Method for preparing genetically modified t cell expressing chimeric antigen receptor

Abstract

To provide a technique for more surely achieving a higher viable cell rate while adopting a transposon method for CAR gene transfer in preparation of CAR-T cells.SOLUTION: Disclosed is a method for preparing a genetically modified T cell that expresses a chimeric antigen receptor, which comprises the following steps (i) to (iv): (i) a step of culturing the genetically modified T cell in a monocyte-depleted T cell-containing cell population; A step of preparing non-proliferative cells holding a viral peptide antigen, which are obtained by performing a culture treatment in the presence of a viral peptide antigen and a treatment for losing proliferation ability; (ii) a step of obtaining genetically modified T cells in which a target antigen-specific chimeric antigen receptor gene is introduced by a transposon method from a T cell-containing cell population subjected to a monocyte-reducing treatment; (iii) a step of mixing and co-culturing the non-proliferative cells prepared in step (i) and the genetically modified T cells obtained in step (ii); and (iv) a step of collecting cells after culture.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 more. [Background technology]

[0002] Genetically modified T cell therapy (CAR therapy) using chimeric antigen receptors (hereinafter referred to as "CARs") is beginning to be applied clinically. CARs are typically single-stranded antibodies. The chain variable region is the extracellular domain, which is followed by the transmembrane region, CD3ζ, and the co-stimulatory signal. CAR-T cells have a structure that links the intracellular domains of molecules that bind to the antigen according to the specificity of the antibody. CAR-T cells are activated by binding to the antigen and damage the target cell (cancer cell, etc.). CAR therapy has advantages such as the relative ease of cell preparation, high cytotoxic activity, and the possibility of long-lasting effects, and is expected to be a new treatment method, especially for intractable cases or cases that are resistant to conventional treatments. In fact, for patients with chemotherapy-resistant acute lymphoblastic leukemia, CARs against the CD19 antigen expressed on the cell surface have been administered to patients. Clinical trials are being conducted in Europe and the United States to introduce genes into peripheral blood T cells collected from the patient, culture them, and then infuse them. Favorable results have been reported, with remission rates of 80-90% (Non-patent documents 1-3). CAR therapy is It is attracting attention 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). [Primary Technology Documents] [Chartered 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 solve the problems of conventional CAR therapy using viral vectors, The use of the transposon method, which is one of the gene modification techniques using viral vectors, is being investigated. The transposon method allows for persistent gene transfer, similar to the viral vector method, but has problems such as lower gene transfer efficiency compared to the viral vector method, and cell damage caused by gene transfer procedures (electroporation and its improved methods, etc.), resulting in reduced cell viability and cell proliferation rates. Patent Document 1 addresses these problems by transforming T cells (genetically modified T cells) after gene transfer procedures into activated T cells carrying viral peptides. However, in the course of the research conducted by the present inventors, it was found that even when this technology was applied, there were cases in which the viability of the cells after culture was extremely 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 have conducted extensive research to solve the above problems. As a result, they have discovered a method for preparing genetically modified T cells that express a chimeric antigen receptor, comprising the following steps (i) to (iv): (i) Monocyte-depleted T cell-containing cell populations were cultured in the presence of viral peptide antigens. (ii) preparing non-proliferating cells carrying viral peptide antigens, which are obtained by subjecting the cells to a culture treatment and a treatment to eliminate proliferation ability; and (iii) preparing non-proliferating cells carrying viral peptide antigens, which are obtained by subjecting the cells to a culture treatment to eliminate proliferation ability and which contain monocyte-depleted T cells. (iii) obtaining genetically modified T cells into which a target antigen-specific chimeric antigen receptor gene has been introduced by transposon method from the cell population; mixing and co-culturing the proliferative cells with the genetically modified T cells obtained in step (ii); It was found that the above-mentioned problem can be solved by (iv) a step of recovering the cells after culture. Based on this finding, the present inventors conducted further research and completed the present invention. That is, the present invention includes 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) monocyte-depleted T cell-containing cell populations were assayed for the presence of viral peptide antigens. providing non-proliferating cells that retain the viral peptide antigen, the non-proliferating cells being obtained by culturing the cells under the conditions described above and then treating the cells to lose their proliferation ability; (ii) from a monocyte-depleted T cell-containing cell population by transposon method; obtaining genetically modified T cells into which a target antigen-specific chimeric antigen receptor gene has been introduced; (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. Between step (iii) and step (iv), the cells after co-culture are expanded into T cells. Item 1. A preparation method according to Item 1, which comprises culturing the cells in the presence of growth factors.

