Method for preparing chimeric antigen receptor cells via electroporation

Abstract

Provided in the present disclosure is a method for preparing chimeric antigen receptor cells via electroporation, which reduces electroporation damage to cells, and improves the proliferation multiple, cell activity and positive rate of the chimeric antigen receptor cells. The method comprises: contacting cells with an auxiliary agent, and then introducing a nucleic acid molecule into the cells via electroporation, wherein the auxiliary agent is insulin and / or an insulin-like growth factor.

Description

A method for preparing chimeric antigen receptor cells by electroporation Technical Field

[0001] The present disclosure relates to the field of cell technology, and more particularly to a method for preparing chimeric antigen receptor cells by electroporation. Background Art

[0002] Cellular immunotherapy has become a new anti-tumor therapy with great potential and significant efficacy, complementing the shortcomings of traditional surgery, radiotherapy, and chemotherapy. Immune cell therapy includes CAR-T immunotherapy, NK immunotherapy, DC immunotherapy, TIL immunotherapy, and others.

[0003] Immune cells generally involve gene editing or gene writing, including activation, transduction, culture and other processes. Viral transfection and electroporation are commonly used transduction methods. The transduction efficiency of viral transfection is low, and improving viral transfection efficiency is the main direction of improvement. CN117402261A discloses an efficient CAR-NK cell preparation scheme. Adding a transduction-promoting reagent (0.51μM BX795) can enhance the transduction efficiency, and the transduction efficiency can reach more than 70%.

[0004] Cell electroporation (also known as electrotransfection or electrotransfection) is a common technique in the field of cell transfection. It involves applying a short pulse of high-voltage electric field to cells, briefly creating channels in the cell membrane that allow extracellular molecules to enter. While electroporation offers high transfection efficiency and a short transfection time, it can cause significant damage to cells, affecting their proliferation and positive rates.

[0005] Therefore, how to reduce cell electroporation damage is a technical problem that needs to be urgently solved in the electroporation process. Summary of the Invention

[0006] The present disclosure aims to provide a method for preparing chimeric antigen receptor cells by electroporation, which reduces cell electroporation damage and improves the proliferation multiple, cell activity and positive rate of chimeric antigen receptor cells. The method comprises: contacting the cells with an adjuvant, and then introducing a nucleic acid molecule into the cells by electroporation; the adjuvant is insulin and / or insulin-like growth factor, and the nucleic acid molecule includes a nucleic acid molecule encoding a chimeric antigen receptor.

[0007] In some embodiments, the concentration of insulin is 10-200 μg / mL, and the concentration of insulin-like growth factor is 1 ng / mL-200 ng / mL.

[0008] In some embodiments, the insulin-like growth factor is selected from the group consisting of: IGF-1, LR3-IGF-1, IGF-2.

[0009] In some embodiments, the cells are isolated from fresh or cryopreserved leukapheresis products; preferably, the cells are CD3+, CD4+ and / or CD8+ T cells.

[0010] In some embodiments, contacting the cell with the adjuvant further comprises contacting the cell with an activating agent for activation.

[0011] In some embodiments, the activator is an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a co-stimulatory molecule on the surface of a cell;

[0012] Preferably, the agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3, more preferably a CD3 antibody;

[0013] Preferably, the agent that stimulates a co-stimulatory molecule 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.

[0014] In some embodiments, the activation time is 1-48 hours, such as 6 hours, 12 hours or 24 hours.

[0015] In some embodiments, the activated culture medium is AIM-V culture medium with or without 5% serum or its substitute; preferably, the culture medium further contains cytokines, such as IL-7 and / or IL-15.

[0016] In some embodiments, the nucleic acid molecule is carried on a non-viral vector.

[0017] In some embodiments, the nucleic acid molecules further comprise a nucleic acid molecule encoding a therapeutic agent, which is an antibody or a cytokine.

[0018] 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.

[0019] In some embodiments, the non-viral vector is a plasmid vector.

[0020] In some embodiments, the non-viral vector is a plasmid vector containing a transposon, wherein the transposon comprises a nucleic acid molecule encoding CAR, and the cell is further contacted with a transposase or a nucleic acid molecule encoding a transposase for electroporation, and the transposon and transposase belong to the same transposon system.

