A method of preparing a chimeric antigen receptor-expressing cell from a cryopreserved cell

Abstract

A method for preparing cells expressing chimeric antigen receptors from cryopreserved cells, the method comprising: (1) thawing cryopreserved cells; (2) activating cells by contacting them with an activator; (3) introducing the cells into the cells by contacting them with a nucleic acid molecule encoding a CAR, the nucleic acid molecule encoding the CAR on a non-viral vector; and (4) harvesting the cells. High-quality CAR-T cells were prepared from cryopreserved PBMCs using an electroporation process, and their performance was comparable to that of CAR-T cells prepared from fresh PBMCs, demonstrating that immunocellular therapy can be achieved through the cryopreservation of PBMCs.

Description

A method for preparing cells expressing chimeric antigen receptors by freezing cells

[0001] This application claims priority to Chinese patent application No. CN202311752578.8 filed on December 19, 2023, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] 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 from frozen cells. Background Art

[0003] 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.

[0004] Generally, CAR-T cells are derived from T cells isolated from a patient's peripheral blood mononuclear cells (PBMCs). These cells are then sorted, activated, and introduced with the CAR gene. The efficacy of CAR-T therapy depends largely on the quality of these T cells. Therefore, the quality of a patient's immune cells is closely related to treatment efficacy. Numerous studies have shown that the composition of a patient's immune cells differs significantly from that of healthy individuals. Healthy individuals may have a greater proportion of CD4+ T cells than patients, and exhibit greater expansion capacity and a wider variety of TCRs during CAR-T culture. TCRs play a crucial role in immune responses. T cells isolated from healthy individuals are less differentiated and better maintain their stem cell-like state in culture. To preserve the properties of T cells and maintain the therapeutic effects of CAR-T, extracting PBMCs from healthy individuals and cryopreserving them for future use is an effective approach.

[0005] There have been reports of using frozen PBMCs to generate CAR-T cells using lentiviral technology, but the production of CAR-T products remains challenging due to high cost, complexity, and significant safety concerns. Non-viral transduction is gaining increasing attention due to its low cost, ease of use, and efficiency comparable to or better than viral transduction. However, there are currently no reports of successful non-viral generation of CAR-T cells using frozen PBMCs.

[0006] Summary of the Invention

[0007] The present invention aims to provide a method for preparing cells expressing chimeric antigen receptors using cryopreserved (ie, cryopreserved) cells by a non-viral delivery technique.

[0008] The present invention provides a method for preparing cells expressing a chimeric antigen receptor from frozen cells, the method comprising: (1) thawing the frozen cells; (2) contacting the cells with an activator for activation; (3) contacting the cells with a nucleic acid molecule encoding a CAR, the nucleic acid molecule encoding the CAR being on a non-viral vector to introduce the nucleic acid molecule into the cells; (4) harvesting the cells. In some embodiments, the frozen cells are cryopreserved blood, leukocyte apheresis products, PBMCs, or T cells (e.g., CD3+, CD4+, and / or CD8+ T cells).

[0009] In some embodiments, the cell expressing the chimeric antigen receptor is a CAR-T cell.

[0010] In some embodiments, cryopreserved cells are revived using serum-free medium, which may contain serum or serum replacement, growth factors, etc., such as 2% FBS, IL-7, IL-15, etc. Cryopreserved cells may also be revived using a resuscitation solution without culture medium, such as a PBS solution containing 0.5% HSA and 2 mM EDTA.

[0011] In some embodiments, cryopreserved cells are thawed using a serum-free medium, which may contain one or more of an apoptosis protein inhibitor and an insulin-transferrin-selenium additive.

[0012] In some embodiments, the action pathway of the apoptosis protein inhibitor includes one or more of p-JAK2, p-STAT3, caspase-3, Bax, and ROCK.

[0013] In some embodiments, the apoptosis protein inhibitor is selected from one or more of kaempferol-3-O-rutinoside, Ac-DEVD-CHO, and RevitaCell.

[0014] In some embodiments, the cells in step (1) are cryopreserved blood, leukapheresis products, or PBMCs, and step (1) further comprises sorting the revived cells by antibody sorting or flow cytometry sorting, such as sorting using CD4 / CD8 magnetic beads. In some embodiments, the cells obtained by the sorting are CD3+ T cells.

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

[0016] In some embodiments, the activator is selected from the group consisting of a CD3 antibody, a CD3 antibody and a CD28 antibody, a CD3 antibody and a 4-1BB antibody, and a CD3 antibody and a 4-1BBL antigen.

[0017] In some embodiments, the activator is a CD3 antibody and a CD28 antibody immobilized on magnetic beads; preferably, the activator is Miltenyi MACS GMP TransAct CD3 / 28 magnetic beads and / or CTSDynabeads CD3 / 28.

[0018] In some embodiments, the final concentration of the activator is 1-20 μg / mL.

[0019] In some embodiments, the concentration ratio of the activator to the immune cells is 1-20 μg / mL: 2.45-2.8×10 8 immune cells, preferably 5-10 μg / mL: 2.5-2.6×10 8 immune cells.

[0020] 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.

[0021] 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, muthsp70-2, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4.

[0022] In some embodiments, the nucleic acid molecule encoding CAR is DNA, and the non-viral vector is a plasmid vector.

[0023] 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.

[0024] 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 (3) is also contacted with a transposase or a nucleic acid molecule encoding a transposase.

[0025] 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.

[0026] In some embodiments, the transposon system is a PB transposon system, a BZ transposon system, or a JL transposon system.

[0027] In some embodiments, the nucleic acid molecule encoding the transposase is DNA or RNA.

[0028] In some embodiments, cells are contacted with a transposase or a nucleic acid molecule encoding a transposase and the cells are transduced by electroporation.

[0029] In some embodiments, the introducing is performed by electroporation.

[0030] In some embodiments, step (3) 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.

[0031] In some embodiments, the amino acid sequence of the JL transposase is shown in SEQ ID NO:5.

[0032] 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).

[0033] 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, I36V, 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, N54K compared with SEQ ID NO: 17; the length of the replicon is ≤800 bp, preferably ≤600 bp or ≤300 bp.

[0034] In a preferred embodiment, the amino acid sequence of the antitoxin protein is shown in any one of SEQ ID NOs: 14-20.

