Methods of producing a population of cells containing immune cells expressing a CAR

Abstract

Disclosed is a method of producing a cell population containing immune cells expressing a chimeric antigen receptor (CAR), the method comprising co-culturing CAR-expressing immune cells and target antigen-expressing cells, the CAR-expressing immune cells being cells in which a CAR gene has been introduced and the target antigen-expressing cells expressing a gene for an antigen targeted by the CAR.

Description

Technical Field

[0001] This disclosure relates to cell populations containing immune cells expressing chimeric antigen receptors (CARs) and methods for producing them. Background Technology

[0002] In recent years, chimeric antigen receptor (CAR)-T cell therapy has been developed as one of the immunotherapies for cancer patients (Non-Patent Literature 1). CAR-T cells are cytotoxic T cells (CTLs) whose T cell receptors (TCRs) are genetically modified, enabling CTLs to directly and selectively recognize tumor cells to exert anti-tumor effects, and have attracted considerable attention as a very promising therapy for refractory tumors. CAR is a general term for a protein that specifically recognizes tumor antigens on its N-terminal side, such as a single-chain antibody (scFv) prepared by modifying the variable region of an antibody into a single-chain amino acid sequence, and has a T cell receptor ζ chain on its C-terminal side. CAR-expressing T cells recognize tumor antigens at their extracellular domains, transmit signals to the T cell via the subsequent ζ chain, and become activated to exert their anti-tumor effects by releasing cell-killing factors such as perforin and granzymes (Non-Patent Literature 1).

[0003] For certain tumors, cancer therapy using CAR-T cells has been approved and put into practical use in Japan, Europe, and the United States. In the field of hematologic malignancies, a phase III clinical trial was conducted for CD19-positive B-lymphocytic tumors, in which CD19-specific CARs were introduced via gene transfer into T cells pre-collected from patients with relapsed acute lymphoblastic leukemia, and the cells were cultured, expanded, and infused into the patients. Molecular biological remission in the bone marrow was then reported in all five patients who received the treatment (Non-Patent Literature 2). Based on this report, two drugs, Tisagen lecleucel (product name: Kymriah®) and Axicabtagene ciloleucel (product name: Yescarta®), have been approved and marketed in Europe and the United States for CD19-positive acute lymphoblastic leukemia and lymphoma. These two drugs have attracted considerable attention as a breakthrough treatment for refractory CD19-positive lymphoblastic leukemia and lymphoma (which have been difficult to cure to date).

[0004] Most clinically used CAR-T cell products are manufactured using gamma retroviruses through genetic modification. The production of these virus-mediated genetically modified cell products involves a highly complex process, including GMP-grade virus production, viral residue testing of the final product, and production in accordance with Cartagena Approval of Type 1 Use in certain countries.

[0005] In order to produce CAR-T cells, in addition to conventional methods using viral vectors, gene modification techniques using non-viral vectors have also been used (Patent Documents 1 and 2). For example, research and development of non-viral genetically modified CAR-T cell therapies are underway, focusing on gene transfer technology that utilizes transposons called piggyBac instead of gamma retroviruses (hereinafter referred to as the "piggyBac transposon-mediated method"). For CAR-T cell production, producing CAR-T cells through non-viral gene modification techniques is a safe and simple method because no virus is used for gene transfer. The ACE method, developed by one of the inventors, Nakazawa et al., is a non-viral genetically modified CAR-T cell culture method (Patent Document 2). This is a pioneering non-viral gene modification method and has overcome the problems of production methods using viral vectors.

[0006] Existing technical documents

[0007] Patent documents

[0008] Patent Document 1: JP2017-22121A

[0009] Patent Document 2: WO2017 / 061615

[0010] Non-patent literature

[0011] Non-patent literature 1: Eshhar Z, Waks T, Gross G, Schindler DG. Specificactivation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors. Proc Natl Acad Sci U SA. 1993;90:720-724.

[0012] Non-patent literature 2: Brentjens RJ, Davila ML, Riviere I, Park J, Wang P, Frattini M, Sadelain M. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci TranslMed. 2013;5:177ra38. Summary of the Invention

[0013] The problem to be solved

[0014] For target molecules in hematologic malignancies such as CD19, conventional methods for producing CAR-T cells can yield clinically usable quality and quantities. However, for target molecules in solid tumors, obtaining a sufficient number of cells for clinical application is challenging. Furthermore, attempts to ensure the required cell quantity for patients through long-term culture or forced activation by co-stimulatory molecules can alter the characteristics of T cells, resulting in reduced cytotoxic activity and easier depletion. Consequently, it is difficult to stably produce a sufficient number and quality of CAR-T cells for clinical application.

[0015] Problem Solving

[0016] The inventors of this invention have conducted diligent research to solve the above problems. They have then discovered that co-culturing CAR-infused immune cells (such as CAR-T cells) with separately prepared target antigen-expressing cells expressing the CAR target antigen not only improves gene transfer efficiency and cell proliferation rate, but also enables the stable culture of immune cells with high cytotoxic activity that are not easily depleted.

[0017] In other words, in one aspect, this disclosure provides a method for producing a cell population containing immune cells expressing a chimeric antigen receptor (CAR), comprising co-culturing immune cells expressing CAR and cells expressing a target antigen of CAR, wherein the immune cells expressing CAR are cells in which the CAR gene has been introduced and the target antigen expressing cells are normal blood cells that have been engineered to express the target antigen.

[0018] In another aspect, this disclosure provides a cell population containing immune cells expressing a chimeric antigen receptor (CAR) produced by methods as defined above.

[0019] In another aspect, this disclosure provides a composition for treating cancer comprising a cell population as defined above.

[0020] The effects of the invention

[0021] According to the present invention, the production efficiency of CAR-expressing immune cells can be increased, and CAR-T cells with high cytotoxic activity can be stably produced. In particular, the production efficiency of CAR-expressing immune cells for solid tumors can be significantly improved, and thus CAR-T cell therapy can be applied to various cancer types. Attached Figure Description

[0022] Figure 1 The CAR gene expression vector and transposase expression vector used in the Examples section are shown.

[0023] Figure 2 The target antigen expression vector used in the Examples section is shown.

[0024] Figure 3 The target antigen expression vector used in the Examples section is shown.

[0025] Figure 4 The results of flow cytometry analysis of HER2-CAR, CD3, PD-1, CCR7 and CD45RA expression in T cells expressing HER2-CAR co-cultured with target antigens expressing HER2 and co-stimulatory molecules (CD80+4-1BBL, CD80 or 4-1BBL) are shown.

[0026] Figure 5 The results of flow cytometry analysis of HER2-CAR, CD3, PD-1, CCR7 and CD45RA expression in T cells expressing HER2-CAR co-cultured with target antigens expressing HER2 and co-stimulatory molecules (CD40 + OX40L, CD40 or OX40L) are shown.

[0027] Figure 6 The results of the first assay show the killing effect of T cells expressing HER2-CAR targeting HER-expressing U2OS cells.

[0028] Figure 7 The results of the second assay (the killing assay) using T cells expressing HER2-CAR to target HER-expressing U2OS cells were shown.

[0029] Figure 8 The results of the killing assays using HER2-CAR-expressing T cells from Example 5, CD19-CAR-expressing T cells from Example 6, and HER2-CAR-expressing T cells from Comparative Example 8 to target HER-expressing U2OS cells are shown. Detailed Implementation

[0030] Unless otherwise stated, the terms used in this disclosure have the meanings commonly understood by those skilled in the art in fields such as organic chemistry, medicine, pharmacy, molecular biology, and microbiology. Below are definitions of some terms used in this disclosure, and these definitions supersede the common understanding found in this disclosure.

[0031] In this disclosure, when a number is accompanied by the term "about," it is intended to include a range of ±10% of that value. For example, "about 20" should include "18-22." The range of numbers includes all numbers between the endpoints and the numbers at the endpoints. The term "about" used for a range applies to both ends of the range. Thus, for example, "about 20-30" should include "18-33."

[0032] In this disclosure, sequence identity refers to the degree of sequence matching between polypeptides or polynucleotides, and it is determined as follows: Two optimally aligned sequences (aligned to maximize amino acid or nucleotide matching) are compared over the sequence regions to be compared. The numerical value (%) of sequence identity is calculated as follows: Identify the identical amino acids or nucleotides present in the two sequences, determine the number of matching sites, divide the number of matching sites by the total number of amino acids or nucleotides in the sequence regions to be compared, and then multiply the resulting value by 100. Examples of algorithms for obtaining optimal alignment and sequence identity include various algorithms commonly available to those skilled in the art (e.g., the BLAST algorithm, the FASTA algorithm). For example, sequence identity can be determined using sequence analysis software such as BLAST or FASTA.

[0033] Chimeric antigen receptor

[0034] Chimeric antigen receptors (also referred to as CARs in this paper) are proteins with structures containing target-specific extracellular domains, transmembrane domains, and intracellular signaling domains, which function as effectors of immune cells from the N-terminus to the C-terminus. The CAR gene is the gene encoding this receptor. Each domain will be described below.

