JP7075595B2 - Thioprine hypersensitive chimeric antigen receptor gene-modified lymphocytes - Google Patents

Description

The present invention relates to genetically modified lymphocytes expressing chimeric antigen receptors (CAR transgenic lymphocytes). More specifically, the present invention relates to CAR gene-introduced lymphocytes whose safety has been enhanced by utilizing a suicide gene system and their uses. This application claims priority based on Japanese Patent Application No. 2016-209066 filed on October 25, 2016, and the entire contents of the patent application are incorporated by reference.

Cell therapy (CAR therapy) using genetically modified lymphocytes (T cells, NK cells, etc.) into which a chimeric antigen receptor (CAR) gene or T cell receptor (TCR) gene has been introduced is effective for refractory cancer. It is expected as a treatment method. In particular, CAR-T cell therapy targeting CD19 has a dramatic therapeutic effect on B-cell tumors. The duration of therapeutic effect and the survival of CAR-T cells in the body are closely related. On / off-target side effects and hypercytokineemia are problems in the administration of genetically modified lymphocytes. In addition, administration of genetically modified lymphocytes produced from allogeneic donors also carries the risk of graft-versus-host disease (GVHD).

By introducing the suicide gene into lymphocytes together with the CAR / TCR gene, it is considered that the above risk can be avoided or mitigated. As suicide genes, herpes simplex virus type 1 thymidine kinase (HSV-TK), CD20, and inducible Caspase9 (iCasp9) are being developed (Non-Patent Documents 1 and 2). All of these suicide gene systems work with a combination of the suicide gene and the corresponding suicide-initiating drug. Since the suicide system usually utilizes an irreversible cell death-inducing pathway, the therapeutic effect that is expected once the suicide system is activated ends at that point. However, when activating the suicide gene system for reversible side effects such as hypercytokinemia and GVHD, only the genetically modified lymphocytes that are abnormally activated by administration of an appropriate amount (small amount) of the suicide activator are removed. It is desirable from the viewpoint of therapeutic effect and medical cost that the function of CAR / TCR gene-modified lymphocytes is maintained again after the side effects are resolved.

The HSV-TK suicide gene is actuated by ganciclovir. Therefore, treated patients cannot use ganciclovir during viral infections. Since the dose, dosage and safety of ganciclovir have been established in humans, it may be possible to regulate the suicidal function of genetically modified lymphocytes by adjusting the dose. However, because the gene product of HSV-TK is a highly immunogenic virus-derived protein, the gene-introduced lymphocytes are eliminated by the patient's immune system and cannot survive for a long time in the body. The CD20 suicide gene is actuated by rituximab. Administration of rituximab also causes B cell depletion as an on-target side effect. Rituximab is also a relatively safe drug with well-established dosage and administration in humans. The transgenic CD20 is not immunogenic. However, since the action of the CD20 suicide gene depends on complement-dependent cellular cytotoxicity and antibody-dependent cellular cytotoxicity, it largely depends on the degree of CD20 expression on the T cell surface and the patient's immune status (presence or absence of effector cells). .. iCasp9 is dimerized by a small molecule compound called CID (chemical inducer of dimerization; AP1903) and induces apoptosis in T cells by activating endogenous Caspase3. Caspase-9 is non-immunogenic, its cell-killing effect is rapid and potent, and it does not depend on the patient's immune status. However, since CID is not a drug, its appropriate dose, dosage and safety are not clear, and it is difficult to regulate suicide function in a dose-dependent manner at this time.

Marin V, Cribioli E, Philip B, Tettamanti S, Pizzitola I, Biondi A, Biagi E, Pule M. Comparison of different suicide-gene strategies for the safety improvement of genetically manipulated T cells. 2012 Dec; 23 (6): 376-86. Jones BS, Lamb LS, Goldman F, Di Stasi A. Improving the safety of cell therapy products by suicide gene transfer. Front Pharmacol. 2014 Nov 27; 5: 254. Relling MV, Hancock ML, Rivera GK, Sandlund JT, Ribeiro RC, Krynetski EY, Pui CH, Evans WE. Mercaptopurine therapy intolerance and heterozygosity at the thiopurine S-methyltransferase gene locus. J Natl Cancer Inst. 1999 Dec 1; 91 (23) ): 2001-8. Relling MV, Hancock ML, Boyett JM, Pui CH, Evans WE. Prognostic importance of 6-mercaptopurine dose intensity in acute lymphoblastic leukemia. Blood. 1999 May 1; 93 (9): 2817-23. Yang JJ, Landier W, Yang W, Liu C, Hageman L, Cheng C, Pei D, Chen Y, Crews KR, Kornegay N, Wong FL, Evans WE, Pui CH, Bhatia S, Relling MV. Inherited NUDT15 variant is a genetic determinant of mercaptopurine intolerance in children with acute lymphoblastic leukemia. J Clin Oncol. 2015 Apr 10; 33 (11): 1235-42. Br. Tanaka Y, Kato M, Hasegawa D, Urayama KY, Nakadate H, Kondoh K, Nakamura K, Koh K, Komiyama T, Manabe A. J Haematol. 2015 Oct; 171 (1): 109-15. Moriyama T, Nishii R, Perez-Andreu V, Yang W, Klussmann FA, Zhao X, Lin TN, Hoshitsuki K, Nersting J, Kihira K, Hofmann U, Komada Y, Kato M, McCorkle R, Li L, Koh K, Najera CR, Kham SK, Isobe T, Chen Z, Chiew EK, Bhojwani D, Jeffries C, Lu Y, Schwab M, Inaba H, Pui CH, Relling MV, Manabe A, Hori H, Schmiegelow K, Yeoh AE, Evans WE , Yang JJ. NUDT15 polymorphisms alter thiopurine metabolism and hematopoietic toxicity. Nat Genet. 2016 Apr; 48 (4): 367-73.

Against this background, the present invention provides a novel suicide gene system that enables the control of post-administration chimeric antigen receptor gene-modified lymphocytes by a highly safe method in order to further advance the clinical application of CAR therapy. Providing is an issue.

In the development of the suicide gene system, the drug (approved drug) whose dose, usage and safety have been established should be selected as the suicide-initiating drug, the suicide gene has no immunogenicity, and the suicide induction mechanism is the patient's. It should be considered that it does not depend on the immune status (effector cells). In addition, if a mechanism to increase the sensitivity of the suicide-initiating drug can be added to the suicide gene, the appearance of side effects due to the suicide-initiating drug itself can be avoided, and the expression of dose-dependent suicide function according to the type and degree of side effects due to CAR therapy can be achieved. It may be possible.

Mercaptopurine (6-MP), one of the thiopurine preparations, is an antimetabolite that mainly acts in the cell cycle S phase (DNA synthesis phase), which is mainly used for the treatment of acute leukemia and chronic myelogenous leukemia. .. In patients with heterozygous or homozygous mutations in the NUDT15 gene or TPMT gene, the activities of NUDT15 and TPMT are reduced, respectively, resulting in significant (high degree of homozygous) leukopenia (Non-Patent Documents 3-7). .. However, in patients with mutations in both genes, the leukocyte count and leukocyte function when the thiopurine preparation is not taken are normal. Focusing on this point, the present inventors can increase the thiopurine sensitivity of the genetically modified lymphocytes by subjecting the genetically modified lymphocytes to a genetic engineering operation that suppresses the expression of the NUDT15 gene or the TPMT gene, and the thiopurine preparation can be used as a suicide-initiating drug. I came up with the idea that a suicide gene system could be constructed. The dosage and usage of 6-MP have been established in pediatric and adult leukemia patients. Furthermore, the tolerated dose of 6-MP in patients with mutations in the NUDT15 gene or TPMT gene has also been reported (Non-Patent Documents 3 to 7). Within the range of proper use, leukopenia is mild in patients without gene mutations, with no or very mild side effects other than leukopenia. Suppression of the expression of the NUDT15 gene or TPMT gene does not lead to the acquisition of immunogenicity. The patient's immune status does not affect the induction of cell death by 6-MP.

Based on the above ideas and considerations, we constructed a new suicide gene system and verified its effect. (See examples below). As a result, experimental results exceeding expectations were obtained, and the effectiveness of the novel suicide gene system was confirmed. Based on the results, the following inventions are provided.