[0035] In some embodiments, the replicon is R6K.

[0036] In some embodiments, the electroporation conditions are as follows: transferring the mixture containing nucleic acid, cells and electroporation solution into an electroporation cup, placing it in a Lonza Nucleofactor 4D or Maxcyte electroporator, and selecting the program numbered FI-115 or Resting T / Expand T4 for electroporation.

[0037] In some embodiments, the method of preparing cells expressing chimeric antigen receptors from frozen cells described in the present invention adopts a rapid CAR-T preparation process.

[0038] The method further satisfies at least any one of the following conditions (a)-(c):

[0039] (a) step (3) is performed together with step (2), or not later than 48, 36, 24, 20, 16, 12, 8, 5, 4, 3, 2 or 1 hour after the start of step (1);

[0040] (b) step (4) is performed no later than 48, 36, 30, 24, 18, 12, 6, 3, 2 or 1 hour after the start of step (3);

[0041] (c) step (4) is performed no later than 72, 60, 48, 36, 30, 24, 20, 18 or 12 hours after the start of step (2).

[0042] In some embodiments, the method of preparing cells expressing a chimeric antigen receptor from cryopreserved cells described herein employs a conventional CAR-T preparation process. The activation time is 12-84 hours, preferably 24-72 hours, and more preferably 24, 48, or 72 hours.

[0043] In some embodiments, the cells from step (4) 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 viable cell number.

[0044] In some embodiments, the method of preparing cells expressing a chimeric antigen receptor from cryopreserved cells of the present invention employs a conventional CAR-T preparation process, and the activation time is 12-84 hours, preferably 24-72 hours, and more preferably 24, 48, or 72 hours.

[0045] In some embodiments, the culture time is at least 3 days, preferably at least 5 days, more preferably 3-13 days.

[0046] In some embodiments, the culture medium is AIM-V medium with or without 5% serum or its substitute. Preferably, the culture medium also contains cytokines, such as IL-7 and / or IL-15. Preferably, the final concentration of IL-7 is 1-25 ng / mL, and the final concentration of IL-15 is 1-25 ng / mL. Preferably, the cytokines are added to the culture medium after 0-6 hours of culture.

[0047] The present invention also provides cells expressing a chimeric antigen receptor prepared by the preparation method of any embodiment.

[0048] The present invention also provides the use of the CAR-expressing cells in the preparation of drugs for treating and / or preventing malignant tumors.

[0049] 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

[0050] Figure 1 is a plasmid map of the P19V21 plasmid.

[0051] Figure 2 is a plasmid map of the MSLN CAR plasmid.

[0052] Figure 3 is a map of the pCpGfree MCS-0637 empty miniplasmid, including the nucleotide sequence of antitoxin 0637 and the R6K replicon.

[0053] Figure 4 shows the map of the pCpGfree MCS-43009 empty vector miniplasmid, including the nucleotide sequence of antitoxin 43009 and the R6K replicon.

[0054] Figures 5-7 illustrate the comparison of cell viability, cell phenotype, and T cell differentiation status between frozen-thawed PBMCs and fresh PBMCs (0M) using the PB transposon system for CAR-T cell production. Data are presented as mean values, with each point representing the data from one individual. Intergroup significance was tested using the Friedman test, with P values ​​≤ 0.05 considered significant. P values ​​are indicated at the top of the graphs. *P ≤ 0.05.

[0055] Figure 8 shows the process of preparing CAR-T using the PB transposon system.

[0056] Figures 9-13 compare cell viability, cell expansion, and the percentage of CD3+ cells, CD4+ to CD8+ T cells, and CAR+ cells in fresh and frozen PBMCs used in the CAR-T cell manufacturing process using the PB transposon system. Data are presented as mean values, with each point representing the data from one individual. Intergroup significance was tested using the Friedman test, with P values ​​≤ 0.05 considered significant. P values ​​are indicated at the top of the graphs. *P ≤ 0.05.

[0057] Figures 14 and 15 show a comparison of T cell differentiation and exhaustion properties of CAR-T cells generated using the PB transposon system and derived from frozen and fresh PBMCs. Data are presented as mean values, with each point representing the data from one individual. Intergroup significance was tested using the Friedman test, with P values ​​≤ 0.05 considered significant. P values ​​are indicated at the top of the graphs. *P ≤ 0.05.

[0058] Figure 16 shows a comparison of cytokine secretion (IL-13, IL-10, IL-6, IL-5, IL-4, IL-12, IFN-γ, and TNF-α) by CAR-T cells prepared using the PB transposon system and fresh and frozen PBMCs stimulated with SKOV3 cells. Data are presented as mean values, with each point representing the data for one individual. Intergroup significance was tested using the Friedman test, with P values ​​≤ 0.05 considered significant. P values ​​are indicated at the top of the graph. *P ≤ 0.05.

[0059] Figure 17 shows the cytotoxicity of CAR-T cells generated using the PB transposon system against SKOV3 cells over 24 hours. CAR-T cells prepared from fresh and frozen PBMCs at different action-target ratios of 4:1 (left) or 2:1 (right) are shown. Data are presented as mean values, with each point representing the data from one individual. Intergroup significance was tested using the Friedman test, with P values ​​≤ 0.05 considered significant. P values ​​are indicated at the top of the graph. *P ≤ 0.05.

[0060] FIG18 shows the cell viability of the rapid CAR-T prepared in Example 9.

[0061] FIG19 shows the changes in cell number fold at different stages of preparing rapid CAR-T in Example 9.

[0062] Figure 20 shows the PBMC phenotype and TCM phenotype before and after sorting of raw material cells for preparing rapid CAR-T in Example 9.

[0063] Figure 21 shows the cell phenotype during the continued culture process of the rapid CAR-T prepared in Example 9.

[0064] FIG22 shows the rapid CAR-TSKOV3 tumor cell incubation for 12 hours prepared in Example 9, and the killing of tumor cells by different effector targets.

[0065] Figure 23 is a map of the plasmid expressing the PD-1 antibody.

[0066] Figure 24 is a plasmid map of P20S23.

[0067] FIG25 shows the CAR-T cell expansion multiples during the preparation process of Example 10.

[0068] FIG26 shows the viability of CAR-T cells prepared in Example 10 on day 11.

[0069] FIG27 shows the stem cell phenotype of the CAR-T cells prepared in Example 10.