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

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

[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 non-proliferating cells added during the co-culture in step (iii) are cultured in the presence of a viral peptide antigen to a T cell-containing cell population that has been subjected to a monocyte reduction treatment. Item 8. The method for preparing cells according to Item 7, wherein the cells are obtained by subjecting the cells to a treatment that causes them to lose their 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 non-proliferating cells and the genetically modified T cells are derived from the same individual. The preparation method according to any one of the above.

[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. Administering a therapeutically effective amount of the genetically modified T cells described in Item 18 to a cancer patient. Cancer treatment, including steps. [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 provides 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) isolating a transposon from the monocyte-depleted T cell-containing cell population; By this method, genetically modified T cells into which a target antigen-specific chimeric antigen receptor gene has been introduced are obtained. (iii) mixing the non-proliferating cells prepared in step (i) with the genetically modified T cells obtained in step (ii) and co-culturing them; (iv) recovering the cells after culturing. The step of

[0033] The method involves the use of virus-specific chimeric antigen receptor gene-modified T cells (hereinafter referred to as "virus This paper describes a method for preparing virus-specific CAR-T cells (referred to as "specific CAR-T cells"), which are expected to be persistent in the body due to stimulation from the viral T cell receptor when used for autologous transplantation. In addition, when used in allogeneic transplantation, it is possible to produce CAR-T cells from the transplant donor by reducing allogeneic immune responses (GVHD), and it is also possible to formulate CAR-T cells from third-party donors. It has been reported that EBV-specific CTL persist in the body for a longer period of time (Pule MA, et al. Nat Med. 2008 Nov;14(11):1264-70.). In addition, a report on a clinical study of third-party derived EBV-specific CTL (Annual Review Blood 2015, published January 2015, Chugai Medical Publishing) showed that the virus-specific cytotoxic T lymphocytes (CTLs) It has been proven to be highly safe.

