JP7789342B2 - Method for preparing genetically modified T cells expressing chimeric antigen receptors - Google Patents

Description

The present invention relates to a method for preparing genetically modified T cells that express chimeric antigen receptors.

Genetically modified T cell therapy (CAR therapy) using chimeric antigen receptors (CARs) is gaining clinical application. CARs typically comprise a single-chain variable region of an antibody as the extracellular domain, coupled to a transmembrane domain, CD3ζ, and the intracellular domain of a molecule that transmits costimulatory signals. Upon antigen binding according to the antibody's specificity, CAR-T cells are activated and attack target cells (e.g., cancer cells). CAR therapy offers advantages such as relatively easy cell preparation, high cytotoxic activity, and potentially durable efficacy. It is particularly promising as a new treatment for refractory or non-resistant cases. Clinical trials in Europe and the United States have demonstrated promising results in patients with chemotherapy-resistant acute lymphoblastic leukemia. CARs targeting the cell surface-expressed CD19 antigen were transfected into peripheral blood T cells collected from patients, cultured, and then infused. Remission rates of 80–90% have been reported (Non-Patent Documents 1–3). CAR therapy is attracting attention in the United States as one of the most promising treatments for intractable cancers.

Traditionally, cells used in CAR therapy (CAR-T cells) have been prepared using viral vectors. However, commonly used retroviruses frequently undergo insertional mutations into proto-oncogenes (gene therapy using hematopoietic stem cells frequently results in leukemia), posing safety concerns. Furthermore, the use of viral vectors necessitates specialized cell culture facilities, which increases the cost of treatment and creates economic issues (Non-Patent Document 4).

International Publication No. 2017/061615

Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR, Teachey DT, Chew A, Hauck B, Wright JF, Milone MC, Levine BL, June CH. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med, 368(16):1509-18. 2013 Maude SL, Frey N, Shaw PA, Aplenc R, Barrett DM, Bunin NJ, Chew A, Gonzalez VE, Zheng Z, Lacey SF, Mahnke YD, Melenhorst JJ, Rheingold SR, Shen A, Teachey DT, Levine BL, June CH, Porter DL, Grupp SA. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med, 371(16):1507-17. 2014 Lee DW, Kochenderfer JN, Stetler-Stevenson M, Cui YK, Delbrook C, Feldman SA, Fry TJ, Orentas R, Sabatino M, Shah NN, Steinberg SM, Stroncek D, Tschernia N, Yuan C, Zhang H, Zhang L, Rosenberg SA, Wayne AS, Mackall CL. T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. Lancet. 2014 Morgan RA. Faster, cheaper, safer, T-cell engineering. J Immunother, 36(1):1-2. 2013

To address the issues with conventional CAR therapy that uses viral vectors, the use of transposon technology, a non-viral vector-based gene modification technique, is being considered. While transposon technology enables persistent gene transfer, similar to viral vector technology, it has issues with lower gene transfer efficiency compared to viral vector technology, and with cell damage during gene transfer procedures (such as electroporation and its improved methods), resulting in reduced cell viability and proliferation rates. Patent Document 1 addresses these issues by co-culturing T cells (genetically modified T cells) after gene transfer with activated T cells carrying viral peptides. However, during the course of the inventor's research, it was discovered that even when this technology was applied, there were cases in which the viable cell rate after culture was significantly low.

The present invention aims to provide a technology for more reliably achieving a higher cell viability rate in the preparation of CAR-T cells while employing the transposon method for CAR gene transfer.

The present inventors conducted extensive research to solve the above-mentioned problems. As a result, they found that the above-mentioned problems can be solved by a method for preparing genetically modified T cells expressing a chimeric antigen receptor, comprising the following steps (i) to (iv): (i) preparing non-proliferating cells that retain a viral peptide antigen by culturing a monocyte-depleted T cell-containing cell population in the presence of a viral peptide antigen and treating it to eliminate its proliferation ability; (ii) obtaining genetically modified T cells into which a target antigen-specific chimeric antigen receptor gene has been introduced by transposon method from the monocyte-depleted T cell-containing cell population; (iii) mixing and co-culturing the non-proliferating cells prepared in step (i) with the genetically modified T cells obtained in step (ii); and (iv) recovering the cells after culture. Based on this finding, the present inventors conducted further research and completed the present invention. Specifically, the present invention encompasses the following aspects.

Item 1. A method for preparing genetically modified T cells that express a chimeric antigen receptor, comprising the following steps (i) to (iv):
(i) preparing non-proliferating cells that retain the viral peptide antigen, which are obtained by subjecting a monocyte-depleted T cell-containing cell population to a culture treatment in the presence of the viral peptide antigen and a treatment to eliminate proliferation ability;
(ii) obtaining genetically modified T cells into which a target antigen-specific chimeric antigen receptor gene has been introduced by a transposon method from the monocyte-depleted T cell-containing cell population;
(iii) mixing and co-culturing the non-proliferating cells prepared in step (i) with the genetically modified T cells obtained in step (ii);
(iv) recovering the cells after culturing.

Item 2. The preparation method described in Item 1, wherein a step of culturing the co-cultured cells in the presence of a T cell growth factor is carried out between steps (iii) and (iv).

Item 3. The preparation method described in Item 1 or 2, wherein the co-culture period in step (iii) is 1 to 21 days.

Item 4. The preparation method described in any one of Items 1 to 3, wherein step (iii) is carried out in the presence of a T cell growth factor.

Item 5. The preparation method described in Item 4, wherein the T cell growth factor includes IL-15.

Item 6. The preparation method described in Item 4 or 5, wherein the T cell growth factors include IL-15 and IL-7.

Item 7. The preparation method described in any one of Items 1 to 6, wherein non-proliferating cells carrying the viral peptide antigen are added during the co-culture in step (iii).

Item 8. The preparation method described in Item 7, wherein the non-proliferating cells added during the co-culture in step (iii) are cells obtained by culturing a monocyte-depleted T cell-containing cell population in the presence of a viral peptide antigen and treating it to eliminate its proliferation ability.

Item 9. The preparation method described in any one of Items 1 to 8, wherein the culture medium for the co-culture in step (iii) contains serum.

Item 10. The preparation method described in Item 9, wherein the serum concentration in the culture medium for co-culture in step (iii) is 1 to 10% (v/v).

Item 11. The preparation method described in any one of Items 1 to 10, wherein step (iii) is initiated within 24 hours of gene transfer in step (ii).

Item 12. The preparation method described in any one of Items 1 to 12, wherein the T cell-containing cell population is peripheral blood mononuclear cells (PBMCs).

