorporated herein by reference.

[0203] In some embodiments, the first functional region and the second functional region in the bispecific antibody are fused via a linker, and the linker is (GGSGG)p or (G4S)mGn, where m, n, and P are each independently a positive integer of 1-10.

[0204] In some embodiments, the bispecific antibody further contains an Fc region and / or a cmyc-his tag; for example, the Fc region is an IgG1, IgG2, IgG3, or IgG4 Fc region.

[0205] In some embodiments, the sequence of the bispecific antibody is shown in any one of SEQ ID NOs: 31-34.

[0206] cytokines

[0207] In some embodiments, steps (1) and / or (2) are performed in a cell culture medium (e.g., serum-free medium) comprising IL-2, IL-15, IL-6, an LSD1 inhibitor, or a MALT1 inhibitor. In some embodiments, steps (1) and (2) are performed in a cell culture medium (e.g., serum-free medium) comprising IL-7, IL-21, or a combination thereof. In some embodiments, steps (1) and / or (2) are performed in a cell culture medium (e.g., serum-free medium) comprising 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 comprising serum replacement (SR).

[0208] Additional Steps

[0209] After the nucleic acid molecules encoding CAR are introduced into the cells, the cells can be cultured for a period of time, or the cells can be harvested directly without culture. In some embodiments, after the nucleic acid molecules encoding CAR are introduced into the cells, the cell suspension is transferred to a new culture medium, which can be a serum-free culture medium, such as AIM-V culture medium + 5% SR.

[0210] In some embodiments, the culture time may be less than 24 hours, such as no longer than 24, 18, 13, 10, 6, 3, 2, or 1 hour. The cells may be harvested and prepared for storage or administration.

[0211] In some embodiments, cell collection is followed by a sorting step (to isolate T cells) and the sorting time is 1-5 hours, such as 1, 2, 3, 4 or 5 hours.

[0212] In some embodiments, the total time from sorting to harvesting of CAR-T cells is 72, 60, 48, 36, 30, 28, 24, 22, 20, 18, 12 or less hours. In an exemplary embodiment, the total time is 6 hours, 22 hours or 28 hours.

[0213] In some embodiments, the total time from contacting the cells with the activator to harvesting the CAR-T cells is 72, 60, 48, 36, 30, 25, 24, 20, 19, 18, 12, 6, 5, 4, 3 or 2 hours or less. In an exemplary embodiment, the total time is 4 hours, 19 hours or 25 hours.

[0214] In some embodiments, the method is performed in a closed system. In some embodiments, the entire process of sorting, activation, transduction, culturing (optional) and harvesting is performed in a closed system.

[0215] Cell phenotype

[0216] In some embodiments, the percentage of naive cells (e.g., naive T cells, such as CD45RO-CCR7+ T cells) in the cells from step (3) (i.e., the harvested cells) differs from the percentage of naive cells (e.g., naive T cells, such as CD45RO-CCR7+ cells) in the cells at the beginning of step (1) by no more than 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, or 12%.

[0217] In some embodiments, the cells of step (3) exhibit a higher percentage (e.g., at least 0.1%, 1%, 5%, 10%, 15%, 20% or more) of CAR-expressing naive cells (e.g., CAR-expressing naive T cells, such as CAR-expressing CD3+CD45RO-CCR7+ T cells) compared to cells prepared by other similar methods, in which 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)).

[0218] 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 beginning of step (1).

[0219] In some embodiments, the percentage of CAR 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 more) than cells prepared by other similar methods, in which 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)).

[0220] 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 more) compared to cells prepared by other similar methods, in which 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)).

[0221] In some embodiments, the cells of step (3) have a greater expansion capacity (e.g., can expand 3-fold, 5-fold, 10-fold, or more at day 5, 10, or 15 in an organoid) than cells prepared by other similar methods, in which 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)).

[0222] In some embodiments, step (3) is initiated no later than 72 hours after initiation of step (4) (e.g., no later than 6, 12, 24, 26, 28, 30, 36, 40, or 48 or 72 hours after initiation of step (4). In some embodiments, the cells of step (3) do not expand, or expand by no more than 5%, 10%, 20%, 30%, 40%, 50%, or 100%, compared to the cells at the end of step (4), e.g., as assessed by viable cell number.

