Selective regulatory gene (SRG) system for genetically modified immune cell therapy

Abstract

The present invention provides a chimeric receptor comprising: a first polypeptide which has (i) a first extracellular region binding to a ligand, (ii) a first transmembrane region, and (iii) a first intracellular region derived from an IL-2 receptor β chain, and in which a mutation is introduced to a tyrosine residue in the first intracellular region; and a second polypeptide which has (iv) a second extracellular region binding to a ligand, (v) a second transmembrane region, and (vi) a second intracellular region derived from an IL-2 receptor γ chain.

Description

Selective Regulatory Gene (SRG) System for Genetically Modified Immune Cell Therapy

[0001] The present invention relates to chimeric receptors that are useful for improving the in vitro proliferation, in vivo proliferation and in vivo persistence of immune cells such as T cells.

[0002] As a therapeutic strategy for tumors, it is expected that T cells that target tumor cells can be produced by introducing into T cells a nucleic acid encoding a chimeric antigen receptor (CAR) that binds to a specific antigen present on the surface of tumor cells, or a nucleic acid encoding a T cell receptor (TCR) that recognizes tumor cells. Here, T cells introduced with a nucleic acid encoding a CAR are called CAR-T cells, and T cells introduced with a nucleic acid encoding a TCR are called TCR-T cells.

[0003] Currently, attempts are being made to develop therapies using CAR-T cells or TCR-T cells targeting many tumor antigens, such as CD19, BCMA, WT1, MART1, gp100, CEA, and mHAG HA-2 antigens. For example, therapy using CD19-specific CAR-T cells has demonstrated remarkable therapeutic effects against B-cell tumors. However, long-term follow-up has revealed tumor recurrence, and the treatment outcomes are far from satisfactory, requiring further innovation. One reason for tumor recurrence after CAR-T cell therapy is said to be the inability of CAR-T cells to persist in the body for long periods of time.

[0004] Interleukin 2 (IL-2) is sometimes used to maintain the proliferation and antitumor activity of exogenously administered T cells in vivo. However, the administration of IL-2 has the problem of inducing side effects. Furthermore, because IL-2 reacts with all T cells that express the IL-2 receptor, it activates not only the administered T cells but also endogenous regulatory T cells, thereby weakening the immune response.

[0005] A known method for overcoming the IL-2 requirement of T cells is to use erythropoietin, a low-toxicity cytokine, as a ligand instead of IL-2. In 1995, Minamoto et al. developed a chimeric receptor comprising the extracellular domain of the erythropoietin receptor and the intracellular domain of the IL-2 receptor subunit β-chain or γ-chain (Non-Patent Document 1).

[0006] In 2020, Campana et al. reported the EpoRm-CAR system, which co-expresses a mutant erythropoietin receptor and a CAR (Non-Patent Document 2). This system aims to enhance the in vivo persistence of T cells co-expressing a mutant erythropoietin receptor and a CAR (hereinafter referred to as EpoRm-CAR-T cells) through erythropoietin stimulation. However, the effectiveness of the EpoRm-CAR system is considered insufficient, for example, because in vitro erythropoietin stimulation resulted in EpoRm-CAR-T cells proliferating only about twice as much as controls, and in in vivo treatment experiments, cancer cell proliferation could not be suppressed unless EpoRm-CAR-T cells were infused into mice prior to inoculation with cancer cells.

[0007] Blood. 1995 Sep 15;86(6):2281-7.Blood. 2020 Feb 27;135(9):668-679.

[0008] As mentioned above, conventional CAR-T therapy has shown unsatisfactory therapeutic outcomes, such as tumor recurrence in long-term follow-up, and further innovations are needed. More specifically, there is still a need for novel methods for enabling T cells administered to patients to persist in the body for a long period of time.

[0009] As a result of extensive research aimed at solving the above problems, the present inventors have discovered a method for selectively controlling the proliferation and survival of T cells in vitro and in vivo, and have completed the present invention.

[0010] That is, the present invention provides: [1] a chimeric receptor comprising: a first polypeptide having (i) a first extracellular region that binds to a ligand, (ii) a first transmembrane region, and (iii) a first intracellular region derived from the IL-2 receptor β chain, wherein one or more tyrosine residues in the first intracellular region have been mutated; and a second polypeptide having (iv) a second extracellular region that binds to a ligand, (v) a second transmembrane region, and (vi) a second intracellular region derived from the IL-2 receptor γ chain; [2] the chimeric receptor according to [1], wherein (iii) the one or more tyrosine residues in the first intracellular region derived from the IL-2 receptor β chain are selected from the group consisting of tyrosine residues at positions 381, 384, and 387 in the amino acid sequence of the IL-2 receptor β chain (RefSeq NP_000869.1); [3] (iii) The chimeric receptor according to [2], wherein one or more tyrosine residues in the first intracellular domain derived from the IL-2 receptor β chain are tyrosine residues corresponding to positions 381, 384, and 387 in the amino acid sequence of the IL-2 receptor β chain (RefSeq NP_000869.1); [4] The chimeric receptor according to any one of [1] to [3], wherein the mutation is a substitution of a tyrosine residue with a phenylalanine residue; [5] The chimeric receptor according to any one of [1] to [4], wherein (i) the first extracellular domain that binds to a ligand and / or (iv) the second extracellular domain that binds to a ligand are extracellular domains derived from the erythropoietin receptor; [6] The chimeric receptor according to any one of [1] to [5], wherein (ii) the first transmembrane domain is a transmembrane domain derived from the IL-2 receptor β chain and (v) the second transmembrane domain is a transmembrane domain derived from the IL-2 receptor γ chain; [7] The chimeric receptor according to any one of [1] to [6], wherein the second polypeptide further comprises an intracellular region derived from CD40; [8] A nucleic acid encoding the chimeric receptor according to any one of [1] to [7]; [9] A vector comprising the nucleic acid according to [8];

[10] The vector according to [9], selected from the group consisting of a plasmid vector, a viral vector, and an artificial chromosome;

[11] The vector according to [9] or

[10] , further comprising a nucleic acid encoding a chimeric antigen receptor or a foreign T cell receptor that recognizes a target cell;

[12] A cell that expresses the chimeric receptor according to any one of [1] to [7];

[13] The cell according to

[12] , further expressing a chimeric antigen receptor or a foreign T cell receptor that recognizes a target cell;

[14] The cell according to

[12] or

[13] , which is an immune cell;

[15] The cell according to

[14] , which is selected from the group consisting of T cells, tumor-infiltrating lymphocytes, NK cells, and NK-T cells;

[16] The cell according to any one of

[12] to

[15] , which is a human-derived cell;

[17] A method for producing a cell that expresses a chimeric receptor, which comprises a step of introducing the nucleic acid according to [8] into a cell;

[18] A method for producing the cell according to

[17] , which comprises a step of introducing the vector according to any one of [9] to

[11] into a cell;

[19] A method for producing cells according to

[17] or

[18] , further comprising a step of isolating and / or expanding cells expressing a chimeric receptor;

[20] A method for producing cells according to any one of

[17] to

[19] , further comprising a step of culturing cells introduced with a nucleic acid encoding a chimeric receptor in the presence of the ligand;

[21] A method for producing cells according to

[20] , further comprising a step of culturing cells introduced with a nucleic acid encoding a chimeric receptor having an extracellular domain of an erythropoietin receptor in the presence of erythropoietin;

[22] A pharmaceutical composition comprising the cells according to any one of

[12] to

[16] as an active ingredient;

[23] The pharmaceutical composition according to

[22] , wherein the cells further express a chimeric antigen receptor or a foreign T cell receptor that recognizes a target cell;

[24] The pharmaceutical composition according to

[23] , wherein the target cell is a tumor cell, and further comprising cells expressing a chimeric antigen receptor or a foreign T cell receptor that recognizes a tumor antigen as an active ingredient;

[25] A method for preventing or treating a disease, characterized by administering the pharmaceutical composition according to any one of

[22] to

[24] to a subject;

[26] A method for reducing target cells in a subject, the method comprising administering to a subject in need thereof the pharmaceutical composition according to any one of

[22] to

[24] .

[0011] The present invention provides a chimeric receptor useful in the field of gene-modified immune cell therapy targeting antigens such as tumor antigens, a nucleic acid encoding the chimeric receptor, a vector containing the nucleic acid, a cell expressing the chimeric receptor, a method for producing the cell, a pharmaceutical composition containing the cell as an active ingredient, and a method for preventing (including prevention of recurrence; the same applies hereinafter) or treating a disease, which comprises administering the pharmaceutical composition to a subject. CAR-T cells or TCR-T cells into which the chimeric receptor of the present invention has been introduced can persist in the body of a patient for a long period of time and exhibit high therapeutic efficacy.

[0012] FIG. 1 shows a method for preparing the plasmid pEX-A2J2 / TEGPEB. FIG. 2 shows a method for preparing the retroviral plasmid pMEI-5 / ZG-SRG_YYY. FIG. 3 shows a method for preparing the retroviral plasmid pMEI-5 / ZG-SRG_FYY. FIG. 4 shows a method for preparing the retroviral plasmid pMEI-5 / ZG-SRG_YFY. FIG. 5 shows a method for preparing the retroviral plasmid pMEI-5 / ZG-SRG_YYF. FIG. 6 shows a method for preparing the retroviral plasmid pMEI-5 / ZG-SRG_FFY. FIG. 7 shows a method for preparing the retroviral plasmid pMEI-5 / ZG-SRG_YFF. FIG. 8 shows a method for preparing the retroviral plasmid pMEI-5 / ZG-SRG_FYF. FIG. 9 shows a method for preparing the retroviral plasmid pMEI-5 / ZG-SRG_FFF. FIG. 10 shows a method for preparing the retroviral plasmid pMEI-5 / SRG_YYY.

[0023] Figure 1 shows a method for preparing the retroviral plasmid pMEI-5 / SRG_FFF.

[0024] Figure 2 shows a method for preparing the retroviral plasmid pMEI-5 / EL-SRG_FFF.

[0025] Figure 3 shows a method for preparing the retroviral plasmid pMEI-5 / FMC28z.

[0026] Figure 4 shows a method for preparing the retroviral plasmid pMEI-5 / FMC28z-SRG_FFF.

[0027] Figure 5 shows a method for preparing the retroviral plasmid pMEI-5 / ZG-EpoRm.

[0028] Figure 6 shows a flow chart for the production and expansion of transduced cells in Example 3-(2).

[0029] Figure 7 shows a growth curve of transduced cells in Example 3-(2).

[0030] Figure 8 shows a growth curve of transduced cells in Example 3-(2).

[0031] Figure 9 shows the ZsGreen1 positivity rate of transduced cells in Example 3-(2).

[0032] Figure 10 shows a growth curve of transduced cells in Example 4.

[0033] Figure 11 shows a growth curve of transduced cells in Example 5.

[0034] Figure 12 shows expansion culture conditions in Example 6.

[0035] Figure 13 shows a growth curve of transduced cells in Example 6. 1 shows a growth curve of transduced cells in Example 6. 2 shows the ZsGreen1 positive rate of transduced cells in Example 6. 3 shows the ZsGreen1 positive rate of transduced cells in Example 6.7-(1) shows the production flow of transduced cells and the measurement flow of in vivo bioluminescence imaging in Example 7-(1). It is a diagram showing in vivo bioluminescence imaging in Example 7-(1). It is a diagram showing a graph in which luminescence intensity is quantified in Example 7-(1). It is a diagram showing the hematocrit value of whole blood in Example 7-(1). It is a diagram showing the production flow of transduced cells and the measurement flow of in vivo bioluminescence imaging in Example 7-(2). It is a diagram showing in vivo bioluminescence imaging in Example 7-(2). It is a diagram showing a graph in which luminescence intensity is quantified in Example 7-(2). It is a diagram showing the hematocrit value of whole blood in Example 7-(2). It is a diagram showing the CAR and SRG (EpoR) positivity rates in Example 8. It is a diagram showing the ratio of CD4 to CD8 in Example 8. It is a diagram showing the ratio of each CD4 and CD8 T cell subset in Example 8. 1 is a diagram showing the ratios of CD4 and CD8 T cell subsets in Example 8. 2 is a diagram showing the measurement of cytotoxic activity in Example 9-(1). 3 is a diagram showing the measurement of IL-2 and IFN-γ production in Example 9-(1). 4 is a diagram showing the measurement of IL-2 and IFN-γ production in Example 9-(1). 5 is a diagram showing the production flow of transduced cells and the measurement flow of in vivo bioluminescence imaging in Example 10. 6 is a diagram showing in vivo bioluminescence imaging in Example 10. 7 is a graph showing the numerical values ​​of luminescence intensity in Example 10. 8 is a diagram showing the Kaplan-Meier curve in Example 10. 9 is a diagram showing a method for preparing the retroviral plasmid pMEI-5 / ZG-SRG_FFF40. 10 is a diagram showing a method for preparing the retroviral plasmid pMEI-5 / SRG_FFF40. 11 is a diagram showing a method for preparing the retroviral plasmid pMEI-5 / EL-SRG_FFF40. 12 is a diagram showing a method for preparing the retroviral plasmid pMEI-5 / FMC28z-SRG_FFF40. 12 is a diagram showing the measurement of cytotoxic activity in Example 12. FIG. 13 is a diagram showing the Kaplan-Meier curve in Example 13.

[0013] The present invention will be described in detail below.

[0014] (1) Chimeric Receptor of the Present Invention The chimeric receptor of the present invention comprises: (i) a first polypeptide having a first extracellular domain that binds to a ligand, (ii) a first transmembrane domain, and (iii) a first intracellular domain derived from the IL-2 receptor β chain, wherein the first intracellular domain has been mutated; and (iv) a second polypeptide having a second extracellular domain that binds to a ligand, (v) a second transmembrane domain, and (vi) a second intracellular domain derived from the IL-2 receptor γ chain.

[0015] The first polypeptide constituting the chimeric receptor of the present invention comprises, from N- to C-terminus, (i) a first extracellular domain capable of binding to a ligand, (ii) a first transmembrane domain, and (iii) a first intracellular domain derived from the IL-2 receptor β chain. The second polypeptide comprises, from N- to C-terminus, (iv) a second extracellular domain capable of binding to a ligand, (v) a second transmembrane domain, and (vi) a second intracellular domain derived from the IL-2 receptor γ chain.

[0016] Furthermore, both peptides may have a signal peptide at their N-terminus. The signal peptide may be, for example, one originally contained in the extracellular domain of one of the polypeptides contained in the chimeric receptor of the present invention as the first or second extracellular domain, or may be one derived from another secretory protein or membrane protein.

[0017] Here, the first extracellular region and the second extracellular region may be different from each other or may be the same. Furthermore, the two extracellular regions may be different regions derived from the same polypeptide. Appropriate regions may be selected from the extracellular regions described below. Furthermore, the first transmembrane region and the second transmembrane region may be different from each other or may be the same. Furthermore, the two transmembrane regions may be different regions derived from the same polypeptide. Appropriate regions may be selected from the transmembrane regions described below.