Item 13. The preparation method described in any one of Items 1 to 13, wherein the T cell-containing cell population is derived from a patient receiving the genetically modified T cells.

Item 14. The preparation method described in any one of Items 1 to 13, wherein the treatment that causes loss of proliferation ability is irradiation.

Item 15. The preparation method according to any one of Items 1 to 14, wherein the transposon method is the PiggyBac transposon method.

Item 16. The preparation method according to any one of Items 1 to 15, wherein the target antigen is CD19, CD22, GD2, B7H3, BCMA, or IGF receptor.

Item 17. The preparation method described in any one of Items 1 to 16, wherein the non-proliferating cells and the genetically modified T cells are derived from the same individual.

Item 18. Genetically modified T cells expressing a chimeric antigen receptor obtained by the preparation method described in any one of Items 1 to 17.

Item 19. A cell preparation comprising a therapeutically effective amount of the genetically modified T cells described in Item 18.

Item 20. A method for treating cancer, comprising administering a therapeutically effective amount of the genetically modified T cells described in Item 18 to a cancer patient.

The present invention provides a technology for more reliably achieving a higher cell viability rate in the preparation of CAR-T cells while employing the transposon method for CAR gene transfer.

The pIRII-CAR.CD19.28z vector (SEQ ID NO: 1) is constructed with the CD19CAR gene flanked by a 5' inverted repeat (5'IR) and a 3' inverted repeat (3'IR). CD19CAR contains a leader sequence (SEQ ID NO: 2), a light chain variable region (VL) (SEQ ID NO: 3), a heavy chain variable region (VH) (SEQ ID NO: 4), an Fc region (CH2, CH3) (SEQ ID NO: 5), the transmembrane and intracellular domains of CD28 (SEQ ID NO: 6), and CD3ζ (SEQ ID NO: 7). Construction of pCMV-piggyBac vector (SEQ ID NO: 8): The piggyBac transposase gene is placed under the control of the CMV immediate early promoter. Figure 9 shows the structure of the pIRII-CAR.CD19_optimized vector (SEQ ID NO: 9), an optimized vector for CAR gene transfer. Compared to the structure of the pIRII-CAR.CD19.28z vector, the Fc region (CH2, CH3) has been deleted.

In this specification, the terms "contain" and "comprise" can be replaced with the more general terms "consist essentially of" or "consist only of."

1. Method for preparing genetically modified T cells that express a chimeric antigen receptor In one aspect, the present invention relates to a method for preparing genetically modified T cells that express a chimeric antigen receptor, the method comprising the following steps (i) to (iv): (i) preparing non-proliferating cells that retain a viral peptide antigen by subjecting a monocyte-depleted T cell-containing cell population to a culture treatment in the presence of a viral peptide antigen and a treatment to eliminate proliferation ability; (ii) obtaining, from the monocyte-depleted T cell-containing cell population, genetically modified T cells into which a target antigen-specific chimeric antigen receptor gene has been introduced by a transposon method; (iii) mixing and co-culturing the non-proliferating cells prepared in step (i) with the genetically modified T cells obtained in step (ii); and (iv) recovering the cells after culture.

This method involves the preparation of virus-specific chimeric antigen receptor gene-modified T cells (hereinafter referred to as "virus-specific CAR-T cells"). Virus-specific CAR-T cells offer important advantages for clinical application, including improved in vivo persistence due to stimulation by viral T cell receptors when used in autologous transplantation, reduced allogeneic immune response (GVHD) when used in allogeneic transplantation, enabling the production of CAR-T cells from transplant donors and potentially the formulation of CAR-T cells from third-party donors. In fact, virus-specific CAR-T cells have been reported to persist in the body for longer periods (Pule MA, et al. Nat Med. 2008 Nov;14(11):1264-70.). Furthermore, a clinical study of third-party-derived EBV-specific CTLs (Annual Review Blood 2015, published January 2015, Chugai Medical Publishing) supports the high safety of virus-specific cytotoxic T cells (CTLs).

The monocyte-depleted T cell-containing cell population used in the present invention can be obtained by subjecting a T cell-containing cell population to monocyte depletion. Preferably, PBMCs (peripheral blood mononuclear cells) collected from peripheral blood can be used as the T cell-containing cell population. Alternatively, mononuclear cells collected from peripheral blood by apheresis can also be used as the "cell population containing T cells." Preferably, the T cell-containing cell population is derived from a patient receiving chimeric antigen receptor gene-modified T cells obtained by the preparation method of the present invention. The monocyte depletion method is not particularly limited as long as it reduces the monocyte ratio (monocyte count/total cell count) in the T cell-containing cell population, and any known method can be used. Examples of monocyte depletion methods include the adhesion method, in which monocytes are adhered to a flask by centrifugation, and the elutriation method. In one embodiment of the present invention, the monocyte ratio after the monocyte depletion treatment is, for example, 1/2 or less, preferably 1/3 or less, more preferably 1/4 or less, and even more preferably 1/5 or less of the monocyte ratio before the monocyte depletion treatment. In one embodiment of the present invention, the monocyte percentage after monocyte reduction treatment is, for example, less than 8%, preferably 5% or less, and more preferably 3% or less. In one embodiment of the present invention, cases requiring monocyte reduction include, for example, cases where the monocyte percentage is 50% or more, preferably 30% or more, even more preferably 20% or more, even more preferably 15% or more, and particularly preferably 10% or more.

When monocyte-reduced PBMCs are used in each step, including steps (i) and (ii), step (i) can be performed using a portion of the monocyte-reduced PBMCs isolated from peripheral blood obtained in a single blood draw, and step (ii) can be performed using another portion (if a second-stage co-culture is performed, then another portion can be used to prepare non-proliferating cells bearing the viral peptide for use in the co-culture). This can reduce the number of blood draws required to practice the present invention, which is extremely advantageous in terms of clinical application, such as reducing the burden on patients.

