Method for producing immune cells

Abstract

Disclosed are a means capable of stably producing TCR-T cells and CAR-T / CAR-NK cells, and a means for treating diseases such as tumors and autoimmune diseases by applying said means. One aspect of the method for producing TCR / CAR / CAAR-expressing immune cells according to the present invention is a method for producing TCR / CAR / CAAR-expressing immune cells by introducing an exogenous receptor gene into pluripotent blood progenitor cells and then inducing differentiation into immune cells. Another aspect of the method for producing TCR / CAR / CAAR-expressing immune cells according to the present invention is a method for producing TCR / CAR / CAAR-expressing immune cells by inducing differentiation of pluripotent blood progenitor cells into immune cells and then introducing an exogenous receptor gene. As the pluripotent blood progenitor cells, induced leukocyte stem (iLS) cells or T / NK progenitor cells can be preferably used.

Description

Method for producing immune cells

[0001] The present invention relates to a method for producing immune cells that express exogenous receptors such as T cell receptors (TCRs) and chimeric antigen receptors (CARs) that specifically recognize a desired antigen, a method for producing an immune cell therapy agent using the method, a method for treating diseases using the immune cells, and a method for producing T / NK precursor cells in large quantities.

[0002] T cells transduced with tumor-specific TCRs or chimeric antigen receptors (CARs) (TCR-T cells, CAR-T cells) and NK cells transduced with CARs (CAR-NK cells) are expected to be new cancer treatment strategies. Methods for producing TCR-T cells, CAR-T cells, and CAR-NK cells for use in immune cell therapy include introducing TCR or CAR genes into T cells / NK cells collected from the patient, T cells / NK cells derived from umbilical cord blood, or known T cell / NK cell lines. Other methods have also been proposed, such as introducing CAR genes into iPS cells and then inducing their differentiation into NK cells to produce CAR-NK cells (Non-Patent Document 1). In commercially available CAR-T therapies (Kymriah®, Yescarta®), CAR-T cells are produced from leukocytes collected from the patient by leukocyte apheresis.

[0003] However, it has been pointed out that TCR-T cells and CAR-T / CAR-NK cells become exhausted within the patient's body and are unable to maintain sufficient cytotoxic activity. From the perspective of sustaining therapeutic effects through repeated cell transplantation as appropriate, it is essential to develop a method for stably producing these tumor-specific immune cells, but such development is insufficient.

[0004] Various techniques for expanding hematopoietic cells while maintaining pluripotency and self-renewal are known. One of these is a method developed by the present inventors' group, disclosed in Non-Patent Documents 2, 3, and Patent Document 1, which can massively expand mouse and human multipotent hematopoietic progenitor cells. In this method, hematopoietic stem and progenitor cells (HSPCs) are transfected with Id3 to inhibit intracellular E2A and then cultured under conditions that induce B cell differentiation. This results in the massive expansion of multipotent hematopoietic progenitor cells with the ability to differentiate into leukocytes (T cells, B cells, myeloid cells, etc.) and self-renewal. The multipotent hematopoietic progenitor cells obtained in this way primarily produce leukocytes when transplanted into the body, and have therefore been named induced leukocyte stem (iLS) cells. It has been shown that iLS cells can be expanded ex vivo for at least several months.

[0005] In addition, the present inventors have reported a method for generating and expanding T / NK progenitor cells (pT / NK cells) capable of differentiating into T cells and NK cells in mouse studies (Non-Patent Documents 2, 4, and 5). In this method, IL-7, SCF, and Flt3-L are added to the culture medium at 10 ng / mL each, and mouse hematopoietic stem / progenitor cells are cultured in the presence of a Notch ligand. This results in the generation of pT / NK cells with self-renewal capacity that maintains pluripotency (the ability to differentiate into T cells, NK cells, and myeloid cells). This method also demonstrates the ability to expand mouse pT / NK cells ex vivo for at least several months.

[0006] Another method developed by another group is known, for example, a method for expanding and culturing mouse hematopoietic stem cells in an undifferentiated state by culturing hematopoietic stem cells in a medium containing polyvinyl alcohol instead of serum albumin (Non-Patent Document 6). This method also allows expansion and culturing of mouse hematopoietic stem cells in an undifferentiated state for more than one month.

[0007] WO 2009 / 113595 A1

[0008] Guozhu Xie et al., EBioMedicine 59 (2020) 102975Ikawa et al. Science, vol.329, pp.93-96, 2 July 2010Ikawa et al. Stem Cell Rep, vol. 5, pp. 716-727, November 10, 2015Ikawa et al. J Exp Med. vol.190, pp.1617-1625, December 6, 1999Ikawa et al. PNAS, vol.98, pp.5164-5169, April 24, 2001Adam C. Wilkinson et al., Nature, vol. 571, pp. 117-121 (2019)

[0009] One object of the present invention is to provide a means for stably producing TCR-T cells and CAR-T / CAR-NK cells. Another object of the present invention is to provide a means for treating diseases using said means. Another object of the present invention is that, in tests in which the methods described in Non-Patent Documents 2, 4, and 5 using mice were applied to human hematopoietic stem cells, it was newly confirmed that there is a limit to the expansion of human T / NK progenitor cells. Therefore, another object of the present invention is to develop a new method that improves the expansion efficiency of human T / NK progenitor cells for application in human immune cell therapy.