[0070] Figure 28 shows the CAR-T cell positive cells and CD4 / CD8 prepared in Example 10.

[0071] FIG29 shows the exhaustion phenotype of CAR-T cells prepared in Example 10. DETAILED DESCRIPTION

[0072] definition

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] The term "transduction" refers to the process of transferring or introducing exogenous nucleic acid into a host cell.

[0078] 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.

[0079] 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.

[0080] 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.).

[0081] The following is an exemplary description of the method herein.

[0082] A method for preparing cells expressing a chimeric antigen receptor from frozen cells, the method comprising: (1) thawing the frozen cells and sorting or not sorting the thawing cells; (2) contacting the cells with an activator for activation; (3) contacting the cells with a nucleic acid molecule encoding a CAR, the nucleic acid molecule encoding the CAR being on a non-viral vector, so as to introduce the nucleic acid molecule into the cells; and (4) harvesting the cells.

[0083] Cell cryopreservation

[0084] Cryopreservation is the process of freezing cells, where cells are obtained from fresh blood, leukocyte apheresis products, or PBMCs from a healthy individual or a cancer patient. After obtaining the cells, they are added to a freezing solution and cryopreserved by direct or programmed cooling.

[0085] The cryopreservation solution can use a combination of cryopreservation solutions known in the art, for example, the cryopreservation solution includes a diluent and a cryoprotectant. The diluent is selected from one or more of compound electrolyte injection, 0.9% NaCl injection, and sodium lactate Ringer's injection. The cryoprotectant is selected from one or more of dextran glucose injection, human albumin solution, dimethyl sulfoxide, ethylene glycol, trehalose, sucrose, and glucose. In a specific embodiment, the cryopreservation solution includes: 40-80% v / v compound electrolyte injection, 5-20% v / v dextran 40 glucose injection, 10-30% v / v human albumin solution, and 5-10% v / v dimethyl sulfoxide. The cryopreservation solution may include other components, such as vitamins, serum, etc. In one embodiment, the concentration of the vitamins is 1-20 mg / ml, preferably 1-15 mg / ml, and more preferably 5-10 mg / ml.

[0086] Cells can be frozen by directly cooling to -80℃ and then storing in a liquid nitrogen tank; or by cooling in sequence at 2-8℃, -20℃, and -80℃, and finally storing in a liquid nitrogen tank.

[0087] The cryopreservation time may be more than 3 months, such as more than 3 months, 6 months, 9 months, 12 months, 2 years, 3 years, 5 years or 10 years.

[0088] Cell recovery

[0089] The general procedure for cell thawing is to remove frozen cells, pre-thaw them, and then add cell thawing solution to the frozen cells. Serum-free medium can be used to thaw frozen cells. This medium may contain serum or serum replacements, growth factors, and other factors, such as 2% FBS, IL-7, and IL-15. Frozen cells can also be thawed using a medium-free thawing solution, such as PBS containing 0.5% HSA and 2 mM EDTA.

[0090] In addition, to promote cell recovery and increase cell viability and activity, other components may be added to the culture medium. For example, the resuscitation solution described in any embodiment of CN201810129440.5 or the culture medium described in any embodiment of CN202311000790.9 may be used. In some embodiments, cryopreserved cells are revived using serum-free culture medium, which may contain one or more of an apoptosis protein inhibitor and an insulin-transferrin-selenium supplement. The apoptosis protein inhibitor targets one or more of p-JAK2, p-STAT3, caspase-3, Bax, and ROCK. The apoptosis protein inhibitor is selected from one or more of kaempferol-3-O-rutinoside, Ac-DEVD-CHO, and RevitaCell. The concentration of kaempferol-3-O-rutinoside is 10 μg / mL to 100 μg / mL, preferably 50 μg / mL to 100 μg / mL. The concentration of Ac-DEVD-CHO is 10-100 μM, preferably 40-60 μM. The dilution ratio of RevitaCell is 50-250 times, preferably 100-200 times.

[0091] Cell sorting

[0092] The frozen cells can be cryopreserved blood, leukocyte apheresis products, PBMCs, T cells or T cells after sorting. The T cells can be CD3+, CD4+ and / or CD8+ T cells. Depending on the type of cells, a sorting or non-sorting step can be included. In some embodiments, T cells are sorted from cryopreserved blood, leukocyte apheresis products or PBMCs. Sorting is performed by antibody sorting or flow cytometry, for example, using CD4 / CD8 magnetic beads for sorting. In some embodiments, the cells obtained by the sorting are CD3+ T cells.

[0093] Cell activation

[0094] Herein, "activator" is used to activate immune cells, which helps to alleviate the cell damage in cell preparation and improve cell viability and survival ratio. In theory, any reagent known in the art that can be used for immune cell activation can be used. In the exemplary embodiment where immune cells are T cells (particularly CD3+ T cells), the activator includes one or more selected from the following: CD3 antibody, CD28 antibody, 4-1BB antibody, 4-1BBL antigen.

[0095] The activation process includes incubating immune cells under conditions where the activator and the cells are in contact. In the incubation mixture, the final concentration of the activator, the concentration of the immune cells, and the ratio of the two are not limited. For example, the activator can be coated on a solid phase carrier, and the final concentration of the coating solution can be 1-20 μg / ml, such as 1, 5, 10, 15 or 20 μg / ml. The concentration ratio of the activator to the immune cells can be 1-20 μg / ml: 2.45-2.8×10 8 The activation temperature is any temperature suitable for the growth of immune cells, preferably 27-45° C., more preferably 37° C. Typically, the activation is performed in an environment containing CO 2 , such as 5% CO 2 .

[0096] The culture medium required for activation incubation can be any commercial or homemade culture medium suitable for growing immune cells (e.g., T cells). In one or more embodiments, activation incubation is performed using AIM-V culture medium with or without 5% serum or its substitute. Preferably, the culture medium also contains cytokines, such as IL-7 and / or IL-15; preferably, the final concentration of IL-7 is 1-50 ng / mL, and the final concentration of IL-15 is 1-50 ng / mL.

