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

Abstract

The present invention provides a chimeric receptor comprising: a first polypeptide having (i) a first extracellular region bound to a ligand, (ii) a first transmembrane region, and (iii) a first intracellular region derived from an IL-2 receptor beta chain, and wherein a mutation is introduced at a tyrosine residue of the first intracellular region; and a second polypeptide having (iv) a second extracellular region bound to a ligand, (v) a second transmembrane region, and (vi) a second intracellular region derived from the IL-2 receptor gamma chain.

Description

Technical Field

[0001] The present invention relates to a chimeric receptor, which can be used to improve the in vitro proliferation ability, in vivo proliferation ability and in vivo survival ability of immune cells such as T cells.

[0002] Background Field

[0003] As a therapeutic strategy for tumors, it is expected that the T cells targeting tumor cells can be produced by introducing nucleic acids encoding a chimeric antigen receptor (CAR) bound to a specific antigen present on the surface of tumor cells or nucleic acids encoding a T cell receptor (TCR) for identifying tumor cells into T cells. In this article, T cells wherein the nucleic acids encoding CAR have been introduced are referred to as CAR-T cells, and T cells wherein the nucleic acids encoding TCR have been introduced are referred to as TCR-T cells.

[0004] Currently, CAR-T cells or TCR-T cell therapies targeting various tumor antigens such as CD19, BCMA, WT1, MART1, gp100, CEA and mHAG HA-2 antigens have been developed. For example, therapy using CD19-specific CAR-T cells has shown significant therapeutic effects on B cell tumors. However, long-term follow-up has revealed tumor recurrence, and the therapeutic effect is far from satisfactory and needs further improvement. One of the reasons for tumor recurrence after treatment with CAR-T cells is that CAR-T cells cannot survive in vivo for a long time.

[0005] Interleukin 2 (hereinafter referred to as IL-2) can be used to maintain the proliferation and anti-tumor activity of exogenously administered T cells in vivo. However, the administration of IL-2 causes side effects. In addition, IL-2 reacts with all T cells expressing IL-2 receptors, activating not only the administered T cells but also endogenous regulatory T cells, thereby weakening the immune response.

[0006] As a known method to overcome the IL-2 requirement of T cells, erythropoietin, a low-toxic cytokine, is used 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 β chain or γ chain of the IL-2 receptor subunit (Non-Patent Document 1).

[0007] In 2020, Campana et al. reported the EpoRm-CAR system, which co-expresses erythropoietin receptor mutants and CAR (non-patent literature 2). The system was developed to enhance the in vivo survival of T cells (hereinafter referred to as EpoRm-CAR-T cells) co-expressing erythropoietin receptor mutants and CAR by erythropoietin stimulation. However, in vitro erythropoietin stimulation causes EpoRm-CAR-T cell proliferation only up to about twice that of control cells. In addition, in vivo treatment experiments show that the proliferation of tumor cells is inhibited only when mice pre-injected with EpoRm-CAR-T cells are inoculated with cancer cells. Therefore, the effectiveness of the EpoRm-CAR system is considered insufficient.

[0008] Prior art literature

[0009] Non-patent literature

[0010] Non-patent document 1: Blood, 1995 Sep 15; 86(6): 2281-7

[0011] Non-patent literature 2: Blood, 2020 Feb 27; 135(9): 668-679 SUMMARY OF THE INVENTION

[0013] Problem to be solved by the invention

[0014] As mentioned above, with conventional CAR-T therapy, long-term follow-up has revealed tumor recurrence, and the treatment results are far from satisfactory. Therefore, further improvements are needed. In particular, a new method is needed to enable the administered T cells to survive in the patient for an extended period of time.

[0015] Solution to the problem

[0016] As a result of intensive studies to solve the above problems, the present inventors found a method for selectively controlling T cell proliferation and survival ex vivo and in vivo, and completed the present invention.

[0017] That is, the present invention relates to:

[0018] [1] A chimeric receptor comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises:

[0019] (i) a first extracellular region that binds to a ligand,

[0020] (ii) the first transmembrane region, and

[0021] (iii) derived from a first intracellular region of the IL-2 receptor β chain, wherein a mutation is introduced at one or more tyrosine residues in said first intracellular region; and

[0022] The second polypeptide comprises:

[0023] (iv) a second extracellular domain that binds to a ligand,

[0024] (v) a second transmembrane region, and

[0025] (vi) derived from the second intracellular domain of the IL-2 receptor γ chain;

[0026] [2] The chimeric receptor according to [1], wherein the one or more tyrosine residues in the first intracellular region derived from the IL-2 receptor β chain described in (iii) are selected from the tyrosine residues at positions 381, 384 and 387 in the amino acid sequence corresponding to the IL-2 receptor β chain (RefSeq NP_000869.1);

[0027] [3] The chimeric receptor according to [2], wherein the one or more tyrosine residues in the first intracellular region derived from the IL-2 receptor β chain described in (iii) are tyrosine residues corresponding to positions 381, 384 and 387 in the amino acid sequence of IL-2 receptor β chain (RefSeq NP_000869.1);

[0028] [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;

[0029] [5] The chimeric receptor according to any one of [1] to [4], wherein the first extracellular region that binds to the ligand of (i) and / or the second extracellular region that binds to the ligand of (iv) is an extracellular region derived from an erythropoietin receptor;

[0030] [6] The chimeric receptor according to any one of [1] to [5], wherein the first transmembrane region of (ii) is a transmembrane region derived from an IL-2 receptor β chain, and the second transmembrane region of (v) is a transmembrane region derived from an IL-2 receptor γ chain;

[0031] [7] The chimeric receptor according to any one of [1] to [6], wherein the second polypeptide further comprises an intracellular region derived from CD40;

[0032] [8] A nucleic acid encoding the chimeric receptor according to any one of [1] to [7];

[0033] [9] A vector comprising the nucleic acid of [8];

[0034]

[10] The vector according to [9], which is selected from a plasmid vector, a viral vector and an artificial chromosome;

[0035]

[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;

[0036]

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

[0037]

[13] The cell according to

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

[0038]

[14] The cell according to

[12] or

[13] , which is an immune cell;

[0039]

[15] The cell according to

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

[0040]

[16] The cell according to any one of

[12] to

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

[0041]

[17] A method for producing a cell expressing a chimeric receptor, the method comprising the step of introducing the nucleic acid according to [8] into the cell;

[0042]

[18] The method according to

[17] , comprising the step of introducing the vector according to any one of [9] to

[11] into a cell

[0043]

[19] The method according to

[17] or

[18] , further comprising the step of isolating and / or amplifying cells expressing the chimeric receptor

[0044]

[20] The method according to any one of

[17] to

[19] , comprising the step of culturing a cell into which a nucleic acid encoding a chimeric receptor has been introduced in the presence of a ligand;

[0045]

[21] The method according to

[20] , comprising the step of culturing cells into which a nucleic acid encoding a chimeric receptor comprising an extracellular domain of an erythropoietin receptor has been introduced in the presence of erythropoietin;

[0046]

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

[12] to

[16] as an active ingredient;

[0047]

[23] The pharmaceutical composition according to

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

[0048]

[24] The pharmaceutical composition according to

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

[0049]

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

[22] to

[24] to a subject; and

[0050]

[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] .

[0051] Effects of the Invention

[0052] The present invention provides chimeric receptors in the field of genetically modified immune cell therapy that can be used for targeting antigens such as tumor antigens, nucleic acids encoding chimeric receptors, vectors containing nucleic acids, cells expressing chimeric receptors, methods for producing cells, pharmaceutical compositions containing cells as active ingredients, and methods characterized by administering pharmaceutical compositions to subjects for preventing (including preventing recurrence, the same below) or treating diseases. CAR-T cells or TCR-T cells into which the chimeric receptors of the present invention have been introduced can survive in patients for a long time and show high therapeutic efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] [ Figure 1 ] Figure 1 The method used to construct plasmid pEX-A2J2 / TEGPEB is shown.

[0055] [ Figure 2 ] Figure 2 The method used to construct the retroviral plasmid pMEI-5 / ZG-SRG_YYY is shown.

[0056] [ Figure 3 ] Figure 3 The method used to construct the retroviral plasmid pMEI-5 / ZG-SRG_FYY is shown.

[0057] [ Figure 4 ] Figure 4 The method used to construct the retroviral plasmid pMEI-5 / ZG-SRG_YFY is shown.

[0058] [ Figure 5 ] Figure 5 The method used to construct the retroviral plasmid pMEI-5 / ZG-SRG_YYF is shown.

[0059] [ Figure 6 ] Figure 6 The method used to construct the retroviral plasmid pMEI-5 / ZG-SRG_FFY is shown.

[0060] [ Figure 7 ] Figure 7 The method used to construct the retroviral plasmid pMEI-5 / ZG-SRG_YFF is shown.

[0061] [ Figure 8 ] Figure 8 The method used to construct the retroviral plasmid pMEI-5 / ZG-SRG_FYF is shown.

[0062] [ Fig. 9 ] Fig. 9 The method used to construct the retroviral plasmid pMEI-5 / ZG-SRG_FFF is shown.

[0063] [ Fig.10 ] Fig.10 The method used to construct the retroviral plasmid pMEI-5 / SRG_YYY is shown.

[0064] [ Fig.11 ] Fig.11 The method used to construct the retroviral plasmid pMEI-5 / SRG_FFF is shown.

[0065] [ Fig.12 ] Fig.12 The method used to construct the retroviral plasmid pMEI-5 / EL-SRG_FFF is shown.

[0066] [ Fig.13 ] Fig.13 The method used to construct the retroviral plasmid pMEI-5 / FMC28z is shown.

[0067] [ Fig.14 ] Fig.14 The method used to construct the retroviral plasmid pMEI-5 / FMC28z-SRG_FFF is shown.

[0068] [ Fig.15 ] Fig.15 The method used to construct the retroviral plasmid pMEI-5 / ZG-EpoRm is shown.

[0069] [ Fig.16 ] Fig.16 A flow chart showing the generation and expansion of transduced cells in Example 3-(2).

[0070] [ Figure 17-1 ] Figure 17-1 The growth curve of the transduced cells in Example 3-(2) is shown.

[0071] [ Figure 17-2 ] Figure 17-2 The growth curve of the transduced cells in Example 3-(2) is shown.

[0072] [ Fig.18 ] Fig.18The ZsGreen1 positive rate in the transduced cells in Example 3-(2) is shown.

[0073] [ Fig.19 ] Fig.19 The growth curve of the transduced cells in Example 4 is shown.

[0074] [ Fig. 20 ] Fig. 20 The growth curve of the transduced cells in Example 5 is shown.

[0075] [ Fig.21 ] Fig.21 The amplification conditions in Example 6 are shown.

[0076] [ Figure 22-1 ] Figure 22-1 The growth curve of the transduced cells in Example 6 is shown.

[0077] [ Figure 22-2 ] Figure 22-2 The growth curve of the transduced cells in Example 6 is shown.

[0078] [ Figure 23-1 ] Figure 23-1 The ZsGreen1 positive rate in the transduced cells in Example 6 is shown.

[0079] [ Figure 23-2 ] Figure 23-2 The ZsGreen1 positive rate in the transduced cells in Example 6 is shown.

[0080] [ Fig.24 ] Fig.24 A flow chart for producing transduced cells and a flow chart for measuring in vivo bioluminescence imaging in Example 7-(1) are shown.

[0081] [ Fig.25 ] Fig.25 In vivo bioluminescence imaging in Example 7-(1) is shown.

[0082] [ Figure 26-1 ] Figure 26-1 A graph showing quantified luminescence intensity in Example 7-(1) is shown.

[0083] [ Figure 26-2 ] Figure 26-2 A graph showing quantified luminescence intensity in Example 7-(1) is shown.

[0084] [ Fig. 27 ] Fig. 27 The hematocrit value of whole blood in Example 7-(1) is shown.

[0085] [ Fig.28 ] Fig.28A flow chart for producing transduced cells and a flow chart for measuring in vivo bioluminescence imaging in Example 7-(2) are shown.

[0086] [ Fig.29 ] Fig.29 In vivo bioluminescence imaging in Example 7-(2) is shown.

[0087] [ Fig.30 ] Fig.30 A graph showing quantified luminescence intensity in Example 7-(2) is shown.

[0088] [ Fig.31 ] Fig.31 The hematocrit value of whole blood in Example 7-(2) is shown.

[0089] [ Fig.32 ] Fig.32 The CAR and SRG (EpoR) positivity rates in Example 8 are shown.

[0090] [ Fig.33 ] Fig.33 The ratio of CD4 to CD8 in Example 8 is shown.

[0091] [ Figure 34-1 ] Figure 34-1 The ratios of CD4 and CD8 T cell subsets in Example 8 are shown.

[0092] [ Figure 34-2 ] Figure 34-2 The ratios of CD4 and CD8 T cell subsets in Example 8 are shown.

[0093] [ Fig.35 ] Fig.35 The measurement of cytotoxic activity in Example 9-(1) is shown.

[0094] [ Figure 36-1 ] Figure 36-1 The measurement results of IL-2 and IFN-γ production in Example 9-(1) are shown.

[0095] [ Figure 36-2 ] Figure 36-2 The measurement results of IL-2 and IFN-γ production in Example 9-(1) are shown.

[0096] [ Fig.37 ] Fig.37 A flow chart for generating transduced cells and a flow chart for measuring in vivo bioluminescence imaging in Example 10 are shown.

[0097] [ Fig.38 ] Fig.38 In vivo bioluminescence imaging in Example 10 is shown.

[0098] [ Fig.39 ] Fig.39 A graph showing quantified luminescence intensity in Example 10.

[0099] [ Fig.40 ] Fig.40 The Kaplan-Meier curve in Example 10 is shown.

[0100] [ Fig.41 ] Fig.41 The method used to construct the retroviral plasmid pMEI-5 / ZG-SRG_FFF40 is shown.

[0101] [ Fig.42 ] Fig.42 The method used to construct the retroviral plasmid pMEI-5 / SRG_FFF40 is shown.