[1] Along with the target antigen-specific chimeric antigen receptor gene, the first nucleic acid construct that produces siRNA that targets the NUDT15 gene in cells and / or the second nucleic acid that produces siRNA that targets the TPMT gene in cells. A method for preparing a genetically modified lymphocyte expressing a chimeric antigen receptor, which comprises the step of introducing a construct into a target cell.
[2] The preparation method according to [1], wherein the introduction of the target antigen-specific chimeric antigen receptor gene, the first nucleic acid construct, and the second nucleic acid construct is performed by the transposon method.
[3] The preparation method according to [2], wherein the transposon method is the piggyBac transposon method.
[4] The target antigen-specific chimeric antigen receptor gene and the first nucleic acid construct and / or the second nucleic acid construct are mounted on the same vector, and the vector is introduced into the target cells [1] to [3]. The preparation method according to any one of the above.
[5] The preparation method according to any one of [1] to [4], wherein the target cell is a T cell.
[6] In cells [1] to [5], siRNAs targeting the NUDT15 gene and / or siRNAs targeting the TPMT gene are produced intracellularly while expressing the chimeric antigen receptor. The genetically modified lymphocyte obtained by the preparation method according to any one of the above items.
[7] A cell preparation containing a therapeutically effective amount of the genetically modified lymphocyte according to [6].
[8] A method for treating cancer, which comprises a step of administering the genetically modified lymphocyte according to [6] to a cancer patient in a therapeutically effective amount.
[9] A method for inducing cell death of the genetically modified lymphocyte, comprising a step of administering a thiopurine preparation to the cancer patient who has received the treatment method according to [8].
[10] Along with a chimeric antigen receptor expression cassette containing a target antigen-specific chimeric antigen receptor gene, a first nucleic acid construct that produces siRNA that targets the NUDT15 gene in cells and / or a siRNA that targets the TPMT gene. A vector carrying a siRNA expression cassette containing a second nucleic acid construct produced intracellularly.
[11] The vector according to [10], wherein the chimeric antigen receptor expression cassette and the siRNA expression cassette have a structure sandwiched between a pair of transposon inverted repeat sequences.
[12] A kit for preparing a genetically modified lymphocyte expressing a chimeric antigen receptor, which comprises the vector according to [11] and a transposase expression vector.
[13] A vector equipped with a chimeric antigen receptor expression cassette containing a target antigen-specific chimeric antigen receptor gene, and
A vector with a siRNA expression cassette containing a first nucleic acid construct that produces siRNAs targeting the NUDT15 gene intracellularly and / or a second nucleic acid construct that produces siRNAs targeting the TPMT gene intracellularly.
A kit for preparing genetically modified lymphocytes expressing a chimeric antigen receptor.
[14] The chimeric antigen receptor expression cassette has a structure sandwiched between a pair of transposon inverted repeat sequences.
The siRNA expression cassette has a structure sandwiched between a pair of transposon inverted repeat sequences.
The preparation kit according to [13], further comprising a transposase expression vector.
[15] A vector equipped with a chimeric antigen receptor first expression cassette containing a target antigen-specific chimeric antigen receptor gene, and
A vector carrying a first siRNA expression cassette containing a first nucleic acid construct that produces siRNA targeting the NUDT15 gene intracellularly,
A vector carrying a second siRNA expression cassette containing a second nucleic acid construct that produces siRNA targeting the TPMT gene intracellularly,
A kit for preparing genetically modified lymphocytes expressing a chimeric antigen receptor.
[16] The chimeric antigen receptor expression cassette has a structure sandwiched between a pair of transposon inverted repeat sequences.
The first siRNA expression cassette has a structure sandwiched between a pair of transposon inverted repeat sequences.
The second siRNA expression cassette has a structure sandwiched between a pair of transposon inverted repeat sequences.
The preparation kit according to [15], further comprising a transposase expression vector.
[17] The preparation kit according to any one of [12], [14], and [16], wherein the transposase is a piggyBac transposase.

pIRII-CAR.CD19-NUDT15KD Vector structure. Leader sequence (SEQ ID NO: 20) and CD19 CAR (light chain variable region (SEQ ID NO: 21), heavy chain variable region (SEQ ID NO: 22), Fc region (SEQ ID NO: 23), transmembrane region of CD28 and intracellular domain (sequence) In addition to the sequence encoding CD3ζ (SEQ ID NO: 25)), a sequence encoding shRNA targeting the NUDT15 gene is loaded. The sequence of the full length of the vector is shown in SEQ ID NO: 10 (6680 bp, No. 1 example) and SEQ ID NO: 11 (6682 bp, No. 2 example). An example of a DNA fragment for RNAi targeting the NUDT15 gene (SEQ ID NO: 13 for No. 1 example, SEQ ID NO: 14 for No. 2 example). It contains a U6 promoter (underlined. SEQ ID NO: 15) and a sequence encoding shRNA (double underlined. No. 1 example is SEQ ID NO: 16 and No. 2 example is SEQ ID NO: 17). Structure of the pIRII-CAR.CD19-TPMTKD vector. Leader sequence (SEQ ID NO: 24) and CD19 CAR (light chain variable region (SEQ ID NO: 21), heavy chain variable region (SEQ ID NO: 22), Fc region (SEQ ID NO: 23), transmembrane region of CD28 and intracellular domain (sequence) In addition to the sequence encoding CD3ζ (SEQ ID NO: 25)), a sequence encoding shRNA targeting the TPMT gene is loaded. The full-length sequence of the vector is shown in SEQ ID NO: 12 (6680 bps). DNA fragment for RNAi targeting the TPMT gene (SEQ ID NO: 18). It contains the U6 promoter (underlined. SEQ ID NO: 15) and the sequence encoding shRNA (double underlined. SEQ ID NO: 19). Comparison of cell numbers on the 14th day of gene transfer. The number of cells is 9.75 × 10 ⁶ for CD19.CAR-NUDT15KD-T cells (NUDT15), 13.65 × 10 ⁶ for CD19.CAR-TPMTKD-T cells (TPMT), and 21.75 × for CD19.CAR-T cells (control). It was 10 ⁶ . Comparison of CAR expression rates on T cells on day 14 of gene transfer. The CAR expression rate was 28% for CD19.CAR-NUDT15KD-T cells (NUDT15), 32% for CD19.CAR-TPMTKD-T cells (TPMT), and 42% for CD19.CAR-T cells (control). .. CAR expression viable cell ratio (%) 72 hours after the addition of 6-mercaptopurine (6-MP). The anti-leukemia (SU / SR cell proliferation inhibitory) effect and 6-MP sensitivity of CAR-T cells were investigated. The anti-leukemic effect (suppression of SU / SR cell proliferation) was 100% for all CAR-T cells (when the 6-MP concentration was 0). On the other hand, the 6-MP sensitivity of CAR-T cells (surviving cell ratio at 50 ng / mL) was 42% for CAR-NUDT15KD-T cells (NUDT15) and 62.5% for CD19.CAR-TPMTKD-T cells (TPMT). , CD19.CAR-T cells (control) was 106.6%.

1. 1. Method for preparing gene-modified lymphocytes expressing chimeric antigen receptor The present invention relates to a method for preparing gene-modified lymphocytes expressing chimeric antigen receptors (CAR gene-introduced lymphocytes). The CAR gene-introduced lymphocytes obtained by the preparation method of the present invention can be used for CAR therapy. In the preparation method of the present invention, the following steps, that is, siRNAs targeting the NUDT15 (nudix hydrolase 15) gene (hereinafter referred to as “NUDT15 siRNA”) together with the target antigen-specific chimeric antigen receptor (CAR) gene are used. Introduce a second nucleic acid construct that produces intracellularly generated first nucleic acid construct and / or siRNA that targets the TPMT (thiopurine S-methyltransferase) gene (hereinafter referred to as "TPMT siRNA") into the target cell. Steps to do. By this step, cells expressing NUDT15 siRNA in addition to the target antigen-specific CAR gene (when the first nucleic acid construct is introduced), cells expressing TPMT siRNA in addition to the target antigen-specific CAR gene (second nucleic acid construct). (When introduced), or cells expressing NUDT15 siRNA and TPMT siRNA in addition to the target antigen-specific CAR gene (when both the first nucleic acid construct and the second nucleic acid construct are introduced) are obtained. Unless otherwise specified, various cells (for example, T cells) in the present specification are human cells.

The CAR gene encodes a chimeric antigen receptor (CAR) that recognizes a particular target antigen. CAR is a structure containing a target-specific extracellular domain, a transmembrane domain, and an intracellular signal domain for the effector function of immune cells. Hereinafter, each domain will be described.

(A) Extracellular domain The extracellular domain exhibits target-specific binding. For example, the extracellular domain contains a scFv fragment of an antitargeted monoclonal antibody. As the monoclonal antibody here, for example, a rodent (mouse, rat, rabbit, etc.) antibody, a human antibody, a humanized antibody, or the like is used. A humanized monoclonal antibody is an antibody in which the structure of a monoclonal antibody of another animal species (for example, mouse or rat) is similar to that of a human antibody, and only the constant region of the antibody is replaced with that of a human antibody. Humanized CDR-grafted antibody (PT Johons et al., Nature 321,522 (1986) in which the chimeric antibody and the portion other than the CDR (complementary determination region) existing in the constant region and the variable region are replaced with those of the human antibody. ))including. A method of selecting a human antibody framework (FR) that is highly homologous to a mouse antibody, a method of producing a highly homologous humanized antibody, and a mouse CDR to a human antibody in order to enhance the antigen-binding activity of a human-type CDR transplanted antibody. Further improved techniques have been developed for the method of substituting amino acids in the FR region after transplantation (US Patent No. 5585089, US Patent No. 5697361, US Patent No. 5693762, US Patent No. 6180370, European Patent No. 451216). , European Patent No. 682040, Patent No. 2828340, etc.), can also be used to produce humanized antibodies.

The scFv fragment is a structure in which a light chain variable region (VL) and a heavy chain variable region (VH) of an immunoglobulin are linked via a linker, and retains the ability to bind to an antigen. As the linker, for example, a peptide linker can be used. A peptide linker is a linker composed of peptides in which amino acids are linearly linked. A typical example of a peptide linker is a linker composed of glycine and serine (GGS linker or GS linker). The GGS linker and the amino acids constituting the GS linker, glycine and serine, have a small size in themselves, and it is difficult for a higher-order structure to be formed in the linker. The length of the linker is not particularly limited. For example, a linker having 5 to 25 amino acid residues can be used. The number of amino acid residues constituting the linker is preferably 8 to 25, more preferably 15 to 20.

