Method for preparing genetically modified T cells expressing chimeric antigen receptors

Abstract

To provide a technique for, in preparation of a CAR-T cell, employing a transposon method in introduction of the CAR gene, and achieving greater viable cell ratio surely.SOLUTION: There is provided a preparation method for preparing a gene modified T cell which expresses a chimeric antigen receptor including the following steps i to iv of: (i) with respect to a T cell-containing cell aggregate which has been subjected to monocyte reduction processing, performing culture processing under presence of a virus peptide antigen, and processing for eliminating a proliferation potency, thereby acquiring and preparing, non-proliferative cells having the virus peptide antigen; (ii) acquiring from the T cell-containing cell aggregate which has been subjected to monocyte reduction processing, gene modified T cells into which a target antigen specific chimeric antigen receptor gene is introduced, by a transposon method; (iii) mixing the non-proliferative cells prepared in the step (i) and the gene modified T cells acquired in the step (ii), thereby co-culturing them; and (iv) collecting the cultured cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for preparing genetically modified T cells that express a chimeric antigen receptor, and other related subjects. [Background technology]

[0002] Genetically engineered 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 transduces 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 long-lasting efficacy. It is particularly promising as a novel therapeutic approach for refractory or resistant to conventional treatments. Clinical trials have been conducted in Europe and the United States for patients with chemotherapy-resistant acute lymphoblastic leukemia. CARs targeting the cell surface-expressed CD19 antigen were transfected into peripheral blood T cells collected from the patient, cultured, and then infused. Favorable results, with 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.

[0003] Traditionally, cells used in CAR therapy (CAR-T cells) have been prepared using viral vectors. However, commonly used retroviruses frequently undergo insertional mutations into protooncogenes (gene therapy using hematopoietic stem cells frequently results in leukemia), raising 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). [Prior art documents] [Patent documents]

[0004]

Patent Document 1

Non-licensed literature

[0005] [Non-licensed document 1] 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 [Non-licensed document 2] 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 [Non-licensed document 3] 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 [Non-patent document 4] Morgan RA. Faster, cheaper, safer, T-cell engineering. J Immunother, 36(1):1-2. 2013 Summary of the Invention [Problem to be solved by the invention]

[0006] To address the issues inherent in conventional CAR therapy using viral vectors, the use of transposon technology, a non-viral vector-based gene modification technique, has been investigated. While transposon technology allows for persistent gene transfer, similar to viral vector technology, it suffers from lower gene transfer efficiency compared to viral vector technology. Furthermore, the gene transfer procedure (such as electroporation and its improved methods) can damage cells, resulting in reduced cell viability and proliferation. Patent Document 1 addresses these issues by co-culturing T cells (genetically modified T cells) after gene transfer with activated T cells carrying a viral peptide. However, during the course of the present inventor's research, it was discovered that even with the application of this technology, there were cases in which the viability of cells after culture was significantly low.

[0007] Therefore, an objective of the present invention is 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 introduction. [Means for solving the problem]

[0008] 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 the cell population 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 cultured cells. Based on this finding, the present inventors conducted further research and completed the present invention. Specifically, the present invention encompasses the following aspects.

[0009] Item 1. 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 the viral peptide antigen, by culturing a monocyte-depleted T cell-containing cell population in the presence of the viral peptide antigen and subjecting the population to a treatment that causes it to lose its 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.

[0010] Item 2. The preparation method according to 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).

[0011] Item 3. The preparation method according to Item 1 or 2, wherein the co-culture period in step (iii) is 1 to 21 days.

[0012] Item 4. The preparation method according to any one of Items 1 to 3, wherein step (iii) is carried out in the presence of a T cell growth factor.

[0013] Item 5. The preparation method according to Item 4, wherein the T cell growth factor comprises IL-15.

[0014] Item 6. The preparation method according to Item 4 or 5, wherein the T cell growth factors include IL-15 and IL-7.

[0015] Item 7. The preparation method according to 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).

[0016] Item 8. The preparation method according to Item 7, wherein the non-proliferating cells added during the co-culture in step (iii) are cells obtained 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.

[0017] Item 9. The preparation method according to any one of Items 1 to 8, wherein the culture medium for the co-culture in step (iii) contains serum.

[0018] Item 10. The preparation method according to Item 9, wherein the serum concentration in the culture medium for the co-culture in step (iii) is 1 to 10% (v / v).