[0021] In one or more embodiments, the transposon system is a PB transposon system, a BZ transposon system, or a JL transposon system.

[0022] In some embodiments, cells are electroporated by contacting with a DNA vector comprising a JL transposon and mRNA encoding a JL transposase, wherein the JL transposon includes a CAR gene expression cassette and terminal inverted repeat sequences located on both sides of the CAR gene expression cassette.

[0023] In some embodiments, the amino acid sequence of the JL transposase is shown in SEQ ID NO: 2.

[0024] In some embodiments, the inverted terminal repeat sequences are set forth in SEQ ID NO: 3 (3' ITR) and SEQ ID NO: 4 (5' ITR).

[0025] 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: 5, or an amino acid sequence having one or more mutations of E24D, I36V, V43I compared with SEQ ID NO: 5; or (2) the amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having one or more mutations of T6I, T43A, K47E, A50S, E5 1D, G52A, N54K compared with SEQ ID NO: 8; the length of the replicon is ≤800bp, preferably ≤600bp or ≤300bp.

[0026] In a preferred embodiment, the amino acid sequence of the antitoxin protein is shown in any one of SEQ ID NOs: 5-11.

[0027] In some embodiments, the replicon is R6K.

[0028] In some embodiments, after the nucleic acid molecule is introduced into the cells by electroporation, the chimeric antigen receptor cells are directly harvested without a culture step.

[0029] In some embodiments, after the nucleic acid molecules are introduced into the cells by electroporation, the step of culturing the chimeric antigen receptor cells is further included, and the chimeric antigen receptor cells are cultured for 1-24 hours, or more than 3 days, such as 5, 9, 12, or 13 days.

[0030] In some embodiments, the culturing of chimeric antigen receptor cells is performed in a serum-free medium containing serum replacement (SR).

[0031] In some embodiments, the culture medium further contains IL-2, IL-15, IL-21, IL-7, IL-6, a LSD1 inhibitor, a MALT1 inhibitor, or a combination thereof.

[0032] The present disclosure also provides chimeric antigen receptor cells prepared by the preparation method of any embodiment.

[0033] The present disclosure also provides use of the chimeric antigen receptor cell in preparing a drug for treating and / or preventing malignant tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is a comparison of CAR-T cell proliferation folds between the Ctrl group and the Insulin group;

[0035] Figure 2 shows the comparison of the number of CAR-T cell CD3+CAR+ positive cells between the Ctrl group and the Insulin group;

[0036] Figure 3 is a plasmid map of the non-antimicroplasmid (0637);

[0037] Figure 4 shows the plasmid map of the non-antimicroplasmid (43009).

[0038] Figure 5 shows the performance evaluation results of CAR-T cells incubated with insulin growth factor before electroporation; Figure 5 A is the CAR-T proliferation multiple, Figure 5 B is the CAR-T cell viability, Figure 5 C is the CD3+CAR+ positivity rate in CAR-T cells, and Figure 5 D is the total number of CD3+CAR+ positive cells.

[0039] Figure 6 shows the performance comparison of CAR-T cells prepared by incubation with insulin and insulin growth factor on the third day of culture after electroporation.

[0040] Figure 7 shows the performance comparison of CAR-T cells prepared by treating cells with insulin in different ways.

[0041] Figure 8 shows the performance comparison of CAR-T cells prepared by treating cells with insulin growth factor in different ways. DETAILED DESCRIPTION

[0042] definition

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] The term "transduction" refers to the process of transferring or introducing exogenous nucleic acid into a host cell.

[0049] 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.

[0050] 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.

[0051] 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.).

[0052] The present disclosure provides a method for preparing chimeric antigen receptor (CAR) cells by electroporation, comprising: contacting cells with an adjuvant, and then introducing a nucleic acid molecule into the cells via electroporation; the adjuvant is insulin and / or insulin-like growth factor, and the nucleic acid molecule includes a nucleic acid molecule encoding a CAR. Contacting the cells with the adjuvant reduces cell damage and significantly increases the proliferation rate and number of positive cells after electroporation.

[0053] The following is an exemplary description of the method herein.

[0054] Cell collection

[0055] The cell can be any cell into which a nucleic acid molecule encoding a chimeric antigen receptor is introduced by electroporation, such as an immune cell. The immune cell is selected from T cells, tumor infiltrating lymphocytes (TIL cells), natural killer (NK) cells or natural killer T (NKT) cells.