[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. Alternatively, PBMCs (peripheral blood mononuclear cells) collected from peripheral blood can be used. In addition, mononuclear cells collected from peripheral blood by apheresis are referred to as "cell populations containing T cells" here. The T cell-containing cell population can be used as a T cell-containing cell population obtained by the preparation method of the present invention. Preferably, the T cells are derived from a patient who will receive the modified T cells from the LA antigen receptor gene. The monocyte reduction treatment reduces the proportion of monocytes (monocytes / total cells) in a T cell-containing cell population. The method is not particularly limited as long as it is a treatment that can reduce the monocyte count, and known methods can be used. Examples of monocyte reduction treatment that can be used include an adhesion method in which monocytes are adhered to a flask by centrifugation, and an elutriation method. In one embodiment of the present invention, the monocyte percentage after monocyte reduction treatment is, for example, 1 / 2 or less, preferably 1 / 2 or less, of the monocyte percentage before monocyte reduction treatment. is 1 / 3 or less, more preferably 1 / 4 or less, and even more preferably 1 / 5 or less. In the method, the monocyte percentage after the monocyte reduction treatment is, for example, less than 8%, preferably 5% or less, more preferably In one embodiment of the present invention, cases requiring monocyte reduction include: Examples of such cases include those in which the monocyte ratio 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 is treated with viral peptides. Before being subjected to a culture treatment in the presence of a T cell antigen and a treatment to eliminate proliferation ability (before step (i)), the T cell-containing cell population can be activated by stimulation with an anti-CD3 antibody and an anti-CD28 antibody. However, since the effects of the present invention can be obtained without such stimulation, it is preferable from the viewpoint of simplicity not to perform such stimulation. Such stimulation can be performed, for example, with an anti-CD3 antibody and an anti-CD28 antibody. In a culture vessel (e.g., a culture dish) whose culture surface is coated with anti-CD28 antibody, for example, for 8 hours to 14 days, The cells are cultured for preferably 1 to 10 days, more preferably 3 to 7 days. The T cells in the population can be stimulated with anti-CD3 and anti-CD28 antibodies. In the stimulation culture with anti-CD3 and anti-CD28 antibodies, it is preferable to culture in the presence of T cell growth factors. This culture enhances the activity of the cells after the stimulation treatment. If the culture period is too short, sufficient activation cannot be expected, and if the culture period is too long, there is a risk of weakening of costimulatory molecules. The cells after culture can be temporarily frozen and stored. In this case, the cells can be thawed at the time of use and subjected to step (i) as is, or they can be stimulated again with anti-CD3 and CD28 antibodies (under the conditions similar to those described above) and then subjected to step (i). Anti-CD3 antibodies (for example, the CD3pure antibody from Miltenyi Biotec) and anti-CD28 antibodies (for example, the CD28pure antibody 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 (for example, Dynabeads T-Activator CD3 / CD28 from VERITAS). It is preferable to use the "OKT3" clone 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 lose their proliferation ability. The order of the culture treatment in the presence of a viral peptide antigen and the treatment to lose their proliferation ability is not particularly limited. Therefore, the proliferation ability may be lost after culture in the presence of a viral peptide antigen, or the proliferation ability may be lost before culture in the presence of a viral peptide antigen. 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 to which the viral peptide antigen has been added 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. The term "viral peptide antigen" as used herein refers to a The term "virus antigen" refers to an epitope peptide or a long peptide containing an epitope that can induce cytotoxic T lymphocytes (CTL) specific to a particular virus. Examples of viral peptide antigens include, but are not limited to, antigen peptides of adenovirus (AdV). (See, for example, WO 2007015540 A1), antigenic peptides of cytomegalovirus (CMV) ( For example, see Japanese Patent Application Laid-Open Nos. 2002-255997, 2004-242599, and 2012-87126), antigen peptides of Epstein-Barr virus (EBV) (for example, see WO 2007049737 A1, (See Patent Application Publication No. 2011-177487 and Patent Publication No. 2006-188513.) 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 (for example, provided by Medical & Biological Laboratories, Inc., Takara Bio, Miltenyi Biotec, etc.). Although it is possible to use one type of antigen peptide, usually two or more types of antigen peptides (antigen peptide mixture) are used. For example, an AdV antigen peptide mixture, a CMV antigen peptide mixture, an EBV antigen peptide mixture, or a combination of two or more of these antigen peptide mixtures is used. Combinations (e.g., AdV antigen peptide mixture, CMV antigen peptide mixture and EBV antigen By using two or more antigen peptides in combination, By this method, multiple T cells with different targets (antigen peptides) can be obtained. It is hoped that the number of patients for whom CAR-T cells obtained through this method are effective will increase (improved coverage). When deciding which antigen peptide to use, the use of the CAR-T cells obtained by the preparation method of the present invention, specifically the disease to be treated and the pathological condition of the patient, should be taken into consideration. For example, in the case of the treatment of relapsed leukemia after hematopoietic stem cell transplantation, the EBV virus antigen peptide mixture may be used alone or in combination with antigen peptide mixtures of other viruses. It is recommended to use them in combination. AdV antigen peptide mixture, CMV antigen peptide mixture, EBV antigen peptide The mixtures are commercially available (for example, PepTivator® AdV5 Hexon, PepTivator® CMV pp65, PepTivator® EBV EBNA-1, PepTivator® EBV BZLF1, and JPT Peptide Technologies, all from Miltenyi Biotec). PepMix™ Collection HCMV, PepMix™ EBV (EBNA1), etc. provided by the Company are readily available.