[0035] (a) Extracellular domain

[0036] The extracellular domain contains an antigen recognition site that exhibits target-specific binding. For example, the extracellular domain may contain an scFv fragment of a monoclonal antibody targeting the target (e.g., a fragment consisting of the amino acid sequence of SEQ ID NO: 1 or 2, or a fragment described in WO2017 / 061615, CN107164338A, WO2016 / 123143, WO2016 / 023253, or JP2018-198601A), or, when the target is a receptor, a ligand that binds to said receptor (e.g., a ligand consisting of the amino acid sequence of SEQ ID NO: 3, or a ligand described in WO2018 / 110374 or WO2018 / 052142). The monoclonal antibody used herein may be, for example, a rodent antibody (such as a mouse, rat, or rabbit), a human antibody, or a humanized antibody. Humanized monoclonal antibodies are antibodies prepared by creating monoclonal antibodies from non-human animals (e.g., mice or rats) with structures similar to those of human antibodies, and include humanized chimeric antibodies in which only the constant region of the antibody is replaced with the constant region of a human antibody, and humanized CDR-transplanted antibodies in which the constant region and portions other than the complementarity-determining region (CDR) in the variable region are replaced with those of the human antibody (PT. Johons et al., Nature 321, 522, 1986). To enhance the antigen-binding activity of humanized CDR-transplantation antibodies, improved techniques have been developed and can be used to manufacture humanized antibodies. These techniques include, for example, selecting a human antibody's frame (FR) region that has high homology with a mouse antibody's frame (FR) region, producing a humanized antibody that has high homology with a mouse antibody, and replacing amino acids in the FR region after transplanting a mouse CDR into a human antibody (see, for example, US5585089B, US5693761B, US5693762B, US6180370B, EP451216B, EP682040B, JP2828340B).

[0037] The scFv fragment has a structure in which the light chain variable region (VL) and heavy chain variable region (VH) of an immunoglobulin are linked by a linker, and it retains the ability to bind antigens. As a linker, a peptide linker can be used, for example. A peptide linker is a linker for a peptide in which amino acids are linearly linked. Examples of peptide linkers include linkers composed of glycine and / or serine (e.g., GGS or GS linkers). Glycine and serine are small in size, thus preventing the linker from forming higher-order structures. The length of the linker is not particularly limited to any specific length. For example, a linker having 5-25 amino acid residues can be used. The length of the linker is preferably 8-25, more preferably 15-20.

[0038] Target antigens can be antigens that are significantly or obviously expressed in tumor cells compared to non-tumor cells. Examples of target antigens include, for example, tumor-associated or tumor-specific antigens such as EPHB4, HER2, EPHA2, EPHB2, EGFR, GD2, phosphatidylinositol proteoglycan-3, 5T4, 8H9, αvβ6 integrin, B-cell maturation antigen (BCMA), B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, κ light chain, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD116, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, ERBB3, ERBB4, FAP, FAR, FBP, fetal AchR, folate receptor α, GD3, HLA-AI MAGE. A1, HLA-A2, IL11Ra, IL13Ra2, KDR, Lambda, Lewis Y, MCSP, mesothelin, MUC1, MUC4, MUC6, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSC1, PSMA, ROR1, Sp17, survivin, TAG72, TEM1, TEM8, VEGF receptor 2, carcinoembryonic antigen, HMW-MAA, VEGF receptor, fibronectin, tendon glycoprotein, or antigens present in the extracellular matrix such as carcinoembryonic antigen (CEA) in necrotic regions of tumors or proteins containing mutations identified through genomic analysis and / or differential expression studies of tumors.

[0039] When the target antigen is a receptor, the receptor's ligand can be used as an antigen recognition site to replace the scFv. For example, the extracellular domains of EFNB2 protein (which is the natural ligand of the EPHB4 receptor) and GM-CSF (which is the ligand of the GM-CSF receptor), as well as Adnectin (which is the ligand of EGFR), IL-11 (which is the ligand of IL11Ra), IL-13 (which is the ligand of IL13Ra2), FSH (which is the ligand of FSHR), T1E (which is the ligand of the ERBB2 family), CD27 (which is the ligand of CD70), DNAM-1 (which is the ligand of ligand-2), NKG2D (which is the ligand of MICA and MICB), and NKp30 (which is the ligand of Gal3) can be used.

[0040] The production method disclosed herein can produce CAR-expressing immune cells in sufficient quantity and quality not only for antigens expressed in blood cells but also for antigens not expressed in such cells, and is particularly suitable for target antigens of solid tumors. Examples of such target antigens include EPHB4, HER2, EPHA2, EPHB2, EGFR, GD2, phosphatidylinositol proteoglycan-3, 5T4, MUC1, MUC4, MUC6, NCAM, EGFR, EGFRvIII, ERBB3, ERBB4, NY-ESO-1, PSCA, PSC1, PSMA, VEGFR receptor 2, carcinoembryonic antigen, HMW-MAA, and VEGF receptor.

[0041] The extracellular domain may contain a leader sequence (signal peptide) that promotes the translocation of CARs to the cell surface. For example, the leader sequence of the GM-CSF receptor can be used as a leader sequence.

[0042] In one embodiment, the extracellular domain comprises, or consists of, an amino acid sequence having 90% or more sequence identity with any of SEQ ID NO: 1-3, preferably SEQ ID NO: 1. In another embodiment, the extracellular domain comprises, or consists of, an amino acid sequence of any of SEQ ID NO: 1-3, preferably SEQ ID NO: 1.

[0043] (b) Transmembrane domain

[0044] The transmembrane domain is located between the extracellular domain and the intracellular signaling domain. As a transmembrane domain, transmembrane domains of CD8, T cell receptor α or β chains, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, GITR, or 4-1BB can be used. The transmembrane domain can also be an artificially constructed peptide. Preferably, the transmembrane domain is the transmembrane domain of CD28 (e.g., a domain consisting of the amino acid sequence of SEQ ID NO: 7 or 8).

[0045] In one embodiment, the transmembrane domain comprises, or is composed of, an amino acid sequence having 90% or more sequence identity with, the amino acid sequence of SEQ ID NO: 7 or 8. In another embodiment, the transmembrane domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 7 or 8.

[0046] (c) Intracellular signaling domains

[0047] Intracellular signaling domains transmit signals essential for immune cells to perform their effector functions. That is, when an extracellular domain binds to a target antigen, the intracellular signaling domain used can transmit the signals necessary to activate the immune cell. The intracellular signaling domain includes a domain for transmitting signals mediated by the TCR complex (for convenience, referred to as the "first domain"), and may further include a domain for transmitting co-stimulatory signals (for convenience, referred to as the "second domain"). Examples of these domains include domains of CD2, CD4, CD5, CD28, CD134, 4-1BB (CD137), GITR, CD27, OX40, HVEM, CD3ζ, FcεRIγ, OX-40, and ICOS. The first domain is preferably a CD3ζ or FcεRIγ domain, more preferably a CD3ζ domain (e.g., a domain consisting of the amino acid sequence of SEQ ID NO: 9). The second structural domain is preferably a CD28, 4-1BB (CD137), CD2, CD4, CD5, CD134, OX-40, or ICOS structural domain, and more preferably a CD28 or 4-1BB structural domain. The first and second structural domains can each be composed of multiple identical or different cascaded structural domains.

[0048] When the intracellular signaling domain comprises a first domain and a second domain, the first and second domains can be connected in any manner, but the second domain is preferably positioned on the transmembrane domain side because it is known that co-stimulation is strongly transmitted under certain conditions when CD3ζ is connected distally. The first and second domains can be connected directly or via a linker. As a linker, for example, a peptide linker can be used. A peptide linker is a linker for peptides in which amino acids are linearly linked, and its structure and characteristics are as described above. The linker connecting the first and second domains can be a linker consisting only of glycine. The length of the linker is not particularly limited to any specific length. For example, a linker having 2-15 amino acid residues can be used.

[0049] In one embodiment, the intracellular signaling domain comprises, or is composed of, an amino acid sequence having 90% or more sequence identity with, the amino acid sequence of SEQ ID NO: 9. In another embodiment, the extracellular domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 9.

[0050] (d) Other components

[0051] Extracellular domains and transmembrane domains can be connected by a spacer domain. The spacer domain facilitates the binding of the CAR to the target antigen. As a spacer domain, the Fc fragment of an antibody or a fragment or derivative thereof, the hinge region of an antibody or a fragment or derivative thereof, the CH2 region of an antibody, the CH3 region of an antibody, an artificial spacer sequence, or a combination thereof (e.g., a domain consisting of the amino acid sequence of any one of SEQ ID NO: 4-6) can be used. For example, the Fc fragment of human IgG (e.g., human IgG1, human IgG4) can be used as a spacer domain. Additionally, a portion of the extracellular domain of CD28 and a portion of the extracellular domain of CD8α can also be used as a spacer domain. The spacer domain can also provide a spacer between the transmembrane domain and the intracellular signaling domain.

[0052] In one embodiment, the spacer domain comprises, or is composed of, an amino acid sequence having 90% or more sequence identity with, any of the amino acid sequences in SEQ ID NO: 4-6. In another embodiment, the spacer domain comprises, or is composed of, the amino acid sequence in SEQ ID NO: 4-6.