[0102] [ Fig.43 ] Fig.43 The method used to construct the retroviral plasmid pMEI-5 / EL-SRG_FFF40 is shown.

[0103] [ Fig.44 ] Fig.44 The method used to construct the retroviral plasmid pMEI-5 / FMC28z-SRG_FFF40 is shown.

[0104] [ Fig.45 ] Fig.45 The measurement of cytotoxic activity in Example 12 is shown.

[0105] [ Fig.46 ] Fig.46 The Kaplan-Meier curve in Example 13 is shown.

[0106] Modes for carrying out the invention

[0107] Hereinafter, the present invention will be described in detail.

[0108] (1) Chimeric receptors of the present invention

[0109] The chimeric receptor of the present invention comprises a first polypeptide and a second polypeptide, the first polypeptide comprising (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 a mutation is introduced into the first intracellular region; and the second polypeptide comprising (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.

[0110] The first polypeptide constituting the chimeric receptor of the present invention comprises, from the N-terminus to the C-terminus, (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. The second polypeptide comprises, from the N-terminus to the C-terminus, (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.

[0111] In addition, both polypeptides may have a signal peptide at their N-terminus. The signal peptide may be, for example, a signal peptide originally derived from the extracellular region of any polypeptide included in the chimeric receptor of the present invention as the first or second extracellular region, or may be a signal peptide derived from other secretory proteins or membrane proteins.

[0112] The first extracellular domain and the second extracellular domain may be different from each other or may be the same. In addition, the two extracellular regions may be different regions derived from the same polypeptide. The two extracellular regions may be appropriately selected from the extracellular regions described below. In addition, the first transmembrane region and the second transmembrane region may be different from each other or may be the same. In addition, the two transmembrane regions may be different regions derived from the same polypeptide. The two transmembrane regions may be appropriately selected from the transmembrane regions described below.

[0113] As used herein, a ligand is a molecule that can be recognized by a specific polypeptide (i.e., an "extracellular region" as described below) or that binds to a specific polypeptide, and is not particularly limited. Examples of ligands include nucleic acids, sugar chains, lipids, peptides, and proteins. For example, when the extracellular region is derived from a receptor, the ligand of the receptor can be used as a ligand in the present invention, and when the extracellular region is an antigen-binding domain of an antibody, the antigen can be used as a ligand in the present invention. 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. Examples of RNA include mRNA. Short-chain nucleic acids are defined as nucleic acids of 2-100 bases, and long-chain nucleic acids are defined as nucleic acids of more than 100 bases.

[0114] 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 factors (SCF) and cell growth factors (FGF, EGF, IGF, HGF, PDGF, NGF, TGF) and T cell activation regulatory factors (PD-L1, PD-L2, CD80, CD86, Ceacam-1, galectin-3, galectin-9, FGL-1, CD112, CD155, HVEM, CD40, OX40L, 4-1BBL, GITRL, CD70, etc.).

[0115] Other examples of ligands include agonists and antagonists of 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, avidins, etc.

[0116] As the ligand in the present invention, a ligand is selected that does not interact or does not show significant interaction with the cell on which the chimeric receptor of the present invention is to be expressed and the cell that can coexist with the cell on which the chimeric receptor of the present invention is to be expressed. For example, when the chimeric receptor is to be expressed on T cells, the ligand is preferably a cytokine whose receptor is not highly expressed on T cells, and more preferably erythropoietin.

[0117] "Ligand-binding extracellular region"

[0118] As used herein, the term "(i) a first extracellular region that binds to a ligand" or "(iv) a second extracellular region that binds to a ligand" (hereinafter collectively referred to as "the extracellular region that binds to a ligand") refers to a region containing a protein molecule or a portion thereof that can bind to a ligand. Examples of the extracellular region that binds to a ligand include an extracellular region derived from a receptor and an antigen-binding domain of an antibody. The extracellular region binds to and interacts with a ligand, whereby ligand specificity is conferred to a cell expressing the chimeric receptor of the present invention.

[0119] The extracellular region is also referred to as an “ectodomain.” As used herein, the term “domain” refers to a region within a polypeptide that folds into a specific structure independently of other regions.

[0120] As used herein, "the extracellular region that binds to a ligand" can be selected from those that can bind to the ligand to be used. Examples of the extracellular region that binds to a ligand include an extracellular region derived from a cytokine receptor, an extracellular region derived from a growth factor receptor, an extracellular region derived from a receptor tyrosine kinase, and an extracellular region derived from a T cell activation regulatory factor receptor, as well as mutants thereof having the same functions as these sequences. In the present invention, an extracellular region derived from a cytokine receptor is preferably used. 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 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 receptor tyrosine kinases include, but are not limited to, EPH receptor A4 (EPHA4) and c-kit. Examples of T cell activation regulator receptors 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, and BTLA.

[0121] Another example of the "extracellular region that binds to a ligand" used in the present invention is the antigen binding domain of an antibody. "Antigen binding domain" refers to the antigen binding portion of an antibody that is capable of binding to an antigen. Examples of antigen binding domains include Fab' fragments, Fab fragments, Fv fragments, and single-chain variable fragments (scFv).

[0122] In addition, the "extracellular region that binds to a ligand" of the present invention can be derived from TCR (TCRα, TCRβ, TCRγ, TCRδ), CD4 extracellular domain, CD8α, CD8β, CD11A, CD11B, CD11C, CD18, CD29, CD49A, CD49B, CD49D, CD49E, CD49F, CD61, CD41 and / or CD51. For example, the CD4 extracellular domain can recognize HIV-infected cells.

[0123] As used herein, the "extracellular region that binds to a ligand" is preferably an extracellular region derived from a receptor, more preferably an extracellular region derived from a cytokine receptor, and most preferably an extracellular region derived from an erythropoietin receptor.

[0124] The "(i) first extracellular region that binds to a ligand" and the "(iv) second extracellular region that binds to a ligand" can be independently selected and can 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" can be an extracellular region derived from an erythropoietin receptor, and the other can be another type of extracellular region, or both the "(i) first extracellular region that binds to a ligand" and the "(iv) second extracellular region that binds to a ligand" can be an extracellular region derived from an erythropoietin receptor.

[0125] "Transmembrane region"

[0126] The transmembrane region is also referred to as a "transmembrane domain" and generally refers to a region of a membrane protein embedded in a cell membrane. As used herein, "(ii) a first transmembrane region" and "(v) a second transmembrane region" (hereinafter collectively referred to as "transmembrane regions") may be derived from naturally occurring polypeptides, or may be artificially designed. Transmembrane regions derived from naturally occurring polypeptides may be obtained from membrane-bound or transmembrane proteins. Examples of transmembrane regions derived from naturally occurring polypeptides include interleukin 1 receptor (IL-1RI), IL-1 receptor accessory protein, 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), on The transmembrane region of 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), TGF-β type II receptor (TβRII), erythropoietin-producing hepatocyte receptor A4 (EPHA4), c-kit, T cell receptor α, T cell receptor β chain, CD3 ζ chain, CD28, CD3 ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154 and GITR. The artificially designed transmembrane region is a polypeptide mainly 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 linker or polypeptide linker, for example a linker of 2-10 amino acids in length, may be placed between the first transmembrane region and the first intracellular region described herein. Preferably, a linker sequence comprising a continuous sequence of glycine-serine is used.

[0127] As used herein, the "(ii) first transmembrane region" contained in the first polypeptide is preferably a transmembrane region derived from the IL-2 receptor β chain. The "(v) second transmembrane region" contained in the second polypeptide is preferably a transmembrane region derived from the IL-2 receptor γ chain.

[0128] "(iii) derived from the first intracellular region of the IL-2 receptor β chain"

[0129] When the extracellular region present in the same molecule binds (interacts) with a ligand, the intracellular region contained in the first polypeptide constituting the chimeric receptor of the present invention is capable of transmitting a signal into the cell.

[0130] As used herein, "IL-2 receptor" means the receptor of IL-2. The natural human IL-2 receptor is composed of three cell membrane surface proteins: α chain (also known as CD25, 55kDa, 251 amino acid residues), β chain (also known as CD122, 75kDa, 525 amino acid residues) and γ chain (also known as CD132, 64kDa, 369 amino acid residues). In nature, the complex of these proteins forms a non-covalent bond with the IL-2 molecule to transmit the signal to the cell. Both β and γ chains belong to the type I interleukin receptor family. The receptor protein forms a dimer to transmit the signal to the cell, and usually they move freely on the cell membrane as a monomer (the so-called fluid mosaic model). However, when the ligand approaches, the receptor protein dimerizes to become able to act as a receptor.

[0131] The source of the IL-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.

[0132] The "(iii) first intracellular region derived from the IL-2 receptor β chain" used in the present invention may be the entire intracellular region of the natural IL-2 receptor β chain, or a portion of the intracellular region of the natural IL-2 receptor β chain. An example of the entire intracellular region of the natural IL-2 receptor β chain is the amino acid sequence from amino acid 266 to 551 in the wild-type human IL-2 receptor β chain amino acid sequence (RefSeq NP_000869.1). Examples of partial intracellular regions of native IL-2 receptor β chain include amino acids 266-541, amino acids 266-531, amino acids 266-521, amino acids 266-511, amino acids 266-501, amino acids 266-491, amino acids 266-481, amino acids 266-471, amino acids 266-461, amino acids 266-451, amino acids 266-441, amino acids 266-431, amino acids 266-421, amino acids 266-411, amino acids 266-401, and amino acids 266-391 in the wild-type human IL-2 receptor β chain amino acid sequence (RefSeq NP_000869.1) (SEQ ID NO:73).

[0133] The "(iii) first intracellular region derived from the IL-2 receptor β chain" used in the present invention is preferably derived from the entire intracellular region of the natural IL-2 receptor β chain, and more preferably derived from the amino acid sequence of amino acids 266-551 in the amino acid sequence of the wild-type human IL-2 receptor β chain (RefSeq NP_000869.1).

[0134] In the "(iii) first intracellular region derived from the IL-2 receptor β chain" used in the present invention, mutations are introduced at 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 replacing at least one amino acid in the amino acid sequence of the intracellular region derived from the IL-2 receptor β chain with another amino acid. Use of the intracellular region into which such mutations have been introduced can achieve efficient cell signaling.

[0135] The position in "(iii) the first intracellular region derived from the IL-2 receptor β chain (RefSeq NP_000869.1)" at which the mutation is introduced is selected from (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 in the amino acid sequence of the non-human IL-2 receptor β chain corresponding to the above-mentioned (1) to (3). The positions in the amino acid sequence of the intracellular region of the non-human IL-2 receptor β chain corresponding to the above-mentioned positions in the amino acid sequence of the intracellular region of the human IL-2 receptor β chain can be easily recognized by those skilled in the art.

[0136] In the "(iii) first intracellular region derived from the IL-2 receptor β chain" used in the present invention, the above-mentioned amino acid residue (tyrosine) is substituted with another amino acid. As long as the desired function of achieving efficient cell signaling is obtained, the amino acid residue after the substitution is not particularly limited. In addition, as long as the desired function is obtained, the amino acid residue after the substitution may be a natural amino acid or an artificial amino acid. Except for a few special amino acids, 20 kinds of amino acids are found in nature, which are divided into several groups based on their structures. For example, there is 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. Since amino acid residues belonging to the same group exhibit similar properties, it is expected that such amino acid residues are interchangeable.

[0137] The mutation introduced into "(iii) the first intracellular region derived from the IL-2 receptor β chain" is preferably substitution of a tyrosine residue with another amino acid belonging to the above-mentioned Group G (phenylalanine, tryptophan), and more preferably substitution of a tyrosine residue with a phenylalanine residue.

[0138] Examples of amino acid substitutions include, but are not limited to, (1) substitution of tyrosine at position 381 with phenylalanine (Y381F), (2) substitution of tyrosine at position 384 with phenylalanine (Y384F), and (3) substitution of tyrosine at position 387 with phenylalanine (Y387F) in the amino acid sequence of human IL-2 receptor β chain (RefSeq NP_000869.1) (SEQID NO:73), and amino acid substitutions corresponding to (1) to (3) above in non-human IL-2 receptor β chain amino acid sequences.

[0139] Preferably, "(iii) the first intracellular region derived from the IL-2 receptor β chain" comprises one or more amino acid substitutions selected from the above-mentioned amino acid substitutions (1) to (3), or one or more amino acid substitutions selected from the amino acid sequence of a non-human IL-2 receptor β chain corresponding to the above-mentioned amino acid substitutions (1) to (3).

[0140] More preferably, "(iii) derived from the first intracellular region of the IL-2 receptor β chain" comprises all three amino acid substitutions (1) to (3) above, or all three amino acid substitutions corresponding to the above (1) to (3) in the amino acid sequence of a non-human IL-2 receptor β chain.

[0141] The IL-2 receptor beta chain is sometimes referred to as "IL-2Rβ" or "IL2Rβ."

[0142] The "(iii) first intracellular region derived from the IL-2 receptor β chain" used in the present invention may include an intracellular region derived from another polypeptide in addition to the intracellular region derived from the IL-2 receptor β chain. Examples of such an 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, and an intracellular region derived from a T cell activation regulatory factor receptor, as well as mutants thereof having the same functions as the above receptors. 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 factor receptor (GHR). Examples of growth factor receptors include, but are not limited to, fibroblast growth factor receptors (FGFR1, FGFR2, FGFR3), epidermal growth factor receptor (EGFR), insulin-like 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 receptor tyrosine kinases include, but are not limited to, erythropoietin-producing hepatocyte receptor A4 (EPHA4) and c-kit. Examples of T cell activation regulatory factor receptors include, but are not limited to, CD27, CD28, CD40, CD40L, GITR, OX-40, ICOS and 4-1BB. For example, an "intracellular region derived from another polypeptide" can be added to, for example, the N-terminus or C-terminus of the intracellular region derived from the β chain of the IL-2 receptor. For example, an "intracellular region derived from another polypeptide" can be fused directly or via a linker to the N-terminus or C-terminus of the intracellular region derived from the β chain of the IL-2 receptor. The linker may, for example, be a peptide linker, such as a peptide linker of 2-10 amino acids in length.