[0019] Item 11. The preparation method according to any one of Items 1 to 10, wherein step (iii) is initiated within 24 hours after gene transfer in step (ii).

[0020] Item 12. The preparation method according to any one of Items 1 to 12, wherein the T cell-containing cell population is peripheral blood mononuclear cells (PBMCs).

[0021] Item 13. The preparation method according to any one of Items 1 to 13, wherein the T cell-containing cell population is derived from a patient who will receive the genetically modified T cells.

[0022] Item 14. The preparation method according to any one of Items 1 to 13, wherein the treatment for eliminating proliferation ability is irradiation.

[0023] Item 15. The preparation method according to any one of Items 1 to 14, wherein the transposon method is the PiggyBac transposon method.

[0024] 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.

[0025] Item 17. The preparation method according to any one of Items 1 to 16, wherein the non-proliferative cells and the genetically modified T cells are derived from the same individual.

[0026] Item 18. Genetically modified T cells expressing a chimeric antigen receptor, obtained by the preparation method according to any one of Items 1 to 17.

[0027] Item 19. A cell preparation comprising a therapeutically effective amount of the genetically modified T cells according to Item 18.

[0028] Item 20. A method for treating cancer, comprising administering a therapeutically effective amount of the genetically modified T cells according to Item 18 to a cancer patient. [Effects of the Invention]

[0029] According to the present invention, a technique can be provided 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. [Brief explanation of the drawings]

[0030] [Figure 1] pIRII-CAR.CD19.28z vector (SEQ ID NO: 1) structure. The CD19CAR gene is 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). [Figure 2] Construction of pCMV-piggyBac vector (SEQ ID NO: 8): The piggyBac transposase gene is placed under the control of the CMV immediate early promoter (CMV immediate early promoter). [Figure 3] 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. DETAILED DESCRIPTION OF THE INVENTION

[0031] In this specification, the expressions "contain" and "comprise" can be replaced with the expressions "consist essentially of" or "consist only of", which are subordinate concepts.

[0032] 1. Method for preparing genetically modified T cells expressing chimeric antigen receptors 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 the 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.

[0033] This method relates to 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 have important advantages for clinical application, such as improved in vivo persistence due to stimulation by viral T cell receptors when used in autologous transplantation, and the potential for the production of CAR-T cells from transplant donors due to reduced allogeneic immune responses (GVHD) when used in allogeneic transplantation. Furthermore, the long-term persistence of virus-specific CAR-T cells has been reported (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).

[0034] 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. The T cell-containing cell population can preferably be PBMCs (peripheral blood mononuclear cells) collected from peripheral blood. Alternatively, monocytes collected from peripheral blood by apheresis, etc., can also be used as the "cell population containing T cells." The T cell-containing cell population is preferably derived from a patient who will receive chimeric antigen receptor gene-modified T cells obtained by the preparation method of the present invention. The monocyte depletion treatment is not particularly limited as long as it can reduce the monocyte ratio (monocyte number / total cell number) in the T cell-containing cell population, and any known method can be used. Examples of monocyte depletion treatment 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, more preferably 20% or more, even more preferably 15% or more, and particularly preferably 10% or more.

[0035] 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 viral peptides to be used 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 and the burden on patients, etc.

[0036] In one embodiment of the present invention, the monocyte-depleted T cell-containing cell population can be stimulated with anti-CD3 and anti-CD28 antibodies to activate it before being cultured in the presence of a viral peptide antigen and subjected to a treatment to eliminate proliferation ability (before step (i)). However, since the effects of the present invention can be achieved without this stimulation, it is preferable to omit this stimulation from the standpoint of simplicity. For example, T cells in 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 using 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. If the culture period is too short, sufficient activation cannot be expected, while if the culture period is too long, there is a risk of attenuation of costimulatory molecules. The cultured cells can be cryopreserved. In this case, the cells can be thawed at the time of use and directly subjected to step (i), or they can be stimulated again with anti-CD3 and CD28 antibodies (under the same conditions as above) and then subjected to step (i). Anti-CD3 antibodies (e.g., the CD3pure antibody available from Miltenyi Biotec) and anti-CD28 antibodies (e.g., the CD28pure antibody available from Miltenyi Biotec) are commercially available and easily 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). The "OKT3" clone is preferably used as the anti-CD3 antibody.