[0018] In the present invention, the ligand is not particularly limited as long as it is a molecule that can be recognized by or bind to a specific polypeptide, i.e., the "extracellular region" described below, and examples thereof include nucleic acids, sugar chains, lipids, peptides, and proteins. If the extracellular region is derived from a receptor, the ligand can be used; if the extracellular region is the antigen-binding domain of an antibody, the antigen can be used as the ligand in the present invention, but the ligand is not limited thereto. Examples of nucleic acids include short-chain nucleic acids, long-chain nucleic acids, single-stranded nucleic acids, double-stranded nucleic acids, DNA, and RNA. Examples of DNA include double-stranded DNA, single-stranded DNA, and cDNA, and examples of RNA include mRNA. Note that short-chain nucleic acids are defined as nucleic acids having 2 to 100 bases, and long-chain nucleic acids are defined as nucleic acids having more than 100 bases.

[0019] Examples of protein ligands include cytokines such as interleukins (IL-1, IL-3, IL-4, IL-6, IL-7, IL-15, IL-21), hematopoietic factors (erythropoietin, thrombopoietin, G-CSF, GM-CSF, etc.), stem cell factor (SCF), and cell growth factors (FGF, EGF, IGF, HGF, PDGF, NGF, TGF), as well as T cell activation regulators (PD-L1, PD-L2, CD80, CD86, Ceacam-1, Galectin-3, Galectin-9, FGL-1, CD112, CD155, HVEM, CD40, OX40L, 4-1BBL, GITRL, CD70, etc.).

[0020] Other examples of ligands include agonists and antagonists for cell membrane receptors, toxins and venoms, viral epitopes, hormones (e.g., opioid hormones, steroid hormones, etc.), peptides, enzymes, enzyme substrates, coenzymes, drugs, lectins, sugars, oligosaccharides, antigens, monoclonal antibodies, cells, bacteria, viruses, avidin, etc.

[0021] In the present invention, the ligand selected does not interact or exhibits no significant interaction with the cells in which the chimeric receptor of the present invention is to be expressed or with cells that may coexist with the cells. For example, in the case of a chimeric receptor to be expressed in T cells, the ligand is preferably a cytokine whose receptor is not highly expressed in T cells, and more preferably erythropoietin.

[0022] "Extracellular domain that binds to a ligand" The "(i) first extracellular domain that binds to a ligand" and "(iv) second extracellular domain that binds to a ligand" (hereinafter collectively referred to as "extracellular domain that binds to a ligand") used in the present invention are domains comprising a proteinaceous molecule or a portion thereof that can bind to a ligand, and include, for example, an extracellular domain derived from a receptor and an antigen-binding domain of an antibody. The extracellular domains bind to and interact with a ligand, thereby conferring ligand specificity to cells expressing the chimeric receptor of the present invention.

[0023] The extracellular region is also called an “extracellular domain.” As used herein, the term “domain” refers to a region within a polypeptide that folds into a specific structure independently of other regions.

[0024] The "extracellular domain that binds to a ligand" used in the present invention may be selected from those capable of binding to the ligand to be used. For example, an extracellular domain derived from a cytokine receptor, an extracellular domain derived from a growth factor receptor, an extracellular domain derived from a receptor tyrosine kinase, an extracellular domain derived from a receptor for a T-cell activation regulator, or any mutant having the same function as any of these sequences may be used. In the present invention, an extracellular domain derived from a cytokine receptor is preferred. Examples of cytokine receptors include, but are not limited to, interleukin 1 receptor (IL-1RI), IL-1 receptor accessory protein (IL-1RAcP), IL-3Rα, IL-3Rβ (common β chain), IL-4Rα, IL-5Rα, IL-6Rα, gp130, IL-7Rα, IL-15Rα, IL-21R, erythropoietin receptor (EpoR), thrombopoietin receptor (TpoR), granulocyte colony-stimulating factor receptor (G-CSFR), granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR), and growth hormone receptor (GHR). Examples of the growth factor receptors include, but are not limited to, FGF receptor 1, FGF receptor 2, FGF receptor 3, EGF receptor, IGF-1 receptor, MET receptor, PDGF receptor, NGF receptor, TGF-β type I receptor (TβRI), and TGF-β type II receptor (TβRII). Examples of the receptor tyrosine kinases include, but are not limited to, EPH receptor A4 (EPHA4) and c-kit. Examples of the receptors for T cell activation regulators include, but are not limited to, PD-1, CTLA-4, TIM-3, LAG-3, CD27, CD28, CD226, CD40L, GITR, OX-40, TIGIT, ICOS, 4-1BB, BTLA, and the like.

[0025] Another example of the "extracellular domain that binds to a ligand" used in the present invention is the antigen-binding domain of an antibody. The term "antigen-binding domain" refers to a portion of an antibody that has antigen-binding activity and is capable of binding to a specific antigen. Examples of antigen-binding domains include Fab' fragments, Fab fragments, Fv fragments, and single-chain variable fragments (scFv).

[0026] Furthermore, the "extracellular domain that binds to a ligand" of the present invention may be derived from TCR (TCRα, TCRβ, TCRγ, TCRδ), CD4 ectodomain, CD8α, CD8β, CD11A, CD11B, CD11C, CD18, CD29, CD49A, CD49B, CD49D, CD49E, CD49F, CD61, CD41, and / or CD51. For example, the CD4 ectodomain can recognize HIV-infected cells.

[0027] In the present invention, the "extracellular domain that binds to a ligand" is preferably an extracellular domain derived from a receptor, more preferably an extracellular domain derived from a cytokine receptor, and most preferably an extracellular domain derived from an erythropoietin receptor.

[0028] The "(i) first extracellular region that binds to a ligand" and the "(iv) second extracellular region that binds to a ligand" are each independently selected and may be the same extracellular region or different types of extracellular regions. For example, one of the "(i) first extracellular region that binds to a ligand" and the "(iv) second extracellular region that binds to a ligand" may be an extracellular region derived from an erythropoietin receptor, and the other may be an extracellular region of another type. Alternatively, both the "(i) first extracellular region that binds to a ligand" and the "(iv) second extracellular region that binds to a ligand" may be extracellular regions derived from an erythropoietin receptor.

[0029] "Transmembrane region" The transmembrane region is also called a "transmembrane domain" and generally refers to a region of a membrane protein that is embedded in the cell membrane. The "(ii) first transmembrane region" and "(v) second transmembrane region" (hereinafter collectively referred to as "transmembrane region") used in the present invention may be derived from a naturally occurring polypeptide or may be artificially designed. A transmembrane region derived from a naturally occurring polypeptide can be obtained from a membrane-bound or transmembrane protein. For example, interleukin 1 receptor (IL-1RI), IL-1 receptor accessory protein (IL-1RAcP), IL-2 receptor α chain, IL-2 receptor β chain, IL-2 receptor γ chain, IL-3Rα, IL-3Rβ (common β chain), IL-4Rα, IL-5Rα, IL-6Rα, gp130, IL-7Rα, IL-15Rα, IL-21R), erythropoietin receptor (EpoR), thrombopoietin receptor, granulocyte colony-stimulating factor receptor (G-CSFR), granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR), growth hormone receptor (GHR), fibroblast growth factor receptor (FGFR1, FGFR2, FGFR3), epithelial growth factor receptor (EGFR), and fibroblast growth factor receptor (FGFR). Transmembrane regions of growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF-1R), MET receptor, platelet-derived growth factor receptor (PDGFR), nerve growth factor receptor (NGFR), TGF-β type I receptor (TβRI), TGF-β type II receptor (TβRII), erythropoietin-producing hepatocyte receptor A4 (EPHA4), c-kit, T cell receptor α, T cell receptor β chain, CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or GITR can be used. Artificially designed transmembrane regions are polypeptides primarily comprising hydrophobic residues such as leucine and valine. For example, a triplet of phenylalanine, tryptophan, and valine can be found at each end of the synthetic transmembrane region. Optionally, a short oligopeptide or polypeptide linker, e.g., a linker 2-10 amino acids in length, can be placed between the first transmembrane region and the first intracellular region described herein.Preferably, a linker sequence having a glycine-serine stretch is used.

[0030] In the present invention, the "(ii) first transmembrane region" contained in the first polypeptide is preferably a transmembrane region derived from the IL-2 receptor β chain. Furthermore, the "(v) second transmembrane region" contained in the second polypeptide is preferably a transmembrane region derived from the IL-2 receptor γ chain.

[0031] "(iii) First intracellular domain derived from IL-2 receptor β chain" The intracellular domain used in the first polypeptide constituting the chimeric receptor of the present invention is capable of transmitting a signal into the cell when the extracellular domain present in the same molecule binds to (interacts with) a ligand.

[0032] As used herein, "IL-2 receptor" refers to a receptor for IL-2. Native human IL-2 receptors are composed of three cell membrane surface proteins: an α chain (also known as CD25, 55 kDa, 251 amino acid residues), a β chain (also known as CD122, 75 kDa, 525 amino acid residues), and a γ chain (also known as CD132, 64 kDa, 369 amino acid residues). In nature, these protein complexes form noncovalent bonds with IL-2 molecules and transmit signals into cells. The β and γ chains belong to the type I interleukin receptor family. Receptor proteins form dimers to transmit signals into cells, but they are usually monomers that move freely on the cell membrane (the so-called fluid mosaic model). However, when a ligand approaches, the receptor proteins form dimers and become functional as receptors.

[0033] The origin of the IL-2 receptor used in the present invention is not particularly limited, and an IL-2 receptor derived from a mammal, such as a human IL-2 receptor, or an IL-2 receptor derived from a non-human mammal such as a monkey, mouse, rat, pig, horse, or dog can be used. The IL-2 receptor used in the present invention is preferably a human IL-2 receptor.

[0034] The "(iii) first intracellular region derived from the IL-2 receptor β chain" used in the present invention can be the entire intracellular region of a naturally occurring IL-2 receptor β chain, or a region derived from a part of the intracellular region of a naturally occurring IL-2 receptor β chain. An example of the entire intracellular region of a naturally occurring IL-2 receptor β chain is the amino acid sequence from positions 266 to 551 in the amino acid sequence of the wild-type human IL-2 receptor β chain (RefSeq NP_000869.1). Further, examples of a part of the intracellular region of the IL-2 receptor β chain include the amino acid sequence of positions 266 to 541, 266 to 531, 266 to 521, 266 to 511, 266 to 501, 266 to 491, 266 to 481, 266 to 471, 266 to 461, 266 to 451, 266 to 441, 266 to 431, 266 to 421, 266 to 411, 266 to 401, and 266 to 391 in the amino acid sequence of the wild-type human IL-2 receptor β chain (RefSeq NP_000869.1) (SEQ ID NO: 73).

[0035] The "(iii) first intracellular domain derived from the IL-2 receptor β chain" used in the present invention is preferably derived from the entire intracellular domain of a naturally occurring IL-2 receptor β chain, and more preferably derived from the amino acid sequence from positions 266 to 551 in the amino acid sequence of the wild-type human IL-2 receptor β chain (RefSeq NP_000869.1).

[0036] The "(iii) first intracellular region derived from the IL-2 receptor β chain" used in the present invention has mutations introduced into one or more tyrosine residues. The "(iii) first intracellular region derived from the IL-2 receptor β chain" used in the present invention is prepared by substituting at least one amino acid with another amino acid in the amino acid sequence of the intracellular region derived from the IL-2 receptor β chain. By utilizing the intracellular region with the mutation introduced, it is possible to transmit signals into cells with high efficiency.

[0037] The position of the mutation to be introduced into "(iii) the first intracellular region derived from the IL-2 receptor β chain" is selected from the group consisting of (1) tyrosine at position 381, (2) tyrosine at position 384, and (3) tyrosine at position 387 in the amino acid sequence of the wild-type human IL-2 receptor β chain (RefSeq NP_000869.1), or is selected from the positions corresponding to the above (1) to (3) in the amino acid sequence of a non-human IL-2 receptor β chain. Those skilled in the art can easily identify the positions in the amino acid sequence of the intracellular region of a non-human IL-2 receptor β chain that correspond to each amino acid position in the amino acid sequence of the intracellular region of the human IL-2 receptor β chain.

[0038] In the "(iii) first intracellular domain derived from the IL-2 receptor β chain" used in the present invention, the amino acid residue (tyrosine) is substituted with another amino acid. The substituted amino acid residue is not particularly limited as long as the desired function of enabling highly efficient intracellular signal transmission is obtained. Furthermore, the substituted amino acid residue may be a natural amino acid or an artificial amino acid as long as the desired function is obtained. With the exception of a few special amino acids, there are 20 naturally occurring amino acids, which are classified into several groups based on their structures. Examples of such amino acids include, but are not limited to, Group A: glycine, alanine; Group B: valine, leucine, isoleucine; Group C: aspartic acid, glutamic acid; Group D: asparagine, glutamine; Group E: serine, threonine; Group F: lysine, arginine, histidine; Group G: phenylalanine, tyrosine, tryptophan; Group H: cysteine, methionine; and Group I: proline. Amino acid residues in the same group exhibit similar properties and are therefore expected to be mutually replaceable.

[0039] The mutation introduced into "(iii) the first intracellular domain derived from the IL-2 receptor β chain" is preferably a substitution of a tyrosine residue with another amino acid belonging to Group G (phenylalanine, tryptophan), and more preferably a substitution of a tyrosine residue with a phenylalanine residue.

[0040] Examples of the amino acid substitution include, but are not limited to, (1) a substitution of tyrosine at position 381 with phenylalanine (Y381F), (2) a substitution of tyrosine at position 384 with phenylalanine (Y384F), and (3) a substitution of tyrosine at position 387 with phenylalanine (Y387F) in the amino acid sequence of the human IL-2 receptor β chain (RefSeq NP_000869.1) (SEQ ID NO: 73), or amino acid substitutions corresponding to the above (1) to (3) in the amino acid sequence of a non-human IL-2 receptor β chain.

[0041] Preferably, the "(iii) first intracellular region derived from the IL-2 receptor β chain" has one or more amino acid substitutions selected from the group consisting of the amino acid substitutions (1) to (3) above, or one or more amino acid substitutions selected from the group of amino acid substitutions corresponding to the above (1) to (3) in the amino acid sequence of a non-human IL-2 receptor β chain.

[0042] More preferably, the "(iii) first intracellular region derived from the IL-2 receptor β chain" is exemplified by one having all three amino acid substitutions (1) to (3) above, or all three amino acid substitutions corresponding to (1) to (3) above in the amino acid sequence of a non-human IL-2 receptor β chain.

[0043] The IL-2 receptor β chain is sometimes referred to as "IL-2Rβ" or "IL2Rβ."