[0143] "(vi) derived from the second intracellular domain of the IL-2 receptor gamma chain"

[0144] The "(vi) second intracellular region derived from the IL-2 receptor γ chain" contained in the second polypeptide constituting the chimeric receptor of the present invention may be the entire intracellular region of the natural IL-2 receptor γ chain, or a portion of the intracellular region of the natural IL-2 receptor γ chain. An example of the entire intracellular region of the natural IL-2 receptor γ chain is the amino acid sequence of amino acids 284-369 in the wild-type human IL-2 receptor γ chain amino acid sequence (RefSeq NP_000197.1) (SEQ ID NO: 74). Examples of partial intracellular regions of the natural IL-2 receptor γ chain include the amino acid sequences of amino acids 284-359, amino acids 284-349, amino acids 284-339, amino acids 284-329, and amino acids 284-319 in the wild-type human IL-2 receptor γ chain amino acid sequence (RefSeq NP_000197.1).

[0145] The "(vi) second intracellular region 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 of amino acids 284-369 in the amino acid sequence of the wild-type human IL-2 receptor γ chain (RefSeq NP_000197.1) (SEQ ID NO: 74).

[0146] The IL-2 receptor gamma chain is also referred to as the "cytokine common gamma chain." The IL-2 receptor gamma chain is sometimes referred to as "IL-2Rγ" or "IL2Rγ."

[0147] The "(vi) second intracellular region derived from the IL-2 receptor γ chain" used in the present invention may include an intracellular region derived from another polypeptide in addition to the intracellular region derived from the IL-2 receptor γ chain. Examples of such an 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, and an intracellular region derived from a T cell activation regulatory factor receptor, as well as mutants thereof having the same functions as the above receptors. 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 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 receptor tyrosine kinases include, but are not limited to, erythropoietin-producing hepatocyte receptor A4 (EPHA4) and c-kit. Examples of T cell activation regulatory factor receptors include, but are not limited to, CD27, CD28, CD40, CD40L, GITR, OX-40, ICOS and 4-1BB. For example, an "intracellular region derived from another polypeptide" may be added, for example, to the N-terminus or C-terminus of the intracellular region derived from the γ chain of the IL-2 receptor, and is preferably added to the C-terminus. For example, the "intracellular region derived from another polypeptide" can be fused directly or via a linker to the N-terminus or C-terminus of the intracellular region derived from the IL-2 receptor γ chain. The linker can be, for example, a peptide linker, such as a peptide linker of 2-10 amino acids in length.

[0148] 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 of amino acids 216-277 in the amino acid sequence of wild-type human CD40 (RefSeq NP_001241.1) (SEQ ID NO: 71).

[0149] In a preferred embodiment of the present invention, the first polypeptide is, for example, a polypeptide comprising amino acids 65-601 of the amino acid sequence of SEQ ID NO: 3, wherein one or more tyrosines selected from tyrosines at positions 431, 434 and 437 are substituted with other amino acids. In a preferred embodiment of the present invention, the second polypeptide is, for example, a polypeptide comprising amino acids 64-396 of the amino acid sequence of SEQ ID NO: 1.

[0150] The present invention provides a precursor of a chimeric receptor. The precursor is characterized by comprising a first polypeptide and a second polypeptide, wherein they are connected to each other via a self-cleaving peptide. More specifically, the chimeric receptor precursor of the present invention is characterized by comprising a first polypeptide and a second polypeptide, wherein they are connected to each other via a self-cleaving peptide, the first polypeptide comprising (i) a first extracellular region that binds to a ligand, (ii) a first transmembrane region, and (iii) a first intracellular region derived from an IL-2 receptor β chain, wherein a mutation is introduced at one or more tyrosine residues in the first intracellular region; and the second polypeptide comprising (iv) a second extracellular region that binds to a ligand, (v) a second transmembrane region, and (vi) a second intracellular region derived from an IL-2 receptor γ chain. Here, the arrangement of the first and second polypeptides is not particularly limited, and may be "first polypeptide-self-cleaving peptide-second polypeptide" or "second polypeptide-self-cleaving peptide-first polypeptide". Further, a linker peptide may be added to one or both ends of the self-cleaving peptide.

[0151] As used herein, the term "self-cleaving peptide" means a peptide sequence with a cleavage activity between two amino acid residues that occur in the peptide sequence itself. Self-cleaving peptides are peptides of 18-22 amino acids in length that induce ribosome jumping during protein translation in cells. Self-cleaving peptides are present in various virus families and share a common motif (DxExNPGP). Self-cleaving peptides inhibit the transferase activity of ribosomes and prevent peptide bonds from forming. Therefore, when the self-cleaving peptide is placed between upstream and downstream genes, translation is destroyed, and therefore, the co-expression of two genes becomes possible.

[0152] For example, in 2A peptides and 2A-like peptides, cleavage occurs between glycine residues and proline residues on these peptides. This occurs due to a "ribosome skipping mechanism", in which the normal peptide bond formation between glycine residues and proline residues does not occur during translation without affecting downstream translation. The ribosome skipping mechanism is known in the art and is used to express multiple proteins encoded by a single messenger RNA (mRNA) molecule.

[0153] Examples of the self-cleaving peptide used in the present invention include P2A peptide derived from porcine teschovirus (PTV), T2A derived from Tachypleus punctatus virus (TaV), F2A derived from foot-and-mouth disease virus (FMDV), and E2A derived from equine rhinitis A virus (ERAV).

[0154] (2) Nucleic Acids of the Present Invention

[0155] The present invention provides a nucleic acid encoding a chimeric receptor of the present invention as described in (1) above. That is, the nucleic acid of the present invention encodes a chimeric receptor comprising a first polypeptide and a second polypeptide, the first polypeptide comprising (i) a first extracellular region that binds to a ligand, (ii) a first transmembrane region, and (iii) a first intracellular region derived from an IL-2 receptor β chain, wherein a mutation is introduced at one or more tyrosine residues in the first intracellular region; and the second polypeptide comprising (iv) a second extracellular region that binds to a ligand, (v) a second transmembrane region, and (vi) a second intracellular region derived from an IL-2 receptor γ chain.

[0156] In a preferred embodiment of the present invention, the nucleic acid encoding the first polypeptide is exemplified by a nucleic acid having a nucleotide sequence encoding a polypeptide comprising amino acids 65-601 of the amino acid sequence of SEQ ID NO:3, wherein one or more tyrosines selected from tyrosines at positions 431, 434 and 437 are substituted with other amino acids. In a preferred embodiment of the present invention, the nucleic acid encoding the second polypeptide is exemplified by a nucleic acid having a nucleotide sequence encoding a polypeptide comprising amino acids 64-396 of the amino acid sequence of SEQ ID NO:1.

[0157] The present invention also provides nucleic acids encoding chimeric receptor precursors.

[0158] The nucleic acid of the present invention is capable of expressing the chimeric receptor of the present invention in a cell. Since the cell expressing the chimeric receptor of the present invention acquires the ability to proliferate in a ligand-dependent manner, the cell can proliferate specifically, that is, the proliferation of the cell can be selectively controlled. Therefore, the nucleic acid of the present invention is referred to as a "selective regulatory gene (SRG)".

[0159] 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. Examples of DNA include double-stranded DNA, single-stranded DNA and cDNA. Examples of RNA include mRNA.

[0160] Nucleic acid of the present invention can be connected with the nucleic acid comprising a promoter sequence so as to express in a cell. The example of a promoter includes a promoter that promotes gene or operably connected construct expression through composition, and a promoter that induces gene or operably connected construct expression through the effect of medicines, etc. (e.g., tetracycline or doxorubicin). In order to effectively transcribe, nucleic acid of the present invention also can be connected with the nucleic acid comprising other regulatory elements cooperating with promoter or transcription start site, such as enhancer sequence or terminator sequence.

[0161] In a certain embodiment, the nucleic acids of the invention are codon optimized for expression in a specific host cell.

[0162] (3) Vector of the present invention

[0163] The present invention provides a vector containing the nucleic acid of the present invention as described in (2) above. That is, the vector of the present invention contains a nucleic acid encoding a chimeric receptor, the chimeric receptor comprising a first polypeptide and a second polypeptide, the first polypeptide comprising (i) a first extracellular region that binds to a ligand, (ii) a first transmembrane region, and (iii) a first intracellular region derived from an IL-2 receptor β chain, wherein a mutation is introduced at one or more tyrosine residues in the first intracellular region; and the second polypeptide comprising (iv) a second extracellular region that binds to a ligand, (v) a second transmembrane region, and (vi) a second intracellular region derived from an IL-2 receptor γ chain.

[0164] The vector of the present invention is capable of expressing the first polypeptide and the second polypeptide in desired cells.

[0165] The embodiment of the carrier of the present invention is illustrated by the following: (a) a carrier containing both a nucleic acid encoding a first polypeptide and a nucleic acid encoding a second polypeptide, and (b) a combination of a carrier containing a nucleic acid encoding a first polypeptide and a carrier containing a nucleic acid encoding a second polypeptide. In the above embodiment (a), the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide can be transcribed and translated by different promoters, or can be transcribed and translated by a single promoter by using an internal ribosome entry site (IRES). In addition, the nucleic acid encoding the polypeptide formed by connecting the first polypeptide and the second polypeptide to each other via a self-cleaving peptide can be transcribed and translated by a single promoter. In the above embodiment (b), the two carriers can be the same type of carrier or different types of carriers.

[0166] The vector that can be used in the present invention is operably connected to a suitable control sequence so as to express the nucleic acid of the present invention in a suitable host cell. Examples of control sequences include promoters for transcribing the nucleic acid of the present invention, optional operator sequences for controlling transcription, sequences encoding ribosome binding sites, enhancers, polyadenylation sequences, and sequences controlling transcription and translation termination. Examples of promoters for controlling transcription in mammalian cells include promoters from mammalian sources (PGK promoter, EF1-α promoter, β-globin promoter, etc.), promoters from viral sources (CMV promoter, SV40 promoter, MMLV-LTR promoter, HIV-LTR promoter, etc.) and artificially constructed promoters (CAG promoter, etc.). In addition, the vector may contain various sequences known to those skilled in the art as needed, such as restriction enzyme cleavage sites, marker genes (selection genes) such as drug resistance genes, signal sequences, leader sequences, etc. These different sequences or sites may be appropriately selected and used by those skilled in the art depending on the type of polypeptide to be expressed, the host cell to be used, culture medium, and other conditions.

[0167] 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 cells (eg, a drug resistance gene, a gene encoding a reporter enzyme, or a gene encoding a fluorescent protein) or a therapeutic gene.

[0168] The therapeutic gene used in the present invention is not particularly limited, as long as it plays a beneficial role in the disease desired to be treated or prevented. Examples of therapeutic genes include polypeptides, ribozymes, antisense RNA, and RNA exhibiting RNA interference activity. Since the present invention produces expression of therapeutic genes in response to the occurrence of diseases such as cancer or viral infection, genes that can be used to eliminate cancer cells or virus-infected cells, such as genes that can exert cytotoxicity, can be used. Examples of therapeutic genes include, but are not limited to, genes encoding CAR, genes encoding TCR, genes encoding polypeptides with protease activity, genes encoding polypeptides with nuclease activity, genes encoding cytokines, and genes involved in nucleic acid metabolism.

[0169] Preferably, the vector of the present invention further contains a nucleic acid encoding a CAR or foreign TCR that recognizes target cells in disease treatment.

[0170] 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. CAR is sometimes referred to as a "chimeric antigen receptor," "chimeric receptor," "T body," or "chimeric immune receptor (CIR)."

[0171] "Antigen binding domain" means a part that can bind to an antigen. A part of an antibody with antigen binding ability can be used as an antigen binding domain. Examples of antigen binding domains include Fab' fragments, Fab fragments, and Fv fragments. Single-chain variable fragments (scFv) are preferably used in the present invention. scFv means a single-chain polypeptide derived from an antibody that retains antigen binding ability. An example of scFv is a polypeptide formed by connecting the Fv region of an immunoglobulin heavy chain (H chain) and the Fv region of an immunoglobulin light chain (L chain) via a spacer sequence by recombinant DNA technology. Various methods for producing scFv are known, and examples thereof include U.S. Patent No. 4,694,778; Science, vol.242, pp.423-442 (1988); Nature, vol.334, p.54454 (1989); and Science, vol.242, pp.1038-1041 (1988). The antigen binding domain is not limited to a polypeptide derived from an antibody. Polypeptides derived from proteins other than antibodies can also be used as antigen binding domains. For example, the entire length or a portion of a protein such as APRIL or GM-CSF can be used as an antigen binding domain.

[0172] By "intracellular domain" is meant any oligo- or polypeptide known to function as a domain that transmits signals leading to activation or inhibition of biological processes within a cell.

[0173] The typical CAR structure consists of scFv, a transmembrane domain and an intracellular domain that activates cells. Known examples of transmembrane domains include transmembrane domains derived from TCR complexes CD3ζ, CD28, CD8α, etc. A preferred example of the intracellular domain is the intracellular domain of TCR complex CD3ζ. CAR with this configuration is referred to as the first generation CAR. CAR-expressing T cells (CAR-T cells) directly recognize the surface antigens of tumor cells without considering how the expression of major histocompatibility complex class I on tumor cells is, and the T cells themselves are activated. Therefore, CAR-expressing T cells can effectively kill tumor cells.

[0174] In order to enhance the T cell activation ability of the first generation CAR, a second generation CAR in which the intracellular domain of T cell co-stimulatory molecules is connected has been developed.Preferred examples of T cell co-stimulatory molecules include the intracellular domain of CD28, the intracellular domain of CD137 (4-1BB) and CD134 (OX40) as members of the tumor necrosis factor (TNF) receptor superfamily, the intracellular domain of interleukin receptors and their modified products, and the intracellular domain of glucocorticoid-induced tumor necrosis factor receptor (GITR).As a further improved type, a third generation CAR in which the intracellular domains of these co-stimulatory molecules are connected in series has been developed, and many CAR molecules targeting various tumor antigens have been reported.The nucleic acid construct of the present invention may contain a sequence encoding any CAR as a desired gene sequence.