[0223] Chimeric antigen receptor

[0224] 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.

[0225] In some embodiments, the signal peptide is selected from the group consisting of a CD8 signal peptide, a CD28 signal peptide, a CD4 signal peptide, and a light chain signal peptide.

[0226] 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, leguman, GD3, CD171, IL-11Ra, PS CA, 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, legumin, 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.

[0227] In some embodiments, the hinge region is selected from the extracellular hinge region of CD8, IgG1 Fc CH2CH3 hinge region, IgD hinge region, the extracellular hinge region of CD28, IgG4 Fc CH2CH3 hinge region and the extracellular hinge region of CD4.

[0228] In some embodiments, the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of 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.

[0229] In some embodiments, the intracellular costimulatory signaling domain comprises an intracellular domain derived from CD28, CD134 / OX40, CD137 / 4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T cell costimulator, and DNAX activating protein 10.

[0230] In some embodiments, the intracellular signaling domain is a CD3ζ intracellular signaling domain or an FcεRIγ intracellular signaling domain.

[0231] In some embodiments, the immune cell is a CAR-T cell targeting mesothelin. The structure of the CAR is as follows: from N-terminus to C-terminus, it contains a CD8α signal peptide, a mesothelin VHH 1444, a CD8α hinge region, a CD28 transmembrane region and an intracellular costimulatory signaling region, and a CD3ζ intracellular signaling domain; the amino acid sequence of the mesothelin VHH 1444 is shown in SEQ ID NO: 36, and the amino acid sequence of the CAR is shown in SEQ ID NO: 37.

[0232] Nucleic acid construct encoding CAR

[0233] 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. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA can be single-stranded or double-stranded.

[0234] The polynucleotide sequences described herein can generally be obtained by PCR amplification. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates to obtain the relevant sequences. Long sequences often require two or more PCR amplifications, followed by splicing the fragments amplified from each amplification into the correct order.

[0235] 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 that are isolated from naturally occurring genes or modified to contain nucleic acid fragments in a manner that does not exist in nature. The term "nucleic acid molecule" mainly refers to a physical nucleic acid molecule, and the term "nucleic acid sequence" mainly refers to a nucleotide sequence on a nucleic acid molecule, but the two terms are 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, and one or more regulatory sequences operably linked to these sequences. The polynucleotide sequences described in the present invention can be manipulated in a variety of ways to ensure the expression of the CAR. Before the nucleic acid construct is inserted into the vector, the nucleic acid construct can be manipulated according to the different or required expression vectors. The technology of using recombinant DNA methods to change polynucleotide sequences is known in the art.

[0236] 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 a gene encoding an extracellular or intracellular polypeptide that is 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, an untranslated region of an mRNA that is important for host cell translation. 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.

[0237] In certain embodiments, the nucleic acid construct is a vector. The term "vector" can transfer a gene sequence to a target cell. Generally, "vector construct," "expression vector," and "gene transfer vector" mean any nucleic acid construct that can direct the expression of a gene of interest and can transfer a gene sequence to a target cell, which can be achieved by genome integration of the entire or partial vector, or by transient or heritable maintenance of the vector as an extrachromosomal element. Therefore, the term includes cloning vectors, expression vectors, and integration vectors. Typically, the polynucleotide sequence of the present invention is operably connected to a promoter, and the construct is incorporated into an expression vector to achieve expression of the polynucleotide sequence of the present invention. The vector can be suitable for replicating and integrating eukaryotic cells. Typical cloning vectors include transcription and translation 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 of which includes one or two or three expression cassettes described in any one of the embodiments herein.

[0238] The polynucleotide sequences of the present invention can be cloned into many types of vectors. For example, they 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, herpes viruses and lentiviruses.

[0239] Typically, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (eg, WO 01 / 96584; WO 01 / 29058; and US Pat. No. 6,326,193).