[0044] The "(iii) first intracellular region derived from an IL-2 receptor β chain" used in the present invention can comprise an intracellular region derived from another polypeptide, in addition to an intracellular region derived from an IL-2 receptor β chain. Examples of the intracellular region derived from another polypeptide include an intracellular region derived from a cytokine receptor, an intracellular region derived from a growth factor receptor, an intracellular region derived from a receptor tyrosine kinase, or an intracellular region derived from a receptor for a T-cell activation regulator, or any variant having the same function as the sequence of any of these. Examples of cytokine receptors include, but are not limited to, interleukin 1 receptor (IL-1RI), IL-1 receptor accessory protein (IL-1RAcP), IL-2 receptor α chain, IL-2 receptor γ chain, IL-3Rα, IL-3Rβ (common β chain), IL-4Rα, IL-5Rα, IL-6Rα, gp130, IL-7Rα, IL-15Rα, IL-21R, erythropoietin receptor (EpoR), thrombopoietin receptor (TpoR), granulocyte colony-stimulating factor receptor (G-CSFR), granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR), and growth hormone receptor (GHR). Examples of the growth factor receptor include, but are not limited to, fibroblast growth factor receptors (FGFR1, FGFR2, FGFR3), epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF-1R), MET receptor, platelet-derived growth factor receptor (PDGFR), nerve growth factor receptor (NGFR), TGF-β type I receptor (TβRI), and TGF-β type II receptor (TβRII). Examples of the receptor tyrosine kinase include, but are not limited to, erythropoietin-producing hepatocyte receptor A4 (EPHA4) and c-kit. Examples of the receptor for the T cell activation regulator include, but are not limited to, CD27, CD28, CD40, CD40L, GITR, OX-40, ICOS, and 4-1BB. The "intracellular region derived from another polypeptide" may be added, for example, to either the N-terminus or C-terminus of the intracellular region derived from the IL-2 receptor β chain.For example, the "intracellular region derived from another polypeptide" may be fused to the N-terminus or C-terminus of the intracellular region derived from the IL-2 receptor β chain directly or via a linker. The linker may be, for example, a peptide linker, e.g., a peptide linker of 2 to 10 amino acids in length.

[0045] "(vi) Second Intracellular Region Derived from IL-2 Receptor γ Chain" The "(vi) second intracellular region derived from IL-2 receptor γ chain" used in the second polypeptide constituting the chimeric receptor of the present invention can be the entire intracellular region of the native IL-2 receptor γ chain, or a portion of the intracellular region of the native IL-2 receptor γ chain. An example of the entire intracellular region of the native IL-2 receptor γ chain is the amino acid sequence from positions 284 to 369 in the amino acid sequence of the wild-type human IL-2 receptor γ chain (RefSeq NP_000197.1) (SEQ ID NO: 74). Furthermore, an example of a portion of the intracellular region of the IL-2 receptor γ chain is the amino acid sequence from positions 284 to 359, 284 to 349, 284 to 339, 284 to 329, or 284 to 319 in the amino acid sequence of the wild-type human IL-2 receptor γ chain (RefSeq NP_000197.1).

[0046] The "(vi) second intracellular domain derived from the IL-2 receptor γ chain" used in the present invention is preferably the entire intracellular domain of the natural IL-2 receptor γ chain, and more preferably the amino acid sequence from positions 284 to 369 in the amino acid sequence of the wild-type human IL-2 receptor γ chain (RefSeq NP_000197.1) (SEQ ID NO: 74).

[0047] The IL-2 receptor γ chain is also called the "cytokine common gamma chain." The IL-2 receptor γ chain is also sometimes referred to as "IL-2Rγ" or "IL2Rγ."

[0048] The "(vi) second intracellular region derived from the IL-2 receptor γ chain" used in the present invention can comprise an intracellular region derived from another polypeptide in addition to an intracellular region derived from the IL-2 receptor γ chain. Examples of the intracellular region derived from another polypeptide include an intracellular region derived from a cytokine receptor, an intracellular region derived from a growth factor receptor, an intracellular region derived from a receptor tyrosine kinase, or an intracellular region derived from a receptor for a T-cell activation regulator, or any variant having the same function as the sequence of any of these. Examples of cytokine receptors include, but are not limited to, interleukin 1 receptor (IL-1RI), IL-1 receptor accessory protein (IL-1RAcP), IL-2 receptor α chain, IL-2 receptor β chain, IL-3Rα, IL-3Rβ (common β chain), IL-4Rα, IL-5Rα, IL-6Rα, gp130, IL-7Rα, IL-15Rα, IL-21R, erythropoietin receptor (EpoR), thrombopoietin receptor (TpoR), granulocyte colony-stimulating factor receptor (G-CSFR), granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR), and growth hormone receptor (GHR). Examples of the growth factor receptors include, but are not limited to, fibroblast growth factor receptors (FGFR1, FGFR2, FGFR3), epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF-1R), MET receptor, platelet-derived growth factor receptor (PDGFR), nerve growth factor receptor (NGFR), TGF-β type I receptor (TβRI), and TGF-β type II receptor (TβRII). Examples of the receptor tyrosine kinases include, but are not limited to, erythropoietin-producing hepatocyte receptor A4 (EPHA4) and c-kit. Examples of the receptors for T cell activation regulators include, but are not limited to, CD27, CD28, CD40, CD40L, GITR, OX-40, ICOS, and 4-1BB. The "intracellular region derived from another polypeptide" may be added to either the N-terminus or C-terminus of the intracellular region derived from the IL-2 receptor γ chain, and is preferably added to the C-terminus.For example, the "intracellular region derived from another polypeptide" may be fused to the N-terminus or C-terminus of the intracellular region derived from the IL-2 receptor γ chain directly or via a linker. The linker may be, for example, a peptide linker, e.g., a peptide linker of 2 to 10 amino acids in length.

[0049] The "intracellular region derived from another polypeptide" used in the present invention is preferably the intracellular region of CD40, and more preferably the amino acid sequence from amino acids 216 to 277 (SEQ ID NO: 71) in the amino acid sequence of wild-type human CD40 (RefSeq NP_001241.1).

[0050] In a preferred embodiment of the present invention, the first polypeptide is exemplified by, but not limited to, a polypeptide having the amino acid sequence of amino acids 65 to 601 of SEQ ID NO: 3 in which one or more tyrosines selected from tyrosines 431, 434, and 437 have been substituted with other amino acids. In a preferred embodiment of the present invention, the second polypeptide is exemplified by, but not limited to, a polypeptide having the amino acid sequence of amino acids 64 to 396 of SEQ ID NO: 1.

[0051] The present invention provides a chimeric receptor precursor. The precursor is characterized by comprising a polypeptide in which a first polypeptide and a second polypeptide are interconnected via a self-cleaving peptide. More specifically, the chimeric receptor precursor of the present invention is characterized by comprising: a first polypeptide having (i) a first extracellular domain capable of binding to a ligand, (ii) a first transmembrane domain, and (iii) a first intracellular domain derived from the IL-2 receptor β chain, wherein a mutation has been introduced into the first intracellular domain; and a second polypeptide having (iv) a second extracellular domain capable of binding to a ligand, (v) a second transmembrane domain, and (vi) a second intracellular domain derived from the IL-2 receptor γ chain, wherein the first and second polypeptides are interconnected via a self-cleaving peptide. The arrangement of the first and second polypeptides is not particularly limited, and may be either "first polypeptide-self-cleaving peptide-second polypeptide" or "second polypeptide-self-cleaving peptide-first polypeptide." Furthermore, the self-cleaving peptide may have a linker peptide attached to one or both ends thereof.

[0052] As used herein, the term "self-cleaving peptide" refers to a peptide sequence with cleavage activity occurring between two amino acid residues within the peptide sequence itself. Self-cleaving peptides are peptides 18 to 22 amino acids long that induce ribosomal skipping during intracellular protein translation. Self-cleaving peptides are found in various virus families and share a consensus motif (DxExNPGP). Self-cleaving peptides inhibit ribosomal transferase activity and suppress peptide bond formation. Therefore, placing a self-cleaving peptide between upstream and downstream genes disrupts translation, allowing for co-expression of two genes.

[0053] For example, in the case of 2A peptides or 2A-like peptides, cleavage occurs between glycine and proline residues on these peptides. This occurs via a "ribosomal skipping mechanism" in which normal peptide bond formation between glycine and proline residues does not occur during translation, without affecting downstream translation. Such ribosomal skipping mechanisms are known in the art and are used for the expression of multiple proteins encoded by a single messenger RNA (mRNA) molecule.

[0054] Self-cleaving peptides that can be used in the present invention include the P2A peptide derived from Porcine teschovirus (PTV), T2A derived from Thosea asigna virus (TaV), F2A derived from foot-and-mouth disease virus (FMDV), and E2A derived from equine rhinitis A virus (ERAV).

[0055] (2) Nucleic Acids of the Present Invention The present invention provides nucleic acids encoding the chimeric receptors of the present invention described above in (1). That is, the nucleic acids of the present invention encode chimeric receptors comprising: (i) a first polypeptide having a first extracellular domain capable of binding to a ligand, (ii) a first transmembrane domain, and (iii) a first intracellular domain derived from the IL-2 receptor β chain, wherein the first intracellular domain has been mutated; and (iv) a second polypeptide having a second extracellular domain capable of binding to a ligand, (v) a second transmembrane domain, and (vi) a second intracellular domain derived from the IL-2 receptor γ chain.

[0056] In a preferred embodiment of the present invention, a nucleic acid encoding a first polypeptide includes, but is not limited to, a nucleic acid having a nucleotide sequence encoding a polypeptide having an amino acid sequence from positions 65 to 601 of the amino acid sequence of SEQ ID NO: 3 in which one or more tyrosines selected from tyrosines at positions 431, 434, and 437 have been substituted with other amino acids. In a preferred embodiment of the present invention, a nucleic acid encoding a second polypeptide includes, but is not limited to, a nucleic acid having a nucleotide sequence encoding a polypeptide having an amino acid sequence from positions 64 to 396 of the amino acid sequence of SEQ ID NO: 1.

[0057] The present invention also provides nucleic acids encoding precursors of chimeric receptors.

[0058] The nucleic acids of the present invention are capable of expressing the chimeric receptors of the present invention in cells. Cells expressing the chimeric receptors of the present invention acquire the ability to proliferate in a ligand-dependent manner, making it possible to specifically proliferate the cells, i.e., selectively regulate cell proliferation. Therefore, the nucleic acids of the present invention are referred to as "selective regulatory genes (SRGs)."

[0059] The type of nucleic acid used in the present invention is not limited, and single-stranded nucleic acid, double-stranded nucleic acid, DNA, and RNA can be used. Of these, DNA can be exemplified by double-stranded DNA, single-stranded DNA, and cDNA, and RNA can be exemplified by mRNA.

[0060] The nucleic acid of the present invention can be linked to a nucleic acid having a promoter sequence so that it can be expressed in cells. Examples of promoters include promoters that constitutively promote the expression of a gene or an operably linked construct, and promoters that induce the expression of a gene or an operably linked construct by the action of a drug (e.g., tetracycline or doxorubicin). The nucleic acid of the present invention can also be linked to a nucleic acid comprising other regulatory elements, such as an enhancer sequence or a terminator sequence, that cooperate with the promoter or transcription initiation site to achieve efficient transcription of the nucleic acid.

[0061] In certain embodiments, the nucleic acids of the invention are codon-optimized for expression in a particular host cell.

[0062] (3) Vector of the Present Invention The present invention provides a vector comprising the nucleic acid of the present invention described above in (2). That is, the vector of the present invention comprises a nucleic acid encoding a chimeric receptor comprising: (i) a first polypeptide having a first extracellular domain capable of binding to a ligand, (ii) a first transmembrane domain, and (iii) a first intracellular domain derived from the IL-2 receptor β chain, wherein the first intracellular domain has been mutated; and (iv) a second polypeptide having a second extracellular domain capable of binding to a ligand, (v) a second transmembrane domain, and (vi) a second intracellular domain derived from the IL-2 receptor γ chain.

[0063] The vector of the present invention can express the first polypeptide and the second polypeptide in a desired cell.

[0064] Examples of vectors of the present invention include (a) a vector containing both a nucleic acid encoding a first polypeptide and a nucleic acid encoding a second polypeptide, and (b) a combination of a vector containing a nucleic acid encoding a first polypeptide and a vector containing a nucleic acid encoding a second polypeptide. In the above-mentioned (a) embodiment, the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide may be transcribed and translated by separate promoters, or may be transcribed and translated by a single promoter using an internal ribosome entry site (IRES). Furthermore, a nucleic acid encoding a polypeptide in which the first polypeptide and the second polypeptide are connected to each other via a self-cleaving peptide may be transcribed and translated by a single promoter. In the above-mentioned (b) embodiment, the two vectors may be the same type or different types.

[0065] Vectors usable in the present invention are operably linked to appropriate control sequences so as to express the nucleic acid of the present invention in appropriate host cells. Control sequences include promoters for transcribing the nucleic acid of the present invention, any operator sequence for controlling transcription, sequences encoding ribosome binding sites, enhancers, polyadenylation sequences, and sequences controlling the termination of transcription and translation. Examples of promoters that control transcription in mammalian cells include mammalian-derived promoters (PGK promoter, EF1-α promoter, β-globin promoter, etc.), virus-derived promoters (CMV promoter, SV40 promoter, MMLV-LTR promoter, HIV-LTR promoter, etc.), and artificially constructed promoters (CAG promoter, etc.). Furthermore, vectors may contain various sequences known to those skilled in the art, such as restriction enzyme cleavage sites, marker genes (selection genes) such as drug resistance genes, signal sequences, leader sequences, etc., as needed. These various sequences or sites can be appropriately selected and used by those skilled in the art depending on the type of polypeptide to be expressed, the host cells used, the culture medium, and other conditions.

[0066] In addition to the nucleic acid of the present invention, the vector of the present invention may contain a gene that can serve as a marker for confirming that the vector has been introduced into a cell (e.g., a drug resistance gene, a gene encoding a reporter enzyme, or a gene encoding a fluorescent protein) or a therapeutic gene.

[0067] The therapeutic gene used in the present invention is not particularly limited, and may be any gene that exerts a favorable effect on a disease for which treatment or prevention is desired, and examples thereof include polypeptides, ribozymes, antisense RNA, and RNA exhibiting RNA interference activity. Since the present invention brings about expression of a therapeutic gene in response to the onset of a disease, for example, carcinogenesis or viral infection, genes useful for eliminating cancer cells or virally infected cells, such as genes that can exert cytotoxicity, can be used. Examples of therapeutic genes include, but are not limited to, genes encoding CARs, genes encoding TCRs, genes encoding polypeptides with protease activity, genes encoding polypeptides with nuclease activity, genes encoding cytokines, and genes involved in nucleic acid metabolism.

[0068] Preferably, the vector of the present invention further comprises a nucleic acid encoding a CAR or a foreign TCR that recognizes target cells in disease treatment.

[0069] A CAR refers to a fusion protein comprising an extracellular domain that binds to an antigen (hereinafter referred to as an antigen-binding domain), a transmembrane domain derived from a polypeptide different from the antigen-binding domain, and at least one intracellular domain. A CAR is also called a "chimeric antigen receptor," "chimeric receptor," "T-body," or "chimeric immune receptor (CIR)."