[0175] As used herein, the term TCR refers to the molecule responsible for T cell antigen recognition function, and is composed of polypeptides such as α chain, β chain, γ chain and δ chain. Wherein, a heterodimer of TCR α chain (TCR α) and TCR β chain (TCR β), or a heterodimer of γ chain and δ chain, and auxiliary molecules such as CD3 complex (including γ, δ, ε and ζ), CD4 or CD8, form TCR.

[0176] In vivo, TCR recognizes peptides (epitopes) presented by target cells (phagocytes, virus-infected cells, cancer cells, etc.) via major histocompatibility complex (MHC), and induces an immune response against target cells. Human MHC is also known as the human histocompatibility leukocyte antigen (HLA) system. HLA is divided into class I and class II; class I includes HLA-A, B, C, E, F, G, H and J, and class II includes HLA-DR, DQ and DP. Like TCR, HLA is formed by a complex of α and β chains and presents epitopes on the cell surface via these chains. HLA class I is present on almost all cells of the body and presents epitopes with a length of about 9 amino acid residues. For example, when virus-infected cells present virus-derived epitopes via HLA, T cells (such as cytotoxic T lymphocytes) with epitope-specific TCR are activated, resulting in an immune response of the body (such as killing virus-infected cells), thereby providing biological defense.

[0177] TCR usually induces an immune response only when it is bound to a complex formed by a specific type of HLA and an antigenic epitope. This restriction is referred to as "HLA restriction". As used herein, the term HLA (or MHC) restriction can be used in conjunction with various peptides, cells, etc. in the case of a TCR, a T cell expressing such a TCR, or a presented antigen-HLA complex. The binding of TCR to an antigenic epitope-HLA complex results in an immune response, and an example of such an immune response is the secretion of cytokines (interferon-γ, TNF-α, IL-2, etc.) by CD8 positive T cells.

[0178] A gene or therapeutic gene that can serve as a marker can be included in the vector in such a manner that it is expressed from a promoter different from the promoter of the nucleic acid encoding the chimeric receptor of the present invention, or from the same promoter as the promoter of the nucleic acid encoding the chimeric receptor of the present invention. In addition, a nucleic acid encoding a fusion polypeptide formed by linking a precursor of the chimeric receptor of the present invention and a polypeptide encoded by a gene that can serve as a marker and / or therapeutic gene via a self-cleaving peptide can be linked to a promoter and carried in a vector.

[0179] The vectors that can be used in the present invention include vectors that are integrated into the host cell genome, vectors that are not integrated into the host cell genome, and free-type vectors that are present in the cytoplasm and replicate autonomously. For example, plasmid vectors, viral vectors, or artificial chromosomes can be used appropriately. Examples of usable viral vectors include retroviral vectors (including tumor retroviral vectors, slow virus 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. The viral vector may preferably be replication-defective so that the virus cannot replicate autonomously in cells infected with the virus. Various viral vectors and methods for producing them are well known to those skilled in the art. Commercially available viral vectors can also be used in the present invention.

[0180] Non-viral vectors can also be used in combination with condensing agents such as liposomes and cationic lipids. In addition, the nucleic acids of the present invention can be introduced into cells by calcium phosphate transfection, DEAE-dextran, electroporation or particle bombardment.

[0181] (4) Cells of the present invention

[0182] The present invention provides a cell expressing the chimeric receptor of the present invention as described in (1) above. That is, the cell of the present invention expresses a chimeric receptor comprising a first polypeptide and a second polypeptide, the first polypeptide comprising (i) a first extracellular region that binds to a ligand, (ii) a first transmembrane region, and (iii) a first intracellular region derived from an IL-2 receptor β chain, wherein a mutation is introduced at one or more tyrosine residues in the first intracellular region; and the second polypeptide comprising (iv) a second extracellular region that binds to a ligand, (v) a second transmembrane region, and (iv) a second intracellular region derived from an IL-2 receptor γ chain.

[0183] The cells of the present invention have the ability to proliferate specifically when stimulated with a ligand. That is, when the cells come into contact with a ligand that binds to a chimeric receptor expressed by the cells in vitro or in vivo, proliferation in the cells begins.

[0184] 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 or dogs can be used. The cells of the present invention are preferably human cells.

[0185] The cell type of the present invention is not particularly limited, and any type of cell can be used.For example, from body fluids, tissues or organs such as blood (peripheral blood, cord blood, etc.) or bone marrow harvest, separation or purification of cells, or by differentiation of the above-mentioned cells or reprogramming the above-mentioned cells to produce pluripotent stem cells (iPS cells) and obtained cells (see, for example, Themeli et al., 2013). Useable cell examples include peripheral blood mononuclear cells (PBMC), immune cells, cord blood mononuclear cells, fibroblasts, adipocyte precursors, hepatocytes, skin keratinocytes, mesenchymal stem cells, adipose stem cells, various cancer cell lines and neural stem cells. Useable cell examples include NK cells and T cells, T cell precursor cells (hematopoietic stem cells, lymphocyte precursor cells, etc.) and cell groups containing the above-mentioned cells. The example of T cell includes CD8 positive T cells, CD4 positive T cells, regulatory T cells, cytotoxic T cells and tumor infiltrating lymphocytes. The cell group containing T cells and T cell precursors includes PBMC. The above-mentioned cells can be obtained from living organisms, can be obtained by amplifying cells obtained from living organisms, or can be established as cell lines. In addition, cells obtained by differentiation from pluripotent stem cells (ES cells, iPS cells, etc.), such as immune cells, can be used as cells of the present invention. The iPS cells produced by T cell clones with the ability to identify and damage specific cells can be differentiated to obtain T cells. The T cells obtained in this way have the same specific cytotoxic activity as the initial T cell clones, and are in a more undifferentiated state. Nucleic acids encoding chimeric receptors of the present invention are introduced into T cells, so that cells that play an excellent therapeutic effect can be easily prepared.

[0186] As used herein, the term "immune cell" refers to the overall cells involved in immune function in vivo, and examples of such cells include hematopoietic stem cells, neutrophils, basophils, macrophages, lymphocytes (B cells and T cells), monocytes, dendritic cells, natural killer (NK) cells and plasma cells. Among them, T cells and NK cells are preferred in the present invention. As used herein, "immune cells" also include "precursor cells of immune cells" that have the ability to differentiate into the above-mentioned immune cells.

[0187] When it is desired to transplant the cells of the present invention or cells differentiated from the cells of the present invention into an organism, it is preferred to introduce the nucleic acid into cells taken from the organism itself or an organism of the same species to be transplanted.

[0188] The cells of the present invention may further express polypeptides (enzymes, antibodies, cytokines, etc.) or nucleic acids (siRNA, etc.) that can be used to treat diseases, preferably CAR or foreign TCR that recognizes target cells.

[0189] As used herein, the term "target cell" refers to a cell that is desired to be reduced or eliminated in a patient, and examples of such cells include tumor cells and cells infected with pathogens. The target cell is preferably a tumor cell, and more preferably a hematopoietic tumor cell or a solid tumor cell.

[0190] The cells of the present invention that further express CAR or foreign TCR that recognizes target cells have the ability to recognize desired target cells and destroy target cells by their cytotoxic activity. Cytotoxic activity can be assessed by known methods. For example, the cytotoxic activity of the cells of the present invention to target cells labeled with radioactive substances, fluorescent substances, etc. can be assessed by measuring the radioactivity or fluorescence intensity from target cells destroyed by the cells of the present invention. Cytotoxic activity can also be detected by measuring the amount of cytokines such as GM-CSF or IFN-γ specifically released from the cells of the present invention or target cells.

[0191] (5) Method for producing cells of the present invention

[0192] The present invention provides a method for producing a cell, the method comprising the step of introducing the nucleic acid of the present invention as described in (2) above into the cell. That is, the method for producing the cell of the present invention comprises introducing into the cell a nucleic acid encoding a chimeric receptor comprising a first polypeptide and a second polypeptide, the first polypeptide comprising (i) a first extracellular region that binds to a ligand, (ii) a first transmembrane region, and (iii) a first intracellular region derived from an IL-2 receptor β chain, wherein a mutation is introduced at one or more tyrosine residues in the first intracellular region; and the second polypeptide comprising (iv) a second extracellular region that binds to a ligand, (v) a second transmembrane region, and (vi) a second intracellular region derived from an IL-2 receptor γ chain.

[0193] This step is performed ex vivo. For example, cells can be generated by transducing cells ex vivo using a viral or non-viral vector containing a nucleic acid of the invention.

[0194] For the cell production method of the present invention, cells derived from mammals, such as human cells, or cells derived from non-human mammals such as monkeys, mice, rats, pigs, cattle or dogs can be used. The cells used in the method of the present invention are not particularly limited, and any of the above cells can be used. The cell can be a cell taken from a living body, a cell obtained by amplifying a cell taken from a living body, or an established cell line. When it is desired to transplant the produced cells or cells differentiated from the produced cells into an organism, it is preferred that the nucleic acid is introduced into a cell taken from the organism itself to be transplanted or an organism of the same type. In addition, the cell can be stimulated before the introduction of the nucleic acid to improve the efficiency of the nucleic acid introduced into the cell, or to enrich a specific type of cell. For example, when PBMCs are cultured in the presence of anti-CD3 antibodies, the proportion of T cells increases. When PBMCs are cultured in the presence of anti-CD3 antibodies and RetroNectin (which is a recombinant fibronectin fragment), a cell population rich in CD8 positive T cells is obtained.

[0195] The nucleic acid encoding the chimeric receptor of the present invention is inserted into a vector, and the vector can be introduced into the cell. As the vector, the vector of the present invention described in (3) above can be used. The method for introducing the vector into the cell is not particularly limited, and an appropriate method can be selected depending on the vector and cell type used.

[0196] In the present invention, non-viral vectors may also be used in combination with adjuvants such as liposomes, cationic lipids or polyethyleneimine. In addition, the nucleic acid of the present invention may be introduced into cells by calcium phosphate transfection, DEAE-dextran, electroporation or particle bombardment.

[0197] Methods for producing various viral vectors and methods for introducing them into cells are also well known to those skilled in the art. For example, for retroviral vectors, appropriate packaging cells are selected depending on the LTR sequence and packaging signal sequence contained in the vector, and the packaging cells can be used to prepare retroviral particles. Examples of packaging cells include PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), GP+E-86 and GP+envAm-12 (U.S. Patent No. 5278056) and Psi-Crip [Proceedings of the National Academy of Sciences, Vol.85, pp.6460-6464 (1988)]. Retroviral particles can also be produced using 293 cells or 293T cells with high transfection efficiency. Retroviral vectors produced based on many types of retroviruses and packaging cells that can be used to package the vectors are widely commercially available from different companies.

[0198] Cells into which the nucleic acid of the present invention has been introduced can be cultured in vitro to increase the number of cells. For cultured cells, a culture medium suitable for cells can be used, and this culture medium can be selected from known or commercially available culture mediums. For example, culture mediums that can be used to culture lymphocytes and other immune cells are known to those skilled in the art, and culture mediums of various compositions are commercially available. There are culture mediums containing serum (e.g., fetal bovine serum; FBS or FCS) of human or animal origin, culture mediums without xenogeneic components, and culture mediums with clear components without unknown components, etc., and such culture mediums can be selected according to purpose. For cell culture, known culture containers (plates, culture dishes, culture bottles, bags, culture tanks, etc.) can be used. Culture conditions can be the same as those used for normal cell culture (e.g., 32-37° C., 5% CO2, etc.). In addition, at appropriate time intervals, cell cultures can be diluted by adding fresh culture medium, or culture medium or culture container can be replaced.

[0199] In the method for producing the cell of the present invention, the proliferation of the cell of the present invention can be promoted by culturing cells in contact with the ligand bound to the chimeric receptor. When a cell group containing the cell of the present invention and other cells is cultured in a culture medium containing a ligand, the cell of the present invention receives a proliferation signal by binding to the ligand and actively proliferates. On the other hand, other cells grow at a normal rate. Therefore, a cell group rich in the cell of the present invention can be obtained. The concentration of the ligand can be appropriately adjusted depending on the type of ligand and cell. For example, in the case of cells expressing a chimeric receptor comprising an extracellular region derived from an erythropoietin receptor, cell proliferation is promoted in a culture medium containing 0.9 IU / mL or more erythropoietin in an erythropoietin concentration-dependent manner. Therefore, the cell of the present invention can be selectively produced. Therefore, the desired highly pure cell group can be obtained without any purification procedure or only with a simple purification procedure. In addition, the cell obtained by the method for producing the cell of the present invention is characterized by having an anti-tumor effect higher than that of the control.

[0200] (6) Pharmaceutical composition of the present invention

[0201] The present invention provides a pharmaceutical composition comprising the cell of the present invention as described in (4) above as an active ingredient. That is, the pharmaceutical composition of the present invention comprises as an active ingredient a cell expressing a chimeric receptor comprising a first polypeptide and a second polypeptide, the first polypeptide comprising (i) a first extracellular region that binds to a ligand, (ii) a first transmembrane region, and (iii) a first intracellular region derived from an IL-2 receptor β chain, wherein a mutation is introduced at one or more tyrosine residues in the first intracellular region; and the second polypeptide comprising (iv) a second extracellular region that binds to a ligand, (v) a second transmembrane region, and (vi) a second intracellular region derived from an IL-2 receptor γ chain.

[0202] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients are well known to those skilled in the art, and examples thereof include phosphate buffered saline (e.g., 0.01M phosphate, 0.138M NaCl, 0.0027M KCl, pH 7.4), aqueous solutions containing inorganic acid salts (e.g., hydrochlorides, hydrobromides, phosphates, sulfates, etc.), saline, ethylene glycol or ethanol solutions, etc., and salts of organic acids (e.g., acetates, propionates, malonates, benzoates, etc.). Auxiliary substances such as wetting agents and emulsifiers and pH buffers may also be used. Pharmaceutically acceptable excipients are described in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991). The pharmaceutical composition may be in any known form suitable for parenteral administration, such as injection or infusion. In addition, formulation additives such as suspending agents, preservatives, stabilizers and / or dispersants, and / or preservatives for extending shelf life during storage may be used.