[0240] 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 transposition vector (or "transposon vector").

[0241] Therefore, in some embodiments, the nucleic acid construct comprises the coding sequence of CAR and 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, wherein the coding sequence of the chimeric antigen receptor and the coding sequence of the transposase are in the expression cassette.

[0242] Pharmaceutical composition

[0243] The present invention also provides cells expressing a chimeric antigen receptor prepared by the preparation method of any embodiment.

[0244] The present invention also provides the use of the CAR-expressing cells in the preparation of drugs for treating or preventing malignant tumors.

[0245] In some embodiments, the tumor is a solid cancer, for example, 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, 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 metastases thereof. In some embodiments, the cancer is a liquid cancer, for example, selected from the group consisting of chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphocytic 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 neoplasms, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), marginal Marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's 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, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, marginal lymph node Primary mediastinal (thymic) large B-cell lymphoma, pediatric 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 unclassifiable lymphoma.

[0246] The cells expressing CAR of the present invention can be administered alone or in combination with diluents and / or other components such as related cytokines or cell groups as a pharmaceutical composition. Briefly, the pharmaceutical composition of the present invention may include cells expressing CAR as described herein, in combination with one or more pharmaceutically or physiologically acceptable adjuvants (e.g., carriers, diluents, or excipients). Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0247] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease.

[0248] When an "immunologically effective amount," "anti-tumor effective amount," "tumor-inhibitory effective amount," or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. The cells can be administered using infusion techniques well known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical art by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0249] The administration of the subject composition can be carried out in any convenient way, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, intravenously or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the CAR-expressing cell composition of the present invention is preferably administered by intravenous injection. The composition of the cell expressing CAR can be directly injected into the tumor, lymph node or infection site.

[0250] The present invention will be described below by way 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, unless otherwise stated, conventional materials and methods in the art.

[0251] Example

[0252] Example 1: CAR-T cell preparation

[0253] 1. Obtain a single sample of white blood cells from the patient, and then use Miltenyi CD4 / CD8 magnetic beads for T cell sorting, 1×10 9 WBCs were added with 200 μL CD4 magnetic beads and 200 μL CD8 magnetic beads and incubated for 30 min. CD4+ T cells and CD8+ T cells were then screened using an XS sorting column.

[0254] 2. After sorting, take 1.05×10 8 Transfer the cell suspension to a flask coated with Miltenyi MACS GMP TransAct CD3 / 28 magnetic beads, add culture medium (AIM-V + 5% SR) to 30 mL / flask, add IL-7 & IL-15 to a final concentration of 25 ng / mL & 25 ng / mL, and incubate at 37°C, 5% CO2 for 1 to 48 hours.

[0255] 3. Group 1 PB D9 is T cells taken after 48 hours of activation, 1*10 7 To each cell group, 320 μg / mL of piggybac mRNA and 84 μg / mL of plasmid P19V21 expressing the MSLN CAR sequence were added. The mixture was transferred to an electroporation cuvette and placed in a Lonza Nucleofactor 4D or Maxcyte electroporator, using the FI-115 or Resting T / Expand T4 program. The electroporated cell suspension was transferred to a T75 culture flask (cultured in AIM-V medium containing 5% SR), mixed thoroughly, and incubated at 37°C, 5% CO2 for 9 days. Cell growth was then observed. The amino acid sequence of piggybac is shown in SEQ ID NO: 39. The plasmid map of plasmid P19V21 is shown in Figure 1, and the sequence is shown in SEQ ID NO: 35. The structure of MSLN CAR is as follows: from N-terminus to C-terminus, it contains CD8α signal peptide, mesothelin VHH No. 1444, CD8α hinge region, CD28 transmembrane region and intracellular co-stimulatory signal region, and CD3ζ intracellular signal domain; the amino acid sequence of mesothelin VHH No. 1444 is shown in SEQ ID NO: 36, and the amino acid sequence of MSLN CAR is shown in SEQ ID NO: 37.