[0053] CAR expression vector

[0054] In this disclosure, CAR-expressing immune cells are prepared by introducing the CAR gene into immune cells using a CAR expression vector. A CAR expression vector is a nucleic acid molecule capable of transporting a nucleic acid molecule encoding a CAR gene into an immune cell. It can be DNA or RNA of any form and origin, and different types of vectors are available. The vector can be a viral vector or a non-viral vector. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, and Sendai virus vectors. Among these, using retroviral vectors, lentiviral vectors, and adeno-associated virus vectors, the target gene incorporated into the vector is integrated into the host chromosome, and stable and long-term expression is anticipated. Each viral vector can be prepared according to conventional methods or by using commercially available kits for this purpose. Examples of non-viral vectors include plasmid vectors, liposome vectors and positively charged liposome vectors (Felgner, PL, Gadek, TR, Holm, M. et al., Proc. Natl. Acad. Sci., 84: 7413-7417, 1987), YAC vectors and BAC vectors.

[0055] CAR expression vectors contain expression units for expressing the CAR gene, which typically include a promoter, the CAR gene, and a polyadenylation (PA) signal. Examples of promoters that can be used in CAR expression cassettes include CMV-IE (a promoter derived from the early cytomegalovirus gene), SV40ori, retroviral LTRSRα, EF1α, and β-actin promoters. Examples of PA signals include PA sequences from SV40, bovine growth hormone, and globulin. The CAR gene is usually attached directly or via another sequence to the 3' end of the promoter, such that the promoter regulates CAR gene expression, and the PA signal is positioned downstream of the CAR gene. The CAR gene is transcribed from such expression units into messenger RNA (mRNA), and the CAR is translated from the mRNA and presented on the cell surface.

[0056] The expression unit may include, for example, genes for detecting gene expression (e.g., reporter genes, cell or tissue-specific genes, or optional marker genes), enhancer sequences for improving expression efficiency, and WRPE sequences.

[0057] The detected genes are used to determine the success or failure and efficiency of CAR expression vector introduction, to detect CAR gene expression or determine CAR gene expression efficiency, or to select or sort cells expressing CAR genes. Examples of genes tested include the neo gene, which confers resistance to neomycin; the npt gene, which confers resistance to kanamycin or other antibiotics (Herrera Estrella, EMBO J. 2 (1983), 987-995); the nptII gene, which confers resistance to kanamycin or other antibiotics (Messing and Vierra, Gene 1 9:259-268 (1982)); the hph gene, which confers resistance to hygromycin (Blochinger and Diggl mann, Mol Cell Bio 4: 2929-2931); and the dhfr gene, which confers resistance to methotrexate (Bourouis et al., EMBO J.2 (7)) (examples of marker genes); the luciferase gene (Giacomin, P1. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996)). 121), β-glucuronidase (GUS) genes, fluorescent proteins such as GFP (Gerdes, FEBS Lett. 389 (1996), 44-47) or their variants (e.g., EGFP, d2EGFP) (examples of reporter genes); and epidermal growth factor receptor (EGFR) genes lacking intracellular domains. The genes to be detected can be linked to CAR genes via, for example, a bicistronic control sequence (e.g., an internal ribosomal recognition sequence (IRES)) or a sequence encoding a self-cleaving peptide. Examples of self-cleaving peptides include the 2A peptide (T2A) derived from the Thosea asigna virus. Different examples of self-cleaving peptides include, but are not limited to, 2A peptides derived from small RNA viruses (F2A), foot-and-mouth disease virus (FMDV) (F2A), equine rhinitis A virus (ERAV) (E2A), and 2A peptides derived from porcine chezinvirus (PTV-1) (P2A), as well as 2A peptides derived from rotavirus, insect virus, AFT virus, or tripanosoma virus.

[0058] immune cells

[0059] In this disclosure, a CAR gene is introduced into immune cells. The immune cells in this disclosure can be T cells (including CD4-positive CD8-negative T cells, CD4-negative CD8-positive T cells, αβ-T cells, γδ-T cells, and NKT cells), B cells, NK cells, monocytes, macrophages, dendritic cells, or combinations thereof. The immune cells can be cells isolated from humans or cells differentiated from cells such as iPS cells, ES cells, or hematopoietic stem cells. Furthermore, the immune cells can be autologous cells or allogeneic cells. In this disclosure, the term "autologous cell" refers to a cell obtained from a subject to whom a cell population produced by the method of this disclosure is to be administered, or a cell derived from such obtained cells. The term "allogeneic cell" means that the cell is not "autologous." Preferably, the immune cells are autologous cells. In one embodiment, the immune cells are lymphocytes (i.e., T cells, B cells, NK cells, or combinations thereof). In another embodiment, the immune cells are T cells. CAR-expressing immune cells can be obtained by transferring a gene into a cell population containing immune cells or their progenitor cells (such as hematopoietic stem cells). For example, CAR-expressing immune cells can be obtained by differentiating cells in which the CAR gene has already been introduced (such as iPS cells, ES cells, or hematopoietic stem cells), or by differentiating cells that have been transformed into iPS cells after the CAR gene has been introduced. In one embodiment, CAR-expressing immune cells are prepared by introducing the CAR gene into blood cells. In this disclosure, the term "blood cell" refers to one or more cells constituting blood and is used to mean a single cell or a cell population containing multiple cells, a cell population consisting of one type of cell, and a cell population containing multiple types of cells. Blood cells are preferably blood cells other than red blood cells and platelets, and such blood cells include immune cells such as lymphocytes and monocytes. Blood cells can be cells isolated from humans or cells differentiated from cells such as iPS cells, ES cells, or hematopoietic stem cells, and can be autologous cells or allogeneic cells, but autologous cells are preferred. In another embodiment, CAR-expressing immune cells are prepared by introducing the CAR gene into PBMCs. PBMCs are preferably autologous PBMCs (i.e., PBMCs collected from a subject to whom the cell population produced by the methods of this disclosure is to be administered). PBMCs can be prepared by conventional methods, for example, by reference to Saha S, Nakazawa Y, Huye LE, Doherty JE, Galvan DL, Rooney CM, Wilson MH. J Vis Exp. 2012 Nov 5;(69): e4235.Unless otherwise stated, any cells described herein (e.g., T cells) are human cells.

[0060] Preparation of CAR-expressing immune cells

[0061] CAR gene expression vectors prepared for gene transfer are introduced into immune cells using conventional methods. In the case of viral vectors, they are introduced into cells via viral infection. In the case of non-viral vectors such as plasmids, conventional methods such as electroporation, liposomes, or calcium phosphate-mediated methods can be used for introduction into cells, with electroporation being the preferred method.

[0062] To improve the efficiency of integration into the host chromosome, gene transfer via transposon-mediated methods is preferred. Transposon-mediated methods are a type of non-viral gene transfer method that integrates a target gene into the host chromosome through a mechanism in which a genome-acting enzyme (transposase) and its specific recognition sequence work together to cause gene translocation. Transposon-mediated methods can be, for example, the piggyBac transposon-mediated method. The piggyBac transposon-mediated method utilizes transposons isolated from insects (Fraser MJ et al., Insect MolBiol. May 1996; 5(2):141-51.; Wilson MH et al., Mol THER January 2007; 15(1):139-45), and it can integrate into mammalian chromosomes very efficiently. The piggyBac transposon-mediated method has been practically used for gene transfer (see, for example, Nakazawa Y et al., J Immunother 32:826-836, 2009; Nakazawa Y et al., J Immunother 6:3-10, 2013).

[0063] Transposon-mediated methods are not limited to those using piggyBac, and can also use transposons such as SleepingBeauty (Ivics Z, Hackett PB, Plasterk RH, Izsvak Z (1997) Cell 91: 501-510.), Frog Prince (Miskey C, Izsvak Z, Plasterk RH, Ivics Z (2003) Nucleic AcidsRes 31: 6873-6881.), Tol1 (Koga A, Inagaki H, Bessho Y, Hori H. Mol Gen Genet. 10 Dec 1995; 249(4):400-5.; Koga A, Shimada A, Kuroki T, Hori H, Kusumi J, Kyono-Hamaguchi Y, Hamaguchi S. J Hum Genet. 2007;52(7):628-35.). Published electronically on June 7, 2007), Tol2 (Koga A, Hori H, Sakaizumi M (2002) Mar Biotechnol 4: 6-11.; Johnson Hamlet MR, Yergeau DA, Kuliyev E, Takeda M, Taira M, Kawakami K, MeadPE (2006) Genesis 44:438-445.; Choo BG, Kondrichin I, Parinov S, Emelyanov A,Go W, Toh WC, Korzh V (2006) BMC Dev Biol 6: 5.).

[0064] Gene transfer via transposon-mediated methods can be a routine procedure. For example, the piggyBac transposon-mediated method can be performed as follows: a vector (transposase plasmid) carrying a gene encoding the piggyBac transposase and a structured vector (transposon plasmid) in which CAR gene expression units are sandwiched between piggyBac inverted repeat sequences are prepared, and these vectors are introduced into target cells using any of a variety of methods such as electroporation, nuclear transfection, lipid transfection, and calcium phosphate-mediated methods.