[0203] (7) Methods for preventing or treating the diseases of the present invention

[0204] The present invention provides a method for preventing or treating a disease, the method comprising administering the cell of the present invention described in (4) above or the pharmaceutical composition of the present invention described in (6) above to a subject. That is, the method for preventing or treating a disease of the present invention comprises administering to a subject a cell expressing a chimeric receptor comprising a first polypeptide and a second polypeptide, the first polypeptide comprising (i) a first extracellular region that binds to a ligand, (ii) a first transmembrane region, and (iii) a first intracellular region derived from an IL-2 receptor β chain, wherein a mutation is introduced at one or more tyrosine residues in the first intracellular region; and the second polypeptide comprising (iv) a second extracellular region that binds to a ligand, (v) a second transmembrane region, and (vi) a second intracellular region derived from an IL-2 receptor γ chain; or a pharmaceutical composition containing the cell as an active ingredient.

[0205] Disease is not particularly limited, as long as the disease is sensitive to cells. Examples of the disease include, but are not limited to, cancer [blood cancer (leukemia, lymphoma, myeloma), solid tumors, etc.], inflammatory diseases / autoimmune diseases (asthma, eczema), hepatitis, and infectious diseases caused by viruses (such as influenza, HIV and others), bacteria and fungi, such as tuberculosis, MRSA, VRE and deep fungal diseases. For example, cells of the present invention are administered for the treatment of a disease, wherein the cells of the present invention have the ability to bind to antigens, i.e., tumor antigens, viral antigens, bacterial antigens, etc., which are possessed by cells that are expected to be reduced or eliminated in the disease. For example, cells of the present invention expressing CARs specific for tumor antigens, or cells of the present invention expressing foreign TCRs that recognize tumor cells, are extremely useful for tumor treatment. In addition, NK cells or cytotoxic T lymphocyte (CTL) clones expressing chimeric receptors of the present invention can also be used to treat diseases on which the cells exert therapeutic effects.

[0206] The cell or pharmaceutical composition can be administered intradermally, subcutaneously, intravenously or in a similar manner, and can be administered systemically or locally to the affected area or the tissue near it. For example, the cell or pharmaceutical composition can be administered locally or subcutaneously. The dosage can be appropriately adjusted depending on the age and weight of the subject to be administered, the method of administration, etc. For this reason, the number of doses and the interval between administration of multiple doses are also considered.

[0207] In the method for preventing or treating the disease of the present invention, a ligand that binds to a chimeric receptor expressed by the cell of the present invention is administered to a subject to which the cell of the present invention has been administered, thereby inducing the in vivo proliferation of the cell of the present invention and / or prolonging the in vivo survival period of the cell of the present invention. The method of administering the ligand is not particularly limited, and an appropriate method is selected in consideration of the distribution of the cell of the present invention in the body, etc. The dosage and administration regimen of the ligand can be appropriately determined in consideration of the cell type and dosage to be combined, the disease type, etc. Example

[0208] Hereinafter, the present invention will be explained in more detail with reference to Examples, but the scope of the present invention is not limited to the Examples.

[0209] Example 1. Preparation of SRG constructs

[0210] (1) pEX-A2J2 / TEG

[0211] Prepare a nucleic acid encoding a seamless fusion polypeptide (T2A-EPOR-IL2Rγ) of the T2A sequence, the polypeptide at positions 2-250 of the amino acid sequence of the human erythropoietin receptor shown in NCBI reference sequence: NP_000112.1 (including the signal sequence and the extracellular domain), and the polypeptide at positions 263-369 of the amino acid sequence of the human IL-2 receptor γ chain (common γ chain) shown in NCBI reference sequence: NP_000197.1 (including the transmembrane domain and the intracellular domain). This is named TEG. The amino acid sequence of the fusion polypeptide is shown in SEQ ID NO: 1. In the amino acid sequence of SEQ ID NO: 1, positions 1-40 correspond to the self-cleaving peptide sequence (Furin-spacer-T2A), positions 41-63 correspond to the signal peptide sequence of the erythropoietin receptor, positions 64-289 correspond to the extracellular domain of the erythropoietin receptor, positions 290-310 correspond to the transmembrane domain of the IL-2 receptor gamma chain, and positions 311-396 correspond to the intracellular domain of the IL-2 receptor gamma chain. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 2. The nucleic acid was synthesized as an artificial gene (T2A and EPOR were codon optimized) and cloned into the pEX-A2J2 vector (manufactured by Eurofins Genomics) to generate the plasmid pEX-A2J2 / TEG.

[0212] (2) pEX-A2J2 / PEB

[0213] A nucleic acid encoding a seamless fusion polypeptide (P2A-EPOR-IL2Rβ) of the P2A sequence, the polypeptide at positions 2-250 of the amino acid sequence of the human erythropoietin receptor as shown in the NCBI reference sequence: NP_000112.1 (including the signal sequence and the extracellular domain), and the polypeptide at positions 241-551 of the amino acid sequence of the human IL-2 receptor β chain as shown in the NCBI reference sequence: NP_000869.1 (including the transmembrane domain and the intracellular domain) was prepared. This was named TEG. The amino acid sequence of the fusion polypeptide is shown in SEQ ID NO:3. In the amino acid sequence of SEQ ID NO:3, positions 1-41 correspond to the self-cleaving peptide sequence (Furin-spacer-P2A), positions 42-64 correspond to the signal peptide sequence of the erythropoietin receptor, positions 65-290 correspond to the extracellular domain of the erythropoietin receptor, positions 291-315 correspond to the transmembrane domain of the IL-2 receptor β chain, and positions 316-601 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. The 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.

[0214] (3) pEX-A2J2 / TEGPEB

[0215] A nucleic acid encoding a seamless fusion polypeptide (T2A-EPOR-IL2Rγ-P2A-EPOR-IL2Rβ) of a fusion polypeptide encoded by TEG (SEQ ID NO: 1) and a fusion polypeptide encoded by PEB (SEQ ID NO: 3) was prepared. This was named TEGPEB. The amino acid sequence of the fusion polypeptide is shown in SEQ ID NO: 5. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 6. Figure 1 As shown, PCR was performed using pEX-A2J2 / TEG, pEX-V_InfRev primer (SEQ ID NO: 7) and pEX-V_InfFwd primer (SEQ ID NO: 8) as templates to prepare a linearized vector for cloning. Next, PCR was performed using pEX-A2J2 / TEG, TSRG1_pEXInfFwd primer (SEQ ID NO: 9) and TSRG1_pEXInfRev primer (SEQ ID NO: 10) as templates to obtain an amplified fragment containing DNA encoding T2A-EPOR-IL2Rγ. Similarly, PCR was performed using pEX-A2J2 / PEB, PSRG2_pEXInfFwd primer (SEQ ID NO: 11) and PSRG2_pEXInfRev primer (SEQ ID NO: 12) as templates to obtain an amplified fragment containing DNA encoding P2A-EPOR-IL2Rβ. These two amplification products were cloned into the linearized vector for cloning described above using In-Fusion HD Cloning Kit (manufactured by TAKARA BIO INC.) to prepare plasmid pEX-A2J2 / TEGPEB.

[0216] (4)pMEI-5 / ZG-SRG_YYY

[0217] A nucleic acid encoding a seamless fusion polypeptide (ZsGreen1-T2A-EPOR-IL2Rγ-P2A-EPOR-IL2Rβ) of ZsGreen1 and a fusion polypeptide (SEQ ID NO:5) encoded by TEGPEB was prepared. This was named ZG-SRG_YYY. The amino acid sequence of the fusion polypeptide is shown in SEQ ID NO:13. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO:14. Figure 2As shown, pMEI-5DNA vector (manufactured by TAKARA BIO INC.) was digested with restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, PCR was performed using pIRES2-ZsGreen1 vector (manufactured by TAKARABIO INC.) as a template, ZGSRG-ZG_M5InfFwd primer (SEQ ID NO: 15) and ZGSRG-ZG_M5InfRev primer (SEQ ID NO: 16) to obtain an amplified fragment containing DNA encoding ZsGreen1. Similarly, PCR was performed using pEX-A2J2 / TEGPEB as a template, ZGSRG-SRG_M5InfFwd primer (SEQ ID NO: 17) and ZGSRG-SRG_M5InfRev primer (SEQ ID NO: 18) to obtain an amplified fragment containing DNA encoding T2A-EPOR-IL2Rγ-P2A-EPOR-IL2Rβ. The two amplification products were cloned into the linearized vector for cloning as described above using the In-Fusion HD cloning kit to prepare the retroviral plasmid pMEI-5 / ZG-SRG_YYY.

[0218] (5)pMEI-5 / ZG-SRG_FYY

[0219] A nucleic acid encoding an amino acid sequence fusion polypeptide in which the tyrosine residue at position 1,058 (corresponding to position 381 in the amino acid sequence of the human IL-2 receptor β chain) in the fusion polypeptide encoded by ZG-SRG_YYY (SEQ ID NO: 13) was replaced with phenylalanine. This was named ZG-SRG_FYY. The amino acid sequence of the fusion polypeptide is shown in SEQ ID NO: 19. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 20. Figure 3As shown, the pMEI-5DNA vector was digested with restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, PCR was performed using pMEI-5 / ZG-SRG_YYY, ZGSRG-ZG_M5InfFwd primer (SEQ ID NO: 15) and FYYmut_Rev primer (SEQ ID NO: 21) as a template to obtain an amplified fragment. Similarly, PCR was performed using pMEI-5 / ZG-SRG_YYY, FYYmut_Fwd primer (SEQID NO: 22) and ZGSRG-SRG_M5InfRev primer (SEQ ID NO: 18) as a template to obtain an amplified fragment. The two amplified products were cloned into the linearized vector for cloning as described above using the In-Fusion HD cloning kit to prepare the retroviral plasmid pMEI-5 / ZG-SRG_FYY.

[0220] (6)pMEI-5 / ZG-SRG_YFY

[0221] A nucleic acid encoding a fusion polypeptide having an amino acid sequence 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) in the fusion polypeptide encoded by ZG-SRG_YYY (SEQ ID NO: 13) was replaced with phenylalanine was prepared. This was named ZG-SRG_YFY. The amino acid sequence of the fusion polypeptide is shown in SEQ ID NO: 23. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 24. Figure 4 As shown, the pMEI-5DNA vector was digested with restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, PCR was performed using pMEI-5 / ZG-SRG_YYY, ZGSRG-ZG_M5InfFwd primer (SEQ ID NO: 15) and YFYmut_Rev primer (SEQ ID NO: 25) as a template to obtain an amplified fragment. Similarly, PCR was performed using pMEI-5 / ZG-SRG_YYY, YFYmut_Fwd primer (SEQ ID NO: 26) and ZGSRG-SRG_M5InfRev primer (SEQ ID NO: 18) as a template to obtain an amplified fragment. The two amplified products were cloned into the linearized vector for cloning as described above using the In-Fusion HD cloning kit to prepare the retroviral plasmid pMEI-5 / ZG-SRG_YFY.

[0222] (7)pMEI-5 / ZG-SRG_YYF

[0223] A nucleic acid encoding a fusion polypeptide having an amino acid sequence in which the tyrosine residue at position 1,064 (corresponding to position 387 in the amino acid sequence of the human IL-2 receptor β chain) in the fusion polypeptide encoded by ZG-SRG_YYY (SEQ ID NO: 13) was replaced with phenylalanine was prepared. This was designated as ZG-SRG_YYF. The amino acid sequence of the fusion polypeptide is shown in SEQ ID NO: 27. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 28. Figure 5 As shown, the pMEI-5DNA vector was digested with restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, PCR was performed using pMEI-5 / ZG-SRG_YYY, ZGSRG-ZG_M5InfFwd primer (SEQ ID NO: 15) and YYFmut_Rev primer (SEQ ID NO: 29) as a template to obtain an amplified fragment. Similarly, PCR was performed using pMEI-5 / ZG-SRG_YYY, YYFmut_Fwd primer (SEQ ID NO: 30) and ZGSRG-SRG_M5InfRev primer (SEQ ID NO: 18) as a template to obtain an amplified fragment. The two amplified products were cloned into the linearized vector for cloning as described above using the In-Fusion HD cloning kit to prepare the retroviral plasmid pMEI-5 / ZG-SRG_YYF.

[0224] (8)pMEI-5 / ZG-SRG_FFY

[0225] A nucleic acid encoding a fusion polypeptide having an amino acid sequence in which the tyrosine residues at positions 1,058 and 1,061 (corresponding to positions 381 and 384 in the amino acid sequence of the human IL-2 receptor β chain) in the fusion polypeptide encoded by ZG-SRG_YYY (SEQ ID NO: 13) were replaced with phenylalanine was prepared. This was designated as ZG-SRG_FFY. The amino acid sequence of the fusion polypeptide is shown in SEQ ID NO: 31. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 32. Figure 6As shown, the pMEI-5DNA vector was digested with restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, PCR was performed using pMEI-5 / ZG-SRG_FYY, ZGSRG-ZG_M5InfFwd primer (SEQ ID NO: 15) and FFYmut_Rev primer (SEQ ID NO: 33) as a template to obtain an amplified fragment. Similarly, PCR was performed using pMEI-5 / ZG-SRG_FYY, FFYmut_Fwd primer (SEQ ID NO: 34) and ZGSRG-SRG_M5InfRev primer (SEQ ID NO: 18) as a template to obtain an amplified fragment. The two amplified products were cloned into the linearized vector for cloning as described above using the In-Fusion HD cloning kit to prepare the retroviral plasmid pMEI-5 / ZG-SRG_FFY.