[0038] By undergoing "treatment to eliminate proliferation ability," T cells that have lost their proliferation ability (non-proliferative The treatment to eliminate proliferation ability is typically radiation, but UV irradiation or chemicals may also be used. An example of radiation 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. Target antigen-specific T cells were transposon-mediated expression of T cell-containing cells in a depleted population. A target chimeric antigen receptor gene is introduced into the target T cell, thereby obtaining a genetically modified T cell. The transposon method is a non-viral gene transfer method. Transposons are a collective term for short genetic sequences that have been conserved throughout evolution and cause gene rearrangement. Gene rearrangement occurs when a genetic enzyme (transposase) pairs with its specific recognition sequence. Examples of transposon methods include the PiggyBac transposon method, which utilizes a transposon isolated from insects (Fraser MJ et al., Insect Mol Biol. 1996 May;5(2):141-51; Wilson MH et al., Mol Ther. 2007 Jan;15(1):139-45), enabling highly efficient integration into mammalian chromosomes. The PiggyBac transposon method has actually been used to introduce a CAR gene (see, for example, Nakazawa Y, et al., J Immunother 32:826-836, 2009; Nakazawa Y et al., J Immunother 6:3-10, 2013, etc.). Transposon methods applicable to the present invention are not limited to those using PiggyBac, and include, for example, those using Sleeping Beauty (Ivics Z, Hackett PB, Plasterk RH, Izsvak Z (1997) Cell 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 7.), Tol2(Koga A, Hori H, Sakaizumi M (2002) Mar Biotechnol 4: 6-11.;Johnson Hamlet MR, Yergeau DA, Kuliyev E, Alternatively, a method using a transposon, such as those described in 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, may be employed.

[0041] The transposon-based introduction procedure can be carried out by a conventional method, and the method is described in previous publications (for example, the PiggyBac transposon method, see Nakazawa Y, et al., J Immunother 32:826-836, 2009, cited above). , Nakazawa Y et al., J Immunother 6:3-10, 2013, or Saha S, Nakazawa Y, Huye LE, Doherty JE, Galvan DL, Rooney CM, Wilson MH. J Vis Exp. 2012 Nov 5;(69):e4235) are useful references. In a preferred embodiment of the present invention, the PiggyBac transposon method is employed. Typically, the PiggyBac transposon method uses a vector (transposase plasmid) carrying a gene encoding the PiggyBac transposase and a vector ( These two vectors are then introduced (transfected) into target cells. Various methods can be used for transfection, including electroporation, nucleofection, lipofection, and calcium phosphate transfection.

[0042] Cells (target cells) into which the CAR gene is introduced include CD4-positive CD8-negative T cells, CD4-negative CD8-positive 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 precursor cells. PBMCs (peripheral blood mononuclear cells) collected from peripheral blood are one of the preferred target cells. That is, in a preferred embodiment, gene transfer is performed on PBMCs that have been subjected to a monocyte reduction treatment. PBMCs may be prepared by a conventional method. For details on the method for preparing PBMCs, see, for example, Saha S, Nakazawa et al. 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) as soon as possible after the gene transfer in step (ii). Specifically, for example, 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 carry out step (i) before step (ii). This allows step (iii) to be started promptly after gene transfer in step (ii).