The target is typically an antigen that is specifically expressed in tumor cells. The term "specific expression" as used herein means that significant or remarkable expression is observed as compared with cells other than tumor, and there is no intention of limiting the expression to those having no expression in cells other than tumor. Examples of target antigens are CD19 antigen, CD20 antigen, GD2 antigen, CD22 antigen, CD30 antigen, CD33 antigen, CD44variant7 / 8 antigen, CD123 antigen, CEA antigen, Her2 / neu antigen, MUC1 antigen, MUC4 antigen, MUC6 antigen, IL. -13 receptor-alpha2, immunoglobulin light chain, PSMA antigen, VEGF receptor2, mesothelin antigen, EGFRvIII, EphA2 antigen, IGFR and the like can be mentioned.

(B) Transmembrane domain The transmembrane domain intervenes between the extracellular domain and the intracellular signaling domain. As the transmembrane domain, a transmembrane domain such as CD28, CD3ε, CD8α, CD3, CD4 or 4-1BB can be used. A transmembrane domain consisting of an artificially constructed polypeptide may be used.

(C) Intracellular signal domain The intracellular signal domain transmits signals necessary for exerting the effector function of immune cells. That is, when the extracellular domain binds to the target antigen, the intracellular signal domain capable of transmitting the signal necessary for the activation of immune cells is used. The intracellular signal domain includes a domain for transmitting a signal via the TCR complex (referred to as "first domain" for convenience) and a domain for transmitting a co-stimulation signal (for convenience, "second domain"). Called) is included. As the first domain, an intracellular domain such as FcεRIγ can be used in addition to CD3ζ. Preferably, CD3ζ is used. Further, as the second domain, the intracellular domain of the co-stimulator molecule is used. Examples of co-stimulatory molecules include CD28, 4-1BB (CD137), CD2, CD4, CD5, CD134, OX-40 or ICOS. Preferably, the intracellular domain of CD28 or 4-1BB is adopted.

The mode of connection between the first domain and the second domain is not particularly limited, but it is preferable that the transmembrane domain is preferably transmitted because it is known that the co-stimulation was strongly transmitted when the CD3ζ was connected distally in the past cases. Place the second domain on the side. Multiple intracellular domains of the same or different species may be linked in a tandem manner to form a first domain. The same applies to the second domain.

The first domain and the second domain may directly link them or may interpose a linker between them. As the linker, for example, a peptide linker can be used. A peptide linker is a linker composed of peptides in which amino acids are linearly linked. The structure, characteristics, etc. of the peptide linker are as described above. However, as the linker here, a linker composed of only glycine may be used. The length of the linker is not particularly limited. For example, a linker having 2 to 15 amino acid residues can be used.

(D) Other factors
A leader sequence (signal peptide) is used to stimulate the secretion of CAR. For example, the leader sequence of the GM-CSF receptor can be used. Further, it is preferable to have a structure in which the extracellular domain and the transmembrane domain are linked via the spacer domain. That is, the CAR of the preferred embodiment comprises a spacer domain between the extracellular domain and the transmembrane domain. The spacer domain is used to promote the binding of CAR to the target antigen. For example, an Fc fragment of human IgG (eg, human IgG1, human IgG4) can be used as a spacer domain. In addition, a part of the extracellular domain of CD28, a part of the extracellular domain of CD8α, and the like can also be used as the spacer domain. A spacer domain can also be provided between the transmembrane domain and the intracellular signal domain.

There have been some reports on experiments and clinical studies using CAR (for example, 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 these reports can be used as a reference to construct the CAR in the present invention.

The first nucleic acid construct that produces NUDT15 siRNA intracellularly and the second nucleic acid construct that produces TPMT siRNA intracellularly are used for suppression of expression by so-called RNAi (RNA interference). In other words, by introducing the first nucleic acid construct and / or the second nucleic acid construct into the target cell, the expression of the target gene (NUDT15, TPMT) can be suppressed by RNAi in the target cell. For convenience of explanation, the first nucleic acid construct and the second nucleic acid construct may be collectively referred to as "construct for siRNA".

RNAi is a sequence-specific post-transcriptional gene suppression process that can be triggered in eukaryotic cells. RNAi for mammalian cells uses short double-stranded RNA (siRNA) with a sequence corresponding to the sequence of the target mRNA. Usually, siRNA is 21 to 23 base pairs. Mammalian cells are known to have two pathways (sequence-specific and sequence-non-specific pathways) that are affected by double-stranded RNA (dsRNA). In the sequence-specific pathway, relatively long dsRNAs are split into short interfering RNAs (ie siRNAs). On the other hand, the sequence non-specific pathway is considered to be triggered by any dsRNA regardless of the sequence as long as it is longer than a predetermined length. In this pathway, dsRNA becomes two enzymes, the active form, PKR, which stops all protein synthesis by phosphorylating the translation initiation factor eIF2, and 2', 5'oligoadenyl, which is involved in the synthesis of RNAase L-activating molecule. Acid synthase is activated. To minimize the progression of this non-specific pathway, it is preferable to use double-stranded RNA (siRNA) shorter than about 30 base pairs (Hunter et al. (1975) J Biol Chem 250: 409-17). See Manche et al. (1992) Mol Cell Biol 12: 5239-48; Minks et al. (1979) J Biol Chem 254: 10180-3; and Elbashir et al. (2001) Nature 411: 494-8. sea bream).

In order to generate target-specific RNAi, siRNA consisting of a sense RNA homologous to a part of the mRNA sequence of the target gene and an antisense RNA complementary thereto may be expressed intracellularly. The first nucleic acid construct and the second nucleic acid construct realize the expression.

SiRNA that targets a specific gene (target gene) is usually a double-stranded RNA in which a sense RNA consisting of a sequence homologous to a continuous region in the mRNA sequence of the gene and an antisense RNA consisting of a complementary sequence thereof are hybridized. RNA. The length of the "continuous region" here is usually 15 to 30 bases long, preferably 18 to 23 bases long, and more preferably 19 to 21 bases long.

It is known that double-stranded RNA having an overhang of several bases at the end exerts a high RNAi effect. Therefore, also in the present invention, it is preferable to adopt siRNA having such a structure. The length of the base forming the overhang is not particularly limited, but is preferably 2 base lengths (for example, TT, UU).

The siRNA can be designed in the usual way. Sequences unique to the target sequence (continuous sequences) are usually used in the design of siRNA. Programs and algorithms for selecting an appropriate target sequence have been developed.

"Nucleic acid construct that produces siRNA intracellularly" refers to a nucleic acid molecule that, when introduced into a cell, produces the desired siRNA (siRNA that causes RNAi against the target gene) by an intracellular process. Typically, a nucleic acid construct is constructed to express an shRNA that is converted to siRNA by a later process and loaded onto a suitable vector. In this way, a vector for siRNA called stem-loop type or short hairpin type (vector in which a sequence encoding shRNA is inserted) or a vector for siRNA called tandem type (sense RNA and antisense RNA are expressed separately). Vector) is obtained. These vectors can be prepared by those skilled in the art according to conventional methods (Brummelkamp TR et al. (2002) Science 296: 550-553; Lee NS et al. (2001) Nature Biotechnology 19: 500-505; Miyagishi. M & Taira K (2002) Nature Biotechnology 19: 497-500; Paddison PJ et al. (2002) Proc. Natl. Acad. Sci. USA 99: 1443-1448; Paul CP et al. (2002) Nature Biotechnology 19: 505-508; Sui G et al. (2002) Proc Natl Acad Sci USA 99 (8): 5515-5520; Paddison PJ et al. (2002) Genes Dev. 16: 948-958 etc.). shRNA has a structure in which sense RNA and antisense RNA are linked via a loop structure (hairpin structure), and the loop structure is cleaved in the cell to form double-stranded siRNA, which brings about the RNAi effect. The length of the loop structure is not particularly limited, but is usually 3 to 23 bases.

The genes whose expression is suppressed in the present invention are the NUDT15 gene and / or the TPMT gene. The sequences of the NUDT15 gene registered in the public database are sequenced with SEQ ID NO: 1 (ACCESSION: NM_018283, DEFINITION: Homo sapiens nudix hydrolase 15 (NUDT15), transcript variant 1.) and SEQ ID NO: 2 (ACCESSION: NM_001304745, DEFINITION: Homo sapiens). The sequence of the TPMT gene is shown in SEQ ID NO: 3 (ACCESSION: NM_000367, DEFINITION: Homo sapiens thiopurine S-methyltransferase (TPMT), mRNA.) In nudix hydrolase 15 (NUDT15), transcript variant 2, mRNA.). Examples of the sequence (sense strand) of NUDT15 siRNA are shown in SEQ ID NO: 4 and SEQ ID NO: 5, and the sequences of shRNA corresponding to these NUDT15 siRNA are shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively. Similarly, an example of the sequence (sense strand) of TPMT siRNA is shown in SEQ ID NO: 8, and the sequence of shRNA corresponding to the TPMT siRNA is shown in SEQ ID NO: 9.