[0226] (9)pMEI-5 / ZG-SRG_YFF

[0227] A nucleic acid encoding a fusion polypeptide having an amino acid sequence in which the tyrosine residues at positions 1,061 and 1,064 (corresponding to positions 384 and 387 in the amino acid sequence of the human IL-2 receptor β chain) in the fusion polypeptide encoded by ZG-SRG_YYY (SEQ ID NO: 13) were replaced with phenylalanine. This was designated ZG-SRG_YFF. The amino acid sequence of the fusion polypeptide is shown in SEQ ID NO: 35. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 36. Figure 7 As shown, the pMEI-5DNA vector was digested with restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, PCR was performed using pMEI-5 / ZG-SRG_YFY, ZGSRG-ZG_M5InfFwd primer (SEQ ID NO: 15) and YFFmut_Rev primer (SEQ ID NO: 37) as a template to obtain an amplified fragment. Similarly, PCR was performed using pMEI-5 / ZG-SRG_YFY, YFFmut_Fwd primer (SEQ ID NO: 38) and ZGSRG-SRG_M5InfRev primer (SEQ ID NO: 18) as a template to obtain an amplified fragment. The two amplified products were cloned into the linearized vector for cloning as described above using the In-Fusion HD cloning kit to prepare the retroviral plasmid pMEI-5 / ZG-SRG_YFF.

[0228] (10)pMEI-5 / ZG-SRG_FYF

[0229] A nucleic acid encoding a fusion polypeptide having an amino acid sequence in which the tyrosine residues at positions 1,058 and 1,064 (corresponding to positions 381 and 387 in the amino acid sequence of the human IL-2 receptor β chain) in the fusion polypeptide encoded by ZG-SRG_YYY (SEQ ID NO: 13) were replaced with phenylalanine. This was designated ZG-SRG_FYF. The amino acid sequence of the fusion polypeptide is shown in SEQ ID NO: 39. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 40. Figure 8 As shown, the pMEI-5DNA vector was digested with restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, PCR was performed using pMEI-5 / ZG-SRG_FYY, ZGSRG-ZG_M5InfFwd primer (SEQ ID NO: 15) and FYFmut_Rev primer (SEQ ID NO: 41) as a template to obtain an amplified fragment. Similarly, PCR was performed using pMEI-5 / ZG-SRG_FYY, FYFmut_Fwd primer (SEQ ID NO: 42) and ZGSRG-SRG_M5InfRev primer (SEQ ID NO: 18) as a template to obtain an amplified fragment. The two amplified products were cloned into the linearized vector for cloning as described above using the In-Fusion HD cloning kit to prepare the retroviral plasmid pMEI-5 / ZG-SRG_FYF.

[0230] (11)pMEI-5 / ZG-SRG_FFF

[0231] A nucleic acid encoding a fusion polypeptide having an amino acid sequence in which the tyrosine residues at positions 1,058, 1,061 and 1,064 (corresponding to positions 381, 384 and 387 in the amino acid sequence of the human IL-2 receptor β chain) in the fusion polypeptide encoded by ZG-SRG_YYY (SEQ ID NO: 13) were replaced with phenylalanine. This was designated ZG-SRG_FFY. The amino acid sequence of the fusion polypeptide is shown in SEQ ID NO: 43. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 44. Fig. 9As shown, the pMEI-5 DNA vector was digested with restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, PCR was performed using pMEI-5 / ZG-SRG_FYY, ZGSRG-ZG_M5InfFwd primer (SEQ ID NO: 15) and FFFmut_Rev primer (SEQ ID NO: 45) as a template to obtain an amplified fragment. Similarly, PCR was performed using pMEI-5 / ZG-SRG_FYY, FFFmut_Fwd primer (SEQ ID NO: 46) and ZGSRG-SRG_M5InfRev primer (SEQ ID NO: 18) as a template to obtain an amplified fragment. The two amplified products were cloned into the linearized vector for cloning as described above using the In-Fusion HD cloning kit to prepare the retroviral plasmid pMEI-5 / ZG-SRG_FFF.

[0232] (12)pMEI-5 / SRG_YYY

[0233] A nucleic acid encoding a fusion polypeptide having an amino acid sequence in which the amino acid sequence of ZsGreen1 and the subsequent T2A sequence are deleted in the fusion polypeptide encoded by ZG-SRG_YYY (SEQ ID NO: 13) was prepared. This was named SRG_YYY. The amino acid sequence of the fusion polypeptide is shown in SEQ ID NO: 47. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 48. Fig.10 As shown, the pMEI-5 DNA vector was digested with restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, PCR was performed using pMEI-5 / ZG-SRG_YYY as a template, SRG_M5InfFwd primer (SEQ ID NO: 49) and ZGSRG-SRG_M5InfRev primer (SEQ ID NO: 18) to obtain an amplified fragment. The amplified product was cloned into the linearized vector for cloning as described above using the In-Fusion HD cloning kit to prepare the retroviral plasmid pMEI-5 / SRG_YYY.

[0234] (13)pMEI-5 / SRG_FFF

[0235] A nucleic acid encoding a fusion polypeptide having an amino acid sequence in which the amino acid sequence of ZsGreen1 and the subsequent T2A sequence are deleted in the fusion polypeptide encoded by ZG-SRG_FFF (SEQ ID NO:43) was prepared. This was named SRG_FFF. The amino acid sequence of the fusion polypeptide is shown in SEQ ID NO:50. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO:51. Fig.11 As shown, the pMEI-5 DNA vector was digested with restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, PCR was performed using pMEI-5 / ZG-SRG_FFF as a template, SRG_M5InfFwd primer (SEQ ID NO: 49) and ZGSRG-SRG_M5InfRev primer (SEQ ID NO: 18) to obtain an amplified fragment. The amplified product was cloned into the linearized vector for cloning as described above using the In-FusionHD cloning kit to prepare the retroviral plasmid pMEI-5 / SRG_FFF

[0236] (14)pMEI-5 / EL-SRG_FFF

[0237] A nucleic acid encoding a seamless fusion polypeptide (Emerald luciferase-T2A-EPOR-IL2Rγ-P2A-EPOR-IL2Rβ) of Emerald luciferase and the fusion polypeptide encoded by SRG_FFF (SEQ ID NO:50) was prepared. This was named EL-SRG_FFF. The amino acid sequence of the fusion polypeptide is shown in SEQ ID NO:52. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO:53. Fig.12 As shown, the pMEI-5 DNA vector was digested with restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, PCR was performed using pELuc-test vector (manufactured by TOYOBO) as a template, ELSRG-EL_M5InfFwd primer (SEQ ID NO: 54) and ELSRG-EL_M5InfRev primer (SEQ ID NO: 55) to obtain an amplified fragment. Similarly, PCR was performed using pMEI-5 / SRG_FFF as a template, ELSRG-SRG_M5InfFwd primer (SEQ ID NO: 56) and ZGSRG-SRG_M5InfRev primer (SEQ ID NO: 18) to obtain an amplified fragment. The two amplified products were cloned into the linearized vector for cloning as described above using the In-Fusion HD cloning kit to prepare the retroviral plasmid pMEI-5 / EL-SRG_FFF.

[0238] (15)pMEI-5 / FMC28z

[0239] Prepare a nucleic acid encoding a polypeptide at positions 1 to 489 of the amino acid sequence of the FMC63-28Z chimeric antigen receptor shown in GenBank: ADM64594.1 (including a signal sequence, an anti-CD19 scFv, a CD28 extracellular domain, a transmembrane domain, an intracellular domain, and a CD3ζ intracellular domain). This is named FMC28z. The amino acid sequence of the fusion polypeptide is shown in SEQ ID NO: 57. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 58. The nucleic acid is synthesized as an artificial gene (codon optimized) and cloned into the pEX-A2J2 vector to prepare the plasmid pEX-A2J2 / FMC28z. Next, as Fig.13 As shown, pEX-A2J2 / FMC28z was digested with restriction enzymes PmlI and BamHI to prepare insert DNA. The insert DNA was then inserted into a linearized pMEI-5 DNA vector obtained by treating with restriction enzymes PmlI and BamHI to prepare a retroviral plasmid pMEI-5 / FMC28z.

[0240] (16)pMEI-5 / FMC28z-SRG_FFF

[0241] A nucleic acid encoding a seamless fusion of the FMC63-28Z chimeric antigen receptor (SEQ ID NO: 57) and the fusion polypeptide encoded by SRG_FFF (SEQ ID NO: 50) was prepared. This was named FMC28z-SRG_FFF. The amino acid sequence of the fusion polypeptide is shown in SEQ ID NO: 59. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 60. Fig.14 As shown, the pMEI-5DNA vector was digested with restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, PCR was performed using pMEI-5 / FMC63-28z, FMC63SRG-FMC_M5InfFwd primer (SEQ ID NO: 61) and FMC63SRG-FMC_M5InfRev primer (SEQ ID NO: 62) as a template to obtain an amplified fragment. Similarly, PCR was performed using pMEI-5 / SRG_FFF, FMC63SRG-SRG_M5InfFwd primer (SEQ ID NO: 63) and ZGSRG-SRG_M5InfRev primer (SEQID NO: 18) as a template to obtain an amplified fragment. The two amplified products were cloned into the linearized vector for cloning as described above using the In-Fusion HD cloning kit to prepare the retroviral plasmid pMEI-5 / FMC28z-SRG_FFF.

[0242] (17)pEX-A2J2 / EpoRm

[0243] A nucleic acid encoding a truncated mutant of the human erythropoietin receptor was prepared, the mutant consisting of a polypeptide at positions 2 to 438 in the amino acid sequence of the human erythropoietin receptor shown in the NCBI reference sequence: NP_000112.1, as described in Blood, Vol. 135(9): 668-679(2020). This was named EpoRm. The amino acid sequence of the polypeptide is shown in SEQ ID NO:64. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO:65. The nucleic acid was synthesized as an artificial gene (codon optimized) and cloned into the pEX-A2J2 vector to prepare the plasmid pEX-A2J2 / EpoRm.

[0244] (18)pMEI-5 / ZG-EpoRm

[0245] A nucleic acid encoding a seamless fusion polypeptide of ZsGreen1 and a truncated mutant of human erythropoietin receptor (SEQ ID NO: 64) was prepared. This was named ZG-EpoRm. The amino acid sequence of the polypeptide is shown in SEQ ID NO: 66. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 67. Fig.15 As shown, the pMEI-5DNA vector was digested with restriction enzymes PmlI and BamHI to prepare a linearized vector for cloning. Next, PCR was performed using pMEI-5 / ZG-SRG_FFF, ZGSRG-ZG_M5InfFwd primer (SEQ ID NO: 15) and ZGEpoRm-ZG_M5InfRev primer (SEQ ID NO: 68) as a template to obtain an amplified fragment. Similarly, PCR was performed using pEX-A2J2 / EpoRm, ZGEpoRm-EpoRm_M5InfFwd primer (SEQ ID NO: 69) and ZGEpoRm-EpoRm_M5InfRev primer (SEQ ID NO: 70) as a template to obtain an amplified fragment. The two amplified products were cloned into the linearized vector for cloning as described above using the In-Fusion HD cloning kit to prepare the retroviral plasmid pMEI-5 / ZG-EpoRm.

[0246] Example 2. Preparation of retroviral vector solution

[0247] Unless specifically stated otherwise, 293T cell lines and PG13 cell lines were cultured in DMEM / F-12 medium supplemented with 10% fetal bovine serum (FBS) and 100 U / mL penicillin-streptomycin.

[0248] The retroviral vectors corresponding to the various retroviral plasmids described in Example 1 were prepared by the procedure described below. First, transient retroviral vector supernatant was prepared using 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_YFF, pMEI-5 / ZG-SRG_Y 293T cells were transfected with 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) and cultured at 37°C in 5% CO2 for 7 hours. The entire medium was replaced with DMEM / F-12 supplemented with sodium butyrate (STEMCELL Technologies Inc.) at a final concentration of 5 mM, and the cells were cultured in a 37°C, 5% CO2 incubator for 16 hours. The entire medium was replaced with DMEM / F-12, and the cells were cultured at 32°C in 5% CO2 for 24 hours. Then, the cells were centrifuged at 2,000 xg 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 retrovirus purification mini kit (ViraTrap, Biomiga, Inc.).

[0249] Next, the PG13 cell line was used as a packaging cell line to produce retroviral vectors. Specifically, PG13 cells seeded in 6-well plates were exposed to purified virus solutions in the presence of 8 μg / mL polybrene and left to stand overnight in an incubator set at 37°C and 5% CO2. The next day, the entire culture medium was replaced with fresh culture medium, and the cells were left to stand in an incubator at 37°C, 5% CO2 for 6 hours. The transduced cells were stripped and all cells were subcultured in T-75 culture flasks. In order to isolate single cell clones by limiting dilution when the cells reached subconfluence, the cells were cultured in an incubator at 37°C, 5% CO2 for 7-10 days. The cells were stripped from the wells by trypsinization, seeded on 12-well plates, and cultured in an incubator at 37°C, 5% CO2 for 3-4 days. The cells were stripped from the wells by trypsin digestion, and 3 / 4 of the cells were resuspended in freezing medium (CELLBANKER; Nippon Zenyaku Kogyo Co., Ltd.), frozen and stored in a -80°C refrigerator. The remaining 1 / 4 cells were inoculated onto a 12-well plate and cultured in an incubator at 37°C, 5% CO2 for 3-4 days. When the cells reached confluence, the culture medium was completely replaced with DMEM / F-12 supplemented with a final concentration of 5mM sodium butyrate. The plate was allowed to stand in an incubator at 37°C, 5% CO2 for 48 hours. After centrifugation at 2,000xg for 10 minutes, the supernatant was collected and filtered through a 0.45μm filter to remove cell debris. Thus, a retroviral vector solution was obtained. A portion of the retroviral vector solution was used to measure the titer [Retroviral Titer Kit (for Real-time PCR), manufactured by TAKARA BIO INC.], and the remainder was dispensed into low-temperature ceramic tubes, frozen and stored in a -80°C refrigerator until use. The viral supernatant titer of each clone was measured and selected [Retroviral Titer Kit (for Real-time PCR)] to establish a clonal line for stably producing retroviral vectors. For a viral titer of 4.0 x 10 9 The retroviral vectors of stable clones were produced at 100 copies / mL or higher, and the cryopreserved cells were thawed and inoculated into T-75 culture flasks and then expanded into T-225 culture flasks. When the cells reached confluence, the culture medium was replaced with DMEM / F-12 supplemented with 5mM (final concentration) sodium butyrate. The retroviral vector solution was collected using the same procedure as above, distributed into low-temperature ceramic tubes, frozen and stored in a -80°C refrigerator until use.