[0037] 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, for example, WO 2007015540 A1), cytomegalovirus (CMV) (see, for example, JP 2002-255997 A, JP 2004-242599 A, JP 2012-87126 A), and Epstein-Barr virus (EBV) (see, for example, 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 commercially available (e.g., provided by Medical & Biological Laboratories, Inc., Takara Bio, Miltenyi Biotec, etc.). Although a single type of antigenic peptide can be used, typically, two or more types of antigenic peptides (antigen peptide mixture) 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, thereby increasing 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, PepTivator (registered trademark) EBV BZLF1 provided by Miltenyi Biotec, and PepMix™ Collection HCMV and PepMix™ EBV (EBNA1) provided by JPT Peptide Technologies), and are easily available.

[0038] By undergoing a "treatment to eliminate proliferation ability," T cells that have lost their proliferation ability (non-proliferative cells) are obtained. The treatment to eliminate proliferation ability is typically radiation exposure, but UV exposure or chemicals may also be used. An example of radiation exposure conditions is treatment using gamma rays at an intensity of 25 Gy to 50 Gy for 15 to 30 minutes.

[0039] The viral peptide-bearing 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-bearing non-proliferating cells can be cryopreserved and thawed for use as viral peptide-bearing non-proliferating cells to be added midway through the co-culture in step (iii).

[0040] In step (ii), genetically modified T cells into which a target antigen-specific chimeric antigen receptor gene has been introduced are obtained by the transposon method. 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 the transposon method, 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 the 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), and enables 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 others. 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 transposons may also be used.

[0041] 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 repeats, and then introducing (transfecting) these two vectors into target cells. Various transfection methods can be used, including electroporation, nucleofection, lipofection, and calcium phosphate precipitation.

[0042] 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. In a preferred embodiment, gene transfer is performed on PBMCs that have been subjected to a monocyte-reducing treatment. PBMCs may be prepared by conventional methods. Regarding the preparation method of PBMCs, 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.

[0043] The cells that have undergone gene transfer manipulation are subjected to co-culture in step (iii), but prior to step (iii), the cells that have undergone gene transfer manipulation may be cultured in the presence of a T cell growth factor (e.g., IL-15 or IL-7). However, from the viewpoint of recovering cell damage caused by the gene transfer manipulation, it is preferable to start step (iii) soon after the 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) after the gene transfer in step (ii). From the same viewpoint, it is preferable to perform step (i) before step (ii). This allows step (iii) to be started promptly after the gene transfer in step (ii).

[0044] 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 described below.

[0045] (a) Extracellular domain The extracellular domain exhibits specific binding to the 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 the 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. Johnson 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 No. 451,216, EP No. 682,040, and JP No. 2,828,340, etc.), and these techniques can also be used to produce humanized antibodies.

[0046] 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 made of a peptide in which amino acids are linked in a linear chain. A typical example of a peptide linker is a linker made of glycine and serine (GGS linker or GS linker). Glycine and serine, which are amino acids 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 containing 5 to 25 amino acid residues can be used. The length of the linker is preferably 8 to 25, more preferably 15 to 20.

[0047] Typically, the target is an antigen that is specifically expressed in tumor cells. Here, "specific expression" refers to a 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, B7-H3, etc.

[0048] (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.

[0049] (c) Intracellular signaling domain The intracellular signaling domain transmits a signal required for immune cell effector function. Specifically, an intracellular signaling domain capable of transmitting a signal required 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 a signal via the TCR complex (for convenience, referred to as the "first domain") and a domain for transmitting a costimulatory signal (for convenience, referred to as the "second domain"). In addition to CD3ζ, intracellular domains such as FcεRIγ can be used as the first domain. CD3ζ is preferably 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.

[0050] The manner in which the first and second domains are linked is not particularly limited, but preferably, the second domain is located closer to the transmembrane domain, since previous studies have shown that linking CD3ζ distally resulted in strong costimulation. The first domain may also be constructed by linking multiple identical or different intracellular domains in tandem. The same applies to the second domain.

[0051] The first domain and the second domain 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 made of a peptide in which amino acids are linked in a linear chain. The structure, characteristics, etc. of the peptide linker are as described above. However, the linker used here may be one made up of only glycine. The length of the linker is not particularly limited. For example, a linker with 2 to 15 amino acid residues can be used.