[0070] An "antigen-binding domain" refers to a portion capable of binding to a certain antigen. A portion of an antibody capable of binding to an antigen can be used as an antigen-binding domain. Examples of antigen-binding domains include Fab' fragments, Fab fragments, and Fv fragments, but single-chain variable region fragments (scFv) are preferred for the present invention. An scFv refers to a single-chain polypeptide derived from an antibody that retains its antigen-binding ability. An example of an scFv is a polypeptide formed by recombinant DNA technology in which the Fv region of an immunoglobulin heavy chain (H chain) and the Fv region of a light chain (L chain) are linked via a spacer sequence. Various methods for producing scFv are known, including those described in U.S. Pat. No. 4,694,778; Science, Vol. 242, pp. 423-442 (1988); Nature, Vol. 334, pp. 54454 (1989); and Science, Vol. 242, pp. 1038-1041 (1988). Furthermore, the antigen-binding domain is not limited to polypeptides derived from antibodies; polypeptides derived from proteins other than antibodies can also be used. For example, the full-length or a portion of a protein such as APRIL or GM-CSF can also be used as the antigen-binding domain.

[0071] "Intracellular domain" means any oligopeptide or polypeptide known to function as a domain that transmits signals that result in the activation or inhibition of biological processes within a cell.

[0072] A typical CAR structure is composed of an scFv, a transmembrane domain, and an intracellular domain that activates cells. Transmembrane domains known to be derived from TCR complexes CD3ζ, CD28, CD8α, and the like are known. The intracellular domain of the TCR complex CD3ζ is preferably used. CARs with such a configuration are called first-generation CARs. CAR-expressing T cells (CAR-T cells) directly recognize surface antigens on tumor cells, regardless of the expression of major histocompatibility complex class I on the tumor cells, and simultaneously activate the T cells themselves, thereby enabling efficient killing of tumor cells.

[0073] Second-generation CARs have been developed in which the intracellular domain of a T cell costimulatory molecule is linked to enhance the T cell activation ability of first-generation CARs. Suitable T cell costimulatory molecules include CD28, the intracellular domain of CD137 (4-1BB) or CD134 (OX40), which are members of the tumor necrosis factor (TNF) receptor superfamily, the intracellular domain of an interleukin receptor and its modified form, and the intracellular domain of the glucocorticoid-induced tumor necrosis factor receptor (GITR). As further improved versions, third-generation CARs have also been developed in which the intracellular domains of these costimulatory molecules are linked in tandem, and many CAR molecules targeting various tumor antigens have been reported. The nucleic acid construct of the present invention may comprise a sequence encoding any of the CARs as the desired gene sequence.

[0074] As used herein, the term "TCR" refers to a molecule responsible for the antigen recognition function of T cells, and is composed of polypeptides such as an α chain, a β chain, a γ chain, and a δ chain. Of these, a heterodimer of a TCR α chain (TCRα) and a TCR β chain (TCRβ), or a heterodimer of a γ chain and a δ chain, forms a TCR together with accessory molecules such as the CD3 complex (including γ, δ, ε, and ζ), CD4, or CD8.

[0075] TCR recognizes peptides (antigen epitopes) presented by target cells (phagocytes, virus-infected cells, cancer cells, etc.) via the major histocompatibility complex (MHC) in the body and initiates an immune response against the target cells. Human MHC is also referred to as the human histocompatibility leukocyte antigen (HLA) system. HLA is classified into class I and class II, with class I including HLA-A, B, C, E, F, G, H, and J, and class II including HLA-DR, DQ, and DP. Like TCR, HLA is also formed by a complex of α and β chains, and presents antigen epitopes on the cell surface via these chains. HLA class I is present on almost all cells in the body and presents antigen epitopes approximately 9 amino acid residues in length. For example, when a virus-infected cell presents a virus-derived antigen epitope via HLA, T cells (e.g., cytotoxic T lymphocytes) having a TCR specific to that antigen epitope are activated, resulting in an immune response (e.g., killing of virus-infected cells) in the body, thereby providing biological defense.

[0076] TCRs can usually initiate an immune response only through binding to a complex formed by a specific type of HLA and an antigen epitope. This restriction is referred to as "HLA restriction." As used herein, the term HLA (or MHC) restriction can be used in combination with various peptides, cells, etc. to refer to TCRs, as well as T cells expressing such TCRs, or the antigen-HLA complexes they present. Binding of TCRs to antigen epitope-HLA complexes results in an immune response, and examples of this immune response include the secretion of cytokines (interferon-γ, TNF-α, IL-2, etc.) by CD8-positive T cells.

[0077] The above-mentioned genes that can serve as markers and therapeutic genes may be carried in a vector so that they are expressed by a promoter different from that of the nucleic acid encoding the chimeric receptor of the present invention, or they may be carried in a vector so that they are expressed by the same promoter as that of the nucleic acid encoding the chimeric receptor of the present invention. Furthermore, a nucleic acid encoding a fusion polypeptide in which a precursor of the chimeric receptor of the present invention and a polypeptide encoded by the gene that can serve as a marker and / or therapeutic gene are connected via a self-cleaving peptide may be linked to a promoter and carried in a vector.

[0078] Vectors that can be used in the present invention include vectors that integrate into the genome of host cells, vectors that do not integrate, and episomal vectors that exist in the cytoplasm and replicate autonomously. For example, plasmid vectors, viral vectors, and artificial chromosomes are suitable. Viral vectors that can be used include retroviral vectors (including oncoretroviral vectors, lentiviral vectors, and pseudotype vectors), adenoviral vectors, adeno-associated virus (AAV) vectors, simian virus vectors, vaccinia virus vectors, Sendai virus vectors, Epstein-Barr virus (EBV) vectors, and HSV vectors. Preferred viral vectors are those that are replication-deficient so that the virus cannot replicate autonomously in infected cells. Various viral vectors and their production methods are well known to those skilled in the art. Commercially available viral vectors may also be used in the present invention.

[0079] Non-viral vectors can also be used in combination with condensing agents such as liposomes and cationic lipids.Furthermore, the nucleic acid of the present invention can be introduced into cells by calcium phosphate transduction, DEAE-dextran, electroporation, or particle bombardment.

[0080] (4) Cells of the Present Invention The present invention provides cells expressing the chimeric receptor of the present invention described above in (1). That is, the cells of the present invention express a chimeric receptor comprising: (i) a first polypeptide having a first extracellular domain capable of binding to a ligand, (ii) a first transmembrane domain, and (iii) a first intracellular domain derived from the IL-2 receptor β chain, wherein the first intracellular domain has been mutated; and (iv) a second polypeptide having a second extracellular domain capable of binding to a ligand, (v) a second transmembrane domain, and (vi) a second intracellular domain derived from the IL-2 receptor γ chain.

[0081] The cells of the present invention have the ability to specifically proliferate upon stimulation with a ligand, i.e., intracellular proliferation is initiated when the cells come into contact with a ligand that binds to the chimeric receptor expressed by the cells, either in vitro or in vivo.

[0082] The origin of the cells of the present invention is not particularly limited, and cells derived from mammals, such as human cells, or cells derived from non-human mammals such as monkeys, mice, rats, pigs, horses, and dogs can be used. The cells of the present invention are preferably human cells.

[0083] The type of cells used in the present invention is not particularly limited, and any cell type can be used. For example, cells collected, isolated, or purified from body fluids, tissues, or organs, such as blood (peripheral blood, umbilical cord blood, etc.) or bone marrow, or cells obtained by differentiating the above cells or reprogramming them to generate pluripotent stem cells (iPS cells) can be used (see, for example, Themeli et al., 2013). Peripheral blood mononuclear cells (PBMCs), immune cells, umbilical cord blood mononuclear cells, fibroblasts, adipocyte precursors, hepatocytes, skin keratinocytes, mesenchymal stem cells, adipose stem cells, various cancer cell lines, or neural stem cells can be used. For example, NK cells, T cells, T cell progenitors (hematopoietic stem cells, lymphocyte precursor cells, etc.), or cell populations containing them can be used. Examples of T cells include CD8+ T cells, CD4+ T cells, regulatory T cells, cytotoxic T cells, and tumor-infiltrating lymphocytes. Cell populations containing T cells and T cell progenitors include PBMCs. The above-mentioned cells may be collected from a living body, obtained by expanding cells collected from a living body, or established as a cell line. Furthermore, cells obtained by differentiation of pluripotent stem cells (ES cells, iPS cells, etc.), such as immune cells, may also be used as the cells of the present invention. iPS cells can be produced from a clone of a T cell that has the ability to recognize and damage specific cells, and then differentiated to obtain T cells. The T cells thus obtained have the same specific cytotoxic activity as the starting T cell clone, but are in a more undifferentiated state. By introducing a nucleic acid encoding the chimeric receptor of the present invention into such T cells, it becomes possible to easily prepare cells that exhibit excellent therapeutic effects.

[0084] As used herein, "immune cells" refers to all cells involved in immune function in the body, and examples include hematopoietic stem cells, neutrophils, basophils, macrophages, lymphocytes (B cells and T cells), monocytes, dendritic cells, natural killer (NK) cells, plasma cells, etc. Of these, T cells and NK cells are preferred for the present invention. Note that "immune cells" as used herein also encompass "immune cell precursor cells" that have the ability to differentiate into the aforementioned immune cells.

[0085] When transplantation of the cells of the present invention or cells differentiated from the cells of the present invention into an organism is desired, it is preferable to introduce the nucleic acid into the organism to be transplanted or into cells taken from an organism of the same species.

[0086] The cells of the present invention may further express a polypeptide (enzyme, antibody, cytokine, etc.) or a nucleic acid (siRNA, etc.) useful for treating a disease, preferably a CAR or a foreign TCR that recognizes a target cell.

[0087] As used herein, the term "target cells" refers to cells desired to be reduced or eliminated in a patient, and examples thereof include tumor cells and cells infected with pathogens. Although not particularly limited, tumor cells are preferred, and hematopoietic tumor cells and solid tumor cells are more preferred.

[0088] The cells of the present invention, which further express a CAR that recognizes a target cell or a foreign TCR, have the ability to recognize desired target cells and destroy the target cells through their cytotoxic activity. This cytotoxic activity can be evaluated by known methods. For example, the cytotoxic activity of the cells of the present invention against target cells labeled with a radioactive substance, a fluorescent substance, or the like can be evaluated by measuring the radioactivity or fluorescence intensity derived from the target cells destroyed by the cells of the present invention. Alternatively, the cytotoxic activity can be detected by measuring the amount of cytokines, such as GM-CSF or IFN-γ, that are specifically released from the cells of the present invention or the target cells.

[0089] (5) Method for Producing a Cell of the Present Invention The present invention provides a method for producing a cell, comprising the step of introducing into a cell the nucleic acid of the present invention described in (2) above. That is, the method for producing a cell of the present invention comprises the step of introducing into a cell a nucleic acid encoding a chimeric receptor comprising: (i) a first polypeptide having a first extracellular domain capable of binding to a ligand, (ii) a first transmembrane domain, and (iii) a first intracellular domain derived from the IL-2 receptor β chain, wherein the first intracellular domain has been mutated; and (iv) a second polypeptide having a second extracellular domain capable of binding to a ligand, (v) a second transmembrane domain, and (vi) a second intracellular domain derived from the IL-2 receptor γ chain.

[0090] This process is carried out ex vivo, for example by transducing cells ex vivo using a viral or non-viral vector containing the nucleic acid of the invention.

[0091] The cell production method of the present invention can use cells derived from mammals, such as humans, or cells derived from non-human mammals, such as monkeys, mice, rats, pigs, cows, and dogs. There are no particular limitations on the cells used in the method of the present invention, and any of the cells described above can be used. The cells may be collected from a living organism, expanded, or established as a cell line. When it is desired to transplant the produced cells or cells differentiated from the cells into a living organism, it is preferable to introduce nucleic acids into cells collected from the living organism itself or from the same species. Furthermore, stimulating cells before nucleic acid introduction can improve the efficiency of nucleic acid introduction into cells or enrich for specific cell types. For example, culturing PBMCs in the presence of an anti-CD3 antibody increases the proportion of T cells. Furthermore, culturing PBMCs in the presence of an anti-CD3 antibody and retronectin, a recombinant fibronectin fragment, can yield a cell population rich in CD8+ T cells.

[0092] A nucleic acid encoding the chimeric receptor of the present invention can be inserted into a vector, and the vector can be introduced into a cell. The vector can be the vector of the present invention described in (3) above. There are no particular limitations on the method for introducing the vector into a cell, and an appropriate method can be selected depending on the vector and cell type used.

[0093] Non-viral vectors can also be used in the present invention when used in combination with auxiliary agents such as liposomes, cationic lipids, polyethyleneimine, etc. Furthermore, the nucleic acids of the present invention can be introduced into cells by calcium phosphate transduction, DEAE-dextran, electroporation, or particle bombardment.

[0094] Methods for producing various viral vectors and methods for introducing them into cells are well known to those skilled in the art. For example, when using a retroviral vector, appropriate packaging cells can be selected based on the LTR sequence and packaging signal sequence of the vector, and retroviral particles can be prepared using these. Examples of packaging cells include PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), GP+E-86, GP+envAm-12 (U.S. Pat. No. 5,278,056), and Psi-Crip [Proceedings of the National Academy of Sciences of the United States of America, Vol. 85, pp. 6460-6464 (1988)]. Retroviral particles can also be produced using 293 cells or 293T cells, which have high transfection efficiency. Retroviral vectors produced based on many types of retroviruses and packaging cells that can be used to package these vectors are widely available commercially from various companies.

[0095] The number of cells into which the nucleic acid of the present invention has been introduced can be expanded by culturing them ex vivo. For cell culture, a medium suitable for the cells may be used, and a medium selected from known or commercially available media can be used. For example, media that can be used for culturing lymphocytes and other immune cells are known to those skilled in the art, and media with various compositions are commercially available. These media include those containing human or animal-derived serum (e.g., fetal bovine serum; FBS or FCS), xeno-free media, and media that do not contain unknown components (defined media), and can be selected according to the purpose. Furthermore, known culture vessels (plates, Petri dishes, flasks, bags, culture tanks, etc.) can be used for cell culture. Furthermore, the culture conditions can be the same as those for ordinary cell culture (e.g., 32-37°C, 5% CO ２ Furthermore, at appropriate time intervals, operations such as diluting the culture by adding fresh medium, exchanging the medium, or exchanging the cell culture vessel can be performed.

[0096] In the method for producing cells of the present invention, the proliferation of the cells can be promoted by culturing the cells of the present invention in contact with a ligand to which the chimeric receptor binds. When a cell population containing the cells of the present invention and other cells is cultured in a medium containing the ligand, the cells of the present invention receive a proliferation signal upon binding to the ligand and proliferate actively. Meanwhile, the other cells proliferate at a normal rate. This allows for a cell population enriched for the cells of the present invention. The concentration of the ligand can be adjusted appropriately depending on the ligand and cell type. Although this is not a limitation of the present invention, in the case of cells expressing a chimeric receptor having an extracellular domain derived from the erythropoietin receptor, erythropoietin concentration-dependent promotion of cell proliferation is observed in a medium containing 0.9 IU / mL or more of erythropoietin. This makes it possible to selectively produce the cells of the present invention, and to obtain a highly purified cell population of interest without purification procedures or with only simple purification procedures. Furthermore, the cells obtained by the method for producing cells of the present invention are characterized by a stronger antitumor effect than the control.