The shRNA itself can also be used as a construct for siRNA. In this case, the vector carrying the CAR gene and the construct for siRNA will be introduced into the target cells.

In a preferred embodiment of the present invention, an expression cassette containing a CAR gene (CAR expression cassette) and an expression cassette containing a first nucleic acid construct and / or a second nucleic acid construct (siRNA expression cassette) are mounted on the same vector. If this configuration is adopted, one type of vector (referred to as "CAR-siRNA vector") may be introduced into the target cell, and the gene transfer operation required for the preparation method of the present invention becomes simple. ..

On the other hand, a vector equipped with a CAR expression cassette (CAR vector) and a vector equipped with an siRNA expression cassette (siRNA vector) may be prepared and these vectors may be introduced into a target cell. The order of introduction is not particularly limited, but the introduction of the CAR vector is preferably preceded. When this order is adopted, it is advisable to select, concentrate or purify the target cells into which the CAR vector has been appropriately introduced, and then introduce the siRNA vector. By doing so, the production efficiency and purity of desired CAR gene-introduced lymphocytes (that is, lymphocytes expressing CAR and / or NUDT15 siRNA and / or TPMT siRNA) can be improved. Currently, various RNAi vectors are available. A vector equipped with an siRNA expression cassette can be constructed using such a known vector. For example, an insert DNA encoding a desired RNA (eg, shRNA) is prepared and then inserted into the cloning site of the vector to obtain an RNAi expression vector. The origin and structure of the vector are not limited as long as it has the function of generating siRNA that exerts RNAi action on the target gene in the cell. As the siRNA vector, a vector equipped with a NUDT15 siRNA expression cassette, a vector equipped with a TPMT siRNA expression cassette, or both, or a vector equipped with a NUDT15 siRNA expression cassette and a TPMT expression cassette is used.

Examples of promoters that can be used for CAR expression cassettes are CMV-IE (cytomegalovirus early gene-derived promoter), SV40ori, retrovirus LTP, SRα, EF1α, β-actin promoter, and the like. The promoter is operably linked to the CAR gene. Here, "the promoter is operably linked to the CAR gene" is synonymous with "the CAR gene is arranged under the control of the promoter", and is usually directly on the 3'end side of the promoter. Alternatively, the CAR gene will be linked via another sequence. A poly A addition signal sequence is placed downstream of the CAR gene. Transcription is terminated by the use of the poly A addition signal sequence. As the poly A addition signal sequence, a poly A addition sequence of SV40, a poly A addition sequence of a bovine-derived growth hormone gene, or the like can be used.

Examples of promoters that can be used for siRNA expression cassettes are the U6 promoter, H1 promoter, tRNA promoter, and the like. These promoters are RNA polymerase III promoters and can be expected to have high expression efficiency.

Even if each of the above vectors (CAR-siRNA vector, CAR vector, siRNA vector) includes a detection gene (reporter gene, cell or tissue-specific gene, selectable marker gene, etc.), enhancer sequence, WRPE sequence, etc. good. The detection gene is used for determining the success or failure and efficiency of the introduction of the expression cassette, detecting the expression of CAR or determining the expression efficiency, and selecting and sorting the cells expressing the CAR gene. On the other hand, the expression efficiency can be improved by using the enhancer sequence. The genes for detection include the neo gene that imparts resistance to neomycin, the npt gene (Herrera Estrella, EMBO J. 2 (1983), 987-995) that imparts resistance to kanamycin, and the nptII gene (Messing & Vierra. Gene 1). 9: 259-268 (1982)), hph gene conferring resistance to neomycin (Blochinger & Diggl mann, Mol Cell Bio 4: 2929-2931), dhfr gene conferring resistance to metatrexate (Bourouis et al. , EMBO J.2 (7)), etc. (marker gene), luciferase gene (Giacomin, P1. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), β -Genes of fluorescent proteins such as glucuronidase (GUS) gene, GFP (Gerdes, FEBS Lett. 389 (1996), 44-47) and its variants (EGFP, d2EGFP, etc.), intracellular domain Genes such as the missing epithelial growth factor receptor (EGFR) gene can be used. The detection gene is linked to the CAR gene via, for example, a bicistronic control sequence (eg, a ribosome internal recognition sequence (IRES)) or a sequence encoding a self-cleaving peptide. An example of a self-cleaving peptide is, but is not limited to, the 2A peptide (T2A) from the Thosea asigna virus. 2A peptide (F2A) derived from foot-and-mouth disease virus (FMDV), 2A peptide (E2A) derived from horse rhinitis A virus (ERAV), 2A peptide (P2A) derived from porcine teschovirus (PTV-1), etc. are known as self-cleaving peptides. Has been done.

Various gene transfer methods can be used for the introduction of the CAR gene, the first nucleic acid construct and the second nucleic acid construct. The gene transfer method is roughly classified into a method using a viral vector and a method using a non-viral vector. The former skillfully utilizes the phenomenon that the virus infects cells, and high gene transfer efficiency can be obtained. As virus vectors, retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, Sendai virus vectors and the like have been developed. Among these, in the retrovirus vector, lentiviral vector, and adeno-associated virus vector, the target gene incorporated into the vector is integrated into the host chromosome, and stable and long-term expression can be expected. Each viral vector can be prepared according to a previously reported method or by using a commercially available dedicated kit. Examples of non-viral vectors include plasmid vectors, liposome vectors, positively charged liposome vectors (Felgner, P.L., Gadek, T.R., Holm, M. et al., Proc. Natl. Acad. Sci., 84: 7413-7417). , 1987), YAC vector, BAC vector can be mentioned.

Preferably, gene transfer is performed by the transposon method. The transposon method is one of the non-viral gene transfer methods. Transposon is a general term for short gene sequences that cause gene translocations that have been conserved during evolution. A pair of gene enzyme (transposase) and its specific recognition sequence causes gene translocation. As the transposon method, for example, the piggyBac transposon method can be used. The PiggyBac transposon method utilizes transposons 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.), Enables highly efficient integration into mammalian chromosomes. The PiggyBac transposon method is actually used for gene transfer (eg, Nakazawa Y, et al., J Immunother 32: 826-836, 2009; Nakazawa Y et al., J Immunother 6: 3-10, 2013, etc. reference). The transposon method applicable to the present invention is not limited to the method using piggyBac, for example, 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 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.) You may decide.

The introduction operation by the transposon method may be performed by a conventional method, and past literature (for example, for the piggyBac transposon method, Nakazawa Y, et al., J Immunother 32: 826-836, 2009, Nakazawa Y et al., J Immunother 6: 3-10, 2013, Saha S, Nakazawa Y, Huye LE, Doherty JE, Galvan DL, Rooney CM, Wilson MH. J Vis Exp. 2012 Nov 5; (69): e4235, Saito S, Nakazawa Y, Sueki A, et al. Anti-leukemic potency of piggyBac-mediated CD19-specific T cells against refractory Philadelphia chromosome-positive acute lymphoblastic leukemia. Cytotherapy. 2014; 16: 1257-69.).

In a preferred embodiment of the present invention, the piggyBac transposon method is adopted. Typically, in the piggyBac transposon method, a vector carrying a gene encoding the piggyBac transposase (transposase plasmid) and a desired nucleic acid construct (CAR expression cassette and / or siRNA expression cassette) are sandwiched between piggyBac reverse repeat sequences. A vector having a structure (transposon plasmid) is prepared, and these vectors are introduced (transfected) into a target cell. Various methods such as electroporation, nucleofection, lipofection, and calcium phosphate method can be used for transfection.

As target cells (cells into which the CAR gene and the first nucleic acid construct and / or the second nucleic acid construct are introduced), CD4-positive CD8-negative T cells, CD4-negative CD8-positive T cells, T cells prepared from iPS cells, αβ- Examples thereof include T cells, γδ-T cells, NK cells, and NKT cells. Various cell populations can be used as long as they contain lymphocytes or progenitor cells as described above. PBMC (peripheral blood mononuclear cells) collected from peripheral blood is one of the preferred target cells. That is, in a preferred embodiment, a gene transfer operation is performed on PBMC. PBMC may be prepared by a conventional method. For the method of preparing PBMC, refer to, for example, Saha S, Nakazawa Y, Huye LE, Doherty JE, Galvan DL, Rooney CM, Wilson MH. J Vis Exp. 2012 Nov 5; (69): e4235. can.

The CAR gene-introduced lymphocytes obtained in the above steps are typically subjected to an activation treatment. For example, stimulation with anti-CD3 antibody and anti-CD28 antibody activates CAR gene-introduced lymphocytes. This treatment usually promotes the survival and proliferation of CAR gene-introduced lymphocytes. For example, by culturing in a culture vessel (for example, a culture dish) coated with an anti-CD3 antibody and an anti-CD28 antibody for 1 to 20 days, preferably 3 to 14 days, and more preferably 5 to 10 days. , Anti-CD3 antibody and anti-CD28 antibody can be stimulated. Anti-CD3 antibody (for example, the trade name CD3pure antibody provided by Miltenyi Biotec) and anti-CD28 antibody (for example, the trade name CD28pure antibody provided by Miltenyi Biotec can be used) are also commercially available. It is easily available. It is also possible to perform the stimulation using magnetic beads coated with anti-CD3 antibody and anti-CD28 antibody (for example, Dynabeads T-Activator CD3 / CD28 provided by VERITAS). It is preferable to use an "OKT3" clone as the anti-CD3 antibody. In order to promote recovery from the damage / disorder caused by the gene transfer operation, the activation treatment is performed not immediately after the gene transfer operation but after about 8 to 48 hours (preferably 16 to 24 hours) have elapsed from the gene transfer operation. It is good to carry out.