[0010] As a result of intensive research, the inventors of the present application have found that TCR- or CAR-expressing immune cells can be prepared efficiently and stably by introducing a TCR gene or a CAR gene into multipotent blood progenitor cells such as artificial leukocyte stem cells or T / NK progenitor cells, amplifying them, and inducing their differentiation into immune cells such as T cells, NK cells, and macrophages; that TCR- or CAR-expressing immune cells can be mass-produced by preparing these multipotent blood progenitor cells using the method for expanding artificial leukocyte stem cells or T / NK progenitor cells developed by the inventors' group; and that TCR- or CAR-expressing immune cells can also be prepared efficiently and stably by differentiating multipotent blood progenitor cells prepared by this expansion method into immune cells and then introducing a TCR or CAR gene into them. Furthermore, previous studies by the present inventors have revealed that in the preparation of mouse T / NK precursor cells, the expansion efficiency can be further improved by increasing the concentrations of IL-7, SCF, and Flt3-L from 10 ng / mL reported in Non-Patent Documents 2, 4, and 5 to approximately 25 to 50 ng / mL. However, in the preparation of human T / NK precursor cells, when these cytokines are used at high concentrations of 10 to 50 ng / mL each, as in the case of mouse cells, the expansion efficiency is only approximately 10 to 50 times higher, and furthermore, a problem has been found in that cell differentiation does not stop at T / NK precursor cells, but many cells are generated that differentiate into NK cells. As a result of intensive research aimed at solving these problems, the inventors have found that reducing the concentrations of SCF, Flt3-L, and IL-7 to less than 10 ng / mL in the process of preparing human T / NK progenitor cells significantly increases the expansion efficiency of human pT / NK cells, and that a 300- to 600-fold expansion is achieved under culture conditions in which TPO or M-CSF is combined, and that human CAR-NK cells prepared from human T / NK progenitor cells produced under these culture conditions have a good antitumor effect. Based on these findings, the inventors have conducted further research and have completed the present invention, which includes the following aspects.

[0011] [1] A method for producing immune cells expressing an exogenous receptor that is a T cell receptor (TCR), a chimeric antigen receptor (CAR), or a chimeric autoantibody receptor (CAAR), the method comprising the steps of: preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells; preparing multipotent blood progenitor cells that express the exogenous receptor by introducing a gene encoding the exogenous receptor into the multipotent blood progenitor cells; and inducing the differentiation of the multipotent blood progenitor cells that express the exogenous receptor into immune cells. [2] A method for producing immune cells expressing an exogenous receptor that is a TCR, a CAR, or a CAAR, the method comprising the steps of: preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells; inducing the differentiation of the multipotent blood progenitor cells into immune cells; and introducing a gene encoding the exogenous receptor into the immune cells, wherein the multipotent blood progenitor cells are induced leukocyte stem cells or T / NK progenitor cells. [3] The method of [1], wherein the multipotent blood progenitor cells are induced leukocyte stem cells or T / NK progenitor cells. [4] The method of [1] or [2], wherein the multipotent blood progenitor cells are induced leukocyte stem cells, and in the step of preparing the multipotent blood progenitor cells, the induced leukocyte stem cells are prepared by culturing a cell population containing hematopoietic stem cells under conditions that induce differentiation into B cells while inhibiting the function of the intracellular transcription factor E2A. [5] The method of [4], wherein the inhibition of E2A function is achieved by forcibly expressing a differentiation inhibitory factor Id in the hematopoietic stem cells. [6] The method of [1] or [2], wherein the multipotent blood progenitor cells are T / NK progenitor cells, and in the step of preparing the multipotent blood progenitor cells, the T / NK progenitor cells are prepared by culturing a cell population containing hematopoietic stem cells in the presence of a Notch ligand. [7] The method of [6], wherein a cell population containing hematopoietic stem cells is cultured in a medium containing SCF, TPO, and G-CSF at concentrations of 100 ng / mL or more each in the absence of a Notch ligand, and then cultured in the presence of a Notch ligand. [8] The method of any one of [1] to [7], wherein the immune cells are T cells, NK cells, macrophages, or B cells. [9] The method of [8], wherein the exogenous receptor is a TCR and the immune cells are T cells.

[10] The method according to [8], wherein the exogenous receptor is a CAR or CAAR, and the immune cell is a T cell, a NK cell, or a macrophage.

[11] The method according to any one of [1] to

[10] , wherein the exogenous receptor is an antigen-specific receptor, and the antigen is a tumor antigen.

[12] A method for producing an immune cell therapy agent for a disease treatable by immune cell therapy (e.g., a tumor or an autoimmune disease), comprising the steps of: preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells; preparing multipotent blood progenitor cells that express an exogenous receptor, which is a TCR or CAR that specifically recognizes a CAAR or a target antigen (e.g., a tumor antigen), by introducing a gene encoding the exogenous receptor into the multipotent blood progenitor cells; preparing T cells (TCR-T, CAR-T, or CAAR-T cells) that express the exogenous receptor by inducing differentiation of the multipotent blood progenitor cells that express the exogenous receptor; stimulating the TCR of the TCR-T, CAR-T, or CAAR-T cells to induce differentiation into CD8-positive cytotoxic TCR-T, CAR-T, or CAAR-T cells; A method comprising the step of preparing CD8-positive cytotoxic TCR-T or CAR-T cells as an immune cell therapy agent for a disease caused by cells expressing the antigen (e.g., a tumor expressing a tumor antigen), or preparing CD8-positive cytotoxic CAAR-T cells as an immune cell therapy agent for an autoimmune disease.

[13] A method for producing an immune cell therapy agent for a disease treatable by immune cell therapy (e.g., a tumor or an autoimmune disease), comprising the steps of: preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells; inducing the differentiation of the multipotent blood progenitor cells into T cells; preparing CD8-positive cytotoxic TCR-T, CAR-T, or CAAR-T cells by TCR stimulation of the T cells and then introducing a gene encoding an exogenous receptor that is a TCR or CAR that specifically recognizes a CAAR or a target antigen (e.g., a tumor antigen); and preparing the CD8-positive cytotoxic TCR-T or CAR-T cells as an immune cell therapy agent for a disease caused by cells expressing the antigen (e.g., a tumor expressing a tumor antigen), or preparing CD8-positive cytotoxic CAAR-T cells as an immune cell therapy agent for an autoimmune disease, wherein the multipotent blood progenitor cells are induced leukocyte stem cells or T / NK progenitor cells.