[0097] The activator 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 activator is a CD3 antibody and a CD28 antibody immobilized on magnetic beads; preferably, the activator is Miltenyi MACS GMP TransAct CD3 / 28 magnetic beads and / or CTSDynabeads CD3 / 28. In some embodiments, the activator is a CD3 antibody, a CD3 antibody and a CD28 antibody, a CD3 antibody and a 4-1BB antibody, or a CD3 antibody and a 4-1BBL antigen immobilized on the container wall; preferably, the container is a T75 flask.

[0098] In the rapid CAR-T preparation process, the activation of step (2) can be performed simultaneously with step (3), or the activation time of step (2) is less than 48, 36, 24, 20, 16, 12, 8, 5, 4, 3, 2 or 1 hour.

[0099] In a common CAR-T preparation process, in a specific embodiment, the sorted T cells are activated by container-coated CD3 antibodies for 24-72 hours and then electroporated, preferably for 48 hours; the sorted T cells are activated by container-coated CD3 antibodies and CD28 antibodies for 24-72 hours and then electroporated, preferably for 48 hours; the sorted T cells are activated by container-coated CD3 antibodies and 4-1BBL antigens for 24-72 hours and then electroporated, preferably for 48 hours; the sorted T cells are activated by container-coated CD3 antibodies and 4-1BB antibodies for 24-72 hours and then electroporated; the sorted T cells are activated by Miltenyi MACS GMP TransAct CD3 / 28 magnetic beads coated with CD3 antibodies and CD28 antibodies for 24-72 hours and then electroporated, preferably for 48-72 hours; the sorted T cells are activated by Dynabeads CD3 / 28 magnetic beads coated with CD3 antibodies and CD28 antibodies for 24-72 hours and then electroporated, preferably for 48-72 hours. In one or more embodiments, the final concentration of Miltenyi MACS GMP TransAct CD3 / 28 magnetic beads is 1 mL / 1×10 8 T cells, 4mL / 1×10 8 T cells, or 8 mL / 1 × 10 8 In one or more embodiments, the ratio of CTS Dynabeads CD3 / 28 magnetic beads to T cells is 1:1.

[0100] Chimeric antigen receptor

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] In some embodiments, the intracellular signaling domain is a CD3ζ intracellular signaling domain or an FcεRIγ intracellular signaling domain.

[0108] 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.

[0109] Plasmid vector

[0110] In some embodiments, the nucleic acid molecule encoding CAR is DNA, and the non-viral vector is a plasmid vector.

[0111] 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.

[0112] 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.

[0113] Transposons and transposases

[0114] In some embodiments, the non-viral vector is a plasmid vector containing a transposon comprising a nucleic acid molecule encoding a CAR.

[0115] 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.

[0116] 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).

[0117] 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).

[0118] 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.

[0119] 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.

[0120] The BZ transposase is a transposase having any one or more of the following mutations compared to SEQ ID NO: 1:

[0121] Q71R\H110R,

[0122] Q71R\Q79R\H110R,

[0123] G216A\Q71R\Q79R\H110R,

[0124] H208V\Q71R\Q79R\H110R、

[0125] H208V\G216A\Q71R\Q79R\H110R、

[0126] F21K\D22A\Q71R\H110R,

[0127] N005S\F21K / D22A\Q71R\Q79R\H110R、

[0128] K120S\N125L\Q71R\Q79R\H110R、

[0129] G216A\H208V\G189A\Q71R\Q79R\H110R、

[0130] G216A\H208V\K251T\Q71R\Q79R\H110R、

[0131] G216A\H208V\K251T\G189A\Q71R\Q79R\H110R、

[0132] G216A\H208V\K251T\G189A\Q138K\Q71R\Q79R\H110R,

[0133] G216A\H208V\K251T\G189A\Q138R\Q71R\Q79R\H110R,

[0134] G216A\H208V\K251T\G189A\K134A\Q71R\Q79R\H110R,

[0135] G216A\H208V\K251T\G189A\Q138K\K134A\Q71R\Q79R\H110R,

[0136] G216A\H208V\K251T\G189A\Q138R\K134A\Q71R\Q79R\H110R,

[0137] G216A\H208V\K251T\G189A\Q138K\V144E\Q71R\Q79R\H110R、

[0138] G216A\H208V\K251T\G189A\Q138K\K137T\Q71R\Q79R\H110R、

[0139] G216A\Q71R\H110R、

[0140] H208V\Q71R\H110R、

[0141] H208V\G216A\Q71R\H110R、

[0142] G216A\H208V\G189A\Q71R\H110R、

[0143] G216A\H208V\K251T\Q71R\H110R、

[0144] G216A\H208V\K251T\G189A\Q71R\H110R、

[0145] G216A\H208V\K251T\G189A\Q138K\Q71R\H110R、

[0146] G216A\H208V\K251T\G189A\Q138R\Q71R\H110R、

[0147] G216A\H208V\K251T\G189A\K134A\Q71R\H110R、

[0148] G216A\H208V\K251T\G189A\Q138K\K134A\Q71R\H110R、

[0149] G216A\H208V\K251T\G189A\Q138R\K134A\Q71R\H110R、

[0150] G216A\H208V\K251T\G189A\Q138K\V144E\Q71R\H110R、

[0151] G216A\H208V\K251T\G189A\Q138K\Q71R\H110R、

[0152] G216A\H208V\K251T\G189A\Q138R\Q71R\H110R、

[0153] G216A\H208V\K251T\G189A\K134A\Q71R\H110R、

[0154] G216A\H208V\K251T\G189A\Q138K\K134A\Q71R\H110R,

[0155] G216A\H208V\K251T\G189A\Q138R\K134A\Q71R\H110R,

[0156] G216A\H208V\K251T\G189A\Q138K\V144E\Q71R\H110R,

[0157] G216A\H208V\K251T\G189A\Q138K\K137T\Q71R\H110R, or

[0158] N005S\F21K / D22A\Q71R\H110R.

[0159] G216A\H208V,

[0160] G216A\H208V\G189A、

[0161] G216A\H208V\K251T,

[0162] G216A\H208V\K251T\G189A、

[0163] G216A\H208V\K251T\G189A\Q138K、

[0164] G216A\H208V\K251T\G189A\Q138R、

[0165] G216A\H208V\K251T\G189A\K134A、

[0166] G216A\H208V\K251T\G189A\Q138K\K134A,

[0167] G216A\H208V\K251T\G189A\Q138R\K134A、

[0168] G216A\H208V\K251T\G189A\Q138K\V144E, or

[0169] G216A\H208V\K251T\G189A\Q138K\K137T.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] In some embodiments, the JL transposon system includes a JL transposase and a JL transposon comprising an ITR sequence recognizable by the JL transposase.