[0256] 4. T cells were collected from group 2 (PB 24h) and group 4 (PB 30h) after activation for 5 and 24h, respectively. 1*10 7For each cell / group, add 320 μg / mL of piggybac mRNA and 84 μg / mL of plasmid P19V21 expressing the MSLN CAR sequence. Transfer the mixture to an electroporation cuvette and place it in a Lonza Nucleofactor 4D or Maxcyte electroporator, using the FI-115 or Resting T / Expand T4 program. Transfer the electroporated cell suspension to a T75 culture flask (using AIM-V medium containing 5% SR), mix thoroughly, and incubate at 37°C, 5% CO2 for no more than 24 hours before harvesting the CAR-T cells.

[0257] Among them, the total preparation cycle of the product in Group 2 is 24 hours, including 3 hours of sorting, 5 hours of activation, 1 hour of electroporation, 13 hours of culture and 2 hours of preparation; the total preparation cycle of the product in Group 4 is 30 hours, including 3 hours of sorting, 24 hours of activation, 1 hour of electroporation and 2 hours of preparation.

[0258] 5. Group 3 JL 24h and Group 5 JL 30h were T cells taken after activation for 5 and 24 hours, 1*10 7 For each cell group, add 5 μg / mL of plasmid expressing MSLN CAR and 2.5 μg / mL of JL enzyme mRNA. Transfer the mixture to an electroporation cuvette and place it in a Lonza Nucleofactor 4D or Maxcyte electroporator, using the FI-115 or Resting T / Expand T4 program. Transfer the electroporated cell suspension to a T75 culture flask (using AIM-V medium containing 5% SR), mix thoroughly, and incubate at 37°C in 5% CO2 for no more than 24 hours before harvesting the CAR-T cells. From cell sorting to CAR-T cell harvesting, the total product preparation cycle for Groups 3 and 5 was 24 hours and 30 hours, respectively, with the duration of each step being the same as for Groups 2 and 4.

[0259] The plasmid map of the MSLN CAR-expressing plasmid is shown in Figure 2, and the sequence is shown in SEQ ID NO: 38. In addition to the transposon, the plasmid backbone includes the R6K replicon and the nucleotide sequence of the antitoxin protein 0637. The nucleotide sequence of the R6K replicon is shown in SEQ ID NO: 23, and the nucleotide sequence of the antitoxin protein 0637 is shown in SEQ ID NO: 21. The structure of the MSLN CAR is the same as in Step 3, and the amino acid sequence of the enzyme is shown in SEQ ID NO: 5.

[0260] 6. Cell viability and cell density were measured using an NC-200 counter after the five culture groups. The cell viability results are shown in Figure 3. All five groups exhibited viabilities exceeding 70%, meeting product quality standards. The 30-hour process produced the highest viability of CAR-T cells (97.4% and 95.1%).

[0261] Example 2: CAR-T positive rate detection

[0262] The cells harvested from the above five groups were tested for CAR+T positive rates. The specific steps are as follows:

[0263] 1. Dissolve 1444-Fc-biotin (i.e., a biotin-labeled fusion protein of VHH 1444 and IgG4 Fc, prepared as described in Example 1 of CN111381020A) and PE-stretavidin (purchased from Shanghai GenScript Biotechnology Co., Ltd.) in PBS to prepare a 100× stock solution at a concentration of 10.0 mg / mL. Dilute the solution 100-fold with PBS before use to obtain a PE-fluorescein-labeled 1444-Fc dilution.

[0264] 2. Take 1×10 of each CAR-T cell prepared in Example 1 6 The cells were centrifuged at 400 g for 5 min, the supernatant medium was discarded, and the cells were resuspended in 1 mL of fresh medium. 1, 2, and 5 μL of the PE-fluorescein-labeled 1444-Fc dilution prepared in step 1 were added to all cells, respectively, and the cells were incubated at 37°C for 1 h.

[0265] 3. Wash each CAR-T cell after incubation in step 2 three times with cold PBS. Resuspend the cells in 1 mL of cold PBS after each wash and centrifuge at 1000 rpm for 3 minutes. After three washes, measure the fluorescence intensity of the cells using flow cytometry, analyze the positive rate, and compare.