[14] A method for producing an immune cell therapy agent for a disease treatable by immune cell therapy (e.g., a tumor or an autoimmune disease), comprising the steps of: preparing multipotent hematopoietic progenitor cells from a cell population containing hematopoietic stem cells; preparing multipotent hematopoietic progenitor cells that express a chimeric receptor, which is a CAR that specifically recognizes a CAAR or a tumor antigen, by introducing a gene encoding the chimeric receptor into the multipotent hematopoietic progenitor cells; preparing NK cells (CAR-NK or CAAR-NK cells) or macrophages (CAR-macrophages or CAAR-macrophages) that express the chimeric receptor by inducing differentiation of the multipotent hematopoietic progenitor cells that express the chimeric receptor into NK cells or macrophages; and preparing the CAR-NK cells or CAR-macrophages as an immune cell therapy agent for a disease caused by cells expressing the antigen (e.g., a tumor that expresses a tumor antigen), or preparing the CAAR-NK cells or CAAR-macrophages as an immune cell therapy agent for an autoimmune disease.

[15] The method described in

[14] , further comprising a step of receptor-stimulating the prepared NK cells or macrophages expressing the chimeric receptor, and preparing the chimeric receptor-expressing NK cells or macrophages after receptor stimulation as the immune cell therapy agent.

[16] A method for producing an immune cell therapy agent for a disease treatable by immune cell therapy (e.g., a tumor or an autoimmune disease), comprising the steps of: preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells; inducing the differentiation of the multipotent blood progenitor cells into NK cells or macrophages; stimulating the NK cells or macrophages with a receptor, and then introducing a gene encoding a chimeric receptor that is a CAAR or a CAR that specifically recognizes a target antigen (e.g., a tumor antigen), thereby preparing NK cells (CAR-NK or CAAR-NK cells) or macrophages (CAR-macrophages or CAAR-macrophages) that express the chimeric receptor; and preparing the CAR-NK cells or CAR-macrophages as an immune cell therapy agent for a disease caused by cells expressing the antigen (e.g., a tumor expressing a tumor antigen), or preparing the CAAR-NK cells or CAAR-macrophages as an immune cell therapy agent for an autoimmune disease, wherein the multipotent blood progenitor cells are induced leukocyte stem cells or T / NK progenitor cells.

[17] A method for treating a patient's disease (e.g., tumor or autoimmune disease) treatable by immune cell therapy, comprising the steps of: preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells; preparing multipotent blood progenitor cells that express an exogenous receptor, which is a TCR or CAR that specifically recognizes a CAAR or a target antigen (e.g., a tumor antigen), by introducing a gene encoding the exogenous receptor into the multipotent blood progenitor cells; inducing the differentiation of the multipotent blood progenitor cells that express the exogenous receptor into T cells, thereby preparing TCR-T, CAR-T, or CAAR-T cells, which are T cells that express the exogenous receptor; TCR-stimulating the TCR-T, CAR-T, or CAAR-T cells to induce differentiation into CD8-positive cytotoxic TCR-T, CAR-T, or CAAR-T cells; A method comprising the step of administering CD8-positive cytotoxic TCR-T or CAR-T cells to a patient having a disease caused by cells expressing the antigen (e.g., a tumor expressing a tumor antigen), or administering CD8-positive cytotoxic CAAR-T cells to a patient with an autoimmune disease.

[18] A method for treating a patient with a disease treatable by immune cell therapy (e.g., a tumor or an autoimmune disease), comprising the steps of: preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells; inducing the differentiation of the multipotent blood progenitor cells into T cells; preparing CD8-positive cytotoxic TCR-T, CAR-T, or CAAR-T cells by TCR-stimulating the T cells and then introducing a gene encoding an exogenous receptor that is a TCR or CAR that specifically recognizes a CAAR or a target antigen (e.g., a tumor antigen); and administering the CD8-positive cytotoxic TCR-T or CAR-T cells to a patient with a disease caused by cells expressing the antigen (e.g., a tumor expressing a tumor antigen), or administering the CD8-positive cytotoxic CAAR-T cells to a patient with an autoimmune disease, wherein the multipotent blood progenitor cells are induced leukocyte stem cells or T / NK progenitor cells.

[19] A method for treating a disease treatable by immune cell therapy in a patient (e.g., a tumor or an autoimmune disease), comprising the steps of: preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells; preparing multipotent blood progenitor cells that express a chimeric receptor, the chimeric receptor being a CAAR or a CAR that specifically recognizes a target antigen (e.g., a tumor antigen), by introducing a gene encoding the chimeric receptor into the multipotent blood progenitor cells; preparing chimeric receptor-expressing immune cells, which are CAR-NK cells, CAAR-NK cells, CAR-macrophages, or CAAR-macrophages, by inducing the differentiation of the chimeric receptor-expressing multipotent blood progenitor cells into NK cells or macrophages; administering the CAR-NK cells or CAR-macrophages to a patient with a disease caused by cells expressing the antigen (e.g., a tumor expressing a tumor antigen), or administering the CAAR-NK cells or CAAR-macrophages to a patient with an autoimmune disease.

[20] The method described in

[19] , further comprising a step of receptor-stimulating the prepared chimeric receptor-expressing immune cells, wherein the receptor-stimulated chimeric receptor-expressing immune cells are administered to the patient.