[0044] The CAR gene encodes a chimeric antigen receptor (CAR) that recognizes a specific target antigen. is a structure that contains a target-specific extracellular domain, a transmembrane domain, and an intracellular signaling domain for immune cell effector function. Each domain is explained 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. The monoclonal antibody used here may be, for example, a rodent antibody (mouse, rat, rabbit, etc.), a human antibody, or a humanized antibody. Human A chimeric monoclonal antibody is an antibody in which the structure of a monoclonal antibody from another animal species (e.g., mouse or rat) is made similar to the structure of a human antibody. It includes human chimeric antibodies in which only the constant region of the antibody is replaced with that of a human antibody, and chimeric antibodies in which the CDRs (complementarity-determining regions) present in the constant region and variable region are replaced with those of a human antibody. This includes humanized CDR-grafted antibodies (PT Jones et al., Nature 321, 522 (1986)), in which the CDR region other than the CDR region is replaced with that of a human antibody. To enhance the antigen-binding activity of humanized CDR-grafted antibodies, methods for selecting a human antibody framework (FR) with high homology to a mouse antibody, methods for producing a highly homologous humanized antibody, and methods for further enhancing the FR region after grafting mouse CDRs onto a human antibody are available. Improved techniques for substituting amino acids have already been developed (U.S. Pat. No. 5,585,089, U ... Patent No. 5693761, U.S. Patent No. 5693762, U.S. Patent No. 6180370, European Patent No. 451216, European (See State Patent No. 682040, Patent No. 2828340, etc.) and can also be used to generate humanized antibodies. can.

[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 signal domain. The transmembrane domain used may be that of CD28, CD3ε, CD8α, CD3, CD4, or 4-1BB. A transmembrane domain consisting of an artificially constructed polypeptide may also be used.

[0049] (c) Intracellular signaling domain The intracellular signaling domain transmits signals necessary for immune cells to exert their effector functions. In other words, when the extracellular domain binds to a target antigen, an intracellular signaling domain is used that can transmit signals necessary for activating immune cells. The intracellular signaling domain includes a domain for transmitting signals via the TCR complex (for convenience, referred to as the "first domain"). The first domain includes a domain for transmitting a costimulatory signal (for convenience, referred to as the "second domain"). In addition to CD3ζ, other intracellular domains such as FcεRIγ are also included as the first domain. Preferably, CD3ζ is used. The intracellular domain of a costimulatory molecule is used. 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 it is preferable that the first and second domains be linked in a manner that has been shown in past cases to have a strong costimulatory effect when CD3ζ is linked distally. The second domain is positioned on the transmembrane domain side. The first domain may be constructed by linking multiple intracellular domains of the same or different types 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 To promote secretion of CAR, a leader sequence (signal peptide) is used. For example, the leader sequence of the GM-CSF receptor can be used. In addition, the extracellular domain and the transmembrane domain can be used. It may be advantageous to have a structure in which the domains are linked 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. In addition, a part of the extracellular domain of CD28 or a part of the extracellular domain of CD8α can be used as the spacer domain. It is also possible to provide a spacer domain 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 (reporter gene, cell- or tissue-specific gene, selectable marker gene, etc.), an enhancer sequence, a WRPE sequence, etc. The detection gene is used to determine the success or efficiency of the introduction of the expression cassette, and to monitor the expression of the CAR gene. It is used for detecting or determining expression efficiency, selecting and separating cells in which the CAR gene is expressed, etc. On the other hand, the use of an enhancer sequence can improve expression efficiency. The detection genes include the neo gene, which confers resistance to neomycin, and the α gene, which confers resistance to kanamycin, etc. The npt gene (Herrera Estrella, EMBO J. 2 (1983), 987-995) and the nptII gene (Messing & Vierra. Gene 19:259-268 (1982)), the hph gene that confers resistance to hygromycin (Blochinger & Digglmann, Mol Cell Bio 4:2929-2931), and metatrexate-responsive dhfr gene (Bourouis et al., EMBO J.2(7)) that confers resistance to reporter 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), epidermal growth factor receptor (EGFR) gene lacking the intracellular domain A gene such as a CAR gene 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 is not limited thereto. Examples of known 2A peptides include the 2A peptide (F2A) derived from foot 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 results in stimulation by non-proliferating cells via costimulatory molecules and viral antigen peptides, activating viral antigen-specific genetically modified T cells and promoting their survival and proliferation. Reproduction is promoted.