[0266] The results are shown in FIG4 , which show that the CAR-T rapidly prepared by the JL transposon system had the highest positive rate (76.11%, 78.34%).

[0267] Example 3: Detection of CAR-T cell differentiation phenotype

[0268] The cells from the five groups above were cultured and subjected to cell differentiation phenotype detection. The specific steps are as follows:

[0269] 1. Prepare 1X working solution by taking lysed red blood cell storage solution and PBS phosphate buffer. Centrifuge the five groups of CAR-T cells prepared in Example 1 at 400g for 5 minutes, discard the upper culture medium, and take 1×10 cells. 6To each tube, add 1 μl of Brilliant Violet 421™ anti-human CD45RO (purchased from Shanghai GenScript Biotechnology Co., Ltd.), 2 μl of PE-labeled CCR7 (purchased from Shanghai GenScript Biotechnology Co., Ltd.), anti-human CD95 (purchased from Shanghai GenScript Biotechnology Co., Ltd.), and 1 μl of APC, respectively, and incubate at 2-8°C in the dark for 15 minutes. To the CAR-T cell sample tube, add 1 μl of PE-stretavidin and incubate at 2-8°C in the dark for 15 minutes.

[0270] 2. Wash each CAR-T cell after incubation in step 2 three times with cold PBS. Resuspend the cells in 1 mL of cold PBS after each wash and centrifuge at 400g for 5 minutes. After three washes, measure the fluorescence intensity of the cells using flow cytometry to analyze the cell differentiation phenotype for comparison.

[0271] The results are shown in Figure 5. Compared with conventional CAR-T cell preparation using PB D9, the rapid CAR-T cell preparation significantly increased the number of poorly differentiated cell subsets (Tnaive: CD45RO-CCR7+ and Tscm: CD45RO+CCR7+CD95+ subsets). In the PB 24h group, the proportion of CD3+CAR+Tnaive cells was 11.34%, and the proportion of CD3+CAR+Tscm cells was 46.10%. In the JL 24h group, the proportion of CD3+CAR+Tnaive cells was 19.23%, and the proportion of CD3+CAR+Tscm cells was 25.41%. In the PB 30h group, the proportion of CD3+CAR+Tnaive cells was 0.16%, and the proportion of CD3+CAR+Tscm cells was 46.34%. In the JL 30h group, the proportion of CD3+CAR+Tnaive cells was 0.40%, and the proportion of CD3+CAR+Tscm cells was 43.60%. In the PB D9 group, the proportion of CD3+CAR+Tnaive cells was 0, and the proportion of CD3+CAR+Tscm cells was 17.58%.

[0272] Example 4: CD4+ and CD8+ ratios in CAR-T positive cells

[0273] The CD4+CAR+ / CD8+CAR+ T cell ratios of the five groups of cells after culture were detected. The specific steps are as follows:

[0274] 1. Prepare 1X working solution by taking lysed red blood cell storage solution and PBS phosphate buffer. Centrifuge each CAR-T cell prepared in Example 1 at 400g for 5 minutes, discard the upper culture medium, and take 1×10 cells. 6For each tube, 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.) and incubate at 2-8°C in the dark for 15 minutes. For the CAR-T cell sample tube, add 1 μl of PE-stretavidin and incubate at 2-8°C in the dark for 15 minutes.

[0275] 2. Wash each CAR-T cell after incubation in step 2 three times with cold PBS. Resuspend the cells in 1 mL of cold PBS after each wash and centrifuge at 400g for 5 minutes. After three washes, measure the fluorescence intensity of the cells using flow cytometry to analyze the cell differentiation phenotype for comparison.

[0276] The results are shown in Figure 6, demonstrating a significant increase in CD4+ T cell subsets in the rapid CAR-T preparation compared to conventional PB D9 preparation. The CD4+ T cell proportion in the PB D9 group was 8.92%. The CD4+ CAR+ T cell proportions in the PB 24h group, the JL 24h group, the JL 24h group, the PB 30h group, and the JL 30h group were 50.45% and 41.09%, respectively.