[21] A method for treating a patient with a disease treatable by immune cell therapy (e.g., a tumor or an autoimmune disease), comprising the steps of: preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells; inducing the differentiation of the multipotent blood progenitor cells into NK cells or macrophages; stimulating the NK cells or macrophages with a receptor, and then introducing a gene encoding a chimeric receptor that is a CAAR or a CAR that specifically recognizes a target antigen (e.g., a tumor antigen), thereby preparing CAR-NK cells, CAAR-NK, CAR-macrophages, or CAAR-macrophages; and administering the CAR-NK cells or CAR-macrophages to a patient with a disease caused by cells expressing the antigen (e.g., a tumor expressing a tumor antigen), or administering the CAAR-NK cells or CAAR-macrophages to a patient with an autoimmune disease, wherein the multipotent blood progenitor cells are induced leukocyte stem cells or T / NK progenitor cells.

[22] A method for treating a patient's disease (e.g., tumor) caused by cells expressing a target antigen, comprising the steps of: preparing CAR-macrophages by the following (1) or (2): (1) preparing multipotent hematopoietic progenitor cells from a cell population containing hematopoietic stem cells, and introducing a gene encoding a CAR that specifically recognizes a target antigen (e.g., a tumor antigen) into the multipotent hematopoietic progenitor cells to prepare multipotent hematopoietic progenitor cells expressing the CAR, and then inducing the differentiation of the CAR-expressing multipotent hematopoietic progenitor cells into macrophages; (2) preparing artificial leukocyte stem cells or T / NK progenitor cells from a cell population containing hematopoietic stem cells, inducing their differentiation into macrophages, and stimulating the receptor, and then introducing the gene encoding the CAR; and preparing CAR-T cells by the following (3) or (4): (3) (3) preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells, introducing a gene encoding the CAR into the multipotent blood progenitor cells to prepare multipotent blood progenitor cells that express the CAR, inducing the differentiation of the CAR-expressing multipotent blood progenitor cells into T cells, and then stimulating the TCR to induce differentiation into CD8-positive cytotoxic TCR-T or CAR-T cells; (4) preparing artificial leukocyte stem cells or T / NK progenitor cells from a cell population containing hematopoietic stem cells, inducing their differentiation into T cells, stimulating the TCR, and then introducing a gene encoding the CAR; and administering a combination of CAR-macrophages and CAR-T cells to a patient with a disease caused by cells expressing the antigen (e.g., a tumor expressing a tumor antigen).

[23] A method for treating a patient's disease (e.g., tumor) caused by cells expressing a target antigen, comprising the steps of: preparing CAR-macrophages by the following steps (1) or (2): (1) preparing multipotent hematopoietic progenitor cells from a cell population containing hematopoietic stem cells, and introducing a gene encoding a CAR that specifically recognizes a target antigen (e.g., a tumor antigen) into the multipotent hematopoietic progenitor cells to prepare multipotent hematopoietic progenitor cells expressing the CAR, and then inducing the differentiation of the CAR-expressing multipotent hematopoietic progenitor cells into macrophages; (2) preparing artificial leukocyte stem cells or T / NK progenitor cells from a cell population containing hematopoietic stem cells, inducing their differentiation into macrophages, and stimulating the receptor, and then introducing the gene encoding the CAR; and preparing CAR-NK cells by the following steps (5) or (6): (5) (2) a method comprising the steps of: (1) preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells, introducing a gene encoding the CAR into the multipotent blood progenitor cells to prepare multipotent blood progenitor cells that express the CAR, and then inducing the differentiation of the CAR-expressing multipotent blood progenitor cells into NK cells; (2) preparing artificial leukocyte stem cells or T / NK progenitor cells from a cell population containing hematopoietic stem cells, inducing their differentiation into NK cells and stimulating their receptors, and then introducing the gene encoding the CAR; and (3) combining CAR-macrophages and CAR-NK cells, and administering the combined CAR-macrophages and CAR-NK cells to a patient with a disease caused by cells expressing the antigen (e.g., a tumor expressing a tumor antigen).

[24] The method described in

[23] , wherein step (5) further comprises inducing the differentiation of the CAR-expressing multipotent blood progenitor cells into NK cells to prepare CAR-NK cells, and then stimulating their receptors.

[25] The method according to any one of

[22] to

[24] , wherein the step (1) further comprises inducing the differentiation of the CAR-expressing multipotent hematopoietic progenitor cells into macrophages to prepare CAR-macrophages, and then receptor-stimulating the CAR-macrophages.

[0012] In the above

[12] to

[25] , preferably, the target antigen is a tumor antigen, and the disease caused by cells expressing the antigen is a tumor expressing the tumor antigen.

[0013] The present invention further provides the following methods, cells, etc.:

[26] A method for producing human T / NK progenitor cells, comprising the step of culturing a cell population containing human hematopoietic stem cells in a medium containing SCF, Flt3-L, and IL-7, each at a concentration of 1 ng / mL or more and less than 10 ng / mL, in the presence of a Notch ligand.

[27] The method of

[26] , wherein the medium further contains at least one of TPO and M-CSF.

[28] The method of any one of

[26] to

[27] , wherein the cell population containing human hematopoietic stem cells is a cell population obtained by culturing a cell population containing human hematopoietic stem cells in a medium containing SCF, TPO, and G-CSF, each at a concentration of 100 ng / mL or more, in the absence of a Notch ligand.

[29] A method for producing human NK cells, comprising the steps of: producing human T / NK precursor cells by the method according to any one of

[26] to

[28] ; and inducing differentiation of human NK cells by culturing the produced human T / NK precursor cells in a medium containing IL-7, Flt3-L, SCF, and IL-15 in the absence of a Notch ligand.

[30] The method for producing immune cells according to claim 6 or 7, wherein the T / NK precursor cells are human T / NK precursor cells, and in the step of preparing multipotent blood precursor cells, the human T / NK precursor cells are produced from a cell population containing human hematopoietic stem cells by the method according to any one of

[26] to

[28] .

[31] The method for producing immune cells according to

[30] , wherein the immune cells are human NK cells, and the step of inducing the differentiation of the multipotent blood progenitor cells into immune cells involves culturing human T / NK precursor cells in a medium containing IL-7, Flt3-L, SCF, and IL-15 in the absence of a Notch ligand to induce the differentiation of the human NK cells.