[0057] The number of non-proliferating cells to be used in co-culture and to obtain genetically modified T cells in step (ii) The ratio of the number of cells in the monocyte-depleted T cell-containing cell population used for The ratio (number of cells / number of cells used to generate genetically modified T cells) is not particularly limited, but is, for example, 0.025 to 2. The ratio is preferably 0.05 to 1, more preferably 0.05 to 0.5, and even more preferably Preferably, it is 0.07 to 0.2.

[0058] This step provides a powerful stimulus for selective expansion of virus-specific CAR-T cells. In order to avoid T cell exhaustion, as a rule, stimulation with anti-CD3 and anti-CD28 antibodies is not added. On the other hand, in order to increase the cell survival rate and proliferation rate, it is recommended to use a culture medium containing a T cell growth factor during co-culture. IL-15 is a preferred T cell growth factor. Preferably, a culture medium containing 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. Commercially available products can also be used. This does not exclude the use of T cell growth factors from animal species other than humans, Typically, T cell growth factors of human origin (which may be recombinant) are used. Growth factors such as human IL-15 and human IL-7 are readily available (for example, from Miltenyi Biotec, R&D Systems, etc.).

[0059] Although a medium containing serum (human serum, fetal bovine serum, etc.) can be used, the use of a serum-free medium makes it possible to prepare cells that are safe for clinical application and have the advantage of being less susceptible to differences in culture efficiency due to differences in serum lots. Specific examples of serum-free media include TexMACS (Miltenyi Biotec), AIM V (registered trademark) (Thermo Fisher Scientific). From the perspective of CAR-T cell proliferation, etc., It is preferable that the co-culture medium in ii) contains serum. In this case, the serum concentration in the co-culture medium is, for example, 0.5 to 10%, preferably 1 to 10%, more preferably 1 to 7% (v / v), and further The concentration is 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 obtained in step (ii) is used. The individual from which the genetically modified T cells are derived (typically, the gene modified T cells obtained by the preparation method of the present invention) It is recommended to use serum collected from patients receiving melamine antigen receptor gene-modified T cells. The basal medium used may be one suitable for culturing T cells, and specific examples include the above-mentioned TexMACS and AIM V (registered trademark). Other culture conditions may be any general conditions suitable for the survival and proliferation of T cells. For example, a CO2 incubator set at 37°C may be used. It can be cultured in a container (CO2 concentration 5%).

[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 effect may not be obtained. However, if the culture period is too long, there is a risk of the cells losing their activity (vitality) and becoming exhausted / fatigued.

[0061] The viral peptide-holding non-proliferating cells may be added during step (iii). Specific examples include adding the viral peptide-holding non-proliferating cells to the culture medium for co-culture, or recovering the cells after co-culture, mixing them with the viral peptide-holding 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-holding 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-holding 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-holding non-proliferating cells are preferably prepared in a T cell-containing cell population that has been subjected to a monocyte-depletion treatment. These cells are obtained by culturing non-proliferating cells carrying viral peptides in the presence of viral peptide antigens and then treating them to lose their proliferation ability. When non-proliferating cells carrying viral peptides are added during step (iii), the timing (the first time, 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. When non-proliferating cells carrying viral peptides 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. The 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, 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. This step allows for efficient This allows for expansion culture and also has the advantage of increasing the cell survival rate. Non-proliferating cells carrying the viral peptide may be added during the expansion culture, or non-proliferating cells carrying the viral peptide may be added during the expansion culture.