[0052] (d) Other elements 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 and a portion of the extracellular domain of CD8α. A spacer domain can also be provided between the transmembrane domain and the intracellular signal domain.

[0053] 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 5:177 ra38, 2013), and the CAR of the present invention can be constructed with reference to these reports.

[0054] In the transposon-based 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.

[0055] The transposon-based plasmid may contain a detection gene (e.g., a reporter gene, a cell- or tissue-specific gene, or a selection marker gene), an enhancer sequence, a WRPE sequence, etc. The detection gene is used to determine the success or efficiency of expression cassette introduction, detect CAR gene expression or determine expression efficiency, select or separate cells in which the CAR gene is expressed, etc. On the other hand, the use of an enhancer sequence improves 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, etc.; the hph gene (Blochinger & Digglmann, Mol Cell Bio 4:2929-2931), which confers resistance to hygromycin; the dhfr gene (Bourouis et al., EMBO J. 2 (7)), which confers resistance to methatrexate (all of these are marker genes); and the 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 epidermal growth factor receptor (EGFR) genes lacking the intracellular domain can be used. The detection gene is linked to the CAR gene, for example, via 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 is not limited to this. Known self-cleaving peptides include the 2A peptide (F2A) derived from hoof disease virus (FMDV), the 2A peptide (E2A) derived from equine rhinitis virus A (ERAV), and the 2A peptide (P2A) derived from porcine teschovirus (PTV-1).

[0056] 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.

[0057] The ratio between the number of non-proliferating cells used in the co-culture and the number of cells in the monocyte-depleted T cell-containing cell population used to obtain the genetically modified T cells in step (ii) (number of non-proliferating cells / number of cells used to produce the 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.

[0058] In this step, stimulation with anti-CD3 antibodies 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. Meanwhile, to increase cell viability and proliferation rates, a culture medium supplemented with a T cell growth factor is preferably used 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 the use of T cell growth factors from non-human animal species is not excluded, 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.).

[0059] Although serum-added media (e.g., human serum, fetal bovine serum) may be used, serum-free media enable the preparation of cells with the advantages of high safety for 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 may be any suitable medium for culturing T cells, such as the above-mentioned TexMACS and AIM V (registered trademark). Other culture conditions may be any suitable medium for the survival and proliferation of T cells, and general culture conditions may be used. For example, the cells may be cultured in a CO2 incubator (CO2 concentration 5%) set at 37°C.

[0060] The period of co-culture 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, whereas if the culture period is too long, there is a risk of a decrease in cell activity (vitality) and cell exhaustion / fatigue.

[0061] The viral peptide-bearing non-proliferating cells may be added during step (iii). Specifically, examples include adding the viral peptide-bearing non-proliferating cells to the culture medium for co-culture, or recovering the cells after co-culture, mixing them with the viral peptide-bearing non-proliferating cells, and then co-culture again. These procedures may be repeated two or more times. By performing stimulation or activation multiple times using the viral peptide-bearing non-proliferating cells in this manner, 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 cells obtained by culturing a monocyte-depleted T cell-containing cell population in the presence of a viral peptide antigen and subjecting it to a treatment to eliminate proliferation ability. When viral peptide-bearing non-proliferating cells are added during step (iii), the timing (the first time when adding 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. When viral peptide-bearing 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.

[0062] In step (iv), the cultured cells are collected by a conventional method, such as by pipetting or centrifugation.

[0063] 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 during the expansion.

[0064] Examples of T cell growth factors that can be used include IL-15 and IL-7. 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) and cell exhaustion / fatigue. The cells may be passaged during the culture. The medium may be changed as needed during the culture. For example, about 1 / 3 to 2 / 3 of the culture medium is changed with new medium every three days.

[0065] 2. Genetically modified T cells expressing chimeric antigen receptors and their uses A further 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" refers to the alleviation (reduction in severity), improvement, remission, or cure (including partial cure) of a disease (disorder) or its symptoms.

[0066] 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, for a single administration, 10 4 ~10 pieces 10 The cell preparation may contain other components, such as dimethyl sulfoxide (DMSO) or serum albumin for the purpose of protecting the cells, antibiotics for the purpose of preventing bacterial contamination, and various components (vitamins, cytokines, growth factors, steroids, etc.) for the purpose of activating, proliferating, or inducing differentiation of the cells.