[32] Cultured human T / NK precursor cells, characterized by being CD122-positive. Preferably, the cultured human T / NK precursor cells further satisfy the condition of expressing one, two, three, four, five, or six surface antigens selected from the group consisting of CD3-negative, CD4-negative, CD8-negative, CD56-negative, CD161-negative, and CD7-positive.

[33] Cultured human NK cells characterized by lower expression of CD16 and KIR, which are indicators of maturity, and lower expression of the exhaustion marker TIGIT, compared to human umbilical cord blood NK cells isolated from human umbilical cord blood, and therefore being younger and less exhausted than the human umbilical cord blood NK cells.

[0014] The method for producing an immune cell therapy agent for a disease treatable by immune cell therapy of the present invention includes the following aspects: [i-1] The method for producing an immune cell therapy agent according to any one of

[12] to

[16] , wherein the multipotent blood progenitor cells are human T / NK progenitor cells, and in the step of preparing the multipotent blood progenitor cells, human T / NK progenitor cells are produced from a cell population containing human hematopoietic stem cells by the method according to any one of

[26] to

[28] . [i-2] A method for producing an immune cell therapy agent according to any one of

[14] to

[16] , wherein the multipotent blood progenitor cells are human T / NK progenitor cells and the immune cells are human NK cells; in the step of preparing the multipotent blood progenitor cells, human T / NK progenitor cells are produced from a cell population containing human hematopoietic stem cells by the method according to any one of

[26] to

[28] ; and in the step of inducing the differentiation of the multipotent blood progenitor cells into immune cells, the human T / NK progenitor cells are cultured in a medium containing IL-7, Flt3-L, SCF, and IL-15 in the absence of a Notch ligand, thereby inducing the differentiation of human NK cells.

[0015] The above-mentioned method of the present invention for treating a patient with a disease treatable by immune cell therapy includes the following aspects: [ii-1] A method for treating a patient with a disease treatable by immune cell therapy according to any one of

[17] to

[21] , wherein the multipotent blood progenitor cells are human T / NK progenitor cells, and in the step of preparing the multipotent blood progenitor cells, human T / NK progenitor cells are produced from a cell population containing human hematopoietic stem cells by the method according to any one of

[26] to

[28] . [ii-2] A method for treating a disease in a patient according to any one of

[19] to

[21] , wherein the multipotent blood progenitor cells are human T / NK progenitor cells, the immune cells are human NK cells, and in the step of preparing the multipotent blood progenitor cells, human T / NK progenitor cells are produced from a cell population containing human hematopoietic stem cells by the method according to any one of

[26] to

[28] , and in the step of inducing the differentiation of the multipotent blood progenitor cells into immune cells, the human T / NK progenitor cells are cultured in a medium containing IL-7, Flt-3L, SCF, and IL-15 in the absence of a Notch ligand, thereby inducing the differentiation of human NK cells.

[0016] The above-mentioned method of the present invention for treating a disease caused by cells expressing a target antigen in a patient includes the following embodiments: [iii-1] The method for treating a disease caused by cells expressing a target antigen in a patient according to any one of

[22] to

[25] , wherein the multipotent blood progenitor cells are human T / NK progenitor cells, and the preparation of multipotent blood progenitor cells from a cell population containing hematopoietic stem cells is carried out by the method according to any one of

[26] to

[28] . [iii-2] The method for treating a disease caused by cells expressing a target antigen in a patient according to

[23] or

[24] , wherein the multipotent blood progenitor cells are human T / NK progenitor cells, the NK cells are human NK cells, and the preparation of multipotent blood progenitor cells from a cell population containing hematopoietic stem cells is carried out by the method according to any one of

[26] to

[28] , and the induction of differentiation into NK cells is carried out by culturing the human T / NK progenitor cells in a medium containing IL-7, Flt3-L, SCF, and IL-15 in the absence of a Notch ligand.

[0017] The present invention enables stable production of TCR / CAR / CAAR-expressing immune cells, such as TCR-T, CAR-T, CAAR-T, CAR-NK, and CAAR-NK cells. The inventors' group developed a method for expanding artificial leukocyte stem cells or T / NK progenitor cells to prepare multipotent blood progenitor cells, enabling stable mass production of TCR / CAR / CAAR-expressing immune cells. TCR / CAR-expressing immune cells produced using antigen-specific receptors that specifically recognize tumor antigens or antigens specific to pathogens such as bacteria and viruses exhibit cytotoxic activity against tumor cells or pathogen-infected cells in patients, and can be used as immune cell therapy agents for the treatment of tumors and infectious diseases. Furthermore, CAAR-expressing immune cells produced using CAARs designed based on any autoantigen exhibit cytotoxic activity against pathogenic B cells that recognize the autoantigen in patients, and can be used as immune cell therapy agents for the treatment of autoimmune diseases caused by the autoantigen or pathogenic B cells. According to the present invention, it is possible to stably mass-produce such immune cell therapy agents, which will greatly contribute to the treatment of diseases such as tumors, infectious diseases, and autoimmune diseases.