[0056] The cells can be fresh or cryopreserved leukocyte apheresis products obtained from an entity. The entity can be a healthy person or a tumor patient. In some embodiments, T cells are isolated from fresh or cryopreserved leukocyte apheresis products (cell sorting), for example, CD3+, CD4+ and / or CD8+ T cells are isolated. The leukocytes include lymphocytes, basophils, neutrophils, eosinophils and monocytes, wherein the monocytes can be PBMCs (peripheral blood mononuclear cells).

[0057] Cell activation

[0058] When cells are in contact with adjuvants (insulin and / or insulin-like growth factor), they are also contacted with an activator for activation. During the activation process, cells are gradually activated. Insulin and / or insulin-like growth factor can better affect cells, thereby improving electroporation protection and reducing the effect of electroporation damage. Activator and insulin and / or insulin-like growth factor can be added to cells simultaneously for activation. In addition, the activator can be added to the cells prior to activation for a certain period of time, and then insulin and / or insulin-like growth factor can be added, or insulin and / or insulin-like growth factor domain cells can be added first and contacted, and then the activator is added. The insulin-like growth factor can be IGF-1, IGF-2, or it can be recombinant insulin-like growth factor, such as human recombinant insulin-like growth factor LR3-IGF-1. The concentration of insulin is 10-200 μg / mL, for example, 10 μg / mL, 25 μg / mL, 50 μg / mL, 150 μg / mL or 200 μg / mL, and the concentration of insulin-like growth factor is 1-200 ng / mL, for example, 1 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 150 ng / mL or 200 ng / mL.

[0059] Compared with not using an adjuvant, the use of an adjuvant can achieve at least one of the following (1)-(3):

[0060] (1) Achieve the same protein expression positive rate with a lower amount of nucleic acid molecules;

[0061] (2) continuous cell proliferation and enhanced killing ability;

[0062] (3) Cells are younger.

[0063] The younger age of cells can be measured using methods known in the art, such as: higher energy, more mitochondria, fewer aging markers (such as less β-galactosidase, less cell cycle checkpoint proteins such as p21, p16), longer telomere length, higher expression of sirt proteins (such as sirt 1-7), etc.

[0064] The activator is an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a co-stimulatory molecule on the surface of the stimulating cell. 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).

[0065] In some embodiments, the agent that stimulates the CD3 / TCR complex is an anti-CD3 antibody.

[0066] 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).

[0067] In some embodiments, the agent that stimulates a co-stimulatory molecule is an agent that stimulates CD28, preferably an anti-CD28 antibody.

[0068] 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.

[0069] 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.

[0070] Plasmid vector

[0071] In some embodiments, the nucleic acid molecule is carried on a non-viral vector.

[0072] In some embodiments, the nucleic acid molecule is selected from one or more of a nucleic acid molecule encoding a CAR, a nucleic acid molecule encoding a therapeutic agent, and a nucleic acid molecule encoding a transposase; preferably a nucleic acid molecule encoding a CAR, and / or a nucleic acid molecule encoding an immunotherapeutic agent.

[0073] In some embodiments, the nucleic acid molecule encoding CAR is DNA, and the non-viral vector is a plasmid vector. The vector generally contains sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The sequence (collectively referred to as "flanking sequences" in certain embodiments) generally includes one or more of the following nucleotide sequences: promoter, one or more enhancer sequences, replication origin, transcription termination sequence, complete intron sequence containing donor and acceptor splicing sites, sequence encoding the leader sequence for polypeptide secretion, ribosome binding site, polyadenylation sequence, multiple linker regions and selectable marker elements for inserting nucleic acids encoding antibodies to be expressed. See, for example, WO 01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193.

[0074] 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.

[0075] Transposons and transposases

[0076] In some embodiments, the non-viral vector is a plasmid vector containing a transposon comprising a nucleic acid molecule encoding a CAR.

[0077] 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.

[0078] 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).