In one aspect of the present invention, before subjecting a monocyte-depleted T cell-containing cell population to culture in the presence of a viral peptide antigen and a treatment to eliminate proliferation ability (before step (i)), the T cell-containing cell population can be stimulated with anti-CD3 and anti-CD28 antibodies for activation. However, since the effects of the present invention can be achieved without this stimulation, it is preferable from the perspective of simplicity to omit this stimulation. For example, T cells within the cell population can be stimulated with anti-CD3 and anti-CD28 antibodies by culturing them in a culture vessel (e.g., a culture dish) whose culture surface has been coated with anti-CD3 and anti-CD28 antibodies for, for example, 8 hours to 14 days, preferably 1 to 10 days, and more preferably 3 to 7 days. In the stimulation culture with anti-CD3 and anti-CD28 antibodies, it is preferable to culture them in the presence of T cell growth factors. This culture enhances the activity of the cells after stimulation. A culture period that is too short may result in insufficient activation, while a culture period that is too long may result in attenuation of costimulatory molecules. The cultured cells can be cryopreserved. In this case, the cells can be thawed and directly subjected to step (i) at the time of use, or they can be stimulated again with anti-CD3 and CD28 antibodies (under the same conditions as above) before being subjected to step (i). Anti-CD3 antibodies (e.g., CD3pure antibody, available from Miltenyi Biotec) and anti-CD28 antibodies (e.g., CD28pure antibody, available from Miltenyi Biotec) are commercially available and readily available. Step stimulation can also be performed using magnetic beads coated with anti-CD3 and anti-CD28 antibodies (e.g., Dynabeads T-Activator CD3/CD28, available from VERITAS). It is preferable to use the "OKT3" clone as the anti-CD3 antibody.

In step (i), "non-proliferating cells retaining viral peptide antigens (on the cell surface)" (hereinafter referred to as "viral peptide-retaining non-proliferating cells") are obtained by subjecting the cells to a culture treatment in the presence of a viral peptide antigen and a treatment to eliminate proliferation ability. The order of the culture treatment in the presence of a viral peptide antigen and the treatment to eliminate proliferation ability is not particularly limited. Therefore, the cells may be cultured in the presence of a viral peptide antigen followed by the loss of proliferation ability, or the cells may be cultured in the presence of a viral peptide antigen after the loss of proliferation ability. Preferably, the former order is adopted, since it is expected that the uptake of viral peptide antigens will be better before the loss of proliferation ability. To culture in the presence of a viral peptide antigen, for example, a medium supplemented with the viral peptide antigen may be used. Alternatively, the viral peptide antigen may be added to the medium during culture. The concentration of the viral peptide antigen added may be, for example, 0.5 μg/ml to 1 μg/ml. The culture period may be, for example, 10 minutes to 5 hours, preferably 20 minutes to 3 hours. As used herein, the term "viral peptide antigen" refers to an epitope peptide or a long peptide containing an epitope that can induce cytotoxic T lymphocytes (CTLs) specific to a particular virus. Examples of viral peptide antigens that can be used include, but are not limited to, antigenic peptides of adenovirus (AdV) (see, e.g., WO 2007015540 A1), cytomegalovirus (CMV) (see, e.g., JP 2002-255997 A, JP 2004-242599 A, JP 2012-87126 A), and Epstein-Barr virus (EBV) (see, e.g., WO 2007049737 A1, JP 2011-177487 A, JP 2006-188513 A). Viral peptide antigens can be prepared by standard methods (e.g., liquid-phase synthesis, solid-phase synthesis) based on sequence information. Some viral peptide antigens are also commercially available (e.g., provided by Medical & Biological Laboratories, Inc., Takara Bio, Miltenyi Biotec, etc.).
Although a single antigenic peptide can be used, typically, two or more antigenic peptides (antigen peptide mixtures) are used. For example, an AdV antigenic peptide mixture, a CMV antigenic peptide mixture, or an EBV antigenic peptide mixture, or a combination of two or more of these antigenic peptide mixtures (e.g., a mixture of an AdV antigenic peptide mixture, a CMV antigenic peptide mixture, and an EBV antigenic peptide mixture), is used. By using two or more antigenic peptides in combination, multiple T cells with different targets (antigen peptides) can be obtained, potentially expanding the number of patients (patients) for whom the CAR-T cells obtained by the preparation method of the present invention are effective (improving coverage). When determining which virus-derived antigenic peptide to use, it is advisable to consider the intended use of the CAR-T cells obtained by the preparation method of the present invention, specifically the disease to be treated and the patient's pathology. For example, when the purpose is to treat relapsed leukemia after hematopoietic stem cell transplantation, an EBV virus antigenic peptide mixture can be used alone or in combination with an antigenic peptide mixture of another virus. AdV antigen peptide mixtures, CMV antigen peptide mixtures, and EBV antigen peptide mixtures are commercially available (e.g., PepTivator (registered trademark) AdV5 Hexon, PepTivator (registered trademark) CMV pp65, PepTivator (registered trademark) EBV EBNA-1, and PepTivator (registered trademark) EBV BZLF1, all provided by Miltenyi Biotec, and PepMix™ Collection HCMV and PepMix™ EBV (EBNA1), all provided by JPT Peptide Technologies, and are easily available).

By undergoing a "treatment to eliminate proliferation ability," T cells that have lost their proliferation ability (non-proliferating cells) are obtained. Treatment to eliminate proliferation ability is typically performed by irradiation, but UV irradiation or chemicals can also be used. One example of radiation conditions is the use of gamma rays at an intensity of 25 Gy to 50 Gy for 15 to 30 minutes.

The viral peptide-carrying non-proliferating cells prepared in step (i) can be maintained in a solution such as a culture medium and used in step (iii). Alternatively, the prepared viral peptide-carrying non-proliferating cells can be frozen and then thawed and used as viral peptide-carrying non-proliferating cells to be added midway through the co-culture in step (iii).

In step (ii), genetically modified T cells are obtained by transposon methodology, into which a target antigen-specific chimeric antigen receptor gene has been introduced. That is, in step (ii), a target antigen-specific chimeric antigen receptor gene is introduced into a monocyte-depleted T cell-containing cell population by transposon methodology, thereby obtaining genetically modified T cells. The transposon method is a non-viral gene transfer method. A transposon is a general term for a short gene sequence that causes gene rearrangement and has been conserved throughout evolution. Gene rearrangement is caused by a pair of a gene enzyme (transposase) and its specific recognition sequence. For example, the PiggyBac transposon method can be used as a transposon method. The PiggyBac transposon method utilizes a transposon isolated from an insect (Fraser MJ et al., Insect Mol Biol. 1996 May;5(2):141-51; Wilson MH et al., Mol Ther. 2007 January;15(1):139-45), enabling highly efficient integration into mammalian chromosomes. The PiggyBac transposon method has actually been used to introduce CAR genes (see, for example, Nakazawa Y, et al., J Immunother 32:826-836, 2009; Nakazawa Y et al., J Immunother 6:3-10, 2013). The transposon method applicable to the present invention is not limited to that using PiggyBac, and examples thereof include Sleeping Beauty (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 Acids Res 31: 6873-6881), Tol1 (Koga A, Inagaki H, Bessho Y, Hori H. Mol Gen Genet. 1995 Dec 10;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. Epub 2007 Jun 1997), and other transposon methods. 7), 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, Mead PE (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), or other transposon-based methods may also be employed.