[0018] This figure illustrates induced leukocyte stem (iLS) cells. iLS cells are multipotent blood progenitor cells with self-renewal capacity and the ability to differentiate into leukocytes such as T cells and B cells. This figure illustrates T / NK progenitor cells (pT / NK cells). CD34+ hematopoietic stem cells are expanded by culturing hematopoietic stem / progenitor cells in the presence of high concentrations of cytokines. These cells are then cultured under Notch signaling stimulation using OP9 / delta-like-1 (DLL1) cells or other media to generate self-renewal pT / NK cells that maintain pluripotency (the ability to differentiate into T cells, NK cells, and myeloid cells). (Top) Schematic diagram of the method for generating iLS cells expressing OTI TCR. (Bottom left) FCM analysis of iLS cells transfected with the OTI TCR gene. OTI TCR-expressing iLS cells (OTI TCR-iLS) with lineage marker (-), human CD25 (+), and GFP (+) were frequently detected. (Bottom right) Graph showing the fold change in proliferation of OTI TCR-iLS cells and control cells (iLS cells spin-infected with an empty retrovirus that does not contain the OTI TCR gene) cultured under iLS cell culture conditions. (A) Schematic of the process for inducing differentiation of T cells (CD4 / CD8 double-positive (DP) cells) from OTI TCR-iLS cells. (B) FCM analysis performed on cells collected 7, 14, and 21 days after the start of induction. DP cells appeared on day 14 of culture, and by day 21 of culture, DP cells were generated at a rate of more than 70%. The percentage and expansion rate of DP cells generated from OTI TCR-iLS cells are shown. The percentage of DP cells was 50-60%, and iLS cells generated approximately 1500-fold more DP cells. Schematic of the process for inducing DP cells generated from OTI TCR-iLS cells to CD8-positive T cells. DP cells were generated using OVA. 257-264By co-culturing with antigen-presenting cells (APCs) pulsed with CD8+ for seven days, over 90% of CD8+ T cells are induced. These CD8+ T cells express the OTI TCR and are mostly effector memory (EM) T cells and central memory (CM) T cells. During this process, the cells are expanded approximately five-fold. Overview of the process for inducing CD8+ T cells from anti-CD19 CAR-expressing iLS cells (anti-CD19 CAR-iLS cells). Anti-CD19 CAR-iLS cells are co-cultured with OP9 / DLL1 cells to induce differentiation into DP cells. DP cells are generated on day 21 of culture. Next, the DP cells are sorted and stimulated with anti-CD3 / CD28 antibodies to induce CD8+ T cells. CD8+ T cells are generated after four days of culture. (A) Overview of the process for evaluating antigen-specific cytokine release from OTI TCR-iLS cell-derived CD8+ T cells (OTI T-iLS cells). (B, C) Cytokine release was examined by intracellular staining of CD8+ T cells derived from OTI TCR-iLS cells stimulated with OVA antigen-presenting cells or PMA + ionomycin. B is a two-parameter histogram of FCM analysis, and C is a graph of this. Unstim. and Unstimul refer to unstimulated cells, APC refer to cells co-cultured with mouse spleen cells that do not present OVA, OVA + APC refer to cells stimulated with OVA antigen-presenting cells, and PMA / Iono refer to cells stimulated with PMA + ionomycin. MFI: median fluorescence intensity. (Top) Outline of the process for evaluating the cytotoxic activity of OTI T-iLS cells. (Bottom) The cytotoxic activity of each effector cell was examined using OVA antigen-transduced B16 melanoma cell line (B16-OVA cells) and Neo gene-transduced B16 melanoma cell line (B16-Neo cells) as target cells, and OTI T-iLS cells and T cells derived from the spleen of OTI Tg mice as effector cells. (Top) Outline of the process for evaluating the antitumor activity of OTI T-iLS cells using B16-OVA cell tumor-bearing mice. (Bottom left) Tumor volume of tumor-bearing mice in each treatment group. (Bottom right) Survival rate of tumor-bearing mice in each treatment group.Untreated, NT: no treatment; IL-2: IL-2 treatment only; OTI-iLS + IL-2: treatment with OTI T-iLS cells and IL-2; OTI-SP + IL-2: treatment with OTI Tg mouse spleen-derived T cells and IL-2. (A) Overview of the process for inducing CAR-NK cells from iLS cells. (B) Schematic diagram of the mouse anti-CD19 CAR sequence of the retroviral vector used in the examples. (C) FCM analysis results of anti-CD19 CAR gene-introduced pT / NK cells (anti-CD19 CAR-pT / NK cells) and anti-CD19 CAR-introduced NK cells after differentiation induction (anti-CD19 CAR-NK cells). This shows that a high percentage (over 70%) of anti-CD19 CAR-NK cells were generated. Cytotoxic activity of anti-CD19 CAR-NK cells against B16 melanoma cells transfected with human CD19 (B16-hCD19 cells) and B16 melanoma cells not transfected with human CD19 (B16 cells). Induction of differentiation of anti-CD19 CAR-iLS cells into macrophages. FCM analysis one week after the start of induction (Day 7) confirmed the generation of CD11b-positive, F4 / 80-positive, MHC class II (IA)-positive macrophages. (Top) Schematic of the process for generating pT / NK cells from human umbilical cord blood CD34-positive cells (hCB-CD34-positive cells) and inducing their differentiation into NK cells. (Bottom) Expression of surface antigen marker molecules was examined by FCM analysis on Days 17 (left) and 25 (center) after the start of culture of hCB-CD34-positive cells. pT / NK cells isolated by cell sorting from the cell population on day 17 of culture, and human umbilical cord blood hematopoietic stem / progenitor cells (hCB-HSPCs) obtained by expanding hCB-CD34+ cells in Stemline II medium, were cultured under NK cell differentiation-inducing conditions, and the numbers of NK cells generated were compared (right). NK cells derived from pT / NK cells were co-cultured with the target cell line, K562 leukemia cell line (K562 cells), or stimulated by treatment with PMA and ionomycin, and cytokine release was analyzed by FCM. Cytotoxic activity of pT / NK cell-derived NK cells and hCB-HSPC-derived NK cells against K562 cells.Schematic diagram of the human anti-CD19 CAR sequence and anti-GD2 CAR sequence of the lentiviral vector used to generate pT / NK cell-derived anti-CD19 CAR-NK cells and anti-GD2 CAR-NK cells (top). FCM analysis of the marker protein Venus confirmed that transgene expression was maintained (bottom). Mock cells were transduced with Venus alone via lentivirus. Antigen-specific cytotoxic activity of pT / NK cell-derived anti-CD19 CAR-NK cells against the NALM6 leukemia cell line (NALM6 cells). The gene transduction efficiency of lentiviruses produced using VSV-G or GALV envelopes into pT / NK cells was compared. pT / NK cells were transduced with a CAR gene-expressing lentivirus produced using a GALV envelope. FCM analysis of the marker protein Venus confirmed that transgene expression was maintained. Mock cells were transduced with Venus alone via lentivirus. CAR-pT / NK cells were transduced with a CAR gene-expressing lentivirus produced using a GALV envelope and induced to differentiate into NK cells, resulting in pT / NK cell-derived NK cells stably expressing the CAR gene. Mock cells were transduced with only Venus via lentivirus. Mouse bone marrow-derived hematopoietic stem / progenitor cells (mouse HSPC, Lineage-Sca1-ckit+: LSK) were cultured on OP9 / DLL1 feeder cells with SCF, IL-7, and Flt3-L at concentrations of 10 ng / ml or 30 ng / ml for two weeks, after which the number of pT / NK cells was counted. The optimal cytokine concentration for generating and expanding pT / NK cells from hCB-CD34+ cells was investigated. The top panel shows an outline of the culture process. The bottom left panel shows the number of pT / NK cells counted after six weeks of culture under cytokine concentrations 1 to 13 listed in Table 1. The bottom right is a graph showing the number of pT / NK cells obtained over time by culture under conditions 1 and 4. (A) Outline of the culture protocol in Example B-2. (B) Proliferation rate of cells from hCB-CD34-positive cells. (C) Results of FCM analysis after NK cell generation. (D) Number of NK cells produced when NK cells are directly induced from hCB-HSPCs and when the pT / NK cell expansion method is used.