[0079] In some embodiments, the cell is further contacted with a transposase or a nucleic acid molecule encoding a transposase for electroporation. In some embodiments, the nucleic acid molecule encoding the transposase is DNA or RNA. In some embodiments, the cell is contacted with a plasmid vector containing a nucleic acid molecule encoding the transposase and a transposon for electroporation. In some embodiments, the cell is contacted with a plasmid vector containing a nucleic acid molecule encoding the transposase and a plasmid vector containing a transposon for electroporation.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] In some embodiments, the JL transposon system includes a JL transposase and a JL transposon comprising an ITR sequence recognizable by the JL transposase.

[0084] In some embodiments, the JL transposase is a mutant transposase of the PS transposase shown in SEQ ID NO: 1, which has one or more of the following mutations compared to the PS transposase shown in SEQ ID NO: 1: 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.

[0085] 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.

[0086] 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 SEQ ID NO: 3 or 4.

[0087] In some embodiments, the transposon system is a PB transposon system, a BZ transposon system, or a JL transposon system.

[0088] 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 disclosure can be referenced to patent application 202310072956., the entire contents of which are incorporated herein by reference.

[0089] 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: 5, or an amino acid sequence having one or more mutations of E24D, I35V, V43I compared to SEQ ID NO: 5; or (2) the amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having one or more mutations of T6I, T43A, K47E, A50S, E51D, G52A, N54K compared to SEQ ID NO: 8; the length of the replicon is ≤800 bp, preferably ≤600 bp or ≤300 bp.

[0090] In some embodiments, the amino acid sequence of the antitoxin protein is as shown in any one of SEQ ID NOs: 5-11.

[0091] In some embodiments, the replicon is selected from ColE1, ColE2, pMB1, pSC101, RSF, R6K, pUC57, RK2, and p15A; preferably R6K or pUC57.

[0092] 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.

[0093] 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: 12 or 13.

[0094] In some embodiments, the nucleotide sequence of the replicon does not contain a CpG motif.

[0095] 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:14.

[0096] In some embodiments, the nucleotide sequence of the microplasmid-free (empty vector) is shown in SEQ ID NO: 15 or 16; the map structure is shown in FIG3 or 4.

[0097] electroporation

[0098] 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, Resting T / Expand T4 or T Cell high Energy for electroporation.

[0099] In some embodiments, after activation and before electroporation, the activated cells are separated from the adjuvant, for example by centrifugation.

[0100] In some embodiments, after activation and before electroporation, for certain types of adjuvants, the activated cells may not be separated from the adjuvant, for example, when the adjuvant is insulin.

[0101] In one or more embodiments, the concentration of activated immune cells in the electroporation mixture is 1×10 3 -1×10 11 / 100μL, for example 1×10 4 -1×10 10 / 100μL, 1×10 5 -1×10 9 / 100μL, 1×10 6 -1×10 8 pcs / 100μL.

[0102] In one or more embodiments, the concentration of the nucleic acid in the electroporation mixture is 1-20 nmol / L, preferably 2-10 nmol / L, more preferably 3.09 nmol / L.

[0103] In some embodiments, the nucleic acid molecule encoding CAR is RNA, such as mRNA, saRNA. The RNA molecule encoding CAR is electroporated into cells and can be used for transient expression of CAR.

[0104] In some embodiments, the cell is contacted with a transposon plasmid vector and a transposase or mRNA encoding a transposase for electroporation, 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.

[0105] In some embodiments, the cell is contacted with a plasmid vector of a transposon for electroporation, wherein the plasmid vector of the transposon comprises a CAR gene expression cassette, ITR sequences 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.

[0106] In some embodiments, the cell is contacted with a plasmid vector of a transposon and a plasmid vector of a transposase for electroporation, wherein the plasmid vector of the transposon comprises a CAR gene expression cassette and an ITR sequence recognizable by a 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.

[0107] In some embodiments, the method comprises contacting cells with a DNA vector comprising a JL transposon and mRNA encoding a JL transposase for electroporation, 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.

[0108] In some embodiments, the amino acid sequence of the JL transposase is shown in SEQ ID NO: 2.

[0109] In some embodiments, the inverted terminal repeat sequences are set forth in SEQ ID NO: 3 (3' ITR) and SEQ ID NO: 4 (5' ITR).

[0110] In some embodiments, the plasmid map of the DNA vector comprising the JL transposon is shown in FIG2 .