Introduction using the transposon method can be performed using standard methods, and previous literature (e.g., for the PiggyBac transposon method, see Nakazawa Y, et al., J Immunother 32:826-836, 2009; Nakazawa Y et al., J Immunother 6:3-10, 2013, as cited above, or Saha S, Nakazawa Y, Huye LE, Doherty JE, Galvan DL, Rooney CM, Wilson MH. J Vis Exp. 2012 Nov 5;(69):e4235) can be used as a reference. In a preferred embodiment of the present invention, the PiggyBac transposon method is used. Typically, the PiggyBac transposon method involves preparing a vector (transposase plasmid) carrying a gene encoding the PiggyBac transposase and a vector (transposon plasmid) carrying a gene encoding the target protein (CAR gene) sandwiched between piggyBac inverted repeat sequences, and then introducing (transfecting) these two vectors into target cells. Various transfection techniques can be used, including electroporation, nucleofection, lipofection, and the calcium phosphate method.

Cells (target cells) into which a CAR gene is introduced include CD4+CD8-T cells, CD4-CD8-T cells, T cells prepared from iPS cells, αβ-T cells, and γδ-T cells. Various cell populations can be used as long as they contain the above-mentioned T cells or progenitor cells. PBMCs (peripheral blood mononuclear cells) collected from peripheral blood are one preferred target cell. That is, in a preferred embodiment, gene transfer is performed on PBMCs that have been treated to reduce the number of monocytes. PBMCs can be prepared using standard methods. For details on PBMC preparation methods, see, for example, Saha S, Nakazawa Y, Huye LE, Doherty JE, Galvan DL, Rooney CM, Wilson MH. J Vis Exp. 2012 Nov 5;(69):e4235.

The cells that have undergone gene transfer are subjected to co-culture in step (iii), but prior to step (iii), the cells that have undergone gene transfer may be cultured in the presence of a T cell growth factor (e.g., IL-15 or IL-7). However, from the perspective of recovering cell damage caused by the gene transfer, it is preferable to start step (iii) soon after gene transfer in step (ii). Specifically, it is preferable to start step (iii) within 24 hours (more preferably within 12 hours, 6 hours, 3 hours, 1 hour, 45 minutes, or 30 minutes) of gene transfer in step (ii). From a similar perspective, it is preferable to perform step (i) before step (ii). This allows step (iii) to be started promptly after gene transfer in step (ii).

The CAR gene encodes a chimeric antigen receptor (CAR) that recognizes a specific target antigen. CARs are structures that contain a target-specific extracellular domain, a transmembrane domain, and an intracellular signaling domain for immune cell effector function. Each domain is explained below.

(a) Extracellular domain. The extracellular domain exhibits specific binding to a target. For example, the extracellular domain comprises an scFv fragment of an anti-target monoclonal antibody. Examples of the monoclonal antibody used herein include rodent (mouse, rat, rabbit, etc.) antibodies, human antibodies, and humanized antibodies. Humanized monoclonal antibodies are antibodies whose structure is similar to that of a human antibody, derived from a monoclonal antibody of another animal species (e.g., mouse or rat). These antibodies include human chimeric antibodies, in which only the constant region of an antibody has been replaced with that of a human antibody, and human CDR-grafted antibodies, in which the portions of the constant region and variable region other than the CDRs (complementarity-determining regions) have been replaced with those of a human antibody (PT. Jones et al., Nature 321, 522 (1986)). To enhance the antigen-binding activity of human CDR-grafted antibodies, improved techniques have already been developed, including methods for selecting human antibody frameworks (FRs) with high homology to mouse antibodies, methods for producing highly homologous humanized antibodies, and methods for substituting amino acids in the FR regions after grafting mouse CDRs into human antibodies (see U.S. Patent Nos. 5,585,089, 5,693,761, 5,693,762, 6,180,370, EP 451,216, EP 682,040, and JP 2,828,340, etc.), and these techniques can also be used to produce humanized antibodies.

An scFv fragment is a structure in which the light chain variable region (VL) and heavy chain variable region (VH) of an immunoglobulin are linked via a linker, and retains antigen-binding ability. For example, a peptide linker can be used as the linker. A peptide linker is a linker consisting of a peptide in which amino acids are linked in a linear chain. Typical examples of peptide linkers are linkers composed of glycine and serine (GGS linker and GS linker). The amino acids glycine and serine that make up the GGS linker and GS linker are small in size and do not easily form higher-order structures within the linker. The length of the linker is not particularly limited. For example, a linker consisting of 5 to 25 amino acid residues can be used. The length of the linker is preferably 8 to 25, and more preferably 15 to 20.

Target antigens typically used are those that are specifically expressed in tumor cells. Here, "specific expression" refers to significant or pronounced expression compared to non-tumor cells, and is not intended to be limited to antigens that are completely unexpressed in non-tumor cells. Examples of target antigens include CD19 antigen, CD20 antigen, GD2 antigen, CD22 antigen, CD30 antigen, CD33 antigen, CD44 variant 7/8 antigen, CEA antigen, Her2/neu antigen, MUC1 antigen, MUC4 antigen, MUC6 antigen, IL-13 receptor-alpha2, immunoglobulin light chain, PSMA antigen, VEGF receptor 2, BCMA, and B7-H3.

(b) Transmembrane domain The transmembrane domain is located between the extracellular domain and the intracellular signaling domain. Examples of the transmembrane domain that can be used include those of CD28, CD3ε, CD8α, CD3, CD4, and 4-1BB. An artificially constructed transmembrane domain made of a polypeptide may also be used.

(c) Intracellular Signaling Domain The intracellular signaling domain transmits signals necessary for immune cell effector function. Specifically, an intracellular signaling domain capable of transmitting signals necessary for immune cell activation upon binding of the extracellular domain to a target antigen is used. The intracellular signaling domain includes a domain for transmitting signals via the TCR complex (for convenience, referred to as the "first domain") and a domain for transmitting costimulatory signals (for convenience, referred to as the "second domain"). Intracellular domains such as CD3ζ and FcεRIγ can be used as the first domain. Preferably, CD3ζ is used. Furthermore, the intracellular domain of a costimulatory molecule is used as the second domain. Examples of costimulatory molecules include CD28, 4-1BB (CD137), CD2, CD4, CD5, CD134, OX-40, and ICOS. Preferably, the intracellular domain of CD28 or 4-1BB is used.