[0174] 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\198K\T129K, R123H, Q136K, K16R , E47K, TQ57-58RR, E32K\T57R\Q58R, T57R, T57K, Q58K, Q58R, S59A, M95L, Y46Q, A8S, T187K, I35V, N199H, N193S, T350S, Q22K, T368 E. N213D, H24R, T150A, H165D, K55R, K73R, L228M, E335S, K159H, V359L, T129Q, H215K, R51K, A84L, Q69E, I284L, K45R, H215E, H215Q, I237V.

[0175] 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.

[0176] 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.

[0177] In some embodiments, the transposon system is a PB transposon system, a BZ transposon system, or a JL transposon system.

[0178] 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.

[0179] 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, 135V, or 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, or N54K compared to SEQ ID NO: 17; the length of the replicon is ≤800 bp, preferably ≤600 bp or ≤300 bp.

[0180] In some embodiments, the amino acid sequence of the antitoxin protein is as shown in any one of SEQ ID NOs: 14-20.

[0181] In some embodiments, the replicon is selected from ColE1, ColE2, pMB1, pSC101, RSF, R6K, pUC57, RK2, and p15A; preferably R6K or pUC57.

[0182] 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.

[0183] 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.

[0184] In some embodiments, the nucleotide sequence of the replicon does not contain a CpG motif.

[0185] 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.

[0186] In some embodiments, the nucleotide sequence of the microplasmid-free (empty vector) is shown in SEQ ID NO: 24 or 25; the map structure is shown in FIG3 or 4 .

[0187] Cell transduction

[0188] Step (3) contacting the cell with a nucleic acid molecule encoding CAR to introduce the nucleic acid molecule into the cell.

[0189] 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.

[0190] In some embodiments, the step (3) 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.

[0191] In some embodiments, the step (3) 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. At this point, the transposon and the nucleic acid molecule encoding the transposase are located on the same plasmid vector.

[0192] In some embodiments, the step (3) 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.

[0193] In some embodiments, step (3) 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.

[0194] In some embodiments, the amino acid sequence of the JL transposase is shown in SEQ ID NO:5.

[0195] 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).

[0196] In some embodiments, the plasmid map of the DNA vector comprising the JL transposon is shown in FIG2 .

[0197] In some embodiments, in step (3), 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.

[0198] In some embodiments, the introduction comprises contacting the cells 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 was 15 μg per 1×10 7 4-5μg cells.

[0199] 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.

[0200] 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 (2) 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.

[0201] In some embodiments, after activation in step (2) 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.

[0202] In some embodiments, the cells may express a therapeutic agent and / or contain a coding sequence for a therapeutic agent, and in step (3), 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.

[0203] In some embodiments, the nucleic acid molecule of the therapeutic agent is also located on a plasmid vector of the transposon.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] In some embodiments, the therapeutic agent is an antibody (eg, a single chain antibody, a single domain antibody, a bispecific antibody) or a cytokine.

[0209] In some embodiments, the therapeutic agent is an immune checkpoint inhibitor.

[0210] 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.

[0211] 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.

[0212] In some embodiments, the sequence of the single-domain antibody targeting PD-1 is shown in any one of SEQ ID NOs: 26-29.

[0213] 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.

[0214] In some embodiments, the sequence of the single-domain antibody targeting CTLA-4 is shown in SEQ ID NO: 30.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] In some embodiments, the sequence of the bispecific antibody is shown in any one of SEQ ID NOs: 31-34.

[0219] cytokines

[0220] In some embodiments, steps (2) and / or (3) 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 (2) and (3) 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 (2) and / or (3) 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).

[0221] Harvesting cells

[0222] After the nucleic acid molecule encoding CAR is introduced into the cells, a cell culture step may be further included before harvesting. The culture may use any conditions and culture medium suitable for the growth of immune cells known in the art. Exemplarily, the electroporated T cells of the present invention are cultured in AIM-V medium with or without 5% serum or its substitute at 37 ° C, 5% CO2. The culture medium may also contain cytokines, such as IL-7 and / or IL-15; preferably, the final concentration of IL-7 is 1-25 ng / mL, and the final concentration of IL-15 is 1-25 ng / mL; preferably, the cytokines are added to the culture medium after 0-6 hours of culture.

[0223] In a rapid CAR-T process, cells are cultured prior to harvesting without substantial expansion, and the culture time is less than 48, 36, 30, 24, 18, 12, 6, 3, 2, or 1 hour. In some embodiments, there is no culture step prior to harvesting cells.

[0224] Step (4) is performed no later than 48, 36, 30, 24, 18, 12, 6, 3, 2 or 1 hour after the start of step (3); or step (4) is performed no later than 72, 60, 48, 36, 30, 24, 20, 18 or 12 hours after the start of step (2).

[0225] In conventional CAR-T technology, the electroporated immune cells can be expanded and cultured for at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, or at least 13 days, preferably 3-13 days.

[0226] In some embodiments, the method is performed in a closed system. In some embodiments, the entire process of sorting, activation, transduction, culturing and harvesting is performed in a closed system.

[0227] Pharmaceutical composition

[0228] The present invention also provides cells expressing a chimeric antigen receptor prepared by the preparation method of any embodiment.

[0229] The present invention also provides the use of the CAR-expressing cells in the preparation of drugs for treating or preventing malignant tumors.

[0230] 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.

[0231] 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.

[0232] 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.

[0233] 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.

[0234] 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.

[0235] 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.