[0065] Preparation of target antigen-expressing cells

[0066] In this disclosure, in addition to CAR-expressing immune cells, normal blood cells engineered to express the target antigen are used as target antigen-expressing cells. Target antigen-expressing cells are cells that have been engineered to express part or all of the target antigen on their cell surface, enabling the CAR introduced into the CAR-expressing immune cells to bind to the target antigen. The target antigen in this disclosure refers to a target antigen recognized by the CAR, and can be a protein, glycan, or glycolipid expressed on the cell surface, enabling the CAR introduced into the immune cells to bind to it. Examples of target antigens include, for example, tumor-associated or tumor-specific antigens targeted by the aforementioned CARs, such as EPHA2, HER2, EPHB2, EPHB4, EGFR, GD2, phosphatidylinositol proteoglycan-3, HER2, 5T4, 8H9, αvβ6 integrin, B-cell maturation antigen (BCMA), B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, κ light chain, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD116, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3 / 4, FAP, FAR, FBP, fetal AchR, folate receptor α, GD2, GD3, HLA-AI MAGE. A1, HLA-A2, IL11Ra, IL13Ra2, KDR, Lambda, Lewis Y, MCSP, mesothelin, MUC1, MUC4, MUC6, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSC1, PSMA, ROR1, Sp17, survivin, TAG72, TEM1, TEM8, VEGF receptor 2, carcinoembryonic antigen, HMW-MAA, VEGF receptor, fibronectin, tendon glycoprotein, or antigens present in the extracellular matrix such as carcinoembryonic antigen (CEA) in necrotic regions of tumors or proteins containing mutations identified through genomic analysis and / or differential expression studies of tumors.

[0067] In one embodiment, the target antigen comprises or consists of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 10, 11, or 16, preferably the amino acid sequence of SEQ ID NO: 10. In another embodiment, the target antigen comprises or consists of an amino acid sequence of SEQ ID NO: 10, 11, or 16, preferably the amino acid sequence of SEQ ID NO: 10.

[0068] As described regarding CAR-expressing immune cells, target antigen-expressing cells can be prepared to express the target antigen by introducing the gene encoding the target antigen into cells using a vector having an expression unit for expressing the target antigen gene. Alternatively, target antigen-expressing cells can also be prepared by preparing mRNA of the target antigen gene and directly introducing said mRNA into cells. Additionally, target antigen-expressing cells can be prepared to express the target antigen by introducing another gene that induces target antigen expression into the cells in place of the gene encoding the target antigen, or by treating the cells with an agent that induces target antigen expression (such as a low molecular weight compound, growth factor, hormone, or cytokine). For example, treatment with sialic acid or a histone deacetylase inhibitor can prepare target antigen-expressing cells expressing GD2. In one embodiment, target antigen-expressing cells are prepared by introducing the target antigen gene into cells, and thus, the target antigen-expressing cells contain a foreign target antigen gene.

[0069] Target antigen-expressing cells can be prepared by introducing a co-stimulatory molecule gene along with a target antigen gene into cells to express the target antigen and the co-stimulatory molecule on the cell surface. Specifically, in one embodiment, the target antigen-expressing cell contains one or more genes for one or more exogenous co-stimulatory molecules. Examples of co-stimulatory molecules include CD40, CD80, 4-1BB ligand (4-1BBL), OX40, OX40L, CD52, CD54, CD70, CD58, CD86, CD95, CD252, CD275, and ligands from the integrin family (e.g., CD49a to CD49h, CD51, CD103, CD41, CD11a to 11c, ITGA9-11, CD18, CD19, CD61, ITGB4-8). In one embodiment, the co-stimulatory molecule is at least one selected from CD40, CD80, 4-1BBL, and OX40L, preferably CD80 and / or 4-1BBL, more preferably CD80 and 4-1BBL.

[0070] In one embodiment, the co-stimulatory molecule comprises or consists of an amino acid sequence having 90% or more sequence identity with any of the amino acid sequences in SEQ ID NO: 12-15. In another embodiment, the co-stimulatory molecule comprises or consists of an amino acid sequence in any of SEQ ID NO: 12-15.

[0071] Costimulatory molecular genes can be introduced as follows: by gene transfer using an expression vector containing a costimulatory molecular gene and a target antigen gene; or by gene transfer simultaneously or separately introducing an expression vector or mRNA of the costimulatory molecule and an expression vector or mRNA of the target antigen, wherein the expression vector or mRNA of the costimulatory molecule is separate from the expression vector or mRNA of the target antigen.

[0072] The cells to be processed to express the target antigen gene for preparing target antigen-expressing cells are normal blood cells (i.e., blood cells excluding cancer cells or cell lines derived therefrom), and are not particularly limited to any of these, but may be cells isolated from humans, cells differentiated from cells such as iPS cells, ES cells, or hematopoietic stem cells, and may be autologous or allogeneic cells, but preferably autologous cells. Blood cells are preferably blood cells other than red blood cells and platelets, and such blood cells include immune cells such as lymphocytes and monocytes. Target antigen-expressing cells may be immune cells such as lymphocytes to which the target antigen gene has been introduced, or cells to which target antigen expression has been induced by introducing another gene or by treating with a reagent. Target antigen-expressing cells may also be cells obtained by introducing the target antigen gene or another gene that induces target antigen expression into progenitor cells such as iPS cells, ES cells, or hematopoietic stem cells, and then differentiating said cells. In a preferred embodiment, target antigen-expressing cells are prepared from PBMCs, preferably by gene transfer into the PBMCs. In one embodiment, target antigen-expressing cells are prepared by introducing the target antigen gene into PBMCs. When using PBMCs, CAR-expressing immune cells with high proliferation efficiency and high quality can be obtained. Furthermore, when using PBMCs, it is possible to efficiently produce CAR-expressing immune cells without introducing co-stimulatory molecular genes. The PBMCs are preferably autologous PBMCs (i.e., PBMCs taken from a subject to whom a cell population produced by the methods of this disclosure is to be administered). When using autologous PBMCs, it is not necessary to remove target antigen-expressing cells to prepare cells to be administered to the patient. Further, both the target antigen-expressing cells and the CAR-expressing immune cells are preferably prepared from the same subject's PBMCs, and more preferably from autologous PBMCs.

[0073] In gene transfer to cells expressing a target antigen, gene expression can be transient or constitutive. Since appropriate and transient stimulation of the CAR and co-stimulatory molecules on the surface of CAR-expressing immune cells is sufficient, and in order to obtain a cell population with a high proportion of CAR-expressing immune cells within a relatively short time period, gene transfer preferably uses a target antigen gene expression vector intended for transient gene expression.

[0074] To obtain a cell population with a high proportion of CAR-expressing immune cells, it is preferable to treat target antigen-expressing cells to lose their proliferative capacity and then co-culture them with CAR-expressing immune cells. The treatment that causes loss of proliferative capacity is typically radiation or ultraviolet irradiation, but can also be reagent treatment. For example, radiation irradiation can be performed by irradiating with gamma rays of intensity 25 to 50 Gy for 15-30 minutes. For example, by setting the dose to 2-400 mJ / cm². 2 Preferred concentration: 6-200 mJ / cm 2 The cells are then subjected to ultraviolet irradiation. Through this treatment, the proliferation of CAR-expressing immune cells becomes dominant, and a sufficient number and quality of cells can be obtained for clinical applications.

[0075] Co-cultivation

[0076] By co-culturing CAR-expressing immune cells and target antigen-expressing cells, the CAR-expressing immune cells proliferate efficiently under antigen stimulation from the target antigen-expressing cells.

[0077] Considering cell recovery and stable transgene expression, CAR-expressing immune cells are preferably cells that have been cultured, for example, for about 8 hours to 2 weeks after the introduction of the CAR gene. Since CAR-expressing immune cells can be depleted through prolonged culture, these cells are more preferably used for co-culture within 8 hours to 1 week, 8 hours to 72 hours, or 24 hours to 72 hours after CAR gene transfer. Following processes for expressing the target antigen (such as gene transfer into cells or cell treatment with reagents), the target antigen-expressing cells should adequately express the target antigen before co-culture begins. For example, preferably, the target antigen-expressing cells have undergone the target antigen expression process 8 hours or more before the start of co-culture.

[0078] The methods of this disclosure may include preparing CAR-expressing immune cells and / or target antigen-expressing cells prior to co-culturing. For example, the methods of this disclosure may include introducing a CAR gene into immune cells and / or expressing a CAR target antigen on normal blood cells. The methods of this disclosure may further include culturing CAR-expressing immune cells and / or target antigen-expressing cells separately.

[0079] The co-cultivation period can be, but is not limited to, 1-21 days, with 1-14 days being preferred.

[0080] The ratio of CAR-expressing immune cells to target antigen-expressing cells at the start of co-culture (CAR-expressing immune cells / target antigen-expressing cells) is not particularly limited, but can be, for example, 0.05:20, preferably 0.1:10, more preferably 0.5:5, wherein the ratio is represented by the total number of cells to which the CAR or target antigen is expressed. When expressed as the number of cells in the culture medium, the cell density during co-culture can be, for example, 1 × 10⁻⁶ cells / year. 6 Cells / mL to 100 × 10⁻⁶ 6 Cells / mL.

[0081] The culture medium used for co-culturing or for preparing immune cells expressing CARs and / or target antigen-expressing cells is not particularly limited, but can be any medium used for routine cell culture, such as RPMI 1640, MEM, X-VIVIO, IMDM, DMEM, DC medium, or OptiMEM. The medium can be one to which serum (such as human serum or fetal bovine serum) is added according to standard methods, or it can be a serum-free medium. Serum-free medium is preferred because it is very safe for clinical applications, and differences in culture efficiency between serum batches are unlikely. Examples of serum-free media include TexMACS. TM (Miltenyi Biotec), AIM V® (ThermoFisher Scientific), and ALyS medium (CellScience & Technology Institute, Inc.). When using serum, autologous serum is preferred, that is, serum collected from an individual from whom CAR-expressing immune cells are derived (more specifically, from a patient to whom the cell population obtained by the production method of this disclosure is administered). The basal medium is a medium suitable for cell culture and may be TexMACS as described above. TM AIM V® or ALyS medium (Cell Science & Technology Institute, Inc.). Other culture conditions are not limited, as long as they are suitable for cell survival and proliferation, and standard conditions can be used. For example, cells can be cultured in a CO2 incubator set at 37°C (CO2 concentration: 5%).