The manner in which the first and second domains are linked is not particularly limited, but preferably, the second domain is positioned closer to the transmembrane domain, as past examples have shown that linking CD3ζ distally results in strong costimulation. The first domain may also be constructed by linking multiple intracellular domains, either identical or different, in tandem. The same applies to the second domain.

The first and second domains may be directly linked, or a linker may be interposed between them. For example, a peptide linker can be used as the linker. A peptide linker is a linker consisting of a peptide in which amino acids are linked in a linear chain. The structure, characteristics, etc. of peptide linkers are as described above. However, a linker consisting only of glycine may also be used here. The length of the linker is not particularly limited. For example, a linker consisting of 2 to 15 amino acid residues can be used.

(d) Other factors
A leader sequence (signal peptide) is used to promote CAR secretion. For example, the leader sequence of the GM-CSF receptor can be used. It may also be advantageous to link the extracellular domain and transmembrane domain via a spacer domain. The spacer domain is used to promote binding between the CAR and the target antigen. For example, the Fc fragment of human IgG (e.g., human IgG1, human IgG4) can be used as the spacer domain. Other spacer domains that can be used include a portion of the extracellular domain of CD28 or a portion of the extracellular domain of CD8α. A spacer domain can also be inserted between the transmembrane domain and the intracellular signal domain.

There have been several reports of experiments and clinical studies using CAR (e.g., Rossig C, et al. Mol Ther 10:5-18, 2004; Dotti G, et al. Hum Gene Ther 20:1229-1239, 2009; Ngo MC, et al. Hum Mol Genet 20 (R1):R93-99, 2011; Ahmed N, et al. Mol Ther 17:1779-1787, 2009; Pule MA, et al. Nat Med 14:1264-1270, 2008; Louis CU, et al. Blood 118:6050-6056, 2011; Kochenderfer JN, et al. Blood 116:4099-4102, 2010; Kochenderfer JN, et al. Blood 119:2709-2720, 2012; Porter DL, et al. N Engl J Med 365:725-733, 2011; Kalos M, et al. Sci Transl Med 3:95ra73, 2011; Brentjens RJ, et al. Blood 118:4817-4828, 2011; Brentjens RJ, et al. Sci Transl Med 5:177ra38, 2013), and the CAR of the present invention can be constructed with reference to these reports.

In the transposon plasmid, a poly(A) addition signal sequence is placed downstream of the CAR gene. Transcription is terminated using the poly(A) addition signal sequence. Examples of poly(A) addition signal sequences that can be used include the SV40 poly(A) addition sequence and the bovine growth hormone gene poly(A) addition sequence.

Transposon-based plasmids may contain detection genes (reporter genes, cell- or tissue-specific genes, selectable marker genes, etc.), enhancer sequences, WRPE sequences, etc. Detection genes are used to determine the success or efficiency of expression cassette introduction, detect CAR gene expression or expression efficiency, select or separate cells in which the CAR gene is expressed, etc. On the other hand, the use of enhancer sequences can improve expression efficiency. Genes for detection include the neo gene, which confers resistance to neomycin; the npt gene (Herrera Estrella, EMBO J. 2 (1983), 987-995) and nptII gene (Messing & Vierra. Gene 1 9:259-268 (1982)), which confers resistance to kanamycin; the hph gene (Blochinger & Digglmann, Mol Cell Bio 4:2929-2931), which confers resistance to hygromycin; and the dhfr gene (Bourouis et al., EMBO J. 2 (7)) (all of these are marker genes), luciferase gene (Giacomin, P1. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), β-glucuronidase (GUS) gene, fluorescent protein genes such as GFP (Gerdes, FEBS Lett. 389 (1996), 44-47) and its variants (EGFP, d2EGFP, etc.) (all of these are reporter genes), and genes such as the epidermal growth factor receptor (EGFR) gene lacking an intracellular domain can be used. The detection gene is linked to the CAR gene via, for example, a bicistronic regulatory sequence (e.g., an internal ribosomal recognition sequence (IRES)) or a sequence encoding a self-cleaving peptide. An example of a self-cleaving peptide is the 2A peptide (T2A) derived from Thosea asigna virus, but this is not limiting. Known self-cleaving peptides include the 2A peptide (F2A) derived from foot disease virus (FMDV), the 2A peptide (E2A) derived from equine rhinitis A virus (ERAV), and the 2A peptide (P2A) derived from porcine teschovirus (PTV-1).

In step (iii), the non-proliferating cells (viral peptide-carrying non-proliferating cells) prepared in step (i) are mixed with the genetically modified T cells obtained in step (ii) and co-cultured. This stimulates the non-proliferating cells via costimulatory molecules and viral antigen peptides, activating the viral antigen-specific genetically modified T cells and promoting their survival and proliferation.

The ratio of the number of non-proliferating cells used in co-culture to the number of cells in the monocyte-depleted T cell-containing cell population used to obtain genetically modified T cells in step (ii) (number of non-proliferating cells/number of cells used to generate genetically modified T cells) is not particularly limited, but is, for example, 0.025 to 2. This ratio is preferably 0.05 to 1, more preferably 0.05 to 0.5, and even more preferably 0.07 to 0.2.

In this step, stimulation with anti-CD3 and anti-CD28 antibodies is generally not performed to selectively expand virus-specific CAR-T cells and to avoid strong stimulation to prevent T cell exhaustion. On the other hand, to increase cell viability and proliferation rates, it is advisable to use a culture medium supplemented with a T cell growth factor during co-culture. IL-15 is a suitable T cell growth factor. Preferably, a culture medium supplemented with IL-7 in addition to IL-15 is used. The amount of IL-15 added is, for example, 5 ng/ml to 10 ng/ml. Similarly, the amount of IL-7 added is, for example, 5 ng/ml to 10 ng/ml. T cell growth factors such as IL-15 and IL-7 can be prepared according to standard methods. Alternatively, commercially available products can be used. While this does not preclude the use of T cell growth factors from non-human animal species, human-derived T cell growth factors (which may be recombinant) are typically used. Growth factors such as human IL-15 and human IL-7 are readily available (for example, provided by Miltenyi Biotec, R&D Systems, etc.).