[0097] (6) Pharmaceutical Composition of the Present Invention The present invention provides a pharmaceutical composition comprising, as an active ingredient, the cells of the present invention described in (4) above. That is, the pharmaceutical composition of the present invention comprises, as an active ingredient, cells expressing a chimeric receptor comprising: (i) a first polypeptide having a first extracellular domain capable of binding to a ligand, (ii) a first transmembrane domain, and (iii) a first intracellular domain derived from the IL-2 receptor β chain, wherein the first intracellular domain has been mutated; and (iv) a second polypeptide having a second extracellular domain capable of binding to a ligand, (v) a second transmembrane domain, and (vi) a second intracellular domain derived from the IL-2 receptor γ chain.

[0098] The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients are well known to those skilled in the art and include, for example, phosphate-buffered saline (e.g., 0.01 M phosphate, 0.138 M NaCl, 0.0027 M KCl, pH 7.4), aqueous solutions containing mineral acid salts such as hydrochloride, hydrobromide, phosphate, and sulfate, physiological saline, glycol or ethanol solutions, and salts of organic acids such as acetate, propionate, malonate, and benzoate. Auxiliaries such as wetting agents or emulsifiers, and pH buffering agents may also be used. Pharmaceutically acceptable excipients are described in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). The composition may be in any known form suitable for parenteral administration, for example, injection or infusion. Furthermore, formulation adjuvants such as suspending agents, preservatives, stabilizers and / or dispersing agents, as well as preservatives for extending the shelf life during storage, may be used.

[0099] (7) The present invention provides a method for preventing or treating a disease, which comprises administering to a subject the cell of the present invention described in (4) above or the pharmaceutical composition of the present invention described in (6) above. That is, the method for preventing or treating a disease of the present invention comprises administering to a subject cells expressing a chimeric receptor comprising: (i) a first polypeptide having a first extracellular domain capable of binding to a ligand, (ii) a first transmembrane domain, and (iii) a first intracellular domain derived from the IL-2 receptor β chain, wherein the first intracellular domain has been mutated; and a second polypeptide having (iv) a second extracellular domain capable of binding to a ligand, (v) a second transmembrane domain, and (vi) a second intracellular domain derived from the IL-2 receptor γ chain; or a pharmaceutical composition comprising the cells as an active ingredient.

[0100] The above-mentioned diseases are not particularly limited as long as they are sensitive to the cells. Examples include, but are not limited to, cancer [blood cancers (leukemia, lymphoma, myeloma, etc.)], inflammatory diseases / autoimmune diseases (asthma, eczema), hepatitis, and infectious diseases caused by viruses such as influenza and HIV, bacteria, or fungi, such as tuberculosis, MRSA, VRE, and deep mycosis. While not particularly limiting the present invention, the cells of the present invention capable of binding to antigens possessed by cells whose reduction or elimination is desired in the above-mentioned diseases, i.e., tumor antigens, viral antigens, bacterial antigens, etc., are administered for the treatment of these diseases. For example, the cells of the present invention expressing a CAR specific to a tumor antigen or the cells of the present invention expressing a foreign TCR that recognizes tumor cells are extremely useful for tumor treatment. Furthermore, NK cells and cytotoxic T lymphocyte (CTL) clones expressing the chimeric receptors of the present invention can also be used to treat diseases for which the respective cells exert a therapeutic effect.

[0101] The cells or pharmaceutical composition can be administered intradermally, subcutaneously, intravenously, or by systemic administration or local administration to the affected area or tissue in the vicinity thereof, for example, subcutaneously. The dosage can be adjusted appropriately depending on the age, weight, administration method, etc. of the subject to be administered. In this case, the number of administrations and the interval between administrations in the case of multiple administrations should also be taken into consideration.

[0102] In the disease prevention or treatment method of the present invention, proliferation of the cells of the present invention in vivo can be induced and / or the survival period of the cells in vivo can be extended by administering a ligand that binds to a chimeric receptor expressed by the cells of the present invention to a subject to which the cells of the present invention have been administered. There are no particular limitations on the method of administering the ligand, and an appropriate method can be selected taking into consideration the distribution of the cells of the present invention in vivo, etc. The dosage and administration schedule of the ligand can also be appropriately determined taking into consideration the type and dosage of cells to be combined, the type of disease, etc.

[0103] The present invention will be explained in more detail with reference to the following examples, but the scope of the present invention is not limited to these examples.

[0104] Example 1 Preparation of SRG Construct (1) pEX-A2J2 / TEG A nucleic acid encoding a fusion polypeptide (T2A-EPOR-IL2Rγ) was prepared by seamlessly fusing the T2A sequence, the polypeptide from positions 2 to 250 of the amino acid sequence of the human erythropoietin receptor (NCBI Reference Sequence: NP_000112.1) (including the signal sequence and extracellular domain), and the polypeptide from positions 263 to 369 of the amino acid sequence of the human IL-2 receptor γ chain (common γ chain) (including the transmembrane domain and intracellular domain) (NCBI Reference Sequence: NP_000197.1). This construct was designated TEG. The amino acid sequence of this fusion polypeptide is shown in SEQ ID NO: 1. In the amino acid sequence of SEQ ID NO: 1, positions 1 to 40 correspond to the self-cleaving peptide sequence (furin-spacer-T2A), positions 41 to 63 correspond to the signal peptide sequence of the erythropoietin receptor, positions 64 to 289 correspond to the extracellular domain of the erythropoietin receptor, positions 290 to 310 correspond to the transmembrane domain of the IL-2 receptor γ chain, and positions 311 to 396 correspond to the intracellular domain of the IL-2 receptor γ chain. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 2. This nucleic acid was synthesized as an artificial gene (T2A and EPOR were codon-optimized) and cloned into the pEX-A2J2 vector (Eurofins Genomics) to prepare the plasmid pEX-A2J2 / TEG.

[0105] (2) pEX-A2J2 / PEB A nucleic acid encoding a fusion polypeptide (P2A-EPOR-IL2Rβ) was constructed by seamlessly fusing the P2A sequence, the polypeptide from positions 2 to 250 of the amino acid sequence of the human erythropoietin receptor (NCBI Reference Sequence: NP_000112.1) (including the signal sequence and extracellular domain), and the polypeptide from positions 241 to 551 of the amino acid sequence of the human IL-2 receptor β chain (NCBI Reference Sequence: NP_000869.1) (including the transmembrane domain and intracellular domain). This polypeptide was designated PEB. The amino acid sequence of this fusion polypeptide is shown in SEQ ID NO: 3. In the amino acid sequence of SEQ ID NO: 3, positions 1 to 41 correspond to the self-cleaving peptide sequence (furin-spacer-P2A), positions 42 to 64 correspond to the signal peptide sequence of the erythropoietin receptor, positions 65 to 290 correspond to the extracellular domain of the erythropoietin receptor, positions 291 to 315 correspond to the transmembrane domain of the IL-2 receptor β chain, and positions 316 to 601 correspond to the intracellular domain of the IL-2 receptor β chain. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 4. This nucleic acid was synthesized as an artificial gene (P2A and EPOR were codon-optimized) and cloned into the pEX-A2J2 vector to prepare the plasmid pEX-A2J2 / PEB.

[0106] (3) pEX-A2J2 / TEGPEB A nucleic acid encoding a fusion polypeptide (T2A-EPOR-IL2Rγ-P2A-EPOR-IL2Rβ) was constructed by seamlessly fusing the fusion polypeptide encoded by TEG (SEQ ID NO: 1) and the fusion polypeptide encoded by PEB (SEQ ID NO: 3). This was designated TEGPEB. The amino acid sequence of this fusion polypeptide is shown in SEQ ID NO: 5. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 6. As shown in Figure 1, a linearized cloning vector was prepared by PCR using pEX-A2J2 / TEG as a template and the pEX-V_InfRev primer (SEQ ID NO: 7) and pEX-V_InfFwd primer (SEQ ID NO: 8). Next, an amplified fragment containing DNA encoding T2A-EPOR-IL2Rγ was obtained by PCR using pEX-A2J2 / TEG as a template and the TSRG1_pEXInfFwd primer (SEQ ID NO: 9) and TSRG1_pEXInfRev primer (SEQ ID NO: 10). Similarly, using pEX-A2J2 / PEB as a template, PCR was performed with the PSRG2_pEXInfFwd primer (SEQ ID NO: 11) and the PSRG2_pEXInfRev primer (SEQ ID NO: 12) to obtain an amplified fragment containing DNA encoding P2A-EPOR-IL2Rβ. These two amplified products were cloned into the linearized cloning vector described above using the In-Fusion HD Cloning Kit (Takara Bio Inc.) to prepare the plasmid pEX-A2J2 / TEGPEB.

[0107] (4) pMEI-5 / ZG-SRG_YYY A nucleic acid encoding a fusion polypeptide (ZsGreen1-T2A-EPOR-IL2Rγ-P2A-EPOR-IL2Rβ) was constructed by seamlessly fusing ZsGreen1 with the TEGPEB-encoded fusion polypeptide (SEQ ID NO: 5). This nucleic acid was designated ZG-SRG_YYY. The amino acid sequence of this fusion polypeptide is shown in SEQ ID NO: 13. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 14. As shown in Figure 2, the pMEI-5 DNA vector (Takara Bio) was digested with the restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, using the pIRES2-ZsGreen1 vector (Takara Bio) as a template, PCR was performed with the ZGSRG-ZG_M5InfFwd primer (SEQ ID NO: 15) and the ZGSRG-ZG_M5InfRev primer (SEQ ID NO: 16) to obtain an amplified fragment containing DNA encoding ZsGreen1. Similarly, using pEX-A2J2 / TEGPEB as a template, PCR was performed with the ZGSRG-SRG_M5InfFwd primer (SEQ ID NO: 17) and the ZGSRG-SRG_M5InfRev primer (SEQ ID NO: 18) to obtain an amplified fragment containing DNA encoding T2A-EPOR-IL2Rγ-P2A-EPOR-IL2Rβ. These two amplified products were cloned into the linearized cloning vector described above using the In-Fusion HD Cloning Kit to generate the retroviral plasmid pMEI-5 / ZG-SRG_YYY.

[0108] (5) pMEI-5 / ZG-SRG_FYY A nucleic acid encoding a fusion polypeptide was prepared in which the tyrosine residue at position 1,058 of the fusion polypeptide (SEQ ID NO: 13) encoded by ZG-SRG_YYY (corresponding to position 381 in the amino acid sequence of the human IL-2 receptor β chain) was replaced with phenylalanine. This nucleic acid was designated ZG-SRG_FYY. The amino acid sequence of this fusion polypeptide is shown in SEQ ID NO: 19. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 20. As shown in Figure 3, the pMEI-5 DNA vector was cleaved with restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, an amplified fragment was obtained by PCR using pMEI-5 / ZG-SRG_YYY as a template and the ZGSRG-ZG_M5InfFwd primer (SEQ ID NO: 15) and the FYYmut_Rev primer (SEQ ID NO: 21). Similarly, an amplified fragment was obtained by PCR using pMEI-5 / ZG-SRG_YYY as a template and the FYYmut_Fwd primer (SEQ ID NO: 22) and ZGSRG-SRG_M5InfRev primer (SEQ ID NO: 18). These two amplified products were cloned into the linearized cloning vector described above using the In-Fusion HD Cloning Kit to prepare the retroviral plasmid pMEI-5 / ZG-SRG_FYY.

[0109] (6) pMEI-5 / ZG-SRG_YFY A nucleic acid encoding a fusion polypeptide was prepared in which the tyrosine residue at position 1,061 (corresponding to position 384 in the amino acid sequence of the human IL-2 receptor β chain) of the fusion polypeptide encoded by ZG-SRG_YYY (SEQ ID NO: 13) was replaced with phenylalanine. This nucleic acid was designated ZG-SRG_YFY. The amino acid sequence of this fusion polypeptide is shown in SEQ ID NO: 23. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 24. As shown in Figure 4, the pMEI-5 DNA vector was cleaved with restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, an amplified fragment was obtained by PCR using pMEI-5 / ZG-SRG_YYY as a template and the ZGSRG-ZG_M5InfFwd primer (SEQ ID NO: 15) and the YFYmut_Rev primer (SEQ ID NO: 25). Similarly, an amplified fragment was obtained by PCR using pMEI-5 / ZG-SRG_YYY as a template and the YFYmut_Fwd primer (SEQ ID NO: 26) and ZGSRG-SRG_M5InfRev primer (SEQ ID NO: 18). These two amplified products were cloned into the linearized cloning vector described above using the In-Fusion HD Cloning Kit to generate the retroviral plasmid pMEI-5 / ZG-SRG_YFY.

[0110] (7) pMEI-5 / ZG-SRG_YYF A nucleic acid encoding a fusion polypeptide was constructed in which the tyrosine residue at position 1,064 of the fusion polypeptide encoded by ZG-SRG_YYY (SEQ ID NO: 13) (corresponding to position 387 in the amino acid sequence of the human IL-2 receptor β chain) was replaced with phenylalanine. This nucleic acid was designated ZG-SRG_YYF. The amino acid sequence of this fusion polypeptide is shown in SEQ ID NO: 27. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 28. As shown in Figure 5, the pMEI-5 DNA vector was cleaved with restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, an amplified fragment was obtained by PCR using pMEI-5 / ZG-SRG_YYY as a template and the ZGSRG-ZG_M5InfFwd primer (SEQ ID NO: 15) and the YYFmut_Rev primer (SEQ ID NO: 29). Similarly, an amplified fragment was obtained by PCR using pMEI-5 / ZG-SRG_YYY as a template and the YYFmut_Fwd primer (SEQ ID NO: 30) and ZGSRG-SRG_M5InfRev primer (SEQ ID NO: 18). These two amplified products were cloned into the linearized cloning vector described above using the In-Fusion HD Cloning Kit to prepare the retroviral plasmid pMEI-5 / ZG-SRG_YYF.

[0111] (8) pMEI-5 / ZG-SRG_FFY A nucleic acid encoding a fusion polypeptide was constructed in which the tyrosine residues at positions 1,058 and 1,061 of the fusion polypeptide (SEQ ID NO: 13) encoded by ZG-SRG_YYY (corresponding to positions 381 and 384 in the amino acid sequence of the human IL-2 receptor β chain) were replaced with phenylalanine. This nucleic acid was designated ZG-SRG_FFY. The amino acid sequence of this fusion polypeptide is shown in SEQ ID NO: 31. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 32. As shown in Figure 6, the pMEI-5 DNA vector was cleaved with restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, an amplified fragment was obtained by PCR using pMEI-5 / ZG-SRG_FYY as a template and the ZGSRG-ZG_M5InfFwd primer (SEQ ID NO: 15) and the FFYmut_Rev primer (SEQ ID NO: 33). Similarly, an amplified fragment was obtained by PCR using pMEI-5 / ZG-SRG_FYY as a template and the FFYmut_Fwd primer (SEQ ID NO: 34) and ZGSRG-SRG_M5InfRev primer (SEQ ID NO: 18). These two amplified products were cloned into the linearized cloning vector described above using the In-Fusion HD Cloning Kit to prepare the retroviral plasmid pMEI-5 / ZG-SRG_FFY.