[0082] T-cell growth factors or activators can be added to the culture medium to aid cell survival and proliferation. Examples of T-cell growth factors include IL-1, IL-2, IL-7, IL-15, and IL-21, and examples of activators include anti-CD3 antibodies and anti-CD28 antibodies. For example, IL-2, anti-CD3 antibodies, and anti-CD28 antibodies can be added to the culture medium during co-culture. These factors are not essential, and especially when using target antigen-expressing cells prepared from PBMCs, clinically applicable CAR-expressing immune cells can be obtained efficiently in a short time without the addition of anti-CD3 antibodies and / or anti-CD28 antibodies. When preparing CAR-expressing immune cells, IL-7 and / or IL-15 can also be added to the culture medium. For example, IL-7 and IL-15 can be added to the culture medium at concentrations of 5 ng / ml to 10 ng / ml, respectively. T-cell growth factors or activators can be prepared according to standard methods, and commercially available products can also be used. T-cell growth factors or activators may be of non-human animal species, but are preferably of human origin (and may be recombinant).

[0083] By co-culturing CAR-expressing immune cells and target antigen-expressing cells, it is possible to obtain a cell population containing a sufficient number and quality of CAR-expressing immune cells for clinical application. Specifically, even when the target antigen is not expressed in blood cells (e.g., in the case of tumor-associated antigens such as HER2 and EPHB4 in solid tumors), co-culturing CAR-expressing immune cells and target antigen-expressing cells provides appropriate stimulation from target antigen-expressing cells to CAR-expressing immune cells and produces a cell population containing a sufficient number of CAR-expressing immune cells with high cytotoxic activity and low depletion rate. The method of this disclosure can efficiently produce cell populations that are expected to be highly effective compared to conventional methods. For example, the cell population obtained by the production method of this disclosure can have a proportion of 20%, 30%, or 40% or more of CAR-expressing immune cells, preferably 40% or more. Additionally, the cell population obtained by the production method of this disclosure has low expression of PD-1, a depletion marker, and for example, the proportion of PD-1-expressing cells among the CAR-expressing immune cells can be less than 10%, preferably less than 5%, more preferably less than 1%. Furthermore, in the cell population obtained by the production method of this disclosure, the proportion of primary cells expressing CAR in the immune cells can be 45%, 50%, 55%, or 60% or more, preferably 60% or more.

[0084] Applications of cell populations

[0085] Cell populations containing CAR-expressing immune cells produced by the methods of this disclosure can be used to treat cancer, particularly cancers that express the target antigen of CAR-expressing immune cells. The cancer can be a solid tumor or a hematologic malignancy. Specific examples of cancer include, but are not limited to, various B-cell lymphomas (e.g., follicular malignant lymphoma, diffuse large B-cell malignant lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20-positive Hodgkin lymphoma), myeloproliferative vegetations, myelodyplastic dysplasia / myeloproliferative vegetations (CMML, JMML, CML, MDS / MPN-UC), myeloproliferative syndromes, acute myeloid leukemia, neuroblastoma, brain tumors, Ewing sarcoma, osteosarcoma, retinoblastoma, small cell lung cancer, non-small cell lung cancer, melanoma, bone and soft tissue sarcoma, kidney cancer, pancreatic cancer, malignant mesothelioma, prostate cancer, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, and colon cancer. In a preferred embodiment, the cancer is a solid tumor. Examples of solid tumors include, for example, neuroblastoma, brain tumors, Ewing sarcoma, osteosarcoma, retinoblastoma, small cell lung cancer, non-small cell lung cancer, melanoma, ovarian cancer, rhabdomyosarcoma, bone and soft tissue sarcoma, kidney cancer, pancreatic cancer, malignant mesothelioma, prostate cancer, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, and colon cancer.

[0086] The cell population of this disclosure is administered at a therapeutically effective dose, which is appropriately determined based on factors such as the subject's age, weight, and symptoms. The subjects of this disclosure are typically human, preferably cancer patients. For example, a dose of 1 × 10⁻⁶ cells per administration may be given. 4 One cell to 1×10 10 The cell population of this disclosure is administered to individuals. The route of administration is not particularly limited, but may include, for example, intravenous injection, intra-arterial injection, portal vein injection, intradermal injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. The cell population of this disclosure may be administered systemically or locally, with local administration including direct injection into a target tissue, organ, or part. The administration plan is appropriately determined based on factors such as the subject's age, weight, and symptoms, and may be a single administration or multiple administrations, either continuously or periodically.

[0087] In addition to the cell population to be administered to a subject, compositions comprising the cell population of this disclosure may contain components such as dimethyl sulfoxide (DMSO) or serum albumin for the purpose of protecting cells, antibiotics for the purpose of preventing bacterial contamination, or any of a variety of components (such as vitamins, cytokines, growth factors, steroids) for the purpose of activating, proliferating, or inducing cell differentiation. The compositions can be prepared by conventional methods.

[0088] Exemplary embodiments of the present invention are described below.

[0089] [1] A method for producing a cell population containing immune cells expressing a chimeric antigen receptor (CAR), comprising co-culturing immune cells expressing CAR and cells expressing a target antigen of CAR, wherein the immune cells expressing CAR are cells in which the CAR gene has been introduced and the target antigen expressing cells are normal blood cells that have been engineered to express the target antigen.

[0090] [2] According to the method of Project 1, the immune cells are lymphocytes.

[0091] [3] According to the method of item 1 or 2, the immune cell is a T cell.

[0092] [4] The method of any one of items 1-3, wherein the target antigen is HER2 or EPHB4.

[0093] [5] The method of any one of items 1-4, wherein the CAR-expressing immune cells are cells prepared by transferring the gene into peripheral blood mononuclear cells (PBMCs).

[0094] [6] The method of any one of items 1-5 further includes preparing immune cells expressing CAR.

[0095] [7] According to the method of Project 6, immune cells expressing CAR are prepared by gene transfer into PBMCs.

[0096] [8] According to the method of item 6 or 7, wherein immune cells expressing CAR are prepared by a piggyBac transposon-mediated method.

[0097] [9] The method of any one of items 1-8, wherein the target antigen expressing cell is a cell in which the target antigen gene has been introduced.

[0098]

[10] According to the method of any one of items 1-9, wherein the target antigen expressing cell is a cell in which one or more genes of one or more co-stimulatory molecules have been introduced.

[0099]

[11] The method of any one of items 1-10, wherein the target antigen expressing cells are cells prepared from PBMCs.

[0100]

[12] According to the method of Project 11, the target antigen-expressing cells are cells prepared by gene transfer into PBMCs.

[0101]

[13] According to the method of Project 12, the gene transfer includes the transfer of the target antigen gene.

[0102]

[14] The method according to any one of items 1-13 further includes preparing target antigen-expressing cells.

[0103]

[15] According to the method of item 14, wherein the target antigen-expressing cells are prepared from PBMCs.

[0104]

[16] According to the method of item 15, wherein the target antigen-expressing cells are prepared by gene transfer into PBMCs.

[0105]

[17] According to the method of item 16, the gene transfer includes the transfer of the target antigen gene.

[0106]

[18] The method according to any one of items 10-17, wherein the one or more co-stimulatory molecules are selected from CD40, CD80, 4-1BBL and OX40L.

[0107]

[19] A population of cells containing immune cells expressing chimeric antigen receptors (CARs) produced by the method according to any one of items 1-18.

[0108]

[20] A composition for treating cancer comprising a cell population according to item 19.

[0109]

[21] According to the composition of item 20, the cancer is a solid tumor.

[0110]

[22] A method for treating cancer, the method comprising administering to a subject a cell population according to item 19.

[0111]

[23] A method for treating cancer, comprising:

[0112] By producing cell populations according to any of the methods in items 1-18, and

[0113] The resulting cell population was then administered to the subject.

[0114]

[24] Cell populations according to item 19 for the treatment of cancer.

[0115]

[25] The cell population of Project 19 is used for the preparation of a drug for the treatment of cancer.

[0116] The invention will be further described below with reference to embodiments. However, the invention is not limited to these embodiments in any sense. Example

[0117] (Example 1)

[0118] Peripheral blood mononuclear cells (PBMCs) were isolated and collected from peripheral blood by hydrostatic centrifugation. Gene transfer was then performed via electroporation into cells measuring 15 × 10⁻⁶ cells. 6 A collection of PBMC cells was introduced into a vector expressing EPHB4-CAR ( Figure 1 , (a)) and transposase expression vector ( Figure 1 (c) To prepare T cells expressing EPHB4-CAR, wherein the EPHB4-CAR has an EPHB4 ligand (SEQ ID NO: 3) as an extracellular domain, a spacer domain (SEQ ID NO: 6), a transmembrane domain (SEQ ID NO: 8), and an intracellular signaling domain (SEQ ID NO: 9). Furthermore, 5 × 10 6 One PBMC was used to introduce a vector expressing EPHB4 (SEQ ID NO: 11) (which is the target molecule of EPHB4-CAR), CD80 (SEQ ID NO: 12), and 4-1BBL (SEQ ID NO: 13) via electroporation. Figure 2 (a) To prepare EPHB4-expressing cells transiently expressing EPHB4. The EPHB4-CAR-expressing T cells and EPHB4-expressing cells prepared in this way were cultured for 1 day after gene transfer, and then the EPHB4-expressing cells were irradiated with ultraviolet light on the first and third days of culture and subjected to 5 × 10⁻⁶ ppm. 6 EPHB4-expressing cells irradiated with ultraviolet light were separated and mixed with T cells expressing EPHB4-CAR. Cells were co-cultured with ALyS culture medium containing IL-7 (10 ng / mL) and IL-15 (5 ng / mL) in the absence of IL-2, anti-CD3 antibody, and anti-CD28 antibody, and were harvested 12 days after the start of culture.