(A) In Example B-3, this shows the cell proliferation rate after 2 weeks of culture of NK cells collected from human umbilical cord blood (CB-NK cells), NK cells collected from human peripheral blood (PB-NK cells), and NK cells derived from human pT / NK cells in MEMα medium supplemented with 10 ng / ml rhIL-15. (B) This shows the expansion rate when mitomycin-treated aAPCs and each NK cell type were mixed at an E:T ratio of 1:2 and the NK cells were expanded in MEMα medium containing 200 IU / ml rhIL-2. In Example B-4, this shows the results of evaluating the expression level of NK cell-associated receptors in NK cells derived from pT / NK cells by FCM analysis (one-parameter histogram). In Example B-4, this shows the results of evaluating the expression level of NK cell-associated receptors in NK cells derived from pT / NK cells by FCM analysis (graph of one-parameter histogram). In Example B-5, this shows the results of comparing the cytotoxic activity of NK cells derived from pT / NK cells against K562 cells with that of PB-NK cells or CB-NK cells. (A) NK cells and K562 cells were mixed at various E:T ratios and co-cultured for 4 hours, followed by FCM analysis of the percentage of dead K562 cells. (B, C) Evaluation of the expression level of effector molecules produced by activated NK cells. B is a two-parameter histogram, and C is a graph of it. In Example B-6, these are results of evaluating the effect of cryopreservation of human pT / NK cell-derived NK cells. (A) Recovery rate of pT / NK cell-derived NK cells after freeze-thawing (number of cells after freezing / number of cells before freezing × 100). (B) Proliferation ability of human pT / NK cell-derived NK cells after freeze-thawing. (C) Cytotoxic activity of freeze-thawed pT / NK cell-derived NK cells against K562 cells. In Example B-7, results of exploring factors that promote the differentiation and proliferation of hCB-HSPCs into pT / NK cells. hCB-HSPCs were cultured on OP9 / DLL1 feeder cells in the presence of 5 ng / ml each of rhIL-7, rhFlt3-L, and rhSCF, and 10 ng / ml of each cytokine or 0.1 mM nicotinamide was added to the culture system, and the expansion rate of pT / NK cells was calculated. In Example B-8, the concentrations at which TPO or M-CSF promote the generation of human pT / NK cells were examined.hCB-HSPCs were cultured on OP9 / DLL1 feeder cells in the presence of 5 ng / ml each of rhIL-7, rhFlt3-L, and rhSCF, and TPO or M-CSF was added at concentrations of 0 to 50 ng / ml to examine the number of pT / NK cells per HSPC. Example B-9 shows the results of evaluating the NK cell differentiation potential of human pT / NK cells expanded with TPO or M-CSF. Example B-10 shows the results of investigating whether TPO is also effective in inducing pT / NK cells from human bone marrow-derived HSPCs (hBM-HSPCs). The top panel shows an outline of the culture protocol. The middle panel shows FCM analysis of pT / NK cells generated and expanded from hBM-HSPCs and NK cells induced from these pT / NK cells. The bottom panel shows the results of comparing the number of NK cells induced from pT / NK cells expanded for 5 weeks using this culture system with and without TPO. In Example B-11, the expression levels of NK cell-associated receptors in pT / NK-derived NK cells induced from hBM-HSPCs were evaluated by FCM analysis (single-parameter histogram). This figure shows the results of comparing the expression of activating receptors, inhibitory receptors, and exhaustion markers in NK cells derived from human T / NK precursor cells (pT / NK-NK cells), human CB-NK cells, and human PB-NK cells induced from hCB-HSPCs. This figure shows RNA-seq data comparing gene expression in human pT / NK-NK cells, human CB-NK cells, and human PB-NK cells induced from hCB-HSPCs. In Example B-12, the cytotoxic activity of pT / NK-derived NK cells induced from hBM-HSPCs against K562 cells was evaluated. NK cells and K562 cells were mixed and cultured at various E:T ratios, and the percentage of dead K562 cells was analyzed by FCM. Identification of specific markers for pT / NK cells (Example B-13). (A) Expression of CD122 (IL-2 receptor β chain) in pT / NK cells was evaluated by FCM analysis. (B) CFSE assay performed to evaluate the proliferation ability of CD122-positive cells. (C) Evaluation of the differentiation ability of CD122-positive cells into T cells and NK cells. Schematic diagram of the CAR sequence of the retroviral vector used to generate anti-CD19 CAR-NK cells and anti-HER2 CAR-NK cells derived from human pT / NK cells, constructed in Example C-1.Generation of anti-CD19 CAR-NK cells and anti-HER2 CAR-NK cells from human pT / NK cell-derived NK cells (Example C-2). (A) Each CAR gene was introduced into human pT / NK cell-derived NK cells using the vector shown in Figure 33. GFP-positive CAR-NK cells were isolated using a cell sorter and further expanded by co-culture with aAPCs. After culture, GFP expression was analyzed by FCM to confirm stable expression of the introduced gene. (B) Antigen-specific cytotoxic activity of anti-CD19 CAR-NK cells or anti-HER2 CAR-NK cells. After co-culture with each target cell at various E:T ratios, the percentage of target cell death was measured by FCM analysis. Introduction of CAR genes into human pT / NK cells (Example C-3). An anti-CD19 CAR gene or anti-HER2 CAR gene was introduced into human pT / NK cells using the vector shown in Figure 33. pT / NK cells expressing each CAR gene were isolated and expanded using GFP expression as an indicator. Stable expression of the transgene was confirmed in the expanded pT / NK cells. Differentiation induction of CAR gene-transduced human pT / NK cells into CAR-NK cells (Example C-4). (A) Scheme for generating CAR-NK cells from CAR gene-transduced pT / NK cells. (B) The obtained CAR-NK cells were expanded by culturing in the presence of aAPC and IL-2. FCM analysis of the expanded CAR-pT / NK cell-derived CAR-NK cells confirmed the generation of CAR-NK cells stably expressing the transgene. (C) CAR-NK cells were generated at a density of 1 x 10 per CAR gene-transduced human pT / NK cells. 4Approximately 100 cells were produced. Evaluation of the cytotoxic activity of CAR gene-transduced pT / NK cell-derived CAR-NK cells against target cells (Example C-5). FCM analysis of representative NK cell-associated receptors was performed on pT / NK cell-derived anti-HER2 CAR-NK cells (pT / NK-CAR-NK), CB-NK-derived anti-HER2 CAR-NK cells (CB-CAR-NK), and PB-NK-derived anti-HER2 CAR-NK cells (PB-CAR-NK) (Example C-6). Cytotoxic activity of CAR gene-transduced pT / NK cell-derived anti-HER2 CAR-NK cells, CB-NK-derived anti-HER2 CAR-NK cells, and PB-NK-derived anti-HER2 CAR-NK cells (Example C-7). Outline of the protocol for evaluating the in vivo antitumor activity of CAR gene-transduced human pT / NK cell-derived CAR-NK cells in Example C-8. Tumor growth in tumor-bearing mice administered with CAR-NK cells was measured by bioluminescence imaging. The top panel shows the time course of tumor growth in tumor-bearing mice administered with CAR-NK cells. The bottom row shows the changes in body weight for each mouse group.