[0250] Example 3. Comparison of proliferation efficiency of cells introduced with SRG constructs

[0251] Hereinafter, nucleic acids encoding the T2A-EPOR-IL2Rγ-P2A-EPOR-IL2Rβ fusion polypeptide (including retroviruses carrying the nucleic acid) are referred to as prototype SRGs, and nucleic acids encoding fusion polypeptides having mutations in the intracellular region of the IL-2 receptor β chain (FYY, YFY, YYF, FFY, YFF, FYF or FFF) are referred to as mutant SRGs.

[0252] (1) Transduction of SRG construct into T cells

[0253] Peripheral blood from volunteers who provided informed consent was collected into BD Vacutainer CPT mononuclear cell collection tubes and centrifuged at 25°C and 1,500 x g for 15 minutes to separate peripheral blood mononuclear cells (PBMC) and plasma. PBMCs were washed twice with CELLOTION (Nippon 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 stored at -80°C until use. Plasma was heat-inactivated in a water bath set at 56°C for 30 minutes and then stored at -80°C until use.

[0254] To activate PBMCs, 5 μg / mL CD3 monoclonal antibody (OKT3) (Thermo Fisher Scientific) and 20 μg / mL RetroNectin (TAKARA BIO INC.) diluted with ACD-A solution (Terumo) were added to a 6-well plate without surface treatment at 1 mL / well, and the plate was placed in an incubator at 37°C, 5% CO2 for 3 hours and at 4°C overnight to prepare an OKT3 / RetroNectin-coated plate. PBMCs were thawed and plated at 2.0 x 10 5 10 cells / mL were suspended in a culture medium [GT-T551 medium (TAKARA BIO INC.) supplemented with 175 IU / mL IL-2 and 0.1% heat-inactivated autologous plasma] and washed three times with DMEM / F-12 medium (without FBS and penicillin-streptomycin). 6.5 mL / well of the cell suspension was added to the OKT3 / RetroNectin-coated plate. 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 PBMC.

[0255] For transduction into T cells, a RetroNectin-conjugated virus transduction method was used [J Biochem. 2001 Sep; 130 (3): 331-4]. In a 6-well plate with an untreated surface, 2 mL / well of a 20 μg / mL RetroNectin solution diluted with PBS was added, and the plate was placed at 4°C. The RetroNectin solution was removed, and 2 mL / well of a blocking solution [phosphate buffered saline (PBS) containing 2% bovine serum albumin (BSA)] was added. The plate was allowed to stand at room temperature for 30 minutes for blocking treatment. The blocking solution was removed, and 2 mL / well of PBS was added to wash the plate once. Then, the PBS was removed. A retroviral vector solution carrying the prototype SRG or the mutant SRG prepared in Example 2 (diluted to 4.0 x 10 9 10 copies / mL) was added to the RetroNectin-coated plate at 4 mL / well. The retrovirus-containing plate was centrifuged at 1,960 x g for 2 hours at 32°C to promote the adsorption of viral particles to RetroNectin. The viral solution was removed from the plate and washed with 2 mL of PBS supplemented with 1% BSA. OKT3 / RetroNectin-activated T cells (diluted to 4.0 x 10 in GT-T551 medium supplemented with 175 IU / mL IL-2 and 0.1% heat-inactivated autologous plasma) were added to the RetroNectin-coated plate at 4 mL / well. The retrovirus-containing plate was centrifuged at 1,960 x g for 2 hours at 32°C to promote the adsorption of viral particles to RetroNectin. The viral solution was removed from the plate and washed with 2 mL of PBS supplemented with 1% BSA. 5 Cells / mL) were added to the virus adsorption plate at 4 mL / well and centrifuged at 1,960 x g for 10 minutes at 32°C. The plate was placed in an incubator at 37°C, 5% CO2 overnight for the first transduction. The transduced T cells were collected the next day and washed with the above medium. After removing the supernatant, the cells were resuspended in 4 mL of medium and added to the virus adsorption plate prepared in the same manner as above. The plate was placed in an incubator set at 37°C and 5% CO2 overnight for the second transduction.

[0256] (2) Comparison of proliferation efficiency of cells transduced with SRG constructs

[0257] like Fig.16 As shown, the transduced T cells were collected the next day and washed twice with a basal medium (GT-T551 medium supplemented with 0.1% heat-inactivated autologous plasma). The transduced cells were resuspended in 20 mL of a 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 and 9.0 IU / mL EPO were added. Figure 17-1 and Figure 17-2Growth curves of cells transfected with prototype SRG and various mutant SRG (ZsGreen1 positive cells) are shown. FFF mutant SRG transduced cells showed EPO-dependent proliferation over a 3-week period after transduction. Fig.18 The ZsGreen1 positive rate is shown. In all transduced cell groups, the addition of EPO induced selective proliferation of transduced cells, and at the end point, a cell population consisting almost entirely of transduced cells was obtained. FFF mutant SRG-transduced cells showed ZsGreen1 positive cell proliferation and EPO concentration-dependent proliferation over a 3-week period after transduction.

[0258] Example 4. Optimization of EPO addition

[0259] T cells stimulated with OKT3 / RetroNectin were transduced with FFF mutant SRG by RetroNectin conjugated virus infection method. T cells that completed the second transduction were collected on the second day (Day 2) and washed twice with basal medium. The 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 and 0, 0.3, 0.9, 3.0, 9.0 or 15 IU / mL EPO were added. Fig.19 The growth curve of transduced cells (ZsGreen1 positive cells) is shown. After sufficient stirring, 100 μL of culture medium is collected, washed twice with CELLOTION, and resuspended in 100 μL PBS. The cell suspension is mixed with 5 μL of a staining solution prepared by diluting 1 mM ethidium homodimer III solution 50 times with PBS, and incubated at room temperature for 15 minutes under light shielding to stain dead cells. Then, 10 μL of reaction solution is transferred to a Countess cell counting chamber slide (Thermo Fisher Scientific), and Countess II FL (Thermo Fisher Scientific) is used to count the number of ZsGreen1 positive and dead cells. ZsGreen1 positive cells proliferate in an EPO concentration-dependent manner within 3 weeks after transduction.

[0260] Example 5. Comparison of the proliferation of transduced cells depending on the amount of gene transfer vector

[0261] T cells stimulated with OKT3 / RetroNectin were transduced with FFF mutant SRG by RetroNectin conjugated virus infection method. The virus concentration at the time of transduction was 2.5x10 3 , 5.0x103 , 1.0x10 4 , 2.0x10 4 and 3.0x10 4 The next day (Day 2), the T cells that completed the second transduction were collected 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 and 0.9 IU / mL EPO were added. Fig. 20 The growth curve of transduced cells (ZsGreen1 positive cells) is shown. After sufficient stirring, 100 μL of culture medium is collected, washed twice with CELLOTION, and resuspended in 100 μL PBS. The cell suspension is mixed with a staining solution (5 μL) prepared by diluting 1 mM ethidium homodimer III solution 50 times with PBS, and incubated at room temperature for 15 minutes under light shielding to stain dead cells. Then, 10 μL of reaction mixture is transferred to a Countess cell counting chamber slide, and CountessII FL is used to count the number of ZsGreen1 positive and dead cells. The proliferation of ZsGreen1 positive cells in a 3-week period after transduction is related to the amount of vector used for transduction.

[0262] Example 6. Proliferation comparison with prior art

[0263] T cells stimulated with OKT3 / RetroNectin were transduced with YYY prototype SRG, FFF mutant SRG, or EpoRm by RetroNectin conjugated virus infection method. T cells that completed the second transduction were collected the next day (Day 2) and washed twice with basal medium. Fig.21As shown, in the culture system containing only IL-2, the transduced cells were resuspended in 30 mL of basal medium supplemented with IL-2 at a final concentration of 175 IU / mL and allowed to stand 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. In the culture system containing a combination of IL-2 and EPO, the transduced cells were resuspended in 30 mL of basal medium supplemented with IL-2 at a final concentration of 175 IU / mL and EPO at a final concentration of 9.0 IU / mL and allowed to stand 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 and EPO at a final concentration of 9.0 IU / mL was added. In the culture system containing only EPO, the transduced cells were resuspended in 20 mL of basal medium supplemented with EPO at a final concentration of 9.0 IU / mL and left to stand 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 EPO at a final concentration of 9.0 IU / mL was added. In addition, as a control, non-transduced T cells were cultured in each of the above culture systems. Figure 22-1 and Figure 22-2 The growth curve of transduced cells (ZsGreen1 positive cells) is shown. After thorough stirring, 100 μL of culture medium was collected, washed twice with CELLOTION, and resuspended in 100 μL PBS. The cell suspension was mixed with 5 μL of a staining solution prepared by diluting 1 mM ethidium homodimer III solution 50 times with PBS, and incubated at room temperature for 15 minutes under light shielding to stain dead cells. Then, 10 μL of the reaction mixture was transferred to a Countess cell counting chamber slide, and the number of ZsGreen1 positive and dead cells was counted using Countess II FL. ZsGreen1 positive cells proliferated in an EPO concentration-dependent manner over a 3-week period after transduction. The ZsGreen1 positive rate measurement results using a flow cytometer are shown in Figure 23-1 and Figure 23-2 In all transduced cell groups, the addition of EPO induced the selective proliferation of transduced cells and at the end point, a cell population consisting almost entirely of transduced cells was obtained. FFF mutant SRG-transduced cells showed proliferation and survival of ZsGreen1-positive cells and EPO concentration-dependent proliferation over a 3-week period after transduction.

[0264] Example 7. EPO-dependent in vivo proliferation of transduced cells infused into immunodeficient mice

[0265] (1) Comparison of in vivo proliferation of cells transduced with SRG constructs at different doses of EPO

[0266] like Fig.24 As shown, T cells stimulated with OKT3 / RetroNectin were transduced with FFF mutant SRG (retroviral vector prepared using pMEI-5 / EL-SRG_FFF) loaded with Emerald luciferase by RetroNectin conjugated virus infection method. T cells that had completed the second transduction were collected on the second day (Day 2) and washed twice with basal medium. The transduced cells were resuspended in 10 mL of basal medium supplemented with 9.0 IU / mL EPO and allowed to stand in an incubator set to 37°C and 5% CO2 (Day 2). On days 4, 7, 9, and 11, an equal volume of basal medium was added, and 9.0 IU / mL EPO was added. On day 14, cells were harvested and washed with CELLOTION. Then, dead cells were removed by density gradient centrifugation using human lymphocyte separation solution (Lympholyte-H; Cedarlane Laboratories Ltd.). The target cell layer was collected and washed twice with CELLOTION. Then, the cells were mixed with a mixture of cell cryopreservation medium CP-1, HSA (Albuminar) and RPMI, and cryopreserved at -80°C until use.

[0267] Immunodeficient NOG tumor-bearing mice were infused with 1x10 6 T cells loaded with SRG-FFF (day 0). 0, 30, 60, 90, 120 and 150 IU / injection of EPO were intraperitoneally administered on days 0, 3, 5, 7, 10, 11 and 14. In vivo bioluminescence imaging of luciferase activity over time after transduced T cell infusion is shown in Fig.25 , and a graph quantifying the luminescence intensity is shown in Figure 26-1 and Figure 26-2 As shown. It was shown that the infused SRG-loaded T cells proliferated in an EPO dose-dependent manner. Fig. 27 As shown, hematocrit values ​​of whole blood collected on day 17 showed an EPO dose-dependent increase.

[0268] (2) Comparison of in vivo proliferation of cells introduced with SRG constructs depending on the time period of EPO administration

[0269] like Fig.28 As shown, immunodeficient NOG tumor-bearing mice were infused with 1x10 6T cells loaded with SRG-FFF (day 0). 90 IU / injection of EPO was given intraperitoneally 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). In vivo bioluminescence imaging of luciferase activity over time after infusion of transduced T cells is shown in Fig.29 , and a graph quantifying the luminescence intensity is shown in Fig.30 As shown. In all conditions, the in vivo proliferation of infused SRG-loaded T cells was EPO dose-dependent. The “twice a week for three weeks” group showed sustained proliferation and survival of the infused cells. Fig.31 As shown, whole blood hematocrit values ​​collected on day 26 showed an EPO-dependent increase.

[0270] Example 8. Measurement of the immunophenotype of transduced T cells

[0271] (1) Transduction of SRG-carrying CAR construct into T cells

[0272] T cells stimulated with OKT3 / RetroNectin were transduced with CAR (retroviral vector prepared using pMEI-5 / FMC28z) or CAR carrying SRG-FFF (retroviral vector prepared using pMEI-5 / FMC28z-SRG_FFF) by RetroNectin conjugated virus infection method. T cells that had completed the second transduction were collected the next day (Day 2) and washed twice with basal medium. Non-transduced 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 allowed to stand in an incubator set to 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. The cells transduced with SRG-carrying CAR were resuspended in 10 mL of basal medium supplemented with EPO at a final concentration of 9.0 IU / mL and left to stand 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, the cells were harvested and washed with CELLOTION. Then, dead cells were removed by density gradient centrifugation using human lymphocyte separation solution (Lympholyte-H). The target cell layer was collected and washed twice with CELLOTION. Then, the cells were mixed with a mixture of cell cryopreservation medium CP-1, HSA (Albuminar) and RPMI and stored at -80 ° C until use.