In order to increase the viability / proliferation rate of cells, it is advisable to use a culture medium to which T cell growth factor has been added during the activation treatment. IL-15 is suitable as the T cell growth factor. Preferably, a culture solution containing IL-7 in addition to IL-15 is used. The amount of IL-15 added is, for example, 1 ng / ml to 20 ng / ml, preferably 5 ng / ml to 10 ng / ml. Similarly, the amount of IL-7 added is, for example, 1 ng / ml to 20 ng / ml, preferably 5 ng / ml to 10 ng / ml. T cell growth factors such as IL-15 and IL-7 can be prepared according to a conventional method. In addition, commercially available products can also be used. Although the use of T cell growth factors in non-human animal species is not excluded, T cell growth factors are usually derived from humans (which may be recombinants). Growth factors such as human IL-15 and human IL-7 can be easily obtained (for example, provided by Miltenyi Biotec, R & D Systems, etc.).

A medium containing serum (human serum, fetal bovine serum, etc.) may be used, but by adopting a serum-free medium, it is highly safe for clinical application and the culture efficiency due to the difference between serum lots is high. It is possible to prepare cells that have the advantage of being less likely to make a difference. Specific examples of serum-free media for lymphocytes are TexMACS ^TM (Miltenyi Biotec) and AIM V® (Thermo Fisher Scientific). When using serum, it is preferable to use self-serum, that is, serum collected from a patient receiving the CAR gene-introduced lymphocyte obtained by the preparation method of the present invention. A medium suitable for lymphocyte culture may be used as the basal medium, and specific examples thereof are TexMACS ^TM and AIM V (registered trademark) described above. Other culture conditions may be any one suitable for the survival and proliferation of lymphocytes, and general ones may be adopted. For example, the cells may be cultured in a CO ₂ incubator (CO ₂ concentration 5%) set at 37 ° C.

After the activation treatment, the cells are collected. The collection operation may be performed by a conventional method. For example, it is collected by pipetting, centrifugation, or the like. In a preferred embodiment, a step of culturing the activated cells in the presence of T cell growth factor is performed prior to the recovery operation. This step enables efficient expansion culture and also has the advantage of increasing cell viability. As the T cell growth factor here, IL-15, IL-7 and the like can be used. As in the case of the activation treatment, the cells may be 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 the number of cells cannot be expected, and if the culture period is too long, the activity (life force) of the cells may decrease, and the cells may be exhausted / fatigued. It may be subcultured in the middle of culturing. In addition, the medium is changed as necessary during the culture. For example, replace about 1/3 to 2/3 of the culture medium with a new medium once every 3 days.

In one aspect, virus-specific chimeric antigen receptor gene-modified T cells (hereinafter referred to as "virus-specific CAR-T cells") are prepared as the CAR gene-introduced lymphocytes of the present invention. Virus-specific CAR-T cells can be expected to improve their sustainability in the body by stimulation from viral T cell receptors when used for autologous transplantation, and further allogeneic immune response (GVHD) when used for allogeneic transplantation. It has important advantages in clinical application, such as the possibility of producing CAR-T from a transplanted donor and the possibility of formulating CAR-T cells from a third-party donor. In fact, virus-specific CAR-T cells have been reported to persist in the body for longer periods of time (Pule MA, et al. Nat Med. 2008 Nov; 14 (11): 1264-70.). In addition, according to a report of a third-party EBV-specific CTL clinical study (Annual Review Blood 2015, published in January 2015, Chugai Medical Co., Ltd.), the safety of virus-specific cytotoxic T cells (CTL) is high. It is backed up.

The preparation method of this embodiment includes the following steps (i) to (iv).
(I) A cell population containing T cells was stimulated with an anti-CD3 antibody and an anti-CD28 antibody, and then cultured in the presence of the viral peptide antigen and treated to lose the proliferative ability to retain the viral peptide antigen. Steps to Prepare Nonproliferative Cells (ii) Along with the target antigen-specific chimeric antigen receptor gene, a first nucleic acid construct that produces an intracellular siRNA that targets the NUDT15 gene and / or a siRNA that targets the TPMT gene. Introducing the second nucleic acid construct generated in the cell into the target cell to obtain the gene-modified T cell (iii) The non-proliferative cell prepared in step (i) and the gene-modified T cell obtained in step (ii). Step of mixing and co-culturing (iv) Step of collecting cells after culturing

In step (i), first, a cell population containing T cells is stimulated with an anti-CD3 antibody and an anti-CD28 antibody to obtain activated T cells. As the "cell population containing T cells", PBMC (peripheral blood mononuclear cells) collected from peripheral blood is preferably used. It is also possible to purify PBMC to increase the content of T cells, or to use mononuclear cells collected from peripheral blood by feresis as the "cell population containing T cells" here.

For example, by culturing in a culture vessel (for example, a culture dish) coated with an anti-CD3 antibody and an anti-CD28 antibody for 3 hours to 3 days, preferably 6 hours to 2 days, and more preferably 12 hours to 1 day. , T cells in the cell population can be stimulated with anti-CD3 antibody and anti-CD28 antibody. Anti-CD3 antibody (for example, the trade name CD3pure antibody provided by Miltenyi Biotec) and anti-CD28 antibody (for example, the trade name CD28pure antibody provided by Miltenyi Biotec can be used) are also commercially available. It is easily available. It is also possible to perform the stimulation in step (i) using magnetic beads coated with anti-CD3 antibody and anti-CD28 antibody (for example, Dynabeads T-Activator CD3 / CD28 provided by VERITAS). It is preferable to use an "OKT3" clone as the anti-CD3 antibody.

After the activated T cells are obtained as described above, the cells are cultured in the presence of a viral peptide antigen and treated to lose their proliferative ability. This gives a non-proliferative "activated T cell holding a viral peptide antigen on the cell surface" (hereinafter referred to as "viral peptide-carrying non-proliferative cell"). The order of the culture in the presence of the viral peptide antigen and the treatment for losing the proliferative ability is not particularly limited. Therefore, the culture may be lost after culturing in the presence of the virus peptide antigen, or may be cultured in the presence of the virus peptide antigen after the loss of the growth ability. Preferably, the former order is adopted because of the expectation that the uptake of the viral peptide antigen will be better before the loss of proliferative capacity.

The "treatment that causes the loss of proliferative capacity" is typically irradiation, but a drug may also be used. An example of irradiation conditions is a treatment using gamma rays at an intensity of 25 Gy to 50 Gy for 15 to 30 minutes.

In order to culture in the presence of the viral peptide antigen, for example, a medium to which the viral peptide antigen is 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 is, for example, 0.5 μg / ml to 1 μg / ml. The culture period is, 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 capable of inducing cytotoxic T cells (CTL) specific to a specific virus. The viral peptide antigen is not limited to these, but is, for example, an adenovirus (AdV) antigen peptide (see, for example, WO 2007015540 A1) and a cytomegalovirus (CMV) antigen peptide (eg, JP-A-2002). -Refer to JP-A-255997, JP-A-2004-242599, JP-A-2012-87126), Epstein-Barr virus (EBV) antigen peptide (eg, WO 2007049737 A1, Japanese Patent Application No. 2011-177487, JP-A. 2006-188513 (see No. 2006-188513), etc. can be used. The viral peptide antigen can be prepared by a conventional method (for example, liquid phase synthesis method, solid phase synthesis method) based on the sequence information. In addition, some viral peptide antigens are commercially available (for example, provided by Medical & Biological Laboratories, 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 or an EBV antigen peptide mixture, or a combination of two or more of these antigen peptide mixtures (for example, an AdV antigen peptide mixture, a CMV antigen peptide mixture and an EBV antigen peptide mixture). (Mixed) is used. By using two or more kinds of antigen peptides in combination, a plurality of activated T cells having different targets (antigen peptides) can be obtained, and the CAR-T cells obtained by the preparation method of the present invention are effective therapeutic targets (patients). ) Can be expected to increase (improvement of coverage). In deciding which virus-derived antigen peptide to use, it is advisable to consider the use of 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. .. For example, for the purpose of treating recurrent leukemia after hematopoietic stem cell transplantation, the antigen-peptide mixture of EBV virus may be used alone or in combination with an antigen-peptide mixture of other viruses. AdV antigen peptide mixture, CMV antigen peptide mixture, and EBV antigen peptide mixture are also commercially available (for example, PepTivator® AdV5 Hexon, PepTivator® CMV pp65, PepTivator (for example, provided by Milteny Biotech). Registered trademarks) EBV EBNA-1, PepTivator (registered trademark) EBV BZLF1, PepMix ^TM Collection HCMV, PepMix ^TM EBV (EBNA1), etc. provided by JPT Peptide Technologies), which can be easily obtained.