[0236] Experimental methods

[0237] Multicolor flow cytometry was used to determine the cell phenotypes of PBMCs and CAR-T cells. Anti-human CD45 (Biolegend), anti-human CD16 (Thermo), anti-human CD3 (Biolegend), and anti-human CD56 (Biolegend) antibodies were used to detect T cell, NK cell, and B cell phenotypes. Anti-human CD3 (BD ​​Biosciences), anti-human CD4 (Biolegend), and anti-human CD8 (Thermo) antibodies were used to assess the purity of enriched T cells. CAR transduction efficiency was assessed using biotin-binding protein from MSLN and streptavidin-PE (BD Biosciences) antibodies. To investigate the differentiation of CAR-T cells during culture, anti-human CD45RO (Biolegend) and anti-human CCR7 (Biolegend) antibodies were used. CAR-T cell exhaustion was assessed using anti-human PD-1 (BD Biosciences), anti-human LAG-3 (Biolegend), and anti-human TIM-3 (BD ​​Biosciences). The cells to be tested were washed in phosphate-buffered saline (PBS) and incubated with the respective antibodies at 4°C for 15 minutes. Subsequently, the cells were washed with 4 mL of PBS and resuspended in 300 μl of PBS. Flow cytometry analysis was performed using a CytoFLEX S instrument from Beckman. The collected data were analyzed using Kaluza analysis software.

[0238] Real-time cell analysis was used to detect the cytotoxicity of CAR-T cells. First, 1×10 cells were seeded per well in an E-96-cell plate (Agilent). 4 target cells. Subsequently, the plate was placed in an xCElligence RTCA SP instrument (Agilent) to monitor the growth of tumor cells. Once the cell index exceeded 1, the following steps were performed. Within 16-24 hours after CAR-T cell recovery, we considered two different effector-target ratios (E:T ratio: 4:1, 2:1) and CAR positivity rates. The corresponding number of positive cells was then inoculated into an E-96-cell plate previously inoculated with tumor cells. Subsequently, the plate was placed in an xCElligence RTCA SP instrument (Agilent) to monitor the cytotoxic effect. After 24 hours, the cytotoxicity data were analyzed.

[0239] For cytokine release assays, 1 × 10 4 Target cells were co-cultured with CAR-T cells at an E:T ratio of 4:1 for 24 hours. Detection of cytokines IL-5, IL-13, IL-2, IL-6, IL-10, IFN-γ, TNF-α, and IL-4 was performed using the LEGENDplex™ Multianalyte Flow Assay Kit manual.

[0240] Example

[0241] Example 1: Freezing, sorting, and recovery of T cells

[0242] 1. 50-60 mL of leukocyte aliquots were collected from four healthy donors using a Spectra Optia instrument and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation. The PBMCs were divided into four fractions. One fraction was immediately generated into CAR-T cells (0M), while the other fractions were cryopreserved in liquid nitrogen using a homemade freezing medium for 3 months (3M), 6 months (6M), and 12 months (12M) prior to CAR-T cell production.

[0243] The frozen cells were revived with EDTA / PBS (containing 5‰ HSA). T cells were sorted using Miltenyi CD4 / CD8 magnetic beads, 1×10 9 WBCs were added with 200μL CD4 magnetic beads and 200μL CD8 magnetic beads and incubated for 30 min, and then CD4+T and CD8+T cells were selected using LS sorting columns.

[0244] 2. After sorting, cells were plated at a density of 1E6 / mL in a culture flask containing culture medium (AIM-V + 5% SR), supplemented with IL-7 & IL-15 (final concentrations of 25 ng / mL & 25 ng / mL) and Miltenyi MACS GMP TransAct CD3 / 28 magnetic beads, and cultured at 37°C, 5% CO2 for 2 days for activation.

[0245] Example 2, Preparation of CAR-T by PB transposon system

[0246] Take the T cells activated in Example 1, 1*10 7 For each cell group, add 320 μg / mL of piggybac mRNA and 90 μg / mL of plasmid P19V21 expressing the MSLNCAR sequence. Transfer the mixture to a 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 supplemented with 5% SR, 25 ng / mL IL-7, and 25 ng / mL IL-15), mix thoroughly, and incubate at 37°C. Replenish the medium every 2-3 days to ensure optimal cell growth conditions. On day 11, wash the cells with saline, add cryopreservation solution, and store in liquid nitrogen.

[0247] The amino acid sequence of the piggybac enzyme is shown in SEQ ID NO: 39. The plasmid map of plasmid P19V21 is shown in Figure 1, and its sequence is shown in SEQ ID NO: 35. The structure of the MSLN CAR comprises, from N-terminus to C-terminus, a CD8α signal peptide, mesothelin VHH 1444, a CD8α hinge region, a CD28 transmembrane region and intracellular costimulatory signaling region, and a CD3ζ intracellular signaling domain. The amino acid sequence of mesothelin VHH 1444 is shown in SEQ ID NO: 36, and the amino acid sequence of the MSLN CAR is shown in SEQ ID NO: 37.

[0248] Example 3, JL transposon system preparation of CAR-T

[0249] Take the T cells activated in Example 1, 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. Exchange the medium every 2-3 days to ensure optimal cell growth conditions. On day 11, wash the cells with saline, add cryopreservation solution, and store in liquid nitrogen.

[0250] 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 Example 2, and the amino acid sequence of the JL enzyme is shown in SEQ ID NO: 5.

[0251] Example 4, JL transposon system preparation of rapid CAR-T

[0252] Take activated T cells, 1*10 7For 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. The total preparation time from cell sorting to CAR-T cell harvesting is 30 hours, including 3 hours of sorting, 24 hours of activation, 1 hour of electroporation, and 2 hours of preparation.

[0253] Example 5, PBMC cell viability and phenotype

[0254] The cell viability of PBMC is a key factor that directly affects the success rate of CAR-T cell manufacturing. The present invention compares the effects of different freezing times on PBMC cell viability. After fresh group (0M), freezing for 3 months (3M), freezing for 6 months (6M) and freezing for 12 months (12M), the survival rates of PBMC were 96.59%, 91.45%, 91.43% and 89.53%, respectively (Figure 5). The survival rate decreased slightly after freezing, but there was no significant difference in cell survival between frozen-thawed PBMCs compared with fresh PBMCs. In addition, the cell phenotypes of PBMCs after different freezing storage times were also compared. PBMC samples were subjected to multicolor flow cytometry analysis to determine the proportion of T cells, B cells, NK cells and the CD4 / CD8 ratio. The results showed that cryopreservation had no significant effect on the proportion of T cells, NK cells, B cells and the CD4 / CD8 ratio (Figure 6). Interestingly, the proportion of NK cells and B cells decreased slightly after freezing. This may be related to the cell state during flow cytometry analysis, but it does not affect the preparation of CAR-T cells obtained by modifying CD3+ T cells. Finally, the CD3+ T cell differentiation status of fresh and frozen-thawed PBMCs was detected by CD45RO and CCR7 staining. The results showed that the proportion of Tn (CD45RO-CCR7+) and Tem (CD45RO+CCR7-) in T cells increased significantly after 12M cryopreservation compared with fresh PBMCs (Figure 7). Studies have shown that Tn and Tem cells can enhance the activation ability, persistence and effector function of CAR-T cells, thereby improving the efficacy of CAR-T cell therapy. In summary, the cryopreservation process itself does not have a negative impact on PBMC cell viability and subsets.