[0111] In some embodiments, cells are electroporated by contacting mRNA encoding transposase and a plasmid containing a transposon. Preferably, the mRNA is used at a dose 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.

[0112] In some embodiments, the cells also incorporate a nucleic acid molecule that is a therapeutic agent.

[0113] In some embodiments, the nucleic acid molecule of the therapeutic agent is also located on a plasmid vector of the transposon.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] In some embodiments, the cell is contacted with a plasmid vector comprising a nucleic acid molecule of a therapeutic agent, a plasmid vector comprising a nucleic acid molecule encoding a CAR, and a transposase or mRNA encoding a transposase and electroporated to simultaneously introduce the nucleic acid molecule encoding the CAR and the nucleic acid molecule of the therapeutic agent into the cell.

[0118] In some embodiments, the therapeutic agent is an antibody (eg, a single chain antibody, a single domain antibody, a bispecific antibody) or a cytokine.

[0119] In some embodiments, the therapeutic agent is an immune checkpoint inhibitor.

[0120] 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.

[0121] 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.

[0122] In some embodiments, the sequence of the single-domain antibody targeting PD-1 is shown in any one of SEQ ID NOs: 17-20.

[0123] 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.

[0124] In some embodiments, the sequence of the single-domain antibody targeting CTLA-4 is shown in SEQ ID NO: 21.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] In some embodiments, the sequence of the bispecific antibody is shown in any one of SEQ ID NOs: 22-25.

[0129] cytokines

[0130] In some embodiments, activation and electroporation are performed in a cell culture medium (e.g., serum-free medium) comprising IL-2, IL-15, IL-6, LSD1 inhibitor, or MALT1 inhibitor. In some embodiments, activation and electroporation are performed in a cell culture medium (e.g., serum-free medium) comprising IL-7, IL-21, or a combination thereof. In some embodiments, activation and electroporation are performed in a cell culture medium (e.g., serum-free medium) comprising IL-2, IL-15, IL-21, IL-7, IL-6, LSD1 inhibitor, MALT1 inhibitor, or a combination thereof. In some embodiments, the cell culture medium is a serum-free medium comprising serum replacement (SR).

[0131] Chimeric antigen receptor

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] In some embodiments, the intracellular signaling domain is a CD3ζ intracellular signaling domain or an FcεRIγ intracellular signaling domain.

[0139] 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: 27, and the amino acid sequence of the CAR is shown in SEQ ID NO: 28.

[0140] Nucleic acid construct encoding CAR

[0141] The present disclosure includes polynucleotide sequences encoding the CAR of the present disclosure. The polynucleotide sequences of the present disclosure can be in the form of DNA or RNA. The DNA forms include cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded.

[0142] 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.

[0143] The present disclosure 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 disclosure 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.

[0144] 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 disclosure. 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 disclosure.

[0145] In certain embodiments, the nucleic acid construct is a vector. The term "vector" is capable of transferring a gene sequence to a target cell. Typically, "vector construct," "expression vector," and "gene transfer vector" refer to any nucleic acid construct capable of directing the expression of a gene of interest and capable of transferring 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 expression of the polynucleotide sequence of the present disclosure is achieved by operably connecting the polynucleotide sequence of the present disclosure to a promoter and incorporating the construct into the expression vector. 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 vector comprising one, two, or three expression cassettes as described in any one of the embodiments herein.

[0146] The polynucleotide sequences of the present disclosure 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.

[0147] 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).

[0148] 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").

[0149] 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.

[0150] Pharmaceutical composition

[0151] The present disclosure also provides cells produced by the production method of any embodiment.

[0152] The present disclosure also provides use of the cells in preparing a drug for treating or preventing malignant tumors.

[0153] 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.

[0154] The cells of the present disclosure can be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as related cytokines or cell populations. Briefly, the pharmaceutical compositions of the present disclosure may include cells 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, and the like; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0155] The pharmaceutical compositions of the present disclosure can be administered in a manner appropriate to 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.

[0156] 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 disclosure 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, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical art by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0157] Administration of the subject compositions can be carried out in any convenient manner, 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 compositions of the present disclosure are administered to the patient by intradermal or subcutaneous injection. In another embodiment, the cell compositions of the present disclosure are preferably administered by intravenous injection. The cell compositions can be injected directly into a tumor, lymph node, or site of infection.