[0112] (9) pMEI-5 / ZG-SRG_YFF A nucleic acid encoding a fusion polypeptide was constructed in which the tyrosine residues at positions 1,061 and 1,064 of the fusion polypeptide (SEQ ID NO: 13) encoded by ZG-SRG_YYY (corresponding to positions 384 and 387 in the amino acid sequence of the human IL-2 receptor β chain) were replaced with phenylalanine. This nucleic acid was designated ZG-SRG_YFF. The amino acid sequence of this fusion polypeptide is shown in SEQ ID NO: 35. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 36. As shown in Figure 7, the pMEI-5 DNA vector was cleaved with restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, an amplified fragment was obtained by PCR using pMEI-5 / ZG-SRG_YFY as a template and the ZGSRG-ZG_M5InfFwd primer (SEQ ID NO: 15) and the YFFmut_Rev primer (SEQ ID NO: 37). Similarly, an amplified fragment was obtained by PCR using pMEI-5 / ZG-SRG_YFY as a template and the YFFmut_Fwd primer (SEQ ID NO: 38) and ZGSRG-SRG_M5InfRev primer (SEQ ID NO: 18). These two amplified products were cloned into the linearized cloning vector described above using the In-Fusion HD Cloning Kit to generate the retroviral plasmid pMEI-5 / ZG-SRG_YFF.

[0113] (10) pMEI-5 / ZG-SRG_FYF A nucleic acid encoding a fusion polypeptide was constructed in which the tyrosine residues at positions 1,058 and 1,064 of the fusion polypeptide (SEQ ID NO: 13) encoded by ZG-SRG_YYY (corresponding to positions 381 and 387 in the amino acid sequence of the human IL-2 receptor β chain) were replaced with phenylalanine. This nucleic acid was designated ZG-SRG_FYF. The amino acid sequence of this fusion polypeptide is shown in SEQ ID NO: 39. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 40. As shown in Figure 8, the pMEI-5 DNA vector was cleaved with restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, an amplified fragment was obtained by PCR using pMEI-5 / ZG-SRG_FYY as a template and the ZGSRG-ZG_M5InfFwd primer (SEQ ID NO: 15) and the FYFmut_Rev primer (SEQ ID NO: 41). Similarly, an amplified fragment was obtained by PCR using pMEI-5 / ZG-SRG_FYY as a template and the FYFmut_Fwd primer (SEQ ID NO: 42) and ZGSRG-SRG_M5InfRev primer (SEQ ID NO: 18). These two amplified products were cloned into the linearized cloning vector described above using the In-Fusion HD Cloning Kit to generate the retroviral plasmid pMEI-5 / ZG-SRG_FYF.

[0114] (11) pMEI-5 / ZG-SRG_FFF A nucleic acid encoding a fusion polypeptide was constructed in which the tyrosine residues at positions 1,058, 1,061, and 1,064 of the fusion polypeptide (SEQ ID NO: 13) encoded by ZG-SRG_YYY (corresponding to positions 381, 384, and 387 in the amino acid sequence of the human IL-2 receptor β chain) were replaced with phenylalanine. This nucleic acid was designated ZG-SRG_FFF. The amino acid sequence of this fusion polypeptide is shown in SEQ ID NO: 43. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 44. As shown in Figure 9, the pMEI-5 DNA vector was cleaved with restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, an amplified fragment was obtained by PCR using pMEI-5 / ZG-SRG_FYY as a template and the ZGSRG-ZG_M5InfFwd primer (SEQ ID NO: 15) and the FFFmut_Rev primer (SEQ ID NO: 45). Similarly, an amplified fragment was obtained by PCR using pMEI-5 / ZG-SRG_FYY as a template and the FFFmut_Fwd primer (SEQ ID NO: 46) and ZGSRG-SRG_M5InfRev primer (SEQ ID NO: 18). These two amplified products were cloned into the linearized cloning vector described above using the In-Fusion HD Cloning Kit to generate the retroviral plasmid pMEI-5 / ZG-SRG_FFF.

[0115] (12) pMEI-5 / SRG_YYY A nucleic acid encoding a fusion polypeptide was prepared by deleting the amino acid sequence of ZsGreen1 and the subsequent T2A from the fusion polypeptide (SEQ ID NO: 13) encoded by ZG-SRG_YYY. This nucleic acid was designated SRG_YYY. The amino acid sequence of this fusion polypeptide is shown in SEQ ID NO: 47. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 48. As shown in Figure 10, the pMEI-5 DNA vector was cleaved with the restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, an amplified fragment was obtained by PCR using pMEI-5 / ZG-SRG_YYY as a template and the SRG_M5InfFwd primer (SEQ ID NO: 49) and the ZGSRG-SRG_M5InfRev primer (SEQ ID NO: 18). This amplified product was cloned into the linearized vector for cloning using the In-Fusion HD Cloning Kit to generate the retroviral plasmid pMEI-5 / SRG_YYY.

[0116] (13) pMEI-5 / SRG_FFF A nucleic acid encoding a fusion polypeptide was prepared by deleting the amino acid sequence of ZsGreen1 and the subsequent T2A from the fusion polypeptide (SEQ ID NO: 43) encoded by ZG-SRG_FFF. This nucleic acid was designated SRG_FFF. The amino acid sequence of this fusion polypeptide is shown in SEQ ID NO: 50. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 51. As shown in Figure 11, the pMEI-5 DNA vector was cleaved with the restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, an amplified fragment was obtained by PCR using pMEI-5 / ZG-SRG_FFF as a template and the SRG_M5InfFwd primer (SEQ ID NO: 49) and the ZGSRG-SRG_M5InfRev primer (SEQ ID NO: 18). This amplified product was cloned into the linearized vector for cloning using the In-Fusion HD Cloning Kit to generate the retroviral plasmid pMEI-5 / SRG_FFF.

[0117] (14) pMEI-5 / EL-SRG_FFF A nucleic acid encoding a fusion polypeptide (Emerald luciferase-T2A-EPOR-IL2Rγ-P2A-EPOR-IL2Rβ) was constructed by seamlessly fusing Emerald luciferase with the fusion polypeptide encoded by SRG_FFF (SEQ ID NO: 50). This nucleic acid was designated EL-SRG_FFF. The amino acid sequence of this fusion polypeptide is shown in SEQ ID NO: 52. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 53. As shown in Figure 12, the pMEI-5 DNA vector was cleaved with the restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, an amplified fragment was obtained by PCR using the pELuc-test vector (manufactured by TOYOBO) as a template and the ELSRG-EL_M5InfFwd primer (SEQ ID NO: 54) and the ELSRG-EL_M5InfRev primer (SEQ ID NO: 55). Similarly, an amplified fragment was obtained by PCR using pMEI-5 / SRG_FFF as a template and the ELSRG-SRG_M5InfFwd primer (SEQ ID NO: 56) and ZGSRG-SRG_M5InfRev primer (SEQ ID NO: 18). These two amplified products were cloned into the linearized cloning vector described above using the In-Fusion HD Cloning Kit to generate the retroviral plasmid pMEI-5 / EL-SRG_FFF.

[0118] (15) pMEI-5 / FMC28z. A nucleic acid encoding a polypeptide consisting of amino acids 1 to 489 of the FMC63-28Z chimeric antigen receptor (GenBank: ADM64594.1) was constructed. This nucleic acid was designated FMC28z. The amino acid sequence of this fusion polypeptide is shown in SEQ ID NO: 57. The nucleotide sequence of this nucleic acid is shown in SEQ ID NO: 58. This nucleic acid was synthesized as an artificial gene (codon-optimized) and cloned into the pEX-A2J2 vector to construct the plasmid pEX-A2J2 / FMC28z. Next, as shown in Figure 13, pEX-A2J2 / FMC28z was cleaved with the restriction enzymes PmlI and BamHI to prepare insert DNA, which was then inserted into the pMEI-5 DNA vector, which had been similarly treated with the restriction enzymes PmlI and BamHI and linearized, to prepare the retroviral plasmid pMEI-5 / FMC28z.

[0119] (16) pMEI-5 / FMC28z-SRG_FFF A nucleic acid encoding a polypeptide seamlessly fused between the FMC63-28Z chimeric antigen receptor (SEQ ID NO: 57) and the fusion polypeptide encoded by SRG_FFF (SEQ ID NO: 50) was constructed. This nucleic acid was designated FMC28z-SRG_FFF. The amino acid sequence of this fusion polypeptide is shown in SEQ ID NO: 59. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 60. As shown in Figure 14, the pMEI-5 DNA vector was cleaved with the restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, an amplified fragment was obtained by PCR using pMEI-5 / FMC63-28z as a template and the FMC63SRG-FMC_M5InfFwd primer (SEQ ID NO: 61) and the FMC63SRG-FMC_M5InfRev primer (SEQ ID NO: 62). Similarly, an amplified fragment was obtained by PCR using pMEI-5 / SRG_FFF as a template and the FMC63SRG-SRG_M5InfFwd primer (SEQ ID NO: 63) and the ZGSRG-SRG_M5InfRev primer (SEQ ID NO: 18). These two amplified products were cloned into the linearized cloning vector described above using the In-Fusion HD Cloning Kit to generate the retroviral plasmid pMEI-5 / FMC28z-SRG_FFF.

[0120] (17) pEX-A2J2 / EpoRm A nucleic acid encoding a truncated mutant of the human erythropoietin receptor (ER) was constructed, consisting of a polypeptide consisting of amino acids 2 to 438 of the ER sequence shown in NCBI Reference Sequence: NP_000112.1, published in Blood, Vol. 135(9):668-679 (2020). This was named EpoRm. The amino acid sequence of this polypeptide is shown in SEQ ID NO: 64. The nucleotide sequence of the ER was also shown in SEQ ID NO: 65. This ER was synthesized as an artificial gene (codon-optimized) and cloned into the pEX-A2J2 vector to construct the plasmid pEX-A2J2 / EpoRm.

[0121] (18) pMEI-5 / ZG-EpoRm A nucleic acid encoding a polypeptide seamlessly fused between ZsGreen1 and a truncated mutant of the human erythropoietin receptor (SEQ ID NO: 64) was constructed. This was designated ZG-EpoRm. The amino acid sequence of this polypeptide is shown in SEQ ID NO: 66. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 67. As shown in Figure 15, the pMEI-5 DNA vector was cleaved with the restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, an amplified fragment was obtained by PCR using pMEI-5 / ZG-SRG_FFF as a template and the ZGSRG-ZG_M5InfFwd primer (SEQ ID NO: 15) and the ZGEpoRm-ZG_M5InfRev primer (SEQ ID NO: 68). Similarly, an amplified fragment was obtained by PCR using pEX-A2J2 / EpoRm as a template and the ZGEpoRm-EpoRm_M5InfFwd primer (SEQ ID NO: 69) and the ZGEpoRm-EpoRm_M5InfRev primer (SEQ ID NO: 70). These two amplified products were cloned into the linearized cloning vector described above using the In-Fusion HD Cloning Kit to prepare the retroviral plasmid pMEI-5 / ZG-EpoRm.

[0122] Example 2 Preparation of Retroviral Vector Solutions Unless otherwise specified, the 293T cell line and the PG13 cell line were cultured in DMEM / F-12 medium supplemented with 10% fetal bovine serum (FBS) and 100 U / mL penicillin-streptomycin.

[0123] Retroviral vectors corresponding to the various retroviral plasmids prepared in Example 1 were prepared using the procedures described below. First, transient retroviral vector supernatants were prepared in 293T cells. Retroviral packaging plasmids (Gag-pol expression plasmid pGP, VSV-G expression plasmid pVSV-G (Uchibori R, et al. Mol Ther Oncolytics. 2018;12:16-25.)) and retroviral plasmids (pMEI-5 / ZG-SRG_YYY, pMEI-5 / ZG-SRG_FYY, pMEI-5 / ZG-SRG_YFY, pMEI-5 / ZG-SRG_YYF, pMEI-5 / ZG-SRG_FFY, pMEI-5 / ZG-SRG_YFF, pMEI-5 / ZG-SRG_FYF, pMEI-5 / ZG-SRG_FFF, pMEI-5 / SRG_YYY, pMEI-5 / SRG_FFF, pMEI-5 / EL-SRG_FFF, pMEI-5 / FMC28z, pMEI-5 / FMC28z-SRG_FFF, or pMEI-5 / ZG-EpoRm) were introduced by the calcium phosphate method and incubated at 37°C, 5% CO2. The cells were cultured for 7 hours under 5% CO2 conditions. The entire medium was replaced with DMEM / F-12 medium supplemented with 5 mM sodium butyrate (STEMCELL Technologies Inc.) at a final concentration of 5 mM, and cultured for 16 hours at 37°C and 5% CO2. The entire medium was replaced with DMEM / F-12 medium, and cultured for 24 hours at 32°C and 5% CO2. After culture, the cells were centrifuged at 2,000 × g for 10 minutes to collect the viral vector supernatant, which was then filtered through a 0.45 μm filter to remove cell debris. The viral supernatant was further purified using a column (Retrovirus Purification Mini Kit, ViraTrap: Biomiga, Inc.).

[0124] Next, retroviral vectors were produced using the PG13 cell line as a packaging cell line. Specifically, PG13 cells seeded in 6-well plates were exposed to the purified virus solution in the presence of 8 μg / mL polybrene and incubated overnight in an incubator set at 37°C and 5% CO2. The following day, the medium was completely replaced and the cells were incubated in an incubator set at 37°C and 5% CO2 for 6 hours. After that, the transduced cells were detached and passaged into T-75 flasks. After reaching subconfluence, the cells were cultured for 7–10 days in an incubator set at 37°C and 5% CO2 for limiting dilution to isolate single-cell clones. Cells were then detached from the wells by trypsinization and seeded into 12-well plates. They were then cultured for 3–4 days in an incubator set at 37°C and 5% CO2. Cells were detached from the wells by trypsinization, and three-quarters of the cells were resuspended in a cryopreservation medium (Cellbanker, manufactured by Nippon Zenyaku Kogyo Co., Ltd.) and frozen in a -80°C freezer. The remaining quarter was seeded into 12-well plates and cultured for 3–4 days in an incubator set at 37°C and 5% CO2. Upon reaching confluence, the medium was replaced with DMEM / F-12 medium supplemented with 5 mM sodium butyrate. The plates were then incubated in an incubator set at 32°C and 5% CO2 for 48 hours. After centrifugation at 2,000 × g for 10 minutes, the supernatant was collected and filtered through a 0.45 μm filter to remove cell debris, resulting in the retroviral vector solution. A portion of the retroviral vector solution was titered (Retrovirus Titer Set (for Real Time PCR), manufactured by Takara Bio Inc.), and the remainder was dispensed into cryovial tubes and frozen in a -80°C freezer until use. The viral supernatant titer of each clone (Retrovirus Titer Set (for Real Time PCR)) was measured and selected to establish a clone line stably producing retroviral vectors. 9For clones stably producing retroviral vectors with a cell count of ≥ 100 copies / mL, thawed cryopreserved cells were seeded in T-75 flasks and then expanded to T-225 flasks. When the cells reached confluence, the medium was replaced with DMEM / F-12 medium supplemented with 5 mM sodium butyrate. The retroviral vector solution was collected using the same procedure as above, dispensed into cryovial tubes, and stored frozen in a -80°C freezer until use.