[0255] Example 6, Viability, Expansion, and Phenotype of CAR-T Cells Prepared with the PB Transposon System

[0256] CAR-T cells are prepared from fresh or cryopreserved PBMCs by CD4 / CD8 magnetic bead separation, 48-hour TransAct TM Activation, and subsequent CAR vector electroporation, and cultured cells for 9 days (Figure 8). First, the present invention tested the effect of cryopreservation on cell viability. Compared with CAR-T cell cultures initiated with fresh PBMCs, frozen-thawed PBMCs initiated CAR-T cells showed no significant difference in cell viability (Figure 9). Previous studies have shown the effect of cryopreservation on cell proliferation. Therefore, we evaluated the proliferation levels of cryopreserved and fresh PBMCs on the first 2 days, day 0 and day 3, day 7, and day 11 of production. Our results showed that cryopreservation reduced the proliferation of non-virally prepared CAR-T cells, but there was no significant difference (Figure 10). Evaluation of the final product phenotype showed that there was no significant difference in average CD3 purity (Figure 11), average CD4:CD8 cell ratio (Figure 12), and average CAR-T transduction efficiency (Figure 13). Therefore, the study showed that the non-viral transposon electroporation process was successful from cryopreserved PBMCs for manufacturing CAR-T cells, and when juxtaposed with fresh PBMCs, there was no significant difference in cell viability, expansion, and cell phenotype.

[0257] Example 7, Differentiation and depletion characteristics of CAR-T cells prepared using the PB transposon system

[0258] The proportion of naive and memory T cells in CAR-T cell products is a key indicator for assessing their function. T cell differentiation status was investigated by staining cells with fluorescently labeled antibodies specific for CD45RO and CCR7. This analysis showed that the fractions of TEM (CD45RO+CCR7-) and TCM (CD45RO+CCR7+) changed little, while the fractions of Tn (CD45RO-CCR7+) and Teff (CD45RO-CCR7-) increased significantly (Figure 14). Further analyses were performed to determine whether freezing affected the persistence of CAR-T cell function. CAR-T cell exhaustion signals, including PD1+, TIM3+, and LAG3+, were examined. The results showed that the proportions of PD1+ and TIM3+ cells were largely unaffected by freezing. Increased LAG3+ levels were observed in CAR-T cells prepared from frozen PBMCs, but the difference was not significant compared with the fresh group (Figure 15). These findings suggest that freezing does not accelerate CAR-T cell exhaustion. Therefore, all these results may indicate that CAR-T preparations from frozen and fresh PBMCs function similarly.

[0259] Example 8, Efficacy and Safety of CAR-T Prepared by PB Transposon System

[0260] The effectiveness and safety of CAR-T cell products are intrinsically linked to the secretion of cytokines. Excessive secretion of cytokines after tumor cell stimulation may trigger a cytokine storm. Therefore, the present invention compared the levels of certain cytokines in CAR-T cell products, including IL-13, IL-10, IL-6, IL-5, IL-4, IL-12, IFN-γ, and TNF-α. The results showed that in the cryopreserved PBMC group, the levels of IL-13, IL-10, IL-4, IFN-γ, and TNF-α were significantly reduced, while the levels of IL-6, IL-5, and IL-12 remained essentially unchanged (Figure 16). Next, the ability of CAR-T cells to kill tumor cells was investigated. CAR-T cells were incubated with SKOV3 tumor cells, and the subsequent growth of tumor cells was monitored by real-time cell analysis. The results showed that there was no significant difference in the cytotoxicity of CAR-T cells from fresh or frozen PBMCs at effector-target ratios of 4:1 and 2:1 (Figure 17). Therefore, all the results showed that CAR-T cells manufactured with frozen PBMCs had no side effects on CAR-T cell cytotoxicity.

[0261] Example 9: Preparation of Rapid CAR-T Using JL Transposon System with Different Cryopreserved Materials

[0262] Fresh blood (FL), fresh PBMC (FP), cryopreserved blood (CL), and cryopreserved PBMC (CP) from donor MS-19 were used as raw materials to verify the effectiveness of fresh and cryopreserved cells in preparing CAR-T cells. Cryopreserved blood and cryopreserved PBMC were resuscitated using EDTA / PBS (containing 5‰ HSA). T cells were sorted using Miltenyi CD4 / CD8 magnetic beads, and 1×10 9 WBCs were added with 200μL CD4 magnetic beads and 200μL CD8 magnetic beads and incubated for 30 min, and then CD4+T and CD8+T cells were selected using LS sorting columns.

[0263] After sorting, cells were plated at a density of 1E6 / mL in a culture flask containing culture medium (AIM-V + 5% SR), supplemented with IL-7 & IL-15 (final concentration of 25 ng / mL & 25 ng / mL) and Miltenyi MACS GMP TransAct CD3 / 28 magnetic beads, and cultured at 37°C, 5% CO2 for 1 day.

[0264] After activation, 1×10 7For 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. Wash the electroporated cell suspension and add freezing medium to harvest the CAR-T cells. The rapidly prepared CAR-T cells were named FL-Fa, FP-Fa, CL-Fa, and CP-Fa.

[0265] The harvested CAR-T cells were cultured for 9 days (D1-D9) in AIM-V medium containing 5% SR for the performance characterization of CAR-T.

[0266] The cell viability results are shown in Figure 18. On the first day of culture, the viability of fresh blood and fresh PBMCs was above 90%, while that of frozen cells was less than 90%. However, as the culture progressed, the difference between the fresh and frozen groups became smaller, with viabilities around 70% on day 3. On day 5 (when cell killing was assessed), the cell viability was slightly below 80%, greater than 90% on day 7, and greater than 95% on day 9.