[0158] Some exemplary embodiments

[0159] 1. A method for preparing chimeric antigen receptor cells by electroporation, comprising: contacting the cells with an adjuvant, and then introducing a nucleic acid molecule into the cells by electroporation; the adjuvant is insulin and / or insulin-like growth factor, and the nucleic acid molecule includes a nucleic acid molecule encoding a chimeric antigen receptor.

[0160] 2. The method according to item 1, wherein the concentration of insulin is 10-200 μg / mL and the concentration of insulin-like growth factor is 1-200 ng / mL.

[0161] 3. The method according to item 1, characterized in that the cells are T cells, preferably, the T cells are CD3+, CD4+ and / or CD8+ T cells.

[0162] 4. The method according to any one of items 1 to 3, wherein the cells are contacted with an adjuvant and further contacted with an activator for activation, wherein the activator is an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates co-stimulatory molecules on the cell surface;

[0163] Preferably, the agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3, more preferably a CD3 antibody;

[0164] Preferably, the agent that stimulates a co-stimulatory molecule 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.

[0165] 5. The method according to any one of items 1 to 4, characterized in that the nucleic acid molecule further comprises a nucleic acid molecule encoding a therapeutic agent, preferably, the therapeutic agent is an antibody or a cytokine.

[0166] 6. The method according to any one of items 1 to 5, characterized in that the nucleic acid molecule is carried on a non-viral vector, the non-viral vector is a plasmid vector containing a transposon, the cells are further contacted with a transposase or a sequence encoding a transposase and electroporated, and the transposon and transposase belong to the same transposon system.

[0167] Preferably, the transposon system is a PB transposon system, a BZ transposon system or a JL transposon system.

[0168] 7. The method according to item 6, characterized in that the plasmid vector is a microtoxin-free 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: 5, or an amino acid sequence having one or more mutations of E24D, I36V, or V43I compared with SEQ ID NO: 5; or (2) the amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having one or more mutations of T6I, T43A, K47E, A50S, E51D, G52A, or N54K compared with SEQ ID NO: 8; the length of the replicon is ≤800 bp, preferably ≤600 bp or ≤300 bp.

[0169] 8. The method according to any one of items 1 to 7, wherein the use of an adjuvant can achieve at least one of the following (1) to (3) compared to not using an adjuvant:

[0170] (1) Achieve the same protein expression positive rate with a lower amount of nucleic acid molecules;

[0171] (2) continuous cell proliferation and enhanced killing ability;

[0172] (3) Cells are younger.

[0173] 9. The method according to any one of items 1 to 8, characterized in that after introducing the nucleic acid molecules into the T cells by electroporation, the step of culturing the CAR-T cells is further included, and the cell culture time is 1 hour to 13 days, preferably 1-24 hours, or more than 3 days, for example 5, 9, 12, or 13 days.

[0174] 10. A chimeric antigen receptor cell produced by the method according to any one of items 1 to 9.

[0175] 11. Use of the chimeric antigen receptor cell according to item 10 in the preparation of a medicament for treating and / or preventing malignant tumors.

[0176] The present disclosure 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 disclosure. The methods and materials used in the examples are, unless otherwise stated, conventional materials and methods in the art.

[0177] Example 1: Preparation of CAR-T cells using insulin culture

[0178] The cells were cultured in CAR-T medium (AIM-V+5%SR+25ng / mL IL7+25ng / mL IL15) and CD4 T cells were isolated from fresh PBMCs using magnetic beads.+ / CD8 + T cells are activated by adding the cells to a culture medium containing TransAct activator.

[0179] The cells in the Insulin group were incubated for 24 hours to activate T cells, and then the activation medium was removed by centrifugation.

[0180] After the treatment, the cells were collected and electroporated using a Maxcyte electroporator. The electroporation process was optimized according to the electroporator manual. 1×10 7 to 4×10 7 For T cells, 8.4ug P19V21 plasmid and 9ug P20S23 plasmid were added to the cell suspension, and 32ug PB mRNA (the amino acid sequence of PB enzyme is shown in SEQ ID NO: 30) was added to the electroporation system. Electroporation was performed using the T Cell high Energy 4 program. After electroporation, the cells were allowed to stand at room temperature for 20 minutes. Afterwards, the T cells were transferred to a 6-well plate containing culture medium and 2.5IU / mL DNase (Benzonase) (which needs to be preheated to 37°C in advance) and placed in an incubator (37°C; 5% CO2) for culture. Half of the medium was changed on the third day after electroporation, and a passage was performed on the fifth day. CAR-T cell preparation was completed on the ninth day. After the CAR-T cell preparation process and completion, cell proliferation and cell surface CAR positivity were detected using NC200 and flow cytometry.