[0067] 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, intraarterial injection, intraportal vein injection, intradermal injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. Local administration may 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 sex, age, weight, pathological condition, etc. of the subject (patient). In addition to a single administration, multiple administrations may be administered continuously or periodically. [Example]

[0068] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0069] (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) Plasmid 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).

[0070] (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, the procedure was as follows: Peripheral blood was collected from each patient, diluted with PBS, and dispensed into 50 ml centrifuge tubes. Ficoll-Paque Premium was layered on top, followed by centrifugation (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 peripheral blood mononuclear cells that had not been subjected to monocyte reduction treatment (hereinafter sometimes referred to as "peripheral blood mononuclear cells (2-1)"). The monocyte percentage (= (monocyte cell 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 12%, and the monocyte percentage among the peripheral blood mononuclear cells from patient 2 was 11.1%.

[0071] (2-2) Preparation of monocyte-reduced 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 left to stand 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 suspended in PBS to obtain monocyte-reduced 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%.

[0072] (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).

[0073] (4) Preparation of activated lymphocytes A PBS solution containing anti-CD3 and anti-CD28 antibodies (anti-CD3 antibody 1 μg / mL, anti-CD28 antibody 1 μg / mL) was dispensed into a 24-well tissue culture plate 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 plate 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.

[0074] (5) Preparation and culture of CAR-T cells using viral peptide-added activated T cells (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.

[0075] Meanwhile, 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). 2.0 x 10 peripheral blood mononuclear cells (2-2) were placed in a 15 mL centrifuge tube. 7 The cells were diluted with PBS, centrifuged, and the supernatant was discarded to form a pellet. The pellet of peripheral blood mononuclear cells was suspended in 100 μL of P3-DNA solution, transferred to a cuvette, and transfected with a gene transfer device (4D-Nucleofector).

[0076] Within 20 minutes after gene transfer, the gene-transfected peripheral blood mononuclear cells were added to the culture vessel containing the 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%).

[0077] <Day 4> Half of the culture medium for the co-culture 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).

[0078] <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).

[0079] <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.

[0080] 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 types of 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%).

[0081] <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.

[0082] <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.

[0083] (6) Preparation and culture of CAR-T cells using viral peptide-added activated T cells (Comparative example: Peripheral blood mononuclear cells (2-1) were used) The same procedure as in (5) above was repeated, 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.

[0084] (7) Results Compared to the case where peripheral blood mononuclear cells that had not been subjected to monocyte reduction treatment (peripheral blood mononuclear cells (2-1)) were used ((6): Comparative Example above), the viability of monocyte-reduced peripheral blood mononuclear cells (peripheral blood mononuclear cells (2-2)) ((5): Example above) was significantly improved (Patient 1: 1.39% (Comparative Example) → 56.6% (Example), Patient 2: 2.97% (Comparative Example) → 85.7% (Example)). Furthermore, gene efficiency for Patient 1 increased from 8.39% (Comparative Example) to 24.6% (Example), and for Patient 2, it was also improved in the Example compared to the Comparative Example.

Claims

1. 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.

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

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

4. The 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.

5. The preparation method according to claim 4, wherein the T cell growth factor comprises IL-15.

6. The preparation method according to claim 4 or 5, wherein the T cell growth factors include IL-15 and IL-7.

7. The preparation method according to 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).

8. The preparation method according to claim 7, wherein the non-proliferating cells added during the co-culture in step (iii) are cells obtained 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 the proliferation ability.

9. 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.

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

11. The preparation method according to any one of claims 1 to 10, wherein step (iii) is initiated within 24 hours after the gene transfer in step (ii).

12. The preparation method according to any one of claims 1 to 11, wherein the T cell-containing cell population is peripheral blood mononuclear cells (PBMCs).

13. The method of any one of claims 1 to 12, wherein the T cell-containing cell population is derived from a patient who will receive the genetically modified T cells.

14. The method according to any one of claims 1 to 13, wherein the treatment for eliminating proliferation ability is irradiation.

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

16. 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.

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

18. 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.

19. A cell preparation comprising a therapeutically effective amount of the cell population of claim 18.

20. The cell preparation according to claim 19, which is a cell preparation for cancer treatment.

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