[0267] The results of CAR-T cell expansion are shown in Figure 19, which shows the changes in cell number multiplication at different stages. The figure shows that the peak of CAR-T cell expansion is late, and the cells have not reached the expansion peak on day 9 (the peak expansion of the conventional process is on day 7). Moreover, from day 3 to day 9, the expansion multiplication rate is well maintained, indicating that the cells have good sustained expansion capacity. In addition, the difference between the fresh group and the frozen group is small, indicating that the frozen cells have the potential for rapid CAR-T preparation.

[0268] During continued culture, cell phenotypes were detected by flow cytometry. The PBMC phenotype and TCM phenotype before and after raw material cell sorting are shown in Figure 20. The figure shows fresh cells or frozen cells. There is no significant difference in cell phenotype before and after sorting. During continued culture, the phenotypes of positive T cells are shown in Figure 21. The results show that the positive rates among the groups are comparable and the change trends are consistent. The positive rate is high on the third day; the positive rate tends to be stable on the fifth day and thereafter; and the CD8 / CD4 ratio is higher than that of the conventional process, that is, the proportion of killer T cells is higher.

[0269] On the fifth day of culture, CAR-T cells were incubated with SKOV3 tumor cells for 12 hours, and subsequent tumor cell growth was monitored by real-time cell analysis using different effector-target ratios (4:1, 2:1, 1:1, and 1:2). The results are shown in Figure 22. The figure shows that there was no significant difference in tumor killing between the groups using fresh or frozen cells.

[0270] In summary, there is little difference in the rapid preparation of CAR-T using fresh blood (FL), fresh PBMC (FP), cryopreserved blood (CL), and cryopreserved PBMC (CP) as raw materials. Therefore, cryopreserved cells can be used for rapid preparation of CAR-T products.

[0271] Example 10: Comparison of CAR-T Preparation Using the PB Transposon System and the JL Transposon System

[0272] Using cryopreserved PBMC (CP) as raw material, the effectiveness of PB transposon system and JL transposon system in preparing CAR-T was verified.

[0273] Cryopreserved PBMCs were revived using EDTA / PBS (containing 5‰ HSA). T cells were sorted using Miltenyi CD4 / CD8 magnetic beads, 1×10 9 WBCs were added with 200μL CD4 magnetic beads and 200μL CD8 magnetic beads and incubated for 30 min, and then CD4+T and CD8+T cells were selected using LS sorting columns.

[0274] After sorting, cells were plated at a density of 1E6 / mL in a culture flask containing culture medium (AIM-V + 5% SR), supplemented with IL-7 & IL-15 (final concentration of 25 ng / mL & 25 ng / mL) and Miltenyi MACS GMP TransAct CD3 / 28 magnetic beads, and cultured at 37°C, 5% CO2 for 2 days for activation.

[0275] After activation, 1×10 7 Add plasmid and transposase mRNA to each cell / group. Transfer the mixture to an electroporation cuvette and place in a Lonza Nucleofactor 4D or Maxcyte electroporator, selecting program FI-115 or Resting T / Expand T4 for electroporation. Transfer the electroporated cell suspension to a T75 culture flask (using AIM-V medium containing 5% SR), mix thoroughly, and incubate at 37°C. Exchange the medium every 2-3 days to ensure optimal cell growth conditions. On day 11, wash the cells with saline, add cryopreservation solution, and store in liquid nitrogen.

[0276] The prepared CAR-T cell was named "Frozen-JL" by adding 50 μg / mL of a plasmid expressing the MSLN CAR, 50 μg / mL of a plasmid expressing the PD-1 antibody, and 50 μg / mL of JL transposase mRNA. The amino acid sequence of the MSLN CAR is shown in SEQ ID NO: 37, the plasmid map of the MSLN CAR plasmid is shown in Figure 2, and the sequence is shown in SEQ ID NO: 38. The map of the plasmid expressing the PD-1 antibody is shown in Figure 23, the sequence is shown in SEQ ID NO: 40, and the sequence of the PD-1 antibody is shown in SEQ ID NO: 29. The amino acid sequence of the JL enzyme is shown in SEQ ID NO: 5.

[0277] The CAR-T prepared by adding 50 μg / mL of piggybac enzyme mRNA, 50 μg / mL of plasmid P19V21 expressing the MSLN CAR sequence, and 50 μg / mL of plasmid P20S23 expressing the PD-1 antibody was named "frozen-PB." The amino acid sequence of the piggybac enzyme is shown in SEQ ID NO: 39, and the plasmid map of P20S23 is shown in Figure 24.

[0278] The expansion folds of CAR-T cells during the production process are shown in Figure 25, and the cell viability on day 11 is shown in Figure 26. These results demonstrate that the JL transposon system exhibits higher expansion efficiency and comparable cell viability when using cryopreserved PBMCs to produce CAR-T cells. The stem cell phenotype of CAR-T cells is shown in Figure 27, demonstrating that the JL transposon system produces CAR-T cells with superior stemness compared to the PB transposon system. The CAR-T cell positive rate and CD4 / CD8 ratio are shown in Figure 28, demonstrating that the CAR-T cells produced using the JL transposon system have a slightly lower CAR positive rate and higher expansion efficiency. The exhaustion phenotype of CAR-T cells is shown in Figure 29, demonstrating that the CAR-T cells produced using the JL transposon system exhibit greater persistence and lower exhaustion (Tim3).

Claims

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

2. The method according to claim 1, characterized in that The cryopreserved cells are cryopreserved blood, leukocyte apheresis products, PBMC or T cells, and the cells expressing chimeric antigen receptors are CAR-T cells.

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

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

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

6. The method according to any one of claims 1 to 5, 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.

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

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

9. The method according to claims 1 to 8, characterized in that in, The method also satisfies at least one or more of the following conditions (a)-(c): (a) step (3) is performed together with step (2) or not later than 48, 36, 24, 20, 16, 12, 8, 5, 4, 3, 2 or 1 hour after the start of step (2), (b) step (4) is performed no later than 48, 36, 30, 24, 18, 12, 6, 3, 2 or 1 hour after the start of step (3); (c) step (4) is performed no later than 72, 60, 48, 36, 30, 24, 20, 18 or 12 hours after the start of step (2).

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

11. A cell expressing a chimeric antigen receptor obtained by the preparation method according to any one of claims 1 to 10.

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