[0273] (2) Immunophenotype of transduced T cells

[0274] Cryopreserved transduced T cells were thawed, stained with monoclonal antibodies specific for CAR (Cytoart, Inc.) and SRG (EpoR) (R&D Systems, Inc.), and stained with monoclonal antibodies specific for CD45RA and CCR7 on CD4-positive or CD8-positive cells (BioLegend, Inc.), and subjected to flow cytometry analysis. Fig.32 The positive rates of CAR and SRG (EpoR) are shown. Fig.33 The ratio of CD4 to CD8 is shown (CAR-T cells are CAR-positive cells, while SRG-loaded CAR-T cells are a population of cells positive for both CAR and SRG). Figure 34-1 and Figure 34-2 The ratio of CD4 and CD8 T cell subsets is shown (CAR-T cells are CAR-positive cells, while SRG-loaded CAR-T cells are a population of cells that are positive for both CAR and SRG). Compared with CAR-T cells, SRG-loaded CAR-T cells maintain CD45RA-positive CCR7-positive T cell subsets.

[0275] Example 9. In vitro cytotoxicity of SRG-loaded CAR-T cells

[0276] (1) Measurement of cytotoxic activity

[0277] The transduced T cells prepared and cryopreserved in Example 8 were thawed and mixed with K562 or K562-CD19 cells at an effector: target ratio of 10:1, 5:1 or 1:1 in a V-bottom 96-well microplate and co-cultured for 18 hours in an incubator at 37°C and 5% CO2. The microplate was centrifuged at 250xg for 10 minutes at room temperature. After the supernatant was dispensed into a flat-bottom 96-well microplate at 100 μL / well, the cytotoxicity was measured using an LDH cytotoxicity detection kit (TAKARA BIO INC.). Fig.35 As shown, although the cytotoxicity of SRG-loaded CAR-T cells was slightly weaker than that of CAR-T cells, SRG-loaded CAR-T cells exhibited cytotoxicity against CD19-positive cells.

[0278] (2) CAR-transduced T cells produce IL-2 and IFN-γ

[0279] The transduced cells prepared and cryopreserved in Example 8 were thawed, mixed with K562 or K562-CD19 cells at a 5:1 effector: target ratio in CTL-Test PLUS medium (Cellular Technology Limited), and transferred to a V-bottom 96-well microplate. The cells were then co-cultured in an incubator at 37°C, 5% CO2 for 18 hours. After centrifugation of the microplate at 250xg for 10 minutes at room temperature, the supernatant was separated and used to measure the production of IL-2 and IFN-γ using the Human ProQuantum Immunoassay Kit (Invitrogen). Figure 36-1 and Figure 36-2 As shown, it was shown that SRG-loaded CAR-T cells activated in response to CD19-positive cells and produced cytokines to the same extent as CAR-T cells.

[0280] Example 10. In vivo anti-tumor effect of SRG-loaded CAR-T cells in tumor-bearing mice

[0281] like Fig.37 As shown, immunodeficient NOG mice were inoculated via tail vein with 1x10 6 NALM-6 cells, a human CD19-positive acute lymphoblastic leukemia cell line, were transduced with the Emerald luciferase gene, and 14 days later, 3x10 6 or 5x10 6 CAR-transduced cells or SRG-loaded CAR-T cells prepared in Example 8 (n=10 each). For mice, 90 IU / injection of EPO was intraperitoneally administered (twice a week for 3 weeks), or no EPO was administered. In vivo bioluminescence imaging of luciferase activity of tumor cells over time after infusion of transduced T cells is shown in Fig.38 , a graph quantifying the luminescence intensity is shown in Fig.39 , and the Kaplan-Meier curve is shown in Fig.40 . SRG-loaded CAR-T cells showed higher anti-tumor activity than CAR-T cells. In addition, the group that received SRG-loaded CAR-T cell infusion in combination with EPO showed stronger anti-tumor activity and better overall survival rate than the other groups.

[0282] Example 11. Preparation of SRG-FFF40 construct

[0283] like Fig.41As shown in FIG. 1 , a retroviral plasmid pMEI-5 / ZG-SRG_FFF40 was prepared so that a nucleotide sequence (SEQ ID NO: 72) encoding a polypeptide at positions 216 to 277 (including the intracellular domain) of the human CD40 amino acid sequence shown in NCBI reference sequence: NP_001241.1 was fused downstream of IL-2Rγ. Fig.42 As shown, the retroviral plasmid pMEI-5 / SRG_FFF40 without ZsGreen was prepared. Fig.43 As shown, the retroviral plasmid pMEI-5 / EL-SRG_FFF40, a version containing Emerald luciferase, was prepared. Fig.44 As shown, a retroviral plasmid containing FMC63-28Z chimeric antigen receptor (SEQ ID NO: 57) was prepared

[0284] pMEI-5 / FMC28z-SRG_FFF40.

[0285] Example 12. In vitro cytotoxicity of CAR-T cells loaded with SRG-FFF40

[0286] The cytotoxic activity of SRG-FFF40-loaded CAR-T cells was measured using the same procedure as described in Example 9-(1). Fig.45 As shown, the cytotoxic activity of CAR-T cells loaded with SRG-FFF40 was stronger than that of CAR-T cells loaded with SRG-FFF and comparable to that of CAR-T cells.

[0287] Example 13. In vivo anti-tumor effect of SRG-FFF40-loaded CAR-T cells in tumor-bearing mice

[0288] The in vivo antitumor effect of SRG-FFF40-loaded CAR-T cells on tumor-bearing mice was measured using the same procedure as in Example 10. The number of cells infused was 5x10 6 cells (n=15 each). Kaplan-Meier curves are shown in Fig.46 . CAR-T cells loaded with SRG-FFF40 showed higher in vivo antitumor activity than CAR-T cells. In addition, the group that received SRG-FFF40-loaded CAR-T cell infusion in combination with EPO administration showed stronger antitumor activity and better overall survival than the other groups.

[0289] Industrial Applicability

[0290] The present invention provides a chimeric receptor comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises (i) a first extracellular region that binds to a ligand, (ii) a first transmembrane region, and (iii) a first intracellular region derived from an IL-2 receptor β chain, wherein a mutation is introduced at one or more tyrosine residues in the first intracellular region; and the second polypeptide comprises (iv) a second extracellular region that binds to a ligand, (v) a second transmembrane region, and (vi) a second intracellular region derived from an IL-2 receptor γ chain. Also provided are nucleic acids encoding chimeric receptors, vectors containing nucleic acids, cells expressing chimeric receptors, methods for producing cells, pharmaceutical compositions comprising cells as active ingredients, and methods for preventing or treating diseases, the methods comprising administering cells or pharmaceutical compositions to a subject. The methods of the present invention are particularly useful for medical applications.

[0291] Sequence Listing Free Text

[0292] SEQ ID NO: 1: TEG amino acid sequence

[0293] SEQ ID NO:2: TEG nucleic acid sequence

[0294] SEQ ID NO:3: PEB amino acid sequence

[0295] SEQ ID NO:4: PEB nucleic acid sequence

[0296] SEQ ID NO:5: TEGPEB amino acid sequence

[0297] SEQ ID NO:6: TEGPEB nucleic acid sequence

[0298] SEQ ID NO:7: pEX-V_InfRev Primer

[0299] SEQ ID NO:8: pEX-V_InfFwd Primer

[0300] SEQ ID NO:9: TSRG1_pEXInfFwd Primer

[0301] SEQ ID NO: 10: TSRG1_pEXInfRev Primer

[0302] SEQ ID NO: 11: PSRG2_pEXInfFwd Primer

[0303] SEQ ID NO: 12: PSRG2_pEXInfRev Primer

[0304] SEQ ID NO: 13: ZG-SRG_YYY amino acid sequence

[0305] SEQ ID NO: 14: ZG-SRG_YYY nucleic acid sequence

[0306] SEQ ID NO: 15: ZGSRG-ZG_M5InfFwd Primer

[0307] SEQ ID NO: 16: ZGSRG-ZG_M5InfRev Primer

[0308] SEQ ID NO: 17: ZGSRG-SRG_M5InfFwd Primer

[0309] SEQ ID NO: 18: ZGSRG-SRG_M5InfRev Primer

[0310] SEQ ID NO: 19: ZG-SRG_FYY amino acid sequence

[0311] SEQ ID NO:20: ZG-SRG_FYY nucleic acid sequence

[0312] SEQ ID NO:21: FYYmut_Rev Primer

[0313] SEQ ID NO:22: FYYmut_Fwd Primer

[0314] SEQ ID NO:23: ZG-SRG_YFY amino acid sequence

[0315] SEQ ID NO:24: ZG-SRG_YFY nucleic acid sequence

[0316] SEQ ID NO:25: YFYmut_Rev Primer

[0317] SEQ ID NO:26: YFYmut_Fwd Primer

[0318] SEQ ID NO:27: ZG-SRG_YYF amino acid sequence

[0319] SEQ ID NO:28: ZG-SRG_YYF nucleic acid sequence

[0320] SEQ ID NO:29: YYFmut_Rev Primer

[0321] SEQ ID NO:30: YYFmut_Fwd Primer

[0322] SEQ ID NO:31: ZG-SRG_FFY amino acid sequence

[0323] SEQ ID NO:32: ZG-SRG_FFY nucleic acid sequence

[0324] SEQ ID NO:33: FFYmut_Rev Primer

[0325] SEQ ID NO:34: FFYmut_Fwd Primer

[0326] SEQ ID NO:35: ZG-SRG_YFF amino acid sequence

[0327] SEQ ID NO:36: ZG-SRG_YFF nucleic acid sequence

[0328] SEQ ID NO:37: YFFmut_Rev Primer

[0329] SEQ ID NO:38: YFFmut_Fwd Primer

[0330] SEQ ID NO:39: ZG-SRG_FYF amino acid sequence

[0331] SEQ ID NO:40: ZG-SRG_FYF nucleic acid sequence

[0332] SEQ ID NO:41: FYFmut_Rev Primer

[0333] SEQ ID NO:42: FYFmut_Fwd Primer

[0334] SEQ ID NO:43: ZG-SRG_FFF amino acid sequence

[0335] SEQ ID NO:44: ZG-SRG_FFF nucleic acid sequence

[0336] SEQ ID NO:45: FFFmut_Rev Primer

[0337] SEQ ID NO:46: FFFmut_Fwd Primer

[0338] SEQ ID NO:47: SRG_YYY amino acid sequence

[0339] SEQ ID NO:48: SRG_YYY nucleic acid sequence

[0340] SEQ ID NO:49: SRG_M5InfFwd Primer

[0341] SEQ ID NO:50: SRG_FFF amino acid sequence

[0342] SEQ ID NO:51: SRG_FFF nucleic acid sequence

[0343] SEQ ID NO:52: EL-SRG_FFF amino acid sequence

[0344] SEQ ID NO:53: EL-SRG_FFF nucleic acid sequence

[0345] SEQ ID NO:54: ELSRG-EL_M5InfFwd Primer

[0346] SEQ ID NO:55: ELSRG-EL_M5InfRev Primer

[0347] SEQ ID NO:56: ELSRG-SRG_M5InfFwd Primer

[0348] SEQ ID NO:57: FMC28z amino acid sequence

[0349] SEQ ID NO:58: FMC28z nucleic acid sequence

[0350] SEQ ID NO:59: FMC28z-SRG_FFF amino acid sequence

[0351] SEQ ID NO:60: FMC28z-SRG_FFF nucleic acid sequence

[0352] SEQ ID NO:61: FMC63SRG-FMC_M5InfFwd Primer

[0353] SEQ ID NO:62: FMC63SRG-FMC_M5InfRev Primers

[0354] SEQ ID NO:63: FMC63SRG-SRG_M5InfFwd Primer

[0355] SEQ ID NO:64: EpoRm amino acid sequence

[0356] SEQ ID NO:65: EpoRm nucleic acid sequence

[0357] SEQ ID NO:66: ZG-EpoRm amino acid sequence

[0358] SEQ ID NO:67: ZG-EpoRm nucleic acid sequence

[0359] SEQ ID NO:68: ZGEpoRm-ZG_M5InfRev Primer

[0360] SEQ ID NO:69: ZGEpoRm-EpoRm_M5InfFwd Primer

[0361] SEQ ID NO:70: ZGEpoRm-EpoRm_M5InfRev Primer

[0362] SEQ ID NO:71: Partial amino acid sequence of CD40

[0363] SEQ ID NO:72: CD40 partial nucleic acid sequence

[0364] SEQ ID NO:73: Wild-type human IL-2 receptor subunit β precursor amino acid sequence

[0365] SEQ ID NO:73: Wild-type human IL-2 receptor subunit γ precursor amino acid sequence

Claims

1. A chimeric receptor comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises: (i) a first extracellular region that binds to a ligand, (ii) the first transmembrane region, and (iii) derived from a first intracellular region of the IL-2 receptor β chain, wherein a mutation is introduced at one or more tyrosine residues in said first intracellular region; and The second polypeptide comprises: (iv) a second extracellular domain that binds to a ligand, (v) a second transmembrane region, and (vi) derived from the second intracellular domain of the IL-2 receptor gamma chain.

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

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

4. The chimeric receptor according to any one of claims 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 claims 1 to 4, wherein the first extracellular region that binds to the ligand of (i) and / or the second extracellular region that binds to the ligand of (iv) is an extracellular region derived from an erythropoietin receptor.

6. The chimeric receptor according to any one of claims 1 to 5, wherein the first transmembrane region of (ii) is a transmembrane region derived from the IL-2 receptor β chain, and the second transmembrane region of (v) is a transmembrane region derived from the IL-2 receptor γ chain.

7. The chimeric receptor of any one of claims 1-6, 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 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 1-7. The cell according to claim 10 , further expressing a chimeric antigen receptor or a foreign T cell receptor that recognizes the target cell.

12. A pharmaceutical composition comprising the cell according to claim 10 or 11 as an active ingredient.

13. A method for preventing or treating a disease, the method comprising administering the pharmaceutical composition according to claim 12 to a subject.

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