Step (ii) corresponds to the gene transfer operation (introduction of CAR gene, first nucleic acid construct and second nucleic acid construct) described above, and various gene transfer methods can be used. The transposon method is preferably adopted. This step yields genetically modified T cells (CAR-T cells).

In step (iii), the non-proliferative cells (virus peptide-retaining non-proliferative cells) prepared in step (i) and the gene-modified T cells obtained in step (ii) are mixed and co-cultured. As a result, stimulation via co-stimulatory molecules and virus antigen peptides by non-proliferative cells is applied, virus antigen-specific gene-modified T cells are activated, and their survival and proliferation are promoted.

The ratio of the number of non-proliferative cells to the number of gene-modified T cells used for co-culture (number of non-proliferative cells / number of gene-modified T cells) is not particularly limited, but is, for example, 0.025 to 0.5.

In principle, this step is an anti-CD3 antibody and an anti-CD28 antibody for reasons such as selectively proliferating virus-specific CAR-T cells, avoiding strong stimulation, and preventing T cell exhaustion / fatigue. Do not add stimulation by. On the other hand, in order to increase the survival rate / proliferation rate of cells, it is advisable to use a culture medium to which T cell growth factor is added at the time of co-culture. IL-15 is suitable as the T cell growth factor. Preferably, a culture solution 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 a conventional method. In addition, commercially available products can also be used. Although the use of T cell growth factors in non-human animal species is not excluded, T cell growth factors are usually derived from humans (which may be recombinants). Growth factors such as human IL-15 and human IL-7 can be easily obtained (for example, provided by Miltenyi Biotec, R & D Systems, etc.).

A medium containing serum (human serum, fetal bovine serum, etc.) may be used, but by adopting a serum-free medium, it is highly safe for clinical application and the culture efficiency due to the difference between serum lots is high. It is possible to prepare cells that have the advantage of being less likely to make a difference. Specific examples of serum-free media for T cells are TexMACS ^TM (Miltenyi Biotec) and AIM V® (Thermo Fisher Scientific). When serum is used, the self-serum, that is, the individual from which the gene-modified T cell obtained in step (ii) is derived (typically, the chimeric antigen receptor gene-modified T cell obtained by the preparation method of the present invention). Serum collected from the patient receiving the administration) should be used. A medium suitable for culturing T cells may be used as the basal medium, and specific examples thereof are TexMACS ^TM and AIM V (registered trademark) described above. Other culture conditions may be any one suitable for survival and proliferation of T cells, and general ones may be adopted. For example, the cells may be cultured in a CO ₂ incubator (CO ₂ concentration 5%) set at 37 ° C.

Viral peptide-carrying non-proliferative cells may be added during step (iii). Alternatively, the cells after co-culture may be collected, mixed with the virus peptide-retaining non-proliferative cells, and then co-cultured again. These operations may be repeated twice or more. In this way, if activation without stimulation using virus peptide-retaining non-proliferative cells is performed multiple times, the induction rate of virus-specific CAR-T cells can be improved, and the number of virus-specific CAR-T cells can be increased. I can expect an increase. The cells prepared again or those prepared in step (i) in which a part of the cells are stored are used as the virus peptide-retaining non-proliferative cells here.

The period of co-culture in the 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, and if the culture period is too long, there is a risk that the activity (life force) of the cells will decrease, and the cells will be exhausted / fatigued.

Prior to co-culture with viral peptide-carrying non-proliferative cells, the genetically modified T cells obtained in step (ii) may be co-cultured with viral peptide-carrying non-proliferative PBMCs (peripheral blood mononuclear cells). .. In this embodiment, the gene-modified T cells obtained in step (ii) and the cells obtained by co-culturing the viral peptide-retaining non-proliferative PBMC, and the viral peptide-retaining non-proliferative cells prepared in step (i). Will be mixed and co-cultured. The viral peptide-retaining non-proliferative PBMCs herein can be prepared by subjecting the PBMCs to culture in the presence of the viral peptide antigen and to a treatment that causes loss of proliferative capacity. Specifically, for example, after radiation-treating PBMC isolated from peripheral blood, the cells are cultured in the presence of a viral peptide antigen to obtain a viral peptide-retaining non-proliferative PBMC. If a part of PBMC isolated from peripheral blood obtained by one blood sampling is used to prepare a virus peptide-retaining non-proliferative PBMC, and a gene-modified T cell is prepared from the other part, The number of blood samplings associated with the implementation of the present invention can be reduced, which is an extremely great advantage in clinical application. In particular, if step (i) is performed using the remaining PBMC to prepare viral peptide-retaining non-proliferative cells (cells used for the second stage co-culture), the necessary three types of cells, that is, , Genetically modified T cells, virus peptide-carrying non-proliferative PBMC used for co-culture with the cells, virus peptide-carrying non-proliferative cells used for the second stage co-culture can be prepared by one blood sampling. Therefore, the burden on the patient in the treatment using the CAR-T cells obtained in the present invention is significantly reduced.

In the step (iv) following the step (iii), the cells after culturing are collected. The collection operation may be performed by a conventional method. For example, it is collected by pipetting, centrifugation, or the like. Between step (iii) and step (iv), a step (expansion culture) may be performed in which the cells after co-culture are cultured in the presence of T cell growth factor. A virus peptide-retaining non-proliferative cell may be added during this expansion culture, or a virus peptide-retaining non-proliferation cell may be added during the expansion culture.

In another embodiment, genetically modified T cells obtained by the same procedure as in step (ii) are co-cultured with viral peptide-carrying non-proliferative PBMC (peripheral blood mononuclear cells), and then an anti-CD28 antibody (anti-CD3 antibody) is used. May be used in combination). Subsequently, after culturing (for example, expanded culture) as necessary, the cells are collected to obtain CAR-T cells (virus-specific CAR-T cells as CAR gene-introduced lymphocytes of the present invention).

2. 2. CAR gene-introduced lymphocytes and their uses The second aspect of the present invention is the genetically modified lymphocytes expressing the chimeric antigen receptor obtained by the preparation method of the present invention (hereinafter, "CAR gene-introduced lymphocytes of the present invention"). ”) And its uses. The CAR gene-introduced lymphocyte of the present invention can be used for the treatment, prevention or amelioration of various diseases (hereinafter referred to as "target diseases") for which CAR therapy is considered to be effective. The representative of the target disease is cancer, but it is not limited to this. Examples of target diseases include various B-cell lymphomas (follicle malignant lymphoma, diffuse malignant lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20-positive Hodgkin lymphoma, etc.), myeloid proliferative tumor, bone marrow. Hypoplastic / myeloid proliferative tumor (CMML, JMML, CML, MDS / MPN-UC), myelodystrophy syndrome, acute myeloid leukemia, neuroblastoma, brain tumor, Ewing's sarcoma, osteosarcoma, retinal blastoma, small lung Cell tumor, melanoma, ovarian cancer, rhabdomyomyoma, kidney cancer, pancreatic cancer, malignant mesoderma, prostate cancer, etc. "Treatment" includes alleviating (mitigating) the symptoms characteristic of the target disease or associated symptoms, preventing or delaying the exacerbation of the symptoms, and the like. "Prevention" means preventing or delaying the onset / delay of a disease (disorder) or its symptoms, or reducing the risk of onset / onset. On the other hand, "improvement" means that the disease (disorder) or its symptom is alleviated (mild), improved, remitted, or cured (including partial cure).

The CAR gene-introduced lymphocyte 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 gene-introduced lymphocyte of the present invention. For example, for a single dose, it contains 1 × 10 ⁴ to 1 × 10 ¹⁰ cells. Various components (vitamins) for the purpose of cell activation, proliferation or differentiation induction, such as dimethylsulfoxide (DMSO) and serum albumin for the purpose of protecting cells, antibiotics for the purpose of preventing bacterial contamination, etc. , Cytokines, growth factors, steroids, etc.) may be contained in the cell preparation.

The administration route of the CAR gene-introduced lymphocyte or cell preparation of the present invention is not particularly limited. For example, it is administered by intravenous injection, intraarterial injection, intramural injection, intradermal injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. Local administration may be used instead of systemic administration. As local administration, direct injection into a target tissue / organ / organ can be exemplified. The administration schedule may be prepared in consideration of the gender, age, body weight, pathological condition, etc. of the subject (patient). In addition to a single dose, multiple doses may be administered continuously or periodically.

In the treatment method using CAR gene-introduced lymphocytes of the present invention, a therapeutically effective amount of CAR gene-introduced lymphocytes is administered to the patient. The CAR gene-introduced lymphocytes of the present invention exhibit the characteristic of being highly sensitive to thiopurine due to its characteristic constitution (the siRNA targeting the NUDT15 gene and / or the siRNA targeting the TPMT gene is produced). Therefore, the administration of the thiopurine preparation enables its control, that is, the induction of specific cell death. For example, when abnormal proliferation or excessive cytokine release is observed after administration of CAR gene-introduced lymphocytes, a thiopurine preparation is administered to the patient to alleviate the side effects. If an appropriate amount of the thiopurine preparation is used, the CAR gene-introduced lymphocytes cannot be completely removed, and after the side effects have subsided, the therapeutic effect of the remaining CAR gene-introduced lymphocytes can be expected to be maintained. In setting the "appropriate amount" here, it is advisable to consider the thiopurine preparation to be used, the disease to be treated, the condition of the patient, and the like. To give a specific example, in the case of application to pediatric patients with acute leukemia, an appropriate amount (appropriate amount) should be 1/20 to 1/2 of the average maximum plasma concentration (88 ng / mL to 195 ng / mL) during maintenance therapy. The amount of thiopurine preparation used) can be set. If serious side effects are observed, it is desirable to administer an amount of thiopurine preparation that can induce cell death to all CAR gene-introduced lymphocytes, aiming for prompt resolution of the side effects.