[0119] (Comparative Example 1)

[0120] PBMCs were collected and EPHB4-CAR-expressing T cells were prepared in the same manner as in Example 1. After irradiating a portion of the collected PBMCs with ultraviolet light, viral peptides (PepTivator CMV pp65, PepTivator AdV5 Hexon, PepTivator EBV EBNA-1, and PepTivator EBV BZLF1, Miltenyi Biotec) were added to the irradiated cells to prepare feeder cells. Additionally, ultraviolet-irradiated Rh30 cells, which are cells from a cancer cell line expressing EPHB4, were prepared. Then, 10 × 10⁻⁶ cells were added to the irradiated cells. 610 T cells expressing EPHB4-CAR and 2×10 6 One feeder cell and 1×10 6 Rh30 cells were mixed and co-cultured with ALyS culture solution containing IL-7 (10 ng / mL) and IL-15 (5 ng / mL) in the presence of anti-CD3 and anti-CD28 antibodies, and were harvested 9 days after the start of culture.

[0121] (Comparative Example 2)

[0122] T cells expressing EPHB4-CAR and Rh30 cells expressing EPHB4, irradiated with ultraviolet light, were prepared in the same manner as in Comparative Example 1. Then, 10 × 10 6 One T cell expressing EPHB4-CAR and 1×10 6 Rh30 cells were mixed and co-cultured for 25 days in ALyS culture solution containing IL-7 (10 ng / mL) and IL-15 (5 ng / mL) in the presence of anti-CD3 and anti-CD28 antibodies, and the cells were collected after culture.

[0123] (Comparative Example 3)

[0124] T cells expressing EPHB4-CAR were prepared in the same manner as in Comparative Example 1. Then, 10 × 10⁻⁶ cells were... 6 T cells expressing EPHB4-CAR were cultured for 25 days in the presence of anti-CD3 and anti-CD28 antibodies with ALyS culture solution containing IL-7 (10 ng / mL) and IL-15 (5 ng / mL), and the cells were collected after culture.

[0125] (Experimental Example 1)

[0126] For the cells obtained in Example 1 and Comparative Examples 1-3, the total number of cells was counted by flow cytometry and the proportion of T cells expressing EPHB4-CAR was analyzed. The results are shown in Table 1.

[0127] [Table 1]

[0128] Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Incubation period (days) 12 9 25 25 <![CDATA[Number of cells before culturing (x 10 6 )]]> 15 10 10 10 <![CDATA[Number of cells after cultivation (x 10 6 )]]> 53 3.4 7.0 5.6 T cells expressing EPHB4-CAR (%) 47.1 1.0 0 0 <![CDATA[Number of T cells expressing EPHB4-CAR (x 10 6 )]]> 25.0 0.03 0 0

[0129] As shown in Table 1, EPHB4-CAR T cells could only be cultured under the conditions of Example 1. These results confirm that the production method, which includes co-culturing EPHB4-expressing cells (engineered to express the target antigen EPHB4 through the introduction of the EPHB4 gene) and T cells expressing EPHB4-CAR, is very useful.

[0130] (Example 2)

[0131] PBMCs were isolated and collected in the same manner as in Example 1. To a 10×10 6 Collected PBMC cells were introduced into a vector expressing HER2-CAR via electroporation. Figure 1 (b) and transposase expression vector ( Figure 1 (c) To prepare T cells expressing HER2-CAR, said HER2-CAR having an anti-HER2scFV (SEQ ID NO: 1) as an extracellular domain, and a spacer domain (SEQ ID NO: 4), a transmembrane domain (SEQ ID NO: 7), and an intracellular signaling domain (SEQ ID NO: 9) as extracellular domains. Furthermore, to 20 × 10 6 One PBMC was introduced via electroporation into an expression vector expressing HER2 (SEQ ID NO: 10) (a target molecule of HER2-CAR), CD80 (SEQ ID NO: 12), and 4-1BBL (SEQ ID NO: 13). Figure 2 (b) To prepare HER2-expressing cells transiently expressing HER2. The HER2-CAR-expressing T cells and HER2-expressing cells prepared in this way were cultured separately for 3 days after gene transfer, and then the HER2-expressing cells were irradiated with ultraviolet light and mixed with the HER2-CAR-expressing T cells. The cells were co-cultured for 11 days in ALyS medium containing IL-7 (10 ng / mL) and IL-15 (5 ng / mL) and 2% artificial serum in the absence of IL-2, anti-CD3 antibody, and anti-CD28 antibody, and were harvested 14 days after the start of culture.

[0132] (Comparative Example 4)

[0133] PBMCs were isolated and collected in the same manner as in Example 1. To a 15×10 6 Collected PBMC cells were introduced into the HER2-CAR expression vector via electroporation. Figure 1 (b) and transposase expression vector ( Figure 1 (c)) To prepare T cells expressing HER2-CAR. Furthermore, under ultraviolet irradiation of 2 × 10⁻⁶ cells... 6 After 15 × 10⁻⁶ PBMCs, viral peptides (PepTivator CMV pp65, PepTivator AdV5 Hexon, PepTivator EBV EBNA-1, and PepTivator EBV BZLF1, Miltenyi Biotec) were added to prepare feeder cells. Then, 15 × 10⁻⁶ PBMCs were... 6HER2-CAR-expressing T cells, thus prepared, were cultured for 7 days on plates in ALyS medium containing IL-7 (10 ng / mL), IL-15 (5 ng / mL), and 2% artificial serum. The target molecule HER2 protein of HER2-CAR was immobilized at 100 μg / mL on these plates and then mixed with feeder cells. The cells were co-cultured for another 7 days and harvested 14 days after the start of culture.

[0134] (Experimental Example 2)

[0135] For the cells obtained in Example 2 and Comparative Example 4, the total number of cells was counted by flow cytometry and the proportion of T cells expressing HER2-CAR was analyzed. The results are shown in Table 2.

[0136] [Table 2]

[0137] Example 2 Comparative Example 4 <![CDATA[Number of cells before cultivation (x 10 6 )]]> 10 15 <![CDATA[Number of cells after cultivation (x 10 6 )]]> 146 11 T cells expressing HER2-CAR (%) 34.1 33.7 <![CDATA[Number of T cells expressing HER2-CAR (x 10 6 )]]> 49.8 3.7

[0138] As shown in Table 2, a sufficient number of HER2-CAR-expressing T cells may be obtained in Example 2 for clinical applications, although the number of HER2-CAR-expressing T cells prepared before the start of culture is less than that in Comparative Example 4. These results confirm that the method of producing HER2-CAR-expressing T cells, which are co-cultured (engineered to express the target antigen HER2 through the introduction of the HER2 gene) and HER2-CAR-expressing T cells, is very useful.

[0139] (Example 3)

[0140] PBMCs were isolated and collected in the same manner as in Example 1. Then, they were directed to a 20×10... 6 The collected PBMC cells were introduced via electroporation into the HER2-CAR expression vector as used in Example 2. Figure 1 (b) and transposase expression vector ( Figure 1 (c) To prepare T cells expressing HER2-CAR. And, 20 × 10 6 A PBMC was introduced via electroporation into an expression vector expressing HER2 (SEQ ID NO: 10) (which is the target molecule of HER2-CAR). Figure 2(c) To prepare HER2-expressing cells transiently expressing HER2 (SEQ ID NO: 10), the HER2-CAR-expressing T cells and HER2-expressing cells prepared in this way were cultured separately for 1 day after gene transfer. The HER2-expressing cells were then irradiated with ultraviolet light and mixed with the HER2-CAR-expressing T cells. The cells were co-cultured in the presence of IL-2, anti-CD3 antibody, and anti-CD28 antibody, and were harvested 14 days after the start of culture.

[0141] (Comparative Example 5)

[0142] HER2-CAR-expressing T cells were prepared in the same manner as in Example 3. Furthermore, instead of the HER2 expression vector, 20 × 10⁶ T cells were injected into the cells. 6 One PBMC was introduced via electroporation into a vector expressing EPHB4 (SEQ ID NO: 11), CD80 (SEQ ID NO: 12), and 4-1BBL (SEQ ID NO: 13). Figure 2 (a) EPHB4-expressing cells were prepared to be used as antigen-presenting cells. The HER2-CAR-expressing T cells and EPHB4-expressing cells prepared in this manner were co-cultured in the same way as in Example 3, and the cells were harvested 14 days after the start of culture.