[0277] Example 5: Proportion of Exhausted Cells in CAR-T Cells

[0278] 1. Prepare 1X working solution by taking lysed red blood cell storage solution and PBS phosphate buffer. Centrifuge each CAR-T cell prepared in Example 1 at 400g for 5 minutes, discard the upper culture medium, and take 1×10 cells. 6 To each tube, 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 at 2-8°C in the dark for 15 minutes. To the CAR-T cell sample tube, add 1 μl of PE-stretavidin (purchased from BD Biosciences Co., Ltd.) and incubate at 2-8°C in the dark for 15 minutes.

[0279] 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 400g for 5 minutes. After three washes, the fluorescence intensity of the cells was detected by flow cytometry, and the cell differentiation phenotype was analyzed and compared. The results are shown in Figure 7, showing that compared with the conventional PB D9 preparation of CAR-T, the proportion of CD8+CAR+PD1+TOX+TIGIT+ exhausted cell subsets in the rapid preparation of CAR-T cells was significantly reduced. The results showed that the proportion of CD8+CAR+PD1+TOX+TIGIT+ cells in the PB D9 group was 46.68%, the proportion of CD8+CAR+PD1+TOX+TIGIT+ cells in the PB 24h group was 25.35%, the proportion of CD8+CAR+PD1+TOX+TIGIT+ cells in the JL 24h group was 30.3%, the proportion of CDS+CAR+PD1+TOX+TIGIT+ cells in the PB 30h group was 18.66%, and the proportion of CD8+CAR+PD1+TOX+TIGIT+ cells in the JL 30h group was 20.66%.

[0280] Example 6: Co-culture of CAR-T cells and organoids

[0281] Each CAR-T cell prepared in Example 1 was co-cultured with thymic mesothelioma organoids to detect the expansion ability of CAR-T cells. The preparation of thymic mesothelioma organoids was based on 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. The specific steps are as follows:

[0282] 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-chilled PBS to remove most of the matrix gel, and resuspend the organoids in culture medium.

[0283] 2. Co-culture of CAR-T cells and tumor organoids: Use culture medium (RPMI1640, 10% FBS, 1% double antibody, 2mM glutamine, 5ng / mL IL-4 and GM-CSF), suspend and adjust to the appropriate concentration, add 5*10 per well of 96-well plate. 5Resuspend the organoid mixture in 100 μL of each CAR-T cell prepared in Example 1 and add equal amounts to a 96-well plate to a total volume of 200 μL. Incubate at 37°C, 5% CO2, and observe daily.

[0284] 3. Cell counts were collected on Day 0 and Day 5. The results, shown in Figure 8, demonstrate that rapid-production CAR-T cells exhibited greater expansion compared to conventional PB D9-based CAR-T cell preparation. The expansion fold for CAR-T cells co-cultured with organoids on Day 5 / Day 0 was 1.97 in the PB D9 group. The expansion fold for CAR-T cells co-cultured with organoids on Day 5 / Day 0 was 10.81 in the JL 30h group.

[0285] Example 7: 6h CAR-T cell preparation and quality evaluation

[0286] 1. Obtain a single sample of white blood cells from the patient, and then use Miltenyi CD4 / CD8 magnetic beads for T cell sorting, 1×10 9 WBCs were added with 200 μL CD4 magnetic beads and 200 μL CD8 magnetic beads and incubated for 30 min, and then CD4+ T cells and CD5+ T cells were selected by XS sorting column.

[0287] 2. After sorting, take 1.05×10 8 Transfer the cell suspension to a flask coated with Miltenyi MACS GMP TransAct CD3 / 28 magnetic beads, add culture medium (AIM-V + 5% SR) to 30 mL / flask, add IL-7 & IL-15 to a final concentration of 25 ng / mL & 25 ng / mL, and incubate at 37°C, 5% CO2 for <1 h.