[0181] The P19V21 plasmid contains the MSLN CAR plasmid. The MSLN CAR structure is as follows: from N-terminus to C-terminus, it contains the CD8α signal peptide, mesothelin VHH 1444, CD8α hinge region, CD28 transmembrane region and intracellular costimulatory signal region, and CD3ζ intracellular signal domain. The amino acid sequence of mesothelin VHH 1444 is shown in SEQ ID NO: 27, and the amino acid sequence of MSLN CAR is shown in SEQ ID NO: 28. The nucleotide sequence of the P19V21 plasmid is shown in SEQ ID NO: 26.

[0182] The above-mentioned P20S23 plasmid is a PD-1 nanobody plasmid, the nucleotide sequence of the plasmid is shown in SEQ ID NO: 30, and the PD-1 nanobody is human immunoglobulin K light chain signal peptide and PD-1VHH in sequence; the amino acid sequence of PD-1VHH is shown in SEQ ID NO: 20.

[0183] On day 9, the CAR-T cell proliferation folds in the Ctrl group were compared with the Insulin group as shown in Figure 1, and the CAR-T cell CD3+CAR+ positive cell results are shown in Figure 2. The three points in the bar graph represent the results of three parallel experiments, and the Ctrl group was standardized for plotting. As can be seen from Figure 1, the cell proliferation fold in the Insulin group on day 9 was about 1.5 times that of the Ctrl group. As can be seen from Figure 2, the CD3+CAR+ positive cells in the Insulin group on day 9 were about 1.7 times that of the Ctrl group. This shows that treating cells with insulin can increase cell proliferation folds and the number of positive cells, that is, it can reduce cell damage.

[0184] Example 2: Preparation of CAR-T cells using insulin growth factor incubation

[0185] According to the method of Example 1, the insulin in the culture medium was replaced with insulin growth factor-1 (IGF-1) to incubate and activate T cells. The incubation time was 6h or 24h, and the concentration of IGF-1 added to the culture medium was 5ng / ml, 25ng / ml or 100ng / ml. After preliminary screening, 5ng / ml incubation for 6h (denoted as IGF1-5-6h group), 25ng / ml incubation for 6h (denoted as IGF1-25-6h group), and 100ng / ml incubation for 24h (denoted as IGF1-100-24h group) can improve cell viability and / or cell expansion ability; the group using only CAR-T culture medium was denoted as ctrl group. The results of CAR-T cell performance evaluation are shown in Figure 5.

[0186] The results showed that adding IGF1 (5 ng / mL) or IGF1 (25 ng / mL) and activated Transact for 6 hours before electroporation effectively increased cell proliferation efficiency by up to 40% and cell viability by up to 15%, and these results were superior to the control group throughout the culture process. Incubating IGF1 (5 ng / mL) or IGF1 (25 ng / mL) and activated Transact for 6 hours effectively increased the CAR-T positive rate by up to 82% and the number of CD3+CAR+ positive cells by up to 1-fold. Incubating IGF1 (100 ng / mL) and activated Transact for 24 hours showed relatively average results.

[0187] Example 3: Comparison of the effects of insulin and insulin growth factor

[0188] On day 3 after electroporation, CAR-T data were compared between insulin (25 μg / mL, 24-hour incubation) and insulin-induced growth factor (IGF) (5 ng / mL, 6-hour incubation), as shown in Figure 6. On day 3, IGF1 outperformed insulin in proliferation rate, cell viability, and total number of positive cells, requiring a shorter treatment time and using a lower concentration.

[0189] Example 4: Insulin and insulin growth factor are treated with different methods

[0190] (1) Insulin

[0191] According to the different timing and treatment methods of insulin addition, patients are divided into the following three groups:

[0192] Insulin-25-24h: Insulin 25ug / ml before electroporation, pre-treatment for 24h.