Although media containing serum (e.g., human serum, fetal bovine serum) may be used, serum-free media allows for the preparation of cells with the advantages of high safety during clinical application and reduced variation in culture efficiency due to differences in serum lots. Specific examples of serum-free media for T cells include TexMACS (Miltenyi Biotec) and AIM V (registered trademark) (Thermo Fisher Scientific). From the perspective of CAR-T cell proliferation, it is preferable that the culture medium for co-culture in step (iii) contains serum. In this case, the serum concentration of the culture medium for co-culture is, for example, 0.5 to 10%, preferably 1 to 10%, more preferably 1 to 7% (v/v), even more preferably 1 to 6% (v/v), even more preferably 1.5 to 5% (v/v), and particularly preferably 1.5 to 3% (v/v). When serum is used, autologous serum, i.e., serum collected from the individual from whom the genetically modified T cells obtained in step (ii) were derived (typically, a patient receiving the chimeric antigen receptor gene-modified T cells obtained by the preparation method of the present invention), is preferably used. The basal medium used may be one suitable for culturing T cells, such as the aforementioned TexMACS and AIM V (registered trademark). Other culture conditions may be any suitable for the survival and proliferation of T cells, and common culture conditions may be used. For example, culture may be performed in a _CO2 incubator (5% _CO2 ) set at 37°C.

The co-culture period in step (iii) is, for example, 1 to 21 days, preferably 5 to 18 days, and more preferably 10 to 14 days. If the culture period is too short, sufficient effects cannot be expected, while if the culture period is too long, there is a risk of a decrease in cell activity (vitality) or cell exhaustion/fatigue.

Viral peptide-bearing non-proliferating cells may be added during step (iii). Specifically, examples include adding viral peptide-bearing non-proliferating cells to the co-culture medium, or recovering cells after co-culture, mixing them with viral peptide-bearing non-proliferating cells, and then co-culturing again. These procedures may be repeated two or more times. By performing stimulation or activation multiple times using viral peptide-bearing non-proliferating cells in this way, it is possible to improve the induction rate of virus-specific CAR-T cells and increase the number of virus-specific CAR-T cells. The viral peptide-bearing non-proliferating cells used here are newly prepared cells or a portion of the cells prepared in step (i) that have been preserved. The viral peptide-bearing non-proliferating cells are preferably obtained by culturing a monocyte-depleted T cell-containing cell population in the presence of a viral peptide antigen and subjecting them to a treatment to eliminate their proliferation ability. When viral peptide-carrying non-proliferating cells are added during step (iii), the timing (or the first timing if added multiple times) is, for example, 1 to 12 days, preferably 2 to 10 days, and more preferably 3 to 9 days from the start of co-culture. Furthermore, when viral peptide-carrying non-proliferating cells are added during step (iii), the ratio of the number of non-proliferating cells added to the number of cells in co-culture (number of non-proliferating cells added/number of cells in co-culture) is not particularly limited, but is, for example, 0.025 to 2. This ratio is preferably 0.1 to 2, more preferably 0.2 to 2, and even more preferably 0.5 to 1.5.

In step (iv), the cultured cells are harvested. The harvesting procedure can be performed by conventional methods, such as pipetting or centrifugation.

In a preferred embodiment, a step of culturing the co-cultured cells in the presence of a T cell growth factor is carried out between steps (iii) and (iv). This step enables efficient expansion and also has the advantage of increasing cell viability. Non-proliferating cells carrying the viral peptide may be added during this expansion, or non-proliferating cells carrying the viral peptide may be added midway through the expansion.

IL-15, IL-7, etc. can be used as T cell growth factors. Preferably, as in step (iii), the cells are cultured in a medium supplemented with IL-15 and IL-7. The culture period is, for example, 1 to 21 days, preferably 5 to 18 days, and more preferably 10 to 14 days. If the culture period is too short, a sufficient increase in cell number cannot be expected, while if the culture period is too long, there is a risk of a decrease in cell activity (vitality) or cell exhaustion/fatigue. The cells may be passaged during the culture. The medium may also be changed as needed during the culture. For example, about 1/3 to 2/3 of the culture medium may be replaced with new medium every three days.

2. Genetically modified T cells expressing chimeric antigen receptors and uses thereof Another aspect of the present invention relates to genetically modified T cells expressing chimeric antigen receptors (hereinafter referred to as "CAR-T cells of the present invention") obtained by the preparation method of the present invention, and uses thereof. The CAR-T cells of the present invention can be used to treat, prevent, or ameliorate various diseases for which CAR therapy is considered effective (hereinafter referred to as "target diseases"). A representative target disease is cancer, but it is not limited to this. Examples of target diseases include various B-cell lymphomas (follicular lymphoma, diffuse lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20-positive Hodgkin's lymphoma, etc.), myeloproliferative neoplasms, myelodysplastic/myeloproliferative neoplasms (CMML, JMML, CML, MDS/MPN-UC), myelodysplastic syndromes, acute myeloid leukemia, neuroblastoma, brain tumors, Ewing's sarcoma, osteosarcoma, retinoblastoma, small cell lung tumor, melanoma, ovarian cancer, rhabdomyosarcoma, kidney cancer, pancreatic cancer, malignant mesothelioma, and prostate cancer. "Treatment" includes alleviating (alleviating) symptoms characteristic of or associated with a target disease, preventing or delaying the worsening of symptoms, etc. "Prevention" means preventing or delaying the onset/onset of a disease (disorder) or its symptoms, or reducing the risk of onset/onset. On the other hand, "improvement" means that a disease (disorder) or its symptoms are alleviated (reduced in severity), improved, remitted, or cured (including partial cure).

The CAR-T cells of the present invention can also be provided in the form of a cell preparation. The cell preparation of the present invention contains a therapeutically effective amount of the CAR-T cells of the present invention. For example, a single administration contains 10 ⁴ to 10 ¹⁰ cells. The cell preparation may contain other components, such as dimethyl sulfoxide (DMSO) or serum albumin for cell protection, antibiotics for preventing bacterial contamination, and various components (vitamins, cytokines, growth factors, steroids, etc.) for cell activation, proliferation, or differentiation induction.

The route of administration of the CAR-T cells or cell preparations of the present invention is not particularly limited. For example, they may be administered by intravenous injection, intra-arterial injection, intraportal vein injection, intradermal injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. Local administration may also be used instead of systemic administration. An example of local administration is direct injection into the target tissue, organ, or tissue. The administration schedule may be determined taking into account the gender, age, weight, and pathological condition of the subject (patient). In addition to a single administration, multiple administrations may be administered continuously or periodically.