[0125] Example 3 Comparison of the proliferation efficiency of SRG construct-transfected cells Hereinafter, a nucleic acid encoding a T2A-EPOR-IL2Rγ-P2A-EPOR-IL2Rβ fusion polypeptide (including retroviruses carrying the same nucleic acid) is referred to as "prototype SRG," and a nucleic acid encoding a fusion polypeptide (FYY, YFY, YYF, FFY, YFF, FYF, and FFF) having a mutation in the intracellular domain of the IL-2 receptor β chain is referred to as "mutated SRG." (1) Transduction of SRG constructs into T cells Peripheral blood was collected from volunteers who provided informed consent into BD Vacutainer CPT mononuclear cell collection tubes and centrifuged at 25°C and 1,500 × g for 15 minutes to separate peripheral blood mononuclear cells (PBMCs) and plasma. PBMCs were washed twice with CELLOTION (Nihon Zenyaku Kogyo Co., Ltd.), mixed with a mixture of cell cryopreservation medium CP-1 (Kyokuto Pharmaceutical Industrial Co., Ltd.), human serum albumin (HSA) (Albuminar; CSL Behring), and RPMI, and frozen at -80°C until use. Plasma was heat-inactivated for 30 minutes in a water bath set at 56°C and then frozen at -80°C until use.

[0126] To activate PBMCs, 5 μg / mL CD3 Monoclonal Antibody (OKT3) (Thermo Fisher Scientific) diluted with ACD-A solution (Terumo) and 20 μg / mL RetroNectin (Takara Bio) were added to an untreated 6-well plate at 1 mL per well. The plate was then left in an incubator at 37°C and 5% CO2 for 3 hours, followed by overnight at 4°C to prepare an OKT3 / RetroNectin-coated plate. Thawed PBMCs were suspended in medium (GT-T551 medium (Takara Bio) supplemented with 175 IU / mL IL-2 and 0.1% heat-inactivated autologous plasma) and incubated at 2.0 × 10 5 The cell suspension was adjusted to 6.5 cells / mL and washed three times with DMEM / F-12 medium (FBS-free, penicillin-streptomycin-free). The cell suspension was added to an OKT3 / RetroNectin-coated plate at 6.5 mL / well. The plate was placed in an incubator at 37°C and 5% CO2 for 4 days to promote the activation and proliferation of T cells in the PBMCs.

[0127] The RetroNectin-conjugated virus infection method (J Biochem. 2001 Sep;130(3):331-4) was used for transduction of T cells. 20 μg / mL RetroNectin diluted with PBS was added to 2 mL / well of a 6-well plate with an untreated surface and left at 4°C. The RetroNectin solution was removed, and 2 mL / well of blocking solution (phosphate-buffered saline (PBS) supplemented with 2% bovine serum albumin (BSA)) was added. The plate was then left to stand at room temperature for 30 minutes for blocking treatment. The blocking solution was removed, and 2 mL / well of PBS was added, followed by washing once and the PBS was removed. The retroviral vector solution (4.0 × 10 in PBS) carrying the prototype SRG and mutant SRG prepared in Example 2 was added to the plate. 9RetroNectin-coated plates were then treated with a 4 mL / well solution of retrovirus-containing T cells (diluted to 175 IU / mL IL-2 and 0.1% inactivated autologous plasma) and incubated for 2 hours at 1,960 × g. The retrovirus-containing plate was then placed in a centrifuge maintained at 32°C and centrifuged at 32°C for 2 hours at 1,960 × g to promote the adsorption of virus particles onto RetroNectin. The virus dilution was removed from the plate and washed with 2 mL of PBS supplemented with 1% BSA. OKT3 / Retronectin-activated T cells (4.0 × 10 cells / well) were cultured in GT-T551 medium supplemented with 175 IU / mL IL-2 and 0.1% inactivated autologous plasma. 5 The virus-adsorbed plate was then loaded with 4 mL of the diluted solution (diluted to 1000 cells / mL) and centrifuged at 32°C and 1,960 x g for 10 minutes. The plate was then placed in an incubator at 37°C and 5% CO2 overnight for the first transduction. The transduced T cells were collected the next day, washed with the medium described above, and after removing the supernatant, resuspended in 4 mL of medium and added to a similarly prepared virus-adsorbed plate. The plate was then placed in an incubator at 37°C and 5% CO2 overnight for the second transduction.

[0128] (2) Comparison of the proliferation efficiency of SRG construct-transfected cells. As shown in Figure 16, transfected T cells were harvested the next day and washed twice with basal medium (GT-T551 medium supplemented with 0.1% heat-inactivated autologous plasma). Transfected cells were resuspended in 20 mL of basal medium supplemented with 9.0 IU / mL erythropoietin (EPO) and placed in an incubator set at 37°C and 5% CO2 (Day 2). On Days 4, 7, 9, 11, 14, 16, and 18, an equal volume of basal medium was added, along with 9.0 IU / mL EPO. Figures 17-1 and 17-2 show the growth curves of cells transfected with the basic SRG and various mutant SRGs (ZsGreen1-positive cells). FFF mutant SRG-transfected cells demonstrated EPO-dependent proliferation for 3 weeks after transfection. Figure 18 shows the ZsGreen1-positive rate. In all transduced cell populations, the addition of EPO induced selective proliferation of the transduced cells, resulting in a cell population composed almost entirely of transduced cells at the endpoint. FFF mutant SRG transduced cells showed proliferation of ZsGreen1-positive cells for 3 weeks after transduction, and this proliferation was EPO concentration-dependent.

[0129] Example 4: Optimization of the Amount of EPO Added. T cells stimulated with OKT3 / Retronectin were transduced with FFF mutant SRG using the RetroNectin-conjugated virus infection method. After the second transduction, T cells were collected the following day (Day 2) and washed twice with basal medium. Transduced cells were resuspended in 20 mL of basal medium supplemented with 0, 0.3, 0.9, 3.0, 9.0, or 15 IU / mL EPO and placed in an incubator set at 37°C and 5% CO2 (Day 2). On Days 4, 7, 9, 11, 14, 16, and 18, an equal volume of basal medium was added, followed by the addition of 0, 0.3, 0.9, 3.0, 9.0, or 15 IU / mL EPO. Figure 19 shows the growth curves of transduced cells (ZsGreen1-positive cells). After thorough mixing, 100 μL of the culture medium was removed, washed twice with CELLOTION, and resuspended in 100 μL of PBS. 5 μL of a staining solution (1 mM Ethidium Homodimer III solution, diluted 50-fold with PBS) was added and mixed. The mixture was incubated at room temperature for 15 minutes in the dark to stain dead cells. 10 μL of the reaction mixture was transferred to Countess Cell Counting Chamber Slides (Thermo Fisher Scientific), and the number of ZsGreen1-positive cells and dead cells was counted using a Countess II FL (Thermo Fisher Scientific). ZsGreen1-positive cells showed EPO concentration-dependent proliferation over a period of 3 weeks after transduction.

[0130] Example 5 Comparison of proliferation of transduced cells depending on the amount of gene transfer vector T cells stimulated with OKT3 / Retronectin were transduced with FFF mutant SRG using the RetroNectin-conjugated virus infection method. The virus concentration at the time of transduction was 2.5x10 3 , 5.0x10 3 , 1.0x10 4 , 2.0x10 4 , 3.0x10 4The second transduction was performed at 100 μL / cell. The T cells after the second transduction were collected the next day (Day 2) and washed twice with basal medium. The transduced cells were resuspended in 20 mL of basal medium supplemented with 9.0 IU / mL EPO and placed in an incubator set at 37°C and 5% CO2 (Day 2). On Days 4, 7, 9, 11, 14, 16, and 18, an equal volume of basal medium was added, along with 9.0 IU / mL EPO. Figure 20 shows the growth curve of transduced cells (ZsGreen1-positive cells). After thorough mixing, 100 μL of the culture medium was removed, washed twice with CELLOTION, and resuspended in 100 μL of PBS. 5 μL of a staining solution (1 mM Ethidium Homodimer III solution, diluted 50-fold with PBS) was added and mixed. The cells were incubated at room temperature for 15 minutes in the dark to stain dead cells. Ten microliters of the reaction mixture was transferred to Countess Cell Counting Chamber Slides, and the number of ZsGreen1-positive cells and dead cells were counted using a Countess II FL. The proliferation of ZsGreen1-positive cells over a 3-week period after transduction was shown to correlate with the amount of vector used for transduction.

[0131] Example 6: Growth Comparison with Prior Art T cells stimulated with OKT3 / Retronectin were transduced with YYY-based SRG, FFF-mutant SRG, or EpoRm using RetroNectin-conjugated virus infection. After the second transduction, T cells were harvested the following day (Day 2) and washed twice with basal medium. As shown in Figure 21, in the IL-2-only culture system, transduced cells were resuspended in 30 mL of basal medium supplemented with IL-2 at a final concentration of 175 IU / mL and placed in an incubator set at 37°C and 5% CO2 (Day 2). On Days 4, 7, 9, 11, 14, 16, and 18, an equal volume of basal medium supplemented with IL-2 at a final concentration of 175 IU / mL was added. For the IL-2 and EPO culture system, transduced cells were resuspended in 30 mL of basal medium supplemented with IL-2 (175 IU / mL) and EPO (9.0 IU / mL) at a final concentration and placed in an incubator set at 37°C with 5% CO2 (Day 2). On Days 4, 7, 9, 11, 14, 16, and 18, an equal volume of basal medium supplemented with IL-2 (175 IU / mL) and EPO (9.0 IU / mL) was added. For the EPO-only culture system, transduced cells were resuspended in 20 mL of basal medium supplemented with EPO (9.0 IU / mL) at a final concentration and placed in an incubator set at 37°C with 5% CO2 (Day 2). On Days 4, 7, 9, 11, 14, 16, and 18, an equal volume of basal medium supplemented with EPO (9.0 IU / mL) was added. In addition, untransduced T cells were cultured under the above conditions as a control. Figures 22-1 and 22-2 show the growth curves of transduced cells (ZsGreen1-positive cells). After thorough mixing, 100 μL of culture medium was removed, washed twice with CELLOTION, and resuspended in 100 μL of PBS. 5 μL of a staining solution prepared by diluting 1 mM Ethidium Homodimer III solution 50-fold with PBS was added and mixed. The mixture was incubated at room temperature for 15 minutes in the dark to stain dead cells. 10 μL of the reaction mixture was transferred to Countess Cell Counting Chamber Slides, and the number of ZsGreen1-positive cells and dead cells was counted using a Countess II FL. ZsGreen1-positive cells showed EPO concentration-dependent proliferation over a period of 3 weeks after transduction.The results of measuring the ZsGreen1 positivity using a flow cytometer are shown in Figures 23-1 and 23-2. In all transduced cell groups, the addition of EPO induced selective proliferation of the transduced cells, resulting in a cell population composed almost entirely of transduced cells at the endpoint. In FFF mutant SRG transduced cells, proliferation and persistence of ZsGreen1 positivity cells were observed over a 3-week period after transduction, in an EPO concentration-dependent manner.

[0132] Example 7: EPO-Dependent In Vivo Growth of Transduced Cells Infused into Immunodeficient Mice (1) Comparison of In Vivo Growth of SRG Construct-Transduced Cells at Different EPO Doses As shown in Figure 24, T cells stimulated with OKT3 / Retronectin were transduced with an Emerald luciferase-carrying FFF mutant SRG (a retroviral vector prepared with pMEI-5 / EL-SRG_FFF) using the RetroNectin-conjugated viral infection method. After the second transduction, T cells were harvested the following day (Day 2) and washed twice with basal medium. Transduced cells were resuspended in 10 mL of basal medium supplemented with 9.0 IU / mL EPO and placed in an incubator set at 37°C and 5% CO2 (Day 2). On Days 4, 7, 9, and 11, an equal volume of basal medium was added, followed by the addition of 9.0 IU / mL EPO. On Day 14, cells were harvested and washed with CELLOTION. Dead cells were then removed by density gradient centrifugation using a human lymphocyte separation solution (Lympholyte-H; Cedarlane Laboratories Ltd.). After collecting the target cell layer and washing it twice with CELLOTION, the cells were mixed in a mixture of cell cryopreservation medium CP-1, HSA (Albuminar), and RPMI, and frozen at -80°C until use.

[0133] Immunodeficient NOG tumor-bearing mice were treated with 1x10 6SRG-FFF-loaded T cells were infused via the tail vein (Day 0). EPO was administered intraperitoneally at 0, 30, 60, 90, 120, and 150 IU / injection on Days 0, 3, 5, 7, 10, 11, and 14, respectively. Figure 25 shows in vivo bioluminescence imaging of luciferase activity over time after transduced T cell infusion, and Figures 26-1 and 26-2 show graphs quantifying luminescence intensity. The infused SRG-loaded T cells demonstrated EPO dose-dependent proliferation. As shown in Figure 27, the hematocrit value of whole blood collected on Day 17 showed an EPO dose-dependent increase.

[0134] (2) Comparison of in vivo proliferation of SRG construct-transfected cells with different EPO administration periods. As shown in Figure 28, 1x10 6 SRG-FFF-loaded T cells were infused via the tail vein (Day 0). EPO was administered intraperitoneally at 90 IU / injection on Days 0, 3, 5, 7, 10, 12, and 14 (three times a week for two weeks) or on Days 0, 3, 7, 10, 14, 17, and 21 (twice a week for three weeks). Figure 29 shows in vivo bioluminescence imaging of luciferase activity over time after transfection of each transduced T cell, and Figure 30 shows a graph of luminescence intensity. In vivo proliferation of the infused SRG-loaded T cells was EPO dose-dependent under all conditions, but the twice-weekly group for three weeks demonstrated sustained proliferation and persistence of the infused cells. As shown in Figure 31, hematocrit values ​​of whole blood collected on Day 26 showed an EPO-dependent increase.