[0288] 3. Take T cells activated for 1 hour, 1*10 7 For each cell / group, add 10 μg / mL of plasmid P19V21 expressing the MSLN CAR sequence and 5 μg / mL of JL enzyme mRNA. Transfer the mixture to an electroporation cuvette and place it in a Lonza Nucleofactor 4D or Maxcyte electroporator, using the FI-115 or Resting T / Expand T4 program. Transfer the electroporated cell suspension to a T75 culture flask (using AIM-V medium containing 5% SR), mix thoroughly, and incubate at 37°C, 5% CO2 for 20 minutes to recover. Wash, harvest, and freeze the CAR-T cells. The total production cycle, from cell sorting to CAR-T cell harvesting, is 6 hours.

[0289] 4. The 6h CAR-T cells prepared above were tested for cell viability and cell density using an NC-200 counter. The cell viability was 94.1%, which met the quality standards of the product; the positive rate detection method was referred to Example 2, and the test result was 21.5%. After cryopreservation and recovery, the cells were cultured in a T75 culture flask (the culture medium was AIM-V culture medium containing 5% SR), mixed, and cultured at 37°C and 5% CO2 for 9 days. The amplification factor of DAY9 / DAY0 was 16.07, which has a high amplification potential. The above results show that the total production time can be shortened to 6h by the preparation method of the present invention.

[0290] Example 8: Specific tumor killing by 30h CAR-T and 6h CAR-T cells

[0291] 1. Target cell preparation: Take healthy Raji cells (passage 1-3) and centrifuge and wash them. Add appropriate amount of RPMI cell culture medium (containing 9% FBS) to adjust the Raji cell density to 1*10 6 cells / mL. Take 2mL of Raji cells in a 15mL centrifuge tube, add 2.5ul of DELFIA BATDA Reagent, mix well, and incubate in a 37±1℃ water bath for 30min. After washing, take 100ul of cells at a density of 1*10 5 Raji cells at a concentration of 10 cells / mL were plated in a V-bottom 96-well plate.

[0292] 2. Effector cell plating: Take 1*10 JL 30h CAR-T cells in Example 1 and 6h CAR-T cells in Example 7 6 -2*10 6 cells / mL were added to a 6-well plate / cell culture flask and placed in a 5±0.5% CO2 incubator at 37±1°C overnight. After sampling and counting, the volume of cell suspension required for the experiment was calculated based on the positive rate of effector cells and the formula. Target suspension volume = (initial effector-target ratio × 10 5 × total target volume) / (positive rate × viable cell density), starting effector-target ratio = 16:1. The starting effector-target ratio cell suspension was serially diluted 2-fold in AIM-V CTS cell culture medium (containing 5% SR) to a minimum effector-target ratio of 1:1. 100 μl of effector cells were sequentially added to the Raji cells in a V-bottom 96-well plate.

[0293] 3. Incubation: Mix the cell mixture using a multichannel pipette to ensure full contact between the effector cells and Raji cells. Place the mixed 96-well V-shaped plate in a 5±0.5% CO2 incubator at 37±1°C and incubate for 3-3.5 hours.

[0294] 4. Reading: After incubation, place the DELFIA Europium Solution in a dark place at room temperature. Centrifuge the entire plate at 500g for 5 minutes. Using a multichannel pipette, remove 20 μl of the supernatant and transfer it to a 96-well plate with a white flat bottom. Add 180 μl of DELFIA Europium Solution to each well, avoiding bubbles. (If bubbles occur, centrifuge briefly at 1500 rpm for 15 seconds.) Incubate the plate on a 96-well microplate mixer for 5 minutes. Within 60 minutes, read the fluorescence using the Multilabel Microplate Detection System in the time-resolved fluorescence DELFIA mode.

[0295] 5. Calculation of Results: % Effector Cell Killing Efficacy = (Fluorescence Value of Test Group - Autofluorescence Value) * 100 / (Maximum Fluorescence Value - Autofluorescence Value). Data results are rounded to one decimal place, where Autofluorescence Value = 100 - (Autofluorescence Value - Background Fluorescence Value) * 100 / (Maximum Fluorescence Value - Background Fluorescence Value). The acceptable level for the background value is ≥ 50%. As shown in Figure 10, the average killing rate of the three CAR-T cell-specific groups was 78.78% after 30 hours, and 81.9% after 6 hours.