The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

(1) Material
(1-1) Antibody: Anti-CD3 antibody (Miltenyi Biotec)
Anti-CD28 antibody (Miltenyi Biotec)
(1-2) Culture solution
TexMACS (serum-free medium) (Miltenyi Biotec)
(1-3) Cytokines
Recombinant human IL-7 (Miltenyi Biotec)
Recombinant human IL-15 (Miltenyi Biotec)
(1-4) Viral peptide mix
PepTivator® CMV pp65-premium grade, human (Miltenyi Biotec)
PepTivator® AdV5 Hexon-premium grade, human (Miltenyi Biotec)
PepTivator® EBV EBNA-1-premium grade, human (Miltenyi Biotec)
PepTivator® EBV BZLF1-premium grade, human (Miltenyi Biotec)
(1-5) Plasmids
pIRII-CAR.CD19.28z vector (Figure 1: expressing CAR)
pIRII-CAR.CD19_optimized vector (Figure 3: Compared to the structure of the pIRII-CAR.CD19.28z vector, the Fc region (CH2, CH3) has been deleted.)
pCMV-piggyBac vector (Figure 2: expressing piggyBac transposase)
(1-6) Cell culture container
24-well uncoated tissue culture plates (Falcon)
24-well tissue culture plate (Falcon)
G-Rex10 (Wilson Wolf).

(2) Preparation of peripheral blood mononuclear cells
(2-1) Preparation of non-monocyte-reduced peripheral blood mononuclear cells
Peripheral blood mononuclear cells were prepared from two patients with B-cell acute lymphoblastic leukemia (Patient 1 and Patient 2). Specifically, peripheral blood was collected from each patient, diluted with PBS, and dispensed into 50 ml centrifuge tubes. Ficoll-Paque Premium was layered on top and centrifuged (540-960 g, 20-30 minutes, 22°C). The mononuclear cell layer was transferred to a 50 ml centrifuge tube and diluted with PBS. The cells were then washed three times with PBS to obtain non-monocyte-reduced peripheral blood mononuclear cells (hereinafter sometimes referred to as "peripheral blood mononuclear cells (2-1)"). The monocyte percentage among the peripheral blood mononuclear cells obtained (= (monocyte cell count/total cell count) × 100) was measured by flow cytometry (using CD14 as a monocyte marker). The monocyte percentage among the peripheral blood mononuclear cells from patient 1 was 12%, and the monocyte percentage among the peripheral blood mononuclear cells from patient 2 was 11.1%.

(2-2) Preparation of Monocyte-Depleted Peripheral Blood Mononuclear Cells. The peripheral blood mononuclear cells obtained in (2-1) above were suspended in culture medium, transferred to a cell stack incubator, and incubated in a _CO2 incubator for 30 minutes. The cell suspension was collected and centrifuged (340 g, 10 minutes, 22°C). The supernatant was discarded, and the pelleted cells were resuspended in PBS to obtain monocyte-depleted peripheral blood mononuclear cells (hereinafter sometimes referred to as "peripheral blood mononuclear cells (2-2)"). The monocyte percentage (= (monocyte count/total cell count) × 100) among the obtained peripheral blood mononuclear cells was measured by flow cytometry (using CD14 as a monocyte marker). The monocyte percentage among the peripheral blood mononuclear cells from patient 1 was 1.17%, and the monocyte percentage among the peripheral blood mononuclear cells from patient 2 was 2.2%.

(3) Preparation of autologous serum Peripheral blood was collected from each of Patients 1 and 2 and collected in serum collection tubes. Serum (supernatant) was collected by centrifugation (2150 g, 5 minutes, 22°C).

(4) Preparation of Activated Lymphocytes: PBS dilutions containing anti-CD3 and anti-CD28 antibodies (anti-CD3 antibody 1 μg/mL, anti-CD28 antibody 1 μg/mL) were dispensed into 24-well tissue culture plates and placed in a _CO₂ incubator for approximately 2 hours. Peripheral blood mononuclear cells (2-1) and peripheral blood mononuclear cells (2-2) were suspended in culture medium supplemented with autologous serum (2% (v/v)) and seeded into the plates after washing the wells with PBS. Culture was initiated in a _CO₂ incubator (37°C, 5% _CO₂ ). IL-15 (5 ng/mL) was added on day 1. On day 4, the culture wells were divided and culture medium supplemented with autologous serum and IL-15 was added. On day 6, half of the culture medium was replaced. On day 7, the cultured cells were harvested and used as activated lymphocytes in the following experiments.

(5) Preparation and culture of CAR-T cells using viral peptide-activated T cell addition (Example: Peripheral blood mononuclear cells (2-2) were used)
<Day 0>
Peripheral blood mononuclear cells (2-2) were suspended in PBS to obtain a cell suspension. Viral peptides (PepTivator CMV pp65, PepTivator AdV5 Hexon, PepTivator EBV EBNA-1, and PepTivator EBV BZLF1, 0.6 nmol each) were added to the cell suspension and incubated at 37°C for 30 minutes. After washing with PBS, the cells were suspended in PBS and transferred to a separation bag. A RAD-SURE 25 Gy was attached and irradiated. After confirming irradiation with the RAD-SURE 25 Gy, the cells were transferred from the separation bag to a 50 ml centrifuge tube. After centrifugation, the supernatant was discarded. The cells were suspended in culture medium supplemented with IL-7 and IL-15 (IL-7: 10 ng/mL, IL-15: 5 ng/mL) and seeded into a 24-well plate.

Separately, the pIRII-CAR.CD19_optimized vector and pCMV-piggyBac vector were mixed with Nucleofector solution (P3 solution) to prepare P3-DNA solution (pIRII-CAR.CD19_optimized vector: 5 μg/100 μL, pCMV-piggyBac vector: 5 μg/100 μL). Peripheral blood mononuclear cells (2-2) were dispensed into 15 mL centrifuge tubes at 2.0 x ¹⁰⁷ cells per tube, diluted with PBS, centrifuged, and the supernatant discarded to form a pellet. The peripheral blood mononuclear cell pellet was suspended in 100 μL of P3-DNA solution, transferred to a cuvette, and transfected using a 4D-Nucleofector.

Within 20 minutes after gene transfer, the gene-transfected peripheral blood mononuclear cells were added to the culture vessel containing the above irradiated cells, and the two cells were mixed (mixed so that the number of peripheral blood mononuclear cells (2-2) used for gene transfer: number of irradiated cells = 10:1), and autologous serum was added to the culture medium (2% (v/v)), and co-culture was initiated in a _CO2 incubator (temperature 37°C, _CO2 5%).

<Day 4>
Half of the co-culture medium was replaced, and autologous serum, IL-7, and IL-15 were added (autologous serum: 2% (v/v), IL-7: 10 ng/mL, IL-15: 5 ng/mL).