As a thiopurine preparation used for inducing cell death of CAR gene-introduced lymphocytes, azathioprine (AZA) such as leukerin and Purinehol containing 6-mercaptopurine (6-MP) as an active ingredient is used as an active ingredient. Examples thereof include imran, azanin, and the like.

3. 3. Vectors and kits for CAR gene-introduced lymphocyte preparation Further aspects of the present invention relate to vectors (CAR gene-introduced lymphocyte preparation vector) and kits (CAR gene-introduced lymphocyte preparation kit) that can be used in the preparation method of the present invention. The CAR gene-introduced lymphocyte preparation vector of the present invention is equipped with a CAR expression cassette and a siRNA expression cassette (that is, a CAR-siRNA vector), and one vector can be used to introduce the two expression cassettes into target cells. to enable. In addition to the CAR gene, the CAR expression cassette contains promoters (CMV-IE, SV40ori, retrovirus LTP, SRα, EF1α, β-actin promoter, etc.) required for the expression of the CAR gene. The siRNA expression cassette includes a construct for siRNA, that is, a nucleic acid construct that produces NUDT15 siRNA intracellularly (first nucleic acid construct), a nucleic acid construct that produces TPMT siRNA intracellularly (second nucleic acid construct), or both. And the promoters (U6 promoter, H1 promoter, tRNA promoter, etc.) required for the expression of the construct for siRNA are included. The vector of the present invention can include a detection gene (reporter gene, cell or tissue-specific gene, selectable marker gene, etc.), enhancer sequence, WRPE sequence, and the like.

Preferably, the vector of the present invention is constructed as a vector used in the transposon method. In this case, typically a structure in which the CAR expression cassette and the siRNA expression cassette are sandwiched between a pair of transposon inverted repeat sequences (eg, 5'side transposon inverted repeat sequence, CAR expression cassette, siRNA expression cassette, 3'side). The vector will be equipped with a transposon (arranged in the order of the inverted repeat sequence).

One aspect of the kit of the present invention is a kit suitable for a method for preparing CAR gene-introduced lymphocytes using a transposon method. The kit includes the CAR-siRNA vector and the transposase expression vector carrying the "CAR expression cassette and siRNA expression cassette" sandwiched between a pair of transposon inverted repeat sequences. The transposase is selected to correspond to a pair of transposon inverted repeats integrated into the CAR-siRNA vector. For example, a combination of piggyBac inverted repeat sequence and piggyBac transposase is adopted.

Another aspect of the kit of the present invention comprises roughly two types of vectors, namely a CAR vector and an siRNA vector. In other words, the kit incorporates a CAR expression cassette and an siRNA expression cassette in separate vectors. When using this kit, for example, the target cell is co-transformed (cotransfected) with a CAR vector and a siRNA vector, or the target cell is transformed with one of the vectors, and then a transformant (vector) is used. Transfection target cells) are transformed with the other vector. As the siRNA vector, a vector equipped with a NUDT15 siRNA expression cassette, a vector equipped with a TPMT siRNA expression cassette, or both, or a vector equipped with a NUDT15 siRNA expression cassette and a TPMT expression cassette is used. The structure that these vectors should have is as described above (see column 1). Each vector is constructed for use in viral gene transfer or non-viral transfer methods. Preferably, each vector is constructed so as to be suitable for gene transfer by the transposon method, which is one of the non-virus transfer methods. That is, the CAR vector is constructed so that the CAR expression cassette has a structure sandwiched between a pair of transposon inverted repeat sequences, and similarly, the siRNA expression cassette has a structure sandwiched between a pair of transposon inverted repeat sequences. Construct a siRNA vector. The kit also includes a transposase expression vector for supplying the transposase. The pair of transposon inverted repeats integrated into the CAR vector and the pair of transposon inverted repeats integrated into the siRNA vector shall be subject to the action of the transposase expressed by the combined transposase expression vector. That is, the transposon and the transposon inverted repeat sequence are configured to correspond to each other.

Reagents, instruments, devices, etc. used for gene transfer operations, reagents, instruments, devices, etc. used for detection and selection of transformants may be included in the kit of the present invention. An instruction manual is usually attached to the kit of the present invention.

The following studies were conducted with the aim of developing a new suicide gene system in CAR therapy.

1. 1. Materials and methods <Preparation of pIRII-CAR.CD19-NUDT15KD vector (Fig. 1)>
(1) Previously reported CD19. CAR expression piggyBac transposon vector (pIRII-CAR.CD19. Saito S, Nakazawa Y, Sueki A, Matsuda K, Tanaka M, Yanagisawa R, Maeda Y, Sato Y, Okabe S, Inukai T, Sugita K, Wilson MH, Rooney CM, Koike K. Anti-leukemic potency of piggyBac-mediated CD19-specific T cells against refractory Philadelphia chromosome-positive acute lymphoblastic leukemia. Cytotherapy. 2014 Sep; 16 (9): 1257-69.) It was cleaved with both the limiting enzymes MunI and ClaI.
(2) A DNA fragment having a shRNA (NUDT15KD) sequence that suppresses the expression of the NUDT15 gene under the U6 promoter and a restriction enzyme recognition sequence (MunI recognition sequence on the 5'side and ClaI recognition sequence on the 3'side) on both sides thereof (Fig.) 2. SEQ ID NO: 13, SEQ ID NO: 14, respectively, including the U6 promoter (SEQ ID NO: 15) and the sequence encoding shRNA (SEQ ID NO: 16, SEQ ID NO: 17) were cleaved with both MunI and ClaI.
(3) The 6341 bp DNA fragment obtained in (1) and the 339 bp (No1.) Or 341 bp (No. 2) DNA fragment obtained in (2) were ligated using T4 DNA ligase, respectively.
(4) Using competent cells, the 6680 bp (No. 1) and 6682 bp (No. 2) circular DNA plasmids obtained in (3) were mass-amplified.
(5) The entire base sequence (SEQ ID NOs: 10 and 11) was confirmed using a sequencer.

<Preparation of pIRII-CAR.CD19-TPMTKD vector (Fig. 3)>
(1) The previously reported CD19.CAR expression piggyBac transposon vector was cleaved with both restriction enzymes MunI and ClaI.
(2) A DNA fragment having an shRNA (TPMTKD) sequence that suppresses the expression of the TPMT gene under the U6 promoter and a restriction enzyme recognition sequence (MunI recognition sequence on the 5'side and ClaI recognition sequence on the 3'side) on both sides thereof (Fig. 4. SEQ ID NO: 18. U6 promoter (SEQ ID NO: 15), including sequence encoding shRNA (SEQ ID NO: 19)) was cleaved with both MunI and ClaI.
(3) The 6341 bp DNA fragment obtained in (1) and the 339 bp DNA fragment obtained in (2) were ligated using T4 DNA ligase.
(4) Using competent cells, the 6680 bp circular DNA plasmid obtained in (3) was amplified in large quantities.
(5) The entire base sequence (SEQ ID NO: 12) was confirmed using a sequencer.

<Preparation of CD19.CAR-NUDT15KD-T cells and CD19.CAR-TPMTKD-T cells>
(1) Mononuclear cells (PBMC) were separated from about 10 ml of peripheral blood using a specific gravity centrifugation method.
(2) Using the electroporation method (Program EO-115) using a combination of 4D-Nucleofector ^TM device and P3 Primary Cell 4D-Nucleofector ^TM X kit (Lonza), pIRII-CAR.CD19-NUDT15KD vector (5 μg) And pCMV-piggyBac vector (5 μg) were introduced into 1x10 ⁷ PBMCs.
(3) Pulsed with the gene-introduced cells obtained in (2) and four types of viral antigen peptides (MACS GMP PepTivator; AdV5 Hexon, CMV pp65, EBV EBNA-1, EBV BZLF1, Miltenyi Biotec, Auburn, CA). 1x10 ⁶ irradiated PBMCs were mixed and filled with 2 ml of Interleukin (IL) -7 (10 ng / ml, Miltenyi) and IL-15 (5 ng / ml, Miltenyi) -added TexMACS medium (Miltenyi). In addition, the anti-CD28 antibody (Miltenyi) was allowed to stand in 1 well of a 24-well culture plate immobilized on a solid phase. Three days after gene transfer, transgenic cells were transferred into 1 well of a non-solidified 24-well culture plate. At that time, 1 ml of IL-7 / IL-15-added TexMACS medium was replaced. Seven days after gene transfer, the transgenic cells were transferred into a G-Rex 10 incubator (Wilson Wolf Manufacturing Inc, New Brighton, MN) filled with 30 ml of IL-7 / IL-15 (5 ng / ml) -added TexMACS medium. .. Cells were harvested 14 days after gene transfer (CD19.CAR-NUDT15KD-T cells, CD19.CAR-TPMTKD-T cells). The expression of CD19 CAR protein was confirmed by flow cytometry using some cells. As control groups, CD19.CAR-T cells (control) and gene-unintroduced T cells (control) into which pIRII-CAR.CD19-SNC1KD, which was obtained by inserting control (SNC1) shRNA into the previously reported pIRII-CAR.CD19 vector, were introduced. mock T cells) were also amplified and cultured in the same manner.