[0064] As the T cell growth factor, IL-15, IL-7, etc. can be used. 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 long, the cell number will not increase sufficiently, and if the culture period is too long, there is a risk of the cells losing their activity (vitality) and becoming exhausted. Subculture may be performed midway through the culture. During culture, the medium should be changed as needed. For example, about 1 / 3 to 2 / 3 of the culture medium should be 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 for the treatment, prevention, or amelioration of 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 malignant lymphoma, diffuse large intestinal lymphoma, and leukemia), and the like. Malignant 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, prostate cancer, etc. "Treatment" includes alleviating (alleviating) symptoms characteristic of the target disease or accompanying symptoms, and preventing or delaying the worsening of symptoms. "Prevention" means to prevent or delay the onset / manifestation of a disease (disorder) or its symptoms, or to reduce the risk of onset / manifestation. On the other hand, "amelioration" means to alleviate (reduce the severity), improve, relieve, or cure (including partial cure) 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 CAR-T cells of the present invention are contained in a therapeutically effective amount. 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 preparation of the present invention is not particularly limited. For example, intravenous injection The drug is administered by intra-arterial injection, intra-portal 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 consideration the sex, age, weight, and pathological condition 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, peripheral blood was collected from the patient, diluted with PBS, and centrifuged to obtain 50 ml of blood. The mononuclear cell layer was transferred to a 50 ml centrifuge tube, diluted with PBS, and washed three times with PBS to obtain non-monocyte-reduced peripheral blood mononuclear cells (hereafter referred to as "peripheral blood mononuclear cells (2-1)"). The percentage of monocytes in the peripheral blood mononuclear cells obtained (= (number of monocytes / total number of cells) × 100) was measured by flow cytometry (using CD14 as a monocyte marker). Meanwhile, the percentage of monocytes in peripheral blood mononuclear cells from patient 1 was 12%, and the percentage of monocytes in 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 a culture medium and cultured in a culture vessel (Cell Stack Culture The cells were transferred to a container and left to stand in a CO2 incubator for 30 minutes. The cell suspension was collected and centrifuged. (340g, 10 minutes, 22℃), then discard the supernatant and suspend the pelleted cells in PBS. Monocyte-reduced peripheral blood mononuclear cells (hereinafter, sometimes referred to as "peripheral blood mononuclear cells (2-2)") The monocyte percentage in the peripheral blood mononuclear cells obtained (= (monocyte cell count / total cell count) × 100) was measured by flow cytometry (using CD14 as a monocyte marker), and found that patient 1 The monocyte percentage in peripheral blood mononuclear cells from patient 1 was 1.17%. The overall rate 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 PBS dilution containing anti-CD3 antibody and anti-CD28 antibody (anti-CD3 antibody 1 μg / mL, anti-CD28 antibody 1 μg / mL) The cells were dispensed into a 24-well tissue culture plate and left to stand in a CO2 incubator for approximately 2 hours. Peripheral blood mononuclear cells (2-1) and peripheral blood mononuclear cells (2-2) were each cultured in the presence of autologous serum (2% (v / v)). The cells were suspended in culture medium and seeded onto the above plates after washing the wells with PBS. Culture was initiated in a CO2 incubator (temperature 37°C, CO2 5%). IL-15 (5 ng / mL) was added on day 1 of culture. On day 4 of culture, the culture wells were divided and culture medium supplemented with autologous serum and IL-15 was added. On day 6 of culture, half of the culture medium was replaced. On day 7 of culture, the cultured cells were collected and used as activated lymphocytes in the following tests.

[0074] (5) Preparation and culture of CAR-T by viral peptide-added activated T cell addition method (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 this cell suspension and incubated at 37°C for 30 minutes. After washing, the cells were suspended in PBS and transferred to a separation bag, and a RAD-SURE 25Gy was attached to irradiate the cells. After confirming irradiation with the RAD-SURE 25Gy, the cells were transferred from the separation bag to a 50ml centrifuge tube. After cardiotomy, the supernatant was discarded and the cells were suspended in a culture medium containing IL-7 and IL-15 (IL-7: 10 ng / mL, IL-15: 5 ng / mL) and seeded onto 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). Peripheral blood mononuclear cells (2-2 ) to 2.0x10 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 of the irradiated cells and the two cells were mixed (the number of peripheral blood mononuclear cells (2-2) used for gene transfer was The number of irradiated cells was mixed at 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 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).