<Day 6>
Half of the co-culture medium was replaced, and autologous serum, IL-7, and IL-15 were added (autologous serum: 2% (v/v), IL-7: 10 ng/mL, IL-15: 5 ng/mL).

<Day 7>
Activated lymphocytes ((4) above) prepared from peripheral blood mononuclear cells (2-2) were suspended in PBS to obtain a cell suspension. Viral peptides (PepTivator CMV pp65, PepTivator AdV5 Hexon, PepTivator EBV EBNA-1, and PepTivator EBV BZLF1, 0.6 nmol each) were added to the cell suspension and incubated at 37°C for 30 minutes. After washing with PBS, the cells were suspended in PBS and transferred to a separation bag. A RAD-SURE 25 Gy was attached and irradiated. After confirming irradiation with the RAD-SURE 25 Gy, the cells were transferred from the separation bag to a 50 ml centrifuge tube. The cells were centrifuged and the supernatant was discarded. The resulting cells were suspended in culture medium and seeded on G-Rex.

Meanwhile, cells were collected from the co-culture medium, suspended in the medium, and added to the culture vessel in which the irradiated cells were seeded, and the two cells were mixed (number of cells collected from the co-culture medium: number of irradiated cells = 1:1). Autologous serum, IL-7, and IL-15 were added to the culture medium (autologous serum: 2% (v/v), IL-7: 10 ng/mL, IL-15: 5 ng/mL), and co-culture was initiated in a _CO2 incubator (temperature 37°C, _CO2 5%).

<Day 10>
Half of the co-culture medium was replaced, and autologous serum, IL-7, and IL-15 were added (autologous serum: 2% (v/v), IL-7: 10 ng/mL, IL-15: 5 ng/mL). Thereafter, the same procedure was repeated depending on the color change of the culture medium.

<Day 14>
Cells were collected from the co-culture medium, and the viability (= (number of viable cells/total number of cells) × 100) and gene transfer efficiency (number of CAR-positive cells/total number of cells) were measured using flow cytometry.

(6) Preparation and culture of CAR-T cells using viral peptide-activated T cell addition (Comparative example: Peripheral blood mononuclear cells (2-1) were used)
The procedure was the same as in (5) above, except that peripheral blood mononuclear cells (2-1) were used instead of peripheral blood mononuclear cells (2-2), and activated lymphocytes prepared from peripheral blood mononuclear cells (2-1) ((4) above) were used instead of activated lymphocytes prepared from peripheral blood mononuclear cells (2-2). On day 14, cells were collected from the co-culture medium, and the viability (= (number of viable cells/total number of cells) × 100) and gene transfer efficiency (number of CAR-positive cells/total number of cells) were measured by flow cytometry.

(7) Results Compared to the use of peripheral blood mononuclear cells that had not been subjected to monocyte reduction treatment (peripheral blood mononuclear cells (2-1)) (see (6): Comparative Example above), the use of peripheral blood mononuclear cells that had been subjected to monocyte reduction treatment (peripheral blood mononuclear cells (2-2)) (see (5): Example above) resulted in a significant improvement in viability (Patient 1: 1.39% (Comparative Example) → 56.6% (Example); Patient 2: 2.97% (Comparative Example) → 85.7% (Example)). Furthermore, the gene efficiency for Patient 1 increased from 8.39% (Comparative Example) to 24.6% (Example), and for Patient 2, it also increased in the Example compared to the Comparative Example.

Claims (20)

A method for preparing a cell population containing genetically modified T cells that express a chimeric antigen receptor, the method comprising the following steps (i) to (iv):
(i) preparing non-proliferating cells that retain the viral peptide antigen, which are obtained by subjecting a T cell-containing cell population, the proportion of monocytes of which has been reduced to 1/2 or less by a monocyte reduction treatment, to a culture treatment in the presence of the viral peptide antigen and a treatment to eliminate proliferation ability;
(ii) obtaining, by transposon method, genetically modified T cells into which a target antigen-specific chimeric antigen receptor gene has been introduced, from a T cell-containing cell population in which the monocyte ratio has been reduced to 1/2 or less by monocyte reduction treatment;
(iii) mixing and co-culturing the non-proliferating cells prepared in step (i) with the genetically modified T cells obtained in step (ii);
(iv) recovering the cells after culturing. The preparation method according to claim 1, wherein a step of culturing the co-cultured cells in the presence of a T cell growth factor is carried out between steps (iii) and (iv). The preparation method according to claim 1 or 2, wherein the co-culture period in step (iii) is 1 to 21 days. The preparation method according to any one of claims 1 to 3, wherein step (iii) is carried out in the presence of a T cell growth factor. The preparation method according to claim 4, wherein the T cell growth factor includes IL-15. The preparation method according to claim 4 or 5, wherein the T cell growth factors include IL-15 and IL-7. The preparation method described in any one of claims 1 to 6, wherein non-proliferating cells carrying the viral peptide antigen are added during the co-culture in step (iii). The preparation method according to claim 7, wherein the non-proliferating cells added during the co-culture in step (iii) are cells obtained by culturing a monocyte-depleted T cell-containing cell population in the presence of a viral peptide antigen and treating it to eliminate its proliferation ability. The preparation method according to any one of claims 1 to 8, wherein the culture medium for the co-culture in step (iii) contains serum. The preparation method according to claim 9, wherein the serum concentration in the culture medium for co-culture in step (iii) is 1 to 10% (v/v). The preparation method described in any one of claims 1 to 10, wherein step (iii) is initiated within 24 hours of gene introduction in step (ii). The preparation method described in any one of claims 1 to 11, wherein the T cell-containing cell population is peripheral blood mononuclear cells (PBMCs). The preparation method described in any one of claims 1 to 12, wherein the T cell-containing cell population is derived from a patient to whom the genetically modified T cells are administered. The preparation method according to any one of claims 1 to 13, wherein the treatment that causes loss of proliferation ability is irradiation. The preparation method according to any one of claims 1 to 14, wherein the transposon method is the PiggyBac transposon method. The preparation method according to any one of claims 1 to 15, wherein the target antigen is CD19, CD22, GD2, B7H3, BCMA, or IGF receptor. The preparation method described in any one of claims 1 to 16, wherein the non-proliferating cells and the genetically modified T cells are derived from the same individual. A cell population comprising genetically modified T cells expressing a chimeric antigen receptor, obtained by the preparation method of any one of claims 1 to 17. A cell preparation comprising a therapeutically effective amount of the cell population of claim 18. The cell preparation according to claim 19, which is a cell preparation for cancer treatment.