[0193] Insulin-25-B&A: Insulin 25ug / ml before electroporation, pre-treatment for 24h, and continued treatment until the third day after electroporation.

[0194] Insulin-25-AF: Only insulin 25ug / ml was used for treatment until day 3 after electroporation.

[0195] The results are shown in FIG7 . Based on the cell proliferation multiples, the number of positive cells, the positive cell rate, and the viability, it can be seen that the most advantageous and stable insulin treatment method is insulin-25-24h.

[0196] (2) Insulin growth factor

[0197] According to the different timing of adding insulin growth factor and the different treatment methods, it is divided into the following three groups:

[0198] IGF1-5-6h: Insulin 25ug / ml before electroporation, pre-treatment for 6h.

[0199] IGF1-5-B&A: Insulin 25ug / ml before electroporation, pre-treatment for 6h, and continued treatment until the 3rd day after electroporation.

[0200] IGF1-5-AF: Only insulin 25ug / ml was used for treatment until day 3 after electroporation.

[0201] The results are shown in FIG8 . According to the cell proliferation multiples, the number of positive cells, the positive cell rate, and the viability, it can be seen that the most advantageous and stable insulin treatment method is insulin-5-6h.

[0202] Partial sequence

Claims

1. A method for preparing chimeric antigen receptor cells by electroporation, characterized in that: include: contacting the cells with an adjuvant and then introducing the nucleic acid molecule into the cells by electroporation; The auxiliary agent is insulin and / or insulin-like growth factor, and the nucleic acid molecule includes a nucleic acid molecule encoding a chimeric antigen receptor.

2. The method according to claim 1, wherein The concentration of the insulin is 10-200 μg / mL, and the concentration of the insulin-like growth factor is 1-200 ng / mL.

3. The method according to claim 1, wherein The cells are T cells, preferably, the T cells are CD3+, CD4+ and / or CD8+ T cells.

4. The method according to any one of claims 1 to 3, wherein When the cells are contacted with the adjuvant, they are also contacted with an activator for activation, wherein the activator is an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates co-stimulatory molecules on the cell surface; Preferably, the agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3, more preferably a CD3 antibody; Preferably, the agent that stimulates a co-stimulatory molecule 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.

5. The method according to any one of claims 1 to 4, characterized in that The nucleic acid molecules also include nucleic acid molecules encoding therapeutic agents, preferably, the therapeutic agent is an antibody or a cytokine.

6. The method according to any one of claims 1 to 5, wherein: The nucleic acid molecule is loaded on a non-viral vector, which is a plasmid vector containing a transposon. The cell is also contacted with a transposase or a sequence encoding a transposase for electroporation. The transposon and the transposase belong to the same transposon system. Preferably, the transposon system is a PB transposon system, a BZ transposon system or a JL transposon system.

7. The method according to claim 6, wherein The plasmid vector is a microantibiotic-free plasmid vector, which contains a nucleotide sequence encoding an antitoxin protein and a replicon; the amino acid sequence of the antitoxin protein contains the following sequence: (1) the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having one or more mutations of E24D, I36V, V43I compared with SEQ ID NO: 5; or (2) the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having one or more mutations of T6I, T43A, K47E, A50S, E51D, G52A, N54K compared with SEQ ID NO: 8; the length of the replicon is ≤800bp, preferably ≤600bp or ≤300bp.

8. The method according to any one of claims 1 to 7, wherein: Compared with not using an adjuvant, the use of an adjuvant can achieve at least one of the following (1)-(3): (1) Achieve the same protein expression positive rate with a lower amount of nucleic acid molecules; (2) continuous cell proliferation and enhanced killing ability; (3) Cells are younger.

9. The method according to any one of claims 1 to 8, wherein After the nucleic acid molecules are introduced into the T cells by electroporation, the step of culturing the CAR-T cells is also included. The cell culture time is 1 hour to 13 days, preferably 1-24 hours, or more than 3 days, for example 5, 9, 12, or 13 days.

10. A chimeric antigen receptor cell prepared by the method according to any one of claims 1 to 9.

11. Use of the chimeric antigen receptor cell according to claim 10 in the preparation of a medicament for treating and / or preventing malignant tumors.

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