[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. A suspension was obtained. Viral peptides (PepTivator CMV pp65, PepTivator AdV5 Hexon, PepTivator EBV EBNA-1, and PepTivator EBV BZLF1, 0.6 nmol each) were added to this 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] On the other hand, cells were collected from the co-culture medium, suspended in the culture medium, and added to the culture vessel in which the irradiated cells were seeded. 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. After adding autologous serum (2% (v / v), IL-7 (10 ng / mL), and IL-15 (5 ng / mL), 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). Ta.

[0082] <Day 14> Cells were collected from the co-culture medium, and the viability (= (number of viable cells / total number of cells) × 100) and The gene transfer efficiency (number of CAR-positive cells / total number of cells) was measured by flow cytometry.

[0083] (6) Preparation and culture of CAR-T by viral peptide-added activated T cell addition method (Comparative example: Peripheral blood mononuclear cells (2-1) were used) Peripheral blood mononuclear cells (2-1) were used instead of peripheral blood mononuclear cells (2-2), and peripheral blood mononuclear cells (2-2) The procedure was the same as in (5) above, except that activated lymphocytes prepared from peripheral blood mononuclear cells (2-1) ((4) above) were used instead of activated lymphocytes prepared from peripheral blood mononuclear cells (2-1). On day 14, cells were collected from the co-culture medium and the viability (= (number of viable cells / total number of cells) × 100) was calculated. The gene transfer efficiency (number of CAR-positive cells / total number of cells) was measured by flow cytometry. .

[0084] (7) Results When peripheral blood mononuclear cells (peripheral blood mononuclear cells (2-1)) that have not been subjected to monocyte reduction treatment are used ( Compared with the above (6): Comparative Example), the monocyte-reduced peripheral blood mononuclear cells (peripheral blood mononuclear cells (2-2 )) (above (5): Example), the viable cell rate was significantly improved (Patient 1: 1.39% (Comparative Example) → 56.6% (Example), Patient 2: 2.97% (Comparative Example) → 85.7% (Example)). In addition, the gene efficiency for Patient 1 was 8.39% (Comparative Example) → 24.6% (Example). For patient 2, the results were also improved in the Example compared to the Comparative Example.

Claims

1. A genetically modified T cell expressing a chimeric antigen receptor, comprising the steps of: Preparation method: (i) monocyte-depleted T cell-containing cell populations are subjected to the detection of viral peptide antigens. a step of preparing non-proliferating cells that retain the viral peptide antigen, the non-proliferating cells being obtained by subjecting the cells to a culture treatment under the conditions of 0.1% CO₂ and a treatment to lose their proliferation ability; (ii) from a monocyte-depleted T cell-containing cell population by transposon method, obtaining genetically modified T cells into which a target antigen-specific chimeric antigen receptor gene has been introduced; (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. Between steps (iii) and (iv), the cells after co-culture are incubated in the presence of a T cell growth factor. The preparation method according to claim 1, wherein the step of culturing is carried out in the presence of

3. 3. The method according to claim 1, wherein the duration of the co-culture in step (iii) is from 1 day to 21 days. Preparation method.

4. 4. The method according to claim 1, wherein step (iii) is carried out in the presence of a T cell growth factor. The preparation method described above.

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 non-proliferating cells added during the co-culture in step (iii) are cultured and proliferated in the presence of a viral peptide antigen to a monocyte-depleted T cell-containing cell population. The method for preparing cells according to claim 7, wherein the cells are obtained by subjecting the cells to a treatment that causes loss of cell function.

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 12, wherein the T cell-containing cell population is peripheral blood mononuclear cells (PBMCs).

13. The preparation method according to any one of claims 1 to 13, wherein the T cell-containing cell population is derived from a patient to whom the genetically modified T cells are administered.

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

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

18. A genetically modified T cell expressing a chimeric antigen receptor, obtained by the preparation method according to any one of claims 1 to 17.

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

20. Administering a therapeutically effective amount of the genetically modified T cells of claim 18 to a cancer patient. Cancer treatments, including:

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