[0143] (Comparative Example 6)

[0144] HER2-CAR-expressing T cells were prepared in the same manner as in Example 3. The HER2-CAR-expressing T cells thus prepared were then mixed with 1×10⁻⁶ cells in the same manner as in Example 3. 6 Co-culture of U2OS cells, which are cells from a HER2-expressing cancer cell line and used as antigen-presenting cells to replace HER2-expressing cells. Cells were harvested 14 days after the start of culture.

[0145] (Example 4)

[0146] PBMCs were isolated and collected in the same manner as in Example 1. Then, they were directed to a 20×10... 6 Collected PBMC cells were introduced via electroporation into the EPHB4-CAR expression vector as used in Example 1. Figure 1 , (a)) and transposase expression vector ( Figure 1 (c) To prepare T cells expressing EPHB4-CAR. And, 20 × 10 6One PBMC was used to introduce expression vectors expressing EPHB4 (SEQ ID NO: 11) (a target molecule of EPHB4-CAR), CD80 (SEQ ID NO: 12), and 4-1BBL (SEQ ID NO: 13) via electroporation to prepare transient EPHB4-expressing cells. The EPHB4-CAR-expressing T cells and EPHB4-expressing cells prepared in this way were cultured separately for 1 day after gene transfer. Then, the EPHB4-expressing cells were irradiated with ultraviolet light and mixed with the EPHB4-CAR-expressing T cells. The cells were co-cultured in the absence of IL-2, anti-CD3 antibody, and anti-CD28 antibody, and harvested 14 days after the start of culture.

[0147] (Comparative Example 7)

[0148] T cells expressing EPHB4-CAR were prepared in the same manner as in Example 4. 20 × 10⁶ cells were then injected into the target cells. 6 One PBMC was introduced via electroporation into a vector expressing CD19 (SEQ ID NO: 16) (instead of EPHB4), CD80 (SEQ ID NO: 12), and 4-1BBL (SEQ ID NO: 13). Figure 2 (d) CD19-expressing cells were prepared to be used as antigen-presenting cells. The EPHB4-CAR-expressing T cells and CD19-expressing cells prepared in this manner were co-cultured in the same way as in Example 4, and the cells were harvested 14 days after the start of culture.

[0149] (Experimental Example 3)

[0150] For the cells obtained in Examples 3 and 4 and Comparative Examples 5-7, the total number of cells was counted by flow cytometry and the proportion of CAR-expressing T cells was analyzed. The results are shown in Table 3.

[0151] [Table 3]

[0152]

[0153] As shown in Table 3, when cancer cells expressing the HER2 target molecule are used as antigen-presenting cells (Comparative Example 6), and when the target antigen expressed by the antigen-presenting cells does not match the CAR-T cell CAR (Comparative Examples 5 and 7), it is impossible to produce a sufficient number of CAR-T cells by culture. Conversely, when cells engineered to express a target antigen that matches the CAR-T cell CAR are used as antigen-presenting cells (Examples 3 and 4), it is possible to produce a sufficient number of CAR-T cells for clinical application.

[0154] (Experimental Example 4)

[0155] To verify the characteristics of CAR-expressing immune cells produced by the production method of this disclosure, CAR-expressing immune cells are produced as follows. First, peripheral blood mononuclear cells (PBMCs) are separated and collected from peripheral blood by specific gravity centrifugation. [The following text appears to be a separate, unrelated section:] ...to 20 × 10... 6 Collected PBMC cells were introduced via electroporation into a HER2-CAR expression vector, as used in Example 2. Figure 1 (b) and transposase expression vector ( Figure 1 (c) To prepare T cells expressing HER2-CAR. Furthermore, to prepare target antigen-expressing cells, 10 × 10⁻⁶ cells were injected. 6 A PBMC was introduced via electroporation to express any one of the following: HER2 (SEQ ID NO: 10) (a target molecule of HER2-CAR) and CD80 (SEQ ID NO: 12), 4-1BBL (SEQ ID NO: 13), CD40 (SEQ ID NO: 14), and the OX40 ligand (SEQ ID NO: 15). Figure 3 , (a)) or two ( Figure 2 (b); Figure 3 The expression vector (b) was used to prepare HER2-expressing cells transiently expressing HER2. The HER2-CAR-expressing T cells and HER2-expressing cells prepared in this way were cultured for 1 day after gene transfer. Then, the HER2-expressing cells were irradiated with ultraviolet light and subjected to 10 × 10⁻⁶ ppm. 6 HER2-expressing cells and 20 × 10 6 A mixture of HER2-CAR-expressing T cells was prepared. Cells were co-cultured in the absence of IL-2, anti-CD3 antibody, and anti-CD28 antibody, and harvested 14 days after the start of culture. The number of cells obtained is shown in Table 4.

[0156] [Table 4]

[0157]

[0158] Regarding the HER2-CAR-expressing T cells thus obtained, the proportion of cells expressing any of the following was analyzed by flow cytometry: T cell marker CD3; introduced HER2-CAR; depletion marker PD-1; and markers for analyzing primordial T cells or central memory T cells, CCR7 and CD45RA. Results showed... Figure 4 and 5 middle.

[0159] Furthermore, cytotoxicity assays were performed using the obtained HER2-CAR-expressing T cells. First, U2OS cells (cells from a HER2-expressing cancer cell line) were injected at a rate of 1 × 10⁻⁶. 4 Cells / well were seeded onto a plate used for a real-time cell analyzer (xCELLigence, ACEA Bioscience, Inc.), allowing the plate to adhere. Next, individual HER2-CAR-expressing T cells were seeded onto the plate at a HER2-CAR-expressing T cell to U2OS cell ratio of 1:2, and the cells were co-cultured for 72 hours to determine the proportion of damaged U2OS cells using the real-time cell analyzer. Subsequently, the HER2-CAR-expressing T cells co-cultured with U2OS cells were added to another well of U2OS cells, and the cells were co-cultured for another 72 hours. Cytotoxicity activity was then measured three times. Results were shown... Figure 6 and 7 middle.

[0160] As in Figure 4 and 5 The results show that HER2-CAR-expressing T cells accounted for 20% or more of the total cell count obtained by the production method of this disclosure, indicating a high proportion of HER2-CAR-expressing T cells. The proportion of cells expressing PD-1 (which is a depletion marker for these T cells) was very small. Furthermore, the proportion of primordial T cells (CD45RA-positive and CCR7-positive) and central memory T cells (CD45RA-negative and CCR7-positive) in the HER2-CAR-expressing T cells was high, totaling at least 45% or more.

[0161] Furthermore, from Figure 6 and 7 The results showed that the HER2-CAR-expressing T cells of this disclosure exhibited excellent cytotoxic activity against HER2-expressing cancer cell lines, and the cells did not become depleted in two consecutive measurements of killing activity, and even had sufficient cytotoxic activity in the second measurement of killing activity.

[0162] These results show that the production method of this disclosure can efficiently produce high-quality cell populations containing a large number of cells with low expression of depletion markers and phenotypes of primordial T cells or central memory T cells, and it is particularly effective in the production of CAR-introduced immune cells for solid tumors.

[0163] (Example 5)

[0164] PBMCs were separated and collected from peripheral blood by gravity centrifugation. Then, they were transferred to 20 × 10⁻⁶ cells / mL. 6Collected PBMC cells were introduced via electroporation into a HER2-CAR expression vector, as used in Example 2. Figure 1 (b) and transposase expression vector ( Figure 1 (c) To prepare T cells expressing HER2-CAR. And, 10 × 10 6 One PBMC was introduced via electroporation to express HER2 (SEQ ID NO: 10) (a target molecule of HER2-CAR), CD80 (SEQ ID NO: 12), and 4-1BBL (SEQ ID NO: 13). Figure 2 The expression vector (d) was used to prepare HER2-expressing cells transiently expressing HER2. The HER2-CAR-expressing T cells and HER2-expressing cells prepared in this way were cultured separately for 1 day after gene transfer, and then the HER2-expressing cells were irradiated with ultraviolet light and mixed with the HER2-CAR-expressing T cells. The cells were co-cultured in the absence of IL-2, anti-CD3 antibody, and anti-CD28 antibody, and harvested 14 days after the start of culture.

[0165] (Example 6)

[0166] PBMCs were separated and collected from peripheral blood by gravity centrifugation. Then, they were transferred to 40 × 10⁻⁶ cells / mL. 6 One PBMC was introduced into the expression vector for CD19-CAR via electroporation. Figure 1 (d) and transposase expression vector ( Figure 1 (c) To prepare T cells expressing CD19-CAR, wherein the CD19-CAR has an anti-CD19scFV (SEQ ID NO: 2) as an extracellular domain, and a spacer domain (SEQ ID NO: 5), a transmembrane domain (SEQ ID NO: 7), and an intracellular signaling domain (SEQ ID NO: 9). Furthermore, 10 × 10 6 One PBMC was introduced via electroporation into expression vectors expressing CD19 (SEQ ID NO: 16) (a target molecule of CD19-CAR), CD80 (SEQ ID NO: 12), and 4-1BBL (SEQ ID NO: 13). Figure 2 (d) To prepare transient CD19-expressing cells, CD19-expressing T cells and CD19-expressing cells were prepared in this way and cultured separately for 1 day after gene transfer. The CD19-expressing cells were then irradiated with ultraviolet light and mixed with the CD19-CAR-expressing T cells. The cells were co-cultured in the absence of IL-2, anti-CD3 antibody, and anti-CD28 antibody, and harvested 14 days after the start of culture.