<Co-culture experiment>
3x10 ^{5 each of CD19.CAR-T cells (control), CD19.CAR-NUDT15KD-T cells, CD19.CAR-TPMTKD-T cells, and 3x10 5 acute} lymphocytic leukemias in 1 well of a 48-well culture plate. (ALL) Cell lines SU / SR and T cells: Leukemia cells were co-cultured in 4 wells under 1: 1 conditions under 1 ml of 10% bovine fetal serum-containing RPMI1640 medium for 5 days. The conditions for T cells alone were also cultured in 4 wells each. On the 2nd day of the start of co-culture, 6-MP was added at a concentration of 0, 5, 10, 50 ng / mL for each culture condition. 72 hours after the addition of 6-MP, cells were collected for each well, the number of viable cells was counted by tripan blue staining, stained with anti-CD3-APC antibody and anti-CD19-PE antibody, and then CD3 positive cells by flow cytometry. The ratio of (T cells) to CD19-positive cells (ALL cells) was measured. At the same time, the expression rate of CD19 CAR in CD3-positive cells was analyzed using F (ab') 2 Fragment Goat anti-human IgG-FITC antibody.

2. 2. result
CD19.CAR expression vectors (pIRII-CAR.CD19-NUDT15KD and pIRII-CAR.CD19-TPMTKD) that knock down the expression of the NUDT15 gene or TPMT gene were constructed (Figs. 1 and 3). Gene transfer of T cells expressing pIRII-CAR.CD19-NUDT15KD (CD19.CAR-NUDT15KD-T cells) and T cells expressing pIRII-CAR.CD19-TPMTKD (CD19.CAR-TPMTKD-T cells) When the number of cells and the expression rate of CAR on the 14th day were confirmed, there was no significant difference from the control (Fig. 5). In addition, as a result of co-culture with the acute lymphocytic leukemia (ALL) cell line SU / SR, CD19.CAR-NUDT15KD-T cells and CD19.CAR-TPMTKD-T cells exert a strong anti-leukemia effect. It was highly sensitive to 6-MP (Fig. 7). In CD19.CAR-NUDT15KD-T cells, cell death was induced in a 6-MP concentration-dependent manner (Fig. 7, left).

3. 3. Consideration
CD19. Suppression of NUDT15 gene or TPMT gene expression in CAR-T cells has been shown to be a suicidal function of CAR-T cells. That is, the effectiveness of the suicide gene system utilizing the suppression of the expression of the NUDT15 gene or the TPMT gene could be confirmed. Its suicide effect is Lafolie P, Bjork O, Hayder S, Ahstrom L, Peterson C. Variability of 6-mercaptopurine pharmacokinetics during oral maintenance therapy of children with acute leukemia. Med Oncol Tumor Pharmacother. 1989; 6 (4): 259-65 .; Hayder S, Lafolie P, Bjork O, Peterson C. 6-mercaptopurine plasma levels in children with acute lymphoblastic leukemia: relation to relapse risk and myelotoxicity. Monit. 1989 Nov; 11 (6): 617-22 .; Kato Y, Matsushita T, Chiba K, Hijiya N, Yokoyama T, Ishizaki T. Dose-dependent kinetics of orally administered 6-mercaptopurine in children with leukemia. J Pediatr 1991 Aug; 119 (2): 311-6 .; Lafolie P, Hayder S, Bjork O, Ahstrom L, Liliemark J, Peterson C. Large interindividual variations in the pharmacokinetics of oral 6-mercaptopurine in maintenance therapy of children with acute It was exhibited at a concentration of about 1/20 to 1/2 of leukaemia and non-Hodgkin lymphoma. Acta Paediatr Scand. 1986 Sep; 75 (5): 797-803.). It is expected that increasing the concentration of 6-MP will induce complete cell death in CD19.CAR-T cells. On the other hand, since 6-MP acts in the S phase of the cell cycle, cells are limited to CD19.CAR-T cells that have overgrown or released excessive cytokines by administering 6-MP at a concentration below the optimum concentration. May induce death. In addition, 6-MP is transferred into the cerebrospinal fluid (Hayder S, Lafolie P, Bjork O, Ahstrom L, Peterson C. 6-Mercaptopurine in cerebrospinal fluid during oral maintenance therapy of children with acute lymphoblastic leukemia. Med Oncol Tumor. Pharmacother. 1988; 5 (3): 187-9.), A novel suicide gene system using 6-MP as a suicide-initiating drug is also effective in treating central nervous system complications of CD19.CAR-T cell therapy. You can expect that.

The present invention provides solutions to the problems of on / off-target side effects, hypercytokine storms, and graft-versus-host disease (GVHD) in CAR therapy. According to the suicide gene system of the present invention, the safety of CAR therapy can be enhanced while maintaining the therapeutic effect.

The present invention is not limited to the description of the embodiments and examples of the above invention. Various modifications are also included in the present invention to the extent that those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of the papers, published patent gazettes, patent gazettes, etc. specified in this specification shall be cited by reference in their entirety.

Claims (17)

Along with the target antigen-specific chimeric antigen receptor gene, the first nucleic acid construct that produces siRNA that targets the human-derived NUDT15 gene in cells and / or the second that produces siRNA that targets the human-derived TPMT gene in cells. A method for in vitro preparation of genetically modified lymphocytes expressing a chimeric antigen receptor, comprising the step of introducing a nucleic acid construct into a target cell. The preparation method according to claim 1, wherein the introduction of the target antigen-specific chimeric antigen receptor gene, the first nucleic acid construct, and the second nucleic acid construct is carried out by the transposon method. The preparation method according to claim 2, wherein the transposon method is a piggyBac transposon method. One of claims 1 to 3, wherein the target antigen-specific chimeric antigen receptor gene and the first nucleic acid construct and / or the second nucleic acid construct are mounted on the same vector, and the vector is introduced into the target cell. The preparation method described in. The preparation method according to any one of claims 1 to 4, wherein the target cell is a T cell. Any of claims 1 to 5, wherein siRNA targeting a human-derived NUDT15 gene and / or siRNA targeting a human-derived TPMT gene is generated intracellularly while expressing a chimeric antigen receptor. The genetically modified lymphocyte obtained by the preparation method described in item 1. A cell preparation containing a therapeutically effective amount of the genetically modified lymphocyte according to claim 6. The cell preparation according to claim 7, which is for the treatment of cancer . A preparation for inducing cell death of the genetically modified lymphocyte, which is used to administer a thiopurine preparation to a cancer patient to whom a therapeutically effective amount of the genetically modified lymphocyte according to claim 6 has been administered. A first nucleic acid construct that intracellularly produces siRNAs targeting the human- derived NUDT15 gene and / or siRNAs targeting the human-derived TPMT gene, along with a chimeric antigen receptor expression cassette containing the target antigen-specific chimeric antigen receptor gene. A vector containing an siRNA expression cassette containing a second nucleic acid construct that produces intracellularly. The vector according to claim 10, wherein the chimeric antigen receptor expression cassette and the siRNA expression cassette have a structure sandwiched between a pair of transposon inverted repeat sequences. A kit for preparing a genetically modified lymphocyte expressing a chimeric antigen receptor, which comprises the vector according to claim 11 and a transposase expression vector. A vector carrying a chimeric antigen receptor expression cassette containing a target antigen-specific chimeric antigen receptor gene, and
A vector with a siRNA expression cassette containing a first nucleic acid construct that produces siRNA that targets the human-derived NUDT15 gene intracellularly and / or a second nucleic acid construct that produces siRNA that targets the human-derived TPMT gene intracellularly. When,
A kit for preparing genetically modified lymphocytes expressing a chimeric antigen receptor. The chimeric antigen receptor expression cassette has a structure sandwiched between a pair of transposon inverted repeat sequences.
The siRNA expression cassette has a structure sandwiched between a pair of transposon inverted repeat sequences.
13. The preparation kit of claim 13, further comprising a transposase expression vector. A vector carrying a chimeric antigen receptor first expression cassette containing a target antigen-specific chimeric antigen receptor gene, and
A vector carrying a first siRNA expression cassette containing a first nucleic acid construct that produces siRNA targeting the human-derived NUDT15 gene in cells.
A vector carrying a second siRNA expression cassette containing a second nucleic acid construct that produces siRNA targeting the human-derived TPMT gene in the cell,
A kit for preparing genetically modified lymphocytes expressing a chimeric antigen receptor. The chimeric antigen receptor expression cassette has a structure sandwiched between a pair of transposon inverted repeat sequences.
The first siRNA expression cassette has a structure sandwiched between a pair of transposon inverted repeat sequences.
The second siRNA expression cassette has a structure sandwiched between a pair of transposon inverted repeat sequences.
The preparation kit according to claim 15, further comprising a transposase expression vector. The preparation kit according to any one of claims 12, 14, and 16, wherein the transposase is a piggyBac transposase.

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