Claims

1. A method for preparing a cell expressing a chimeric antigen receptor, characterized in that: The preparation method comprises: (1) contacting cells with an activator for activation; (2) contacting cells with a nucleic acid molecule encoding CAR, wherein the nucleic acid molecule encoding CAR is on a non-viral vector, so as to introduce the nucleic acid molecule into the cells; (3) harvesting the cells; The method further satisfies at least one or more of the following conditions (a) to (c): (a) step (2) is performed 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); (b) step (3) is performed 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 performed no later than 72, 60, 48, 36, 30, 24, 20, 18, 12, 6, 5, 4, 3 or 2 hours after the start of step (1).

2. The preparation method according to claim 1, characterized in that The nucleic acid molecule encoding CAR is DNA, and the non-viral vector is a plasmid vector; Or the nucleic acid molecule encoding 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, and the transposon contains a nucleic acid molecule encoding CAR. In step (2), the cell is also contacted with a transposase or a nucleic acid molecule encoding a transposase; The transposon and transposase belong to the same transposon system. Preferably, the transposon system is selected from the group consisting of: Tol1 transposon system, Tol2 transposon system, Frog Prince transposon system, Minos transposon system, Hsmar1 transposon system, Helaiser 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 variants or derivatives of the above transposon systems. More preferably, the transposon system is a PB transposon system, a BZ transposon system or a JL transposon system.

4. The preparation method according to claim 1, characterized in that: The introduction was performed by electroporation.

5. The preparation method according to any one of claims 1 to 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 surface of stimulating cells; 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 co-stimulatory 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 to 5, characterized in that: The cells in step (3) also satisfy at least any one of the following conditions ①-⑤: ① The cells of step (3) show a higher percentage (e.g., at least 0.1%, 1%, 5%, 10%, 15%, 20% or more) of CAR-expressing initial cells (e.g., CAR-expressing initial T cells, such as CAR-expressing CD3+CD45RO-CCR7+T cells) compared to cells prepared by other similar methods, wherein 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)); ② Compared with the percentage of stem cell memory T cells (e.g., CD45RO+CCR7+CD95+T cells) in the cells at the beginning of step (1), the percentage of stem cell memory T cells (e.g., CD45RO+CCR7+CD95+T cells) in the cells of step (3) increases; ③ Compared with cells prepared by other similar methods, the percentage of CAR stem cell memory T cells (e.g., CD3+CD45RO+CCR7+CD95+T cells expressing CAR) in the cells of step (3) is higher (e.g., at least 1%, 5%, 10%, 15%, 20%, 30% or more), in which 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)); ④ Compared with cells prepared by other similar methods, 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 more), in which 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)); ⑤ The cells in step (3) have a higher expansion capacity (for example, they can expand 3 times, 5 times, 10 times or more on day 5, 10 or 15 in the organoid) compared to cells prepared by other similar methods, in which step (3) is performed more than 72 hours after the start of step (1) (for example, 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 to 6, characterized in that: In the step (2), after the nucleic acid molecule is introduced into the cell, the step of culturing the cell is not included; Or in the step (2), after introducing the nucleic acid molecule into the cell, the step of culturing the cell is further included, and The cells are cultured for no longer than 24, 18, 13, 10, 6, 3, 2 or 1 hour.

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

9. The preparation method according to any one of claims 1 to 8, characterized in that: The method further comprises the step (4) before the step (1): obtaining fresh or cryopreserved blood, leukocyte apheresis product or PBMC from an entity; Preferably, step (4) further comprises isolating T cells from fresh or cryopreserved blood, leukapheresis products or PBMCs; More preferably, step (4) further comprises isolating CD3+, CD4+ and / or CD8+ T cells from fresh or cryopreserved blood, leukapheresis products or PBMCs.

10. The preparation method according to claim 9, characterized in that: Step (3) is performed no later than 72 hours after the start of step (4) (eg, no 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 to 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 to 11, characterized in that: 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.

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

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

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