[0135] Example 8: Measurement of Immunophenotype of Transduced T Cells (1) Transduction of SRG-Equipped CAR Constructs into T Cells. T cells stimulated with OKT3 / Retronectin were transduced with CAR (retroviral vector prepared with pMEI-5 / FMC28z) or SRG-FFF-equipped CAR (retroviral vector prepared with pMEI-5 / FMC28z-SRG_FFF) using the RetroNectin-conjugated viral infection method. After the second transduction, T cells were collected the following day (Day 2) and washed twice with basal medium. Non-plasma cells and CAR-transduced cells were suspended in 30 mL of basal medium supplemented with IL-2 at a final concentration of 175 IU / mL and placed in an incubator set at 37°C and 5% CO2 (Day 2). On Days 4, 7, 9, 11, 14, 16, and 18, an equal volume of basal medium supplemented with IL-2 at a final concentration of 175 IU / mL was added. Cells transduced with SRG-carrier were resuspended in 10 mL of basal medium supplemented with EPO at a final concentration of 9.0 IU / mL and placed in an incubator set at 37°C and 5% CO2 (Day 2). On Days 4, 7, 9, and 11, an equal volume of basal medium supplemented with 9.0 IU / mL EPO was added. On Day 14, cells were harvested and washed with CELLOTION. Dead cells were then removed by density gradient centrifugation using human lymphocyte separation solution (Lympholyte-H). After harvesting the target cell layer, the cells were washed twice with CELLOTION and mixed in a mixture of cell cryopreservation medium CP-1, HSA (Albuminar), and RPMI, and frozen at -80°C until use.

[0136] (2) Immunophenotype of Transduced T Cells. Cryopreserved transduced T cells were thawed and stained with CAR (Cytoart, Inc.) and SRG (EpoR) (R&D Systems, Inc.)-specific antibodies. CD45RA and CCR7 on CD4+ or CD8+ cells were stained with specific monoclonal antibodies (BioLegend, Inc.), and flow cytometry analysis was performed. Figure 32 shows the CAR and SRG (EpoR) positivity rates. Figure 33 shows the CD4 and CD8 ratios (CAR-T cells are CAR-positive cells, and SRG-loaded CAR-T cells are a population of cells positive for both CAR and SRG). Figures 34-1 and 34-2 show the ratios of CD4 and CD8 T cell subsets (CAR-T cells are CAR-positive cells, and SRG-loaded CAR-T cells are a population of cells positive for both CAR and SRG). SRG-loaded CAR-T cells maintained the CD45RA-positive CCR7-positive T cell subset compared with CAR-T cells.

[0137] Example 9: In Vitro Cytotoxicity of SRG-Loaded CAR-T Cells (1) Measurement of Cytotoxic Activity The transduced T cells prepared and cryopreserved in Example 8 were thawed and mixed with K562 or K562-CD19 cells in a V-bottom 96-well microplate at effector:target ratios of 10:1, 5:1, or 1:1. The cells were then co-cultured for 18 hours in an incubator set at 37°C and 5% CO2. The microplate was centrifuged at 250 × g and room temperature for 10 minutes, and the supernatant was dispensed into a flat-bottom 96-well microplate at 100 μL / well. Cytotoxic activity was measured using an LDH Cytotoxicity Detection Kit (Takara Bio Inc.). As shown in Figure 35, the cytotoxic activity of SRG-loaded CAR-T cells was slightly weaker than that of CAR-T cells, but they still exhibited cytotoxic activity against CD19-positive cells.

[0138] (2) IL-2 and IFN-γ Production by CAR-Transduced T Cells. The transduced cells prepared and cryopreserved in Example 8 were thawed, mixed with K562 or K562-CD19 cells at an effector:target ratio of 5:1 in CTL-Test PLUS Medium (Cellular Technology Limited), transferred to a V-bottom 96-well microplate, and co-cultured for 18 hours in an incubator set at 37°C and 5% CO2. The microplate was centrifuged at 250 × g and room temperature for 10 minutes, and the supernatant was collected and assayed for IL-2 and IFN-γ production using a Human ProQuantum Immunoassay Kit (Invitrogen). As shown in Figures 36-1 and 36-2, SRG-loaded CAR-T cells were activated in response to CD19-positive cells and produced cytokines at levels comparable to those of CAR-T cells.

[0139] Example 10 In vivo antitumor effect of SRG-loaded CAR-T cells on tumor-bearing mice As shown in Figure 37, 1x10 cells of the human CD19-positive acute lymphoblastic leukemia cell line NALM-6 transduced with the Emerald luciferase gene were injected into the tail vein of immunodeficient NOG mice. 6 14 days later, 3x10 CAR-transduced cells or SRG-loaded CAR-T cells prepared in Example 8 were inoculated. 6 pcs or 5x10 6 Each transduced T cell was infused into the tumor cells (n = 10 per group). 90 IU / injection of EPO was administered intraperitoneally (twice a week for 3 weeks). Alternatively, EPO was not administered. Figure 38 shows in vivo bioluminescence imaging of luciferase activity in tumor cells over time after transduction of each transduced T cell, Figure 39 shows a graph quantifying the luminescence intensity, and Figure 40 shows a Kaplan-Meier curve. SRG-loaded CAR-T cells exhibited higher in vivo antitumor activity than CAR-T cells. Furthermore, the group receiving SRG-loaded CAR-T cells in combination with EPO administration exhibited stronger antitumor activity and a better overall survival rate than the other groups.

[0140] Example 11: Preparation of SRG-FFF40 Construct As shown in Figure 41, the retroviral plasmid pMEI-5 / ZG-SRG_FFF40 was constructed so that the nucleotide sequence (SEQ ID NO: 72) encoding the polypeptide (including the intracellular domain) from positions 216 to 277 of the amino acid sequence of human CD40 (NCBI Reference Sequence: NP_001241.1) (SEQ ID NO: 71) was fused downstream of IL-2Rγ. Also, as shown in Figure 42, a version of the retroviral plasmid pMEI-5 / SRG_FFF40 that does not contain ZsGreen was constructed. Furthermore, as shown in Figure 43, a version of the retroviral plasmid pMEI-5 / EL-SRG_FFF40 that contains Emerald luciferase was constructed. Furthermore, as shown in Figure 44, a version of the retroviral plasmid pMEI-5 / FMC28z-SRG_FFF40 that contains the FMC63-28Z chimeric antigen receptor (SEQ ID NO: 57) was constructed.

[0141] Example 12 In vitro cytotoxicity of SRG-FFF40-loaded CAR-T cells The cytotoxic activity of SRG-FFF40-loaded CAR-T cells was measured using the same procedure as in Example 9-(1). As shown in Figure 45, the intensity of the cytotoxic activity of SRG-FFF40-loaded CAR-T cells was stronger than that of SRG-FFF-loaded CAR-T cells and equivalent to that of CAR-T cells.

[0142] Example 13 In vivo antitumor effect of SRG-FFF40-loaded CAR-T cells on cancer-bearing mice The in vivo antitumor effect of SRG-FFF40-loaded CAR-T cells on cancer-bearing mice was measured using the same procedure as in Example 10. The cell infusion dose was 5x10 6 The number of subjects was 15 (n = 15 for each group). Kaplan-Meier curves are shown in Figure 46. SRG-FFF40-loaded CAR-T cells exhibited higher in vivo antitumor activity than CAR-T cells. Furthermore, the SRG-FFF40-loaded CAR-T cell infusion group combined with EPO administration exhibited stronger antitumor activity and a better overall survival rate than the other groups.

[0143] The present invention provides a chimeric receptor comprising: a first polypeptide having (i) a first extracellular domain capable of binding to a ligand, (ii) a first transmembrane domain, and (iii) a first intracellular domain derived from the IL-2 receptor β chain, with a mutation introduced into the first intracellular domain; and a second polypeptide having (iv) a second extracellular domain capable of binding to a ligand, (v) a second transmembrane domain, and (vi) a second intracellular domain derived from the IL-2 receptor γ chain. Also provided are a nucleic acid encoding the chimeric receptor, a vector comprising the nucleic acid, a cell expressing the chimeric receptor, a method for producing the cell, a pharmaceutical composition comprising the cell as an active ingredient, and a method for preventing or treating a disease, comprising administering the cell or the pharmaceutical composition to a subject. The methods of the present invention are particularly useful for applications in the medical field.

[0144] SEQ ID NO: 1: TEG amino acid sequence SEQ ID NO: 2: TEG nucleic acid sequence SEQ ID NO: 3: PEB amino acid sequence SEQ ID NO: 4: PEB nucleic acid sequence SEQ ID NO: 5: TEGPEB amino acid sequence SEQ ID NO: 6: TEGPEB nucleic acid sequence SEQ ID NO: 7: pEX-V_InfRev primer SEQ ID NO: 8: pEX-V_InfFwd primer SEQ ID NO: 9: TSRG1_pEXInfFwd primer SEQ ID NO: 10: TSRG1_pEXInfRev primer SEQ ID NO: 11: PSRG2_pEXInfFwd primer SEQ ID NO: 12: PSRG2_pEXInfRev primer SEQ ID NO: 13: ZG-SRG_YYY amino acid sequence SEQ ID NO: 14: ZG-SRG_YYY nucleic acid sequence SEQ ID NO: 15: ZGSRG-ZG_M5InfFwd primer SEQ ID NO: 16: ZGSRG-ZG_M5InfRev primer SEQ ID NO: 17: ZGSRG-SRG_M5InfFwd primer SEQ ID NO: 18: ZGSRG-SRG_M5InfRev primer SEQ ID NO: 19: ZG-SRG_FYY amino acid sequence SEQ ID NO: 20: ZG-SRG_FYY nucleic acid sequence SEQ ID NO: 21: FYYmut_Rev primer SEQ ID NO: 22: FYYmut_Fwd primer SEQ ID NO: 23: ZG-SRG_YFY amino acid sequence SEQ ID NO: 24: ZG-SRG_YFY nucleic acid sequence SEQ ID NO: 25: YFYmut_Revprimer SEQ ID NO: 26: YFYmut_Fwd primer SEQ ID NO: 27: ZG-SRG_YYF amino acid sequence SEQ ID NO: 28: ZG-SRG_YYF nucleic acid sequence SEQ ID NO: 29: YYFmut_Rev primer SEQ ID NO: 30: YYFmut_Fwd primer SEQ ID NO: 31: ZG-SRG_FFY amino acid sequence SEQ ID NO: 32: ZG-SRG_FFY nucleic acid sequence SEQ ID NO: 33: FFYmut_Rev primer SEQ ID NO: 34: FFYmut_Fwd primer SEQ ID NO: 35: ZG-SRG_YFF amino acid sequence SEQ ID NO: 36: ZG-SRG_YFF nucleic acid sequence SEQ ID NO: 37: YFFmut_Rev primer SEQ ID NO: 38: YFFmut_Fwd primer SEQ ID NO: 39: ZG-SRG_FYF amino acid sequence SEQ ID NO: 40: ZG-SRG_FYF nucleic acid sequence SEQ ID NO: 41: FYFmut_Rev primer SEQ ID NO: 42: FYFmut_Fwd primer SEQ ID NO: 43: ZG-SRG_FFF amino acid sequence SEQ ID NO: 44: ZG-SRG_FFF nucleic acid sequence SEQ ID NO: 45: FFFmut_Rev primer SEQ ID NO: 46: FFFmut_Fwd primer SEQ ID NO: 47: SRG_YYY amino acid sequence SEQ ID NO: 48: SRG_YYY nucleic acid sequence SEQ ID NO: 49: SRG_M5InfFwd primer SEQ ID NO: 50: SRG_FFF amino acidsequence SEQ ID NO: 51: SRG_FFF nucleic acid sequence SEQ ID NO: 52: EL-SRG_FFF amino acid sequence SEQ ID NO: 53: EL-SRG_FFF nucleic acid sequence SEQ ID NO: 54: ELSRG-EL_M5InfFwd primer SEQ ID NO: 55: ELSRG-EL_M5InfRev primer SEQ ID NO: 56: ELSRG-SRG_M5InfFwd primer SEQ ID NO: 57: FMC28z amino acid sequence SEQ ID NO: 58: FMC28z nucleic acid sequence SEQ ID NO: 59: FMC28z-SRG_FFF amino acid sequence SEQ ID NO: 60: FMC28z-SRG_FFF nucleic acid sequence SEQ ID NO: 61: FMC63SRG-FMC_M5InfFwd primer SEQ ID NO: 62: FMC63SRG-FMC_M5InfRev primer SEQ ID NO: 63: FMC63SRG-SRG_M5InfFwd primer SEQ ID NO: 64: EpoRm amino acid sequence SEQ ID NO: 65: EpoRm nucleic acid sequence SEQ ID NO: 66: ZG-EpoRm amino acid sequence SEQ ID NO: 67: ZG-EpoRm nucleic acid sequence SEQ ID NO: 68: ZGEpoRm-ZG_M5InfRev primer SEQ ID NO: 69: ZGEpoRm-EpoRm_M5InfFwd primer SEQ ID NO: 70: ZGEpoRm-EpoRm_M5InfRev primer SEQ ID NO: 71: CD40 partial amino acid sequence SEQ ID NO: 72: CD40 partial nucleic acid sequence SEQ IDNO: 73: Wild-type human IL-2 receptor subunit beta precursor amino acid sequence SEQ ID NO: 74: ild-type human IL-2 receptor subunit gamma precursor amino acid sequence

Claims

1. (i) a first extracellular domain that binds to a ligand; (ii) a first transmembrane domain, and (iii) a first intracellular domain derived from the IL-2 receptor β chain and wherein one or more tyrosine residues in the first intracellular region have been mutated; and (iv) a second extracellular domain that binds to a ligand; (v) a second transmembrane domain, and (vi) a second intracellular domain derived from the IL-2 receptor γ chain a second polypeptide having A chimeric receptor comprising:

2. (iii) The chimeric receptor according to claim 1, wherein the one or more tyrosine residues in the first intracellular domain derived from the IL-2 receptor β chain are selected from the group consisting of tyrosine residues corresponding to positions 381, 384, and 387 in the amino acid sequence of the IL-2 receptor β chain shown in SEQ ID NO: 73 (RefSeq NP_000869.1).

3. (iii) The chimeric receptor according to claim 2, wherein the one or more tyrosine residues in the first intracellular domain derived from the IL-2 receptor β chain are tyrosine residues corresponding to positions 381, 384, and 387 in the amino acid sequence of the IL-2 receptor β chain shown in SEQ ID NO: 73 (RefSeq NP_000869.1).

4. The chimeric receptor of claim 1, wherein the mutation is a substitution of a tyrosine residue with a phenylalanine residue.

5. The chimeric receptor of claim 1, wherein (i) the first extracellular domain that binds to a ligand and / or (iv) the second extracellular domain that binds to a ligand is an extracellular domain derived from an erythropoietin receptor.

6. The chimeric receptor according to claim 1, wherein (ii) the first transmembrane region is a transmembrane region derived from the IL-2 receptor β chain, and (v) the second transmembrane region is a transmembrane region derived from the IL-2 receptor γ chain.

7. The chimeric receptor of claim 1 , wherein the second polypeptide further comprises an intracellular domain derived from CD40.

8. A nucleic acid encoding the chimeric receptor according to any one of claims 1 to 7.

9. A vector comprising the nucleic acid of claim 8.

10. A cell expressing the chimeric receptor according to any one of claims 1 to 7.

11. The cell of claim 10, further expressing a chimeric antigen receptor or a foreign T cell receptor that recognizes a target cell.

12. A pharmaceutical composition comprising the cells of claim 10 as an active ingredient.