[0167] (Comparative Example 8)

[0168] PBMCs were separated and collected from peripheral blood by gravity centrifugation. Then, 0.125 × 10⁻⁶ ppm were added. 6 Each PBMC was stimulated with anti-CD3 and anti-CD28 antibodies and used with a retroviral vector having the same HER2-CAR expression unit as in Example 2. Figure 3 (c) Gene transfer was performed. Cells were cultured in the absence of IL-2 and harvested 14 days after the start of culture.

[0169] (Experimental Example 5)

[0170] Killing assays were performed using CAR-expressing T cells obtained in Examples 5, 6, and Comparative Example 8. First, U2OS cells (cells of a HER2-expressing cancer cell line) were seeded at 5000 cells / well on plates used in a real-time cell analyzer (xCELLigence, ACEA Bioscience, Inc.), allowing adhesion to the plate. Then, CAR-expressing T cells obtained in Examples 5, 6, and Comparative Example 8 were seeded on the plates, such that the ratio of CAR-expressing T cells to U2OS cells was 4:1. The cells were co-cultured for 100 hours, and the proportion of damaged U2OS cells was measured using a real-time cell analyzer. Results were shown... Figure 8 middle.

[0171] As in Figure 8 The results showed that, 72 hours after the start of co-culture, the HER2-CAR-expressing T cells of Example 5 damaged 95% or more of the HER2-expressing U2OS cells, while the cytotoxicity of the HER2-CAR-expressing T cells of Comparative Example 8 was approximately 65%. Furthermore, in the CD19-CAR-expressing T cells of Example 6, approximately 30% non-specific cytotoxicity against HER2-expressing U2OS cells was observed. Therefore, the CAR-dependent specific cytotoxic activity is considered weak in the CAR-T cells prepared in Comparative Example 8. From these results, it is concluded that the HER2-CAR-expressing T cells obtained by the production method of this disclosure possess strong CAR-specific cytotoxic activity.

[0172] (Example 7)

[0173] PBMCs were isolated and collected from healthy subjects. Then, they were directed to 20 × 10⁻⁶ cells / mL. 6 Collected PBMC cells were introduced via electroporation into a HER2-CAR expression vector, as used in Example 2. Figure 1 (b) and transposase expression vector ( Figure 1(c) To prepare T cells expressing HER2-CAR. And, 20 × 10 6 A PBMC was introduced via electroporation into an expression vector expressing HER2 (SEQ ID NO:10) (which is the target molecule of HER2-CAR). Figure 2 (c) To prepare HER2-expressing cells transiently expressing HER2 (SEQ ID NO:10), the HER2-CAR-expressing T cells and HER2-expressing cells prepared in this manner were cultured separately for 1 day after gene transfer. The HER2-expressing cells were then irradiated with ultraviolet light and mixed with the HER2-CAR-expressing T cells. The cells were co-cultured in the absence of IL-2, anti-CD3 antibody, and anti-CD28 antibody, and harvested 14 days after the start of culture.

[0174] (Comparative Example 9)

[0175] In the same manner as in Example 7, to 10×10 6 HER2-CAR-expressing T cells were prepared by introducing HER2-CAR expression vectors and transposase expression vectors into PBMCs via electroporation. Then, 3 × 10⁶ T cells were collected from the same healthy subjects. 6 Each PBMC was irradiated with ultraviolet light and mixed with HER2-CAR-expressing T cells thus prepared. The cells were co-cultured in the same manner as in Example 7 and harvested 14 days after the start of culture.

[0176] (Experimental Example 6)

[0177] For the cells obtained in Example 7 and Comparative Example 9, respectively, the total number of cells was counted by flow cytometry and the proportion of CAR-expressing T cells was analyzed. The results are shown in Table 5.

[0178] [Table 5]

[0179]

[0180] As shown in Table 5, when cells are simply co-cultured with PBMCs (Comparative Example 9), it is impossible to produce a sufficient number of CAR-T cells through culture. In contrast, the production method of the present invention can produce a sufficient number of CAR-T cells for clinical applications (Example 7).

[0181] (Example 8)

[0182] PBMCs were isolated and collected from healthy subjects. Then, they were directed to 17 × 10⁻⁶ cells / mL. 6 Collected PBMC cells were introduced via electroporation into a HER2-CAR expression vector, as used in Example 2. Figure 1(b) and transposase expression vector ( Figure 1 (c) To prepare T cells expressing HER2-CAR. And, 17 × 10 6 A PBMC was introduced via electroporation into an expression vector expressing HER2 (SEQ ID NO:10) (which is the target molecule of HER2-CAR). Figure 2 (c) To prepare HER2-expressing cells transiently expressing HER2 (SEQ ID NO:10), the HER2-CAR-expressing T cells and HER2-expressing cells prepared in this manner were cultured separately for 1 day after gene transfer. The HER2-expressing cells were then irradiated with ultraviolet light and mixed with the HER2-CAR-expressing T cells. The cells were co-cultured in the absence of IL-2, anti-CD3 antibody, and anti-CD28 antibody, and harvested 14 days after the start of culture.

[0183] (Comparative Example 10)

[0184] In the same manner as in Example 8, to 17×10 6 HER2-CAR-expressing T cells were prepared by introducing HER2-CAR expression vectors and transposase expression vectors into PBMCs via electroporation. Furthermore, in addition to using 17 × 10⁻⁶ PBMCs... 6 HER2-expressing cells were prepared by introducing the HER2 expression vector into K562 cells (non-HER2-expressing) in the same manner as PBMCs. The HER2-expressing cells thus prepared were irradiated with ultraviolet light and mixed with T cells expressing HER2-CAR. The cells were co-cultured in the same manner as in Example 8 and harvested 14 days after the start of culture.

[0185] (Comparative Example 11)

[0186] In the same manner as in Example 8, to 17×10 6 HER2-CAR-expressing T cells were prepared by introducing HER2-CAR expression vectors and transposase expression vectors into PBMCs via electroporation. Furthermore, in addition to using 10 × 10⁶ PBMCs... 6 HER2-expressing cells were prepared by introducing the HER2 expression vector into Rh30 cells (expressing HER2) in the same manner as PBMCs. The HER2-expressing cells thus prepared were irradiated with ultraviolet light and mixed with T cells expressing HER2-CAR. The cells were co-cultured in the same manner as in Example 8 and harvested 14 days after the start of culture.

[0187] (Experimental Example 6)

[0188] For the cells obtained in Example 8 and Comparative Examples 10 and 11, respectively, the total number of cells was counted by flow cytometry and the proportion of CAR-expressing T cells was analyzed. The results are shown in Table 6.

[0189]

[0190] As shown in Table 6, compared to the cells obtained in the comparative examples using K562 and Rh30 cells, HER2-expressing cells (prepared by expressing the target antigen HER2 in PBMCs) yielded 5-fold or more of cells. Therefore, the method of this disclosure was found to exhibit excellent cell productivity and the ability to produce a sufficient number of cells required for clinical applications. Specifically, when cells prepared from PBMCs were used as HER2-expressing cells, the proportion of CD8-positive cells was 58.7%, more than double that obtained using K562 or Rh30 cells, and the number of CAR-positive, CD8-positive cells obtained was more than 10-fold. It was found that CD8-positive CAR-T cells can be primarily produced.

[0191] Furthermore, in terms of quality, when cells prepared from PBMCs were used as HER2 expression cells, PD-1 expression in CAR-positive cells was extremely low at 0.7%, compared to when K562 or Rh30 cells were used. The proportion of CD45RA and CCR7 positive primary cells was also 62.8%, which is more than 1.5 times higher, indicating that the cells were young and not depleted and could produce high-quality CAR-T cells.

[0192] Industrial applicability

[0193] According to this disclosure, the cell proliferation rate can be increased in the production of CAR-expressing immune cells, and CAR-T cells with high cytotoxic activity can be stably produced. In particular, the production efficiency of CAR-expressing immune cells for solid tumors can be significantly improved, and therefore CAR-T cell therapy can be applied to a variety of cancer types.

Claims

1. A method for producing a cell population containing T cells expressing chimeric antigen receptors (CARs) to be administered to a subject, characterized in that, This includes co-culturing T cells expressing CAR and cells expressing the target antigen of CAR. The T cells expressing CAR are cells that have been introduced with the CAR gene and have been prepared by gene transfer of the CAR gene into autologous peripheral blood mononuclear cells (PBMCs). The cells expressing the target antigen are cells that have been introduced with the target antigen gene and have been prepared by gene transfer of the target antigen gene into autologous PBMCs.

2. The method according to claim 1, wherein the target antigen is HER2 or EPHB4.

3. The method according to claim 1 or 2, wherein the method further comprises preparing T cells expressing CAR.

4. The method according to claim 3, characterized in that, CAR-expressing T cells were prepared using a piggyBac transposon-mediated method.

5. The method according to claim 1 or 2, wherein the target antigen-expressing cell is a cell that has been introduced into one or more genes of one or more co-stimulatory molecules.

6. The method according to claim 1 or 2, wherein the method further comprises preparing target antigen-expressing cells.

7. The method according to claim 5, wherein the one or more co-stimulatory molecules are selected from CD40, CD80, 4-1BBL, and OX40L.

8. A cell population containing T cells expressing chimeric antigen receptor (CAR) produced by the method according to any one of claims 1-7.

9. A composition for treating cancer, said composition comprising the cell population according to claim 8.

10. The composition according to claim 9, wherein the cancer is a solid tumor.

Images