[0019] In the present invention, "immune cell therapy" refers to a method for treating a disease by utilizing the biological defense function of immune cells to reduce or eliminate disease-causing cells or tissues. Representative immune cell therapies include CAR immune cell therapy, which treats tumors by using immune cells expressing CARs that specifically recognize an antigen expressed in cells contained in tumor tissue to reduce or eliminate cells expressing the antigen. Other immune cell therapies include immunotherapy targeting B cell-specific antigens for the treatment of autoimmune diseases, and immune cell therapy targeting bacterial or viral antigens to reduce or eliminate bacterially or virally infected cells for the treatment of bacterial or viral infections. "Diseases caused by cells expressing target antigens" include various diseases such as tumors, autoimmune diseases, bacterial infections, and viral infections. The term "cells expressing a target antigen" encompasses not only cells expressing the target antigen from their own nuclear or mitochondrial genomes, but also cells expressing the target antigen via bacteria or viruses that have infected the cells.

[0020] In the present invention, the term "exogenous receptor" refers to a T cell receptor (TCR), a chimeric antigen receptor (CAR), or a chimeric autoantibody receptor (CAAR). In the present specification, "TCR, CAR, or CAAR" may be abbreviated as "TCR / CAR / CAAR." The term "cells expressing an exogenous receptor" includes TCR / CAR / CAAR-expressing cells (cells such as multipotent blood progenitor cells, the same applies hereinafter), TCR / CAR-expressing cells, TCR / CAAR-expressing cells, CAR / CAAR-expressing cells, TCR-expressing cells, CAR-expressing cells, and CAAR-expressing cells. Furthermore, in the present specification, the term "antigen-specific receptor" refers to a TCR or CAR. In the present specification, "TCR or CAR" may be abbreviated as "TCR / CAR." The term "cells expressing an antigen-specific receptor" includes TCR / CAR-expressing cells, TCR-expressing cells, and CAR-expressing cells. As used herein, expressions such as "TCR or CAR that specifically recognizes a tumor antigen," "tumor treatment using the same," and the like describe one embodiment of the use of the TCR / CAR-expressing cells of the present invention. TCR / CAR-expressing cells that target an antigen expressed in cells other than tumor cells can be used to treat diseases that are treated by reducing or eliminating cells that express the antigen (e.g., autoimmune diseases, bacterial infections, etc.). Therefore, even inventions that include the above descriptions can be applied to diseases other than tumors. Furthermore, in the present invention, the term "chimeric receptor" means a CAR or CAAR. In the present specification, "CAR or CAAR" may be written as "CAR / CAAR." The term "cells expressing a chimeric receptor" includes CAR / CAAR-expressing cells, CAR-expressing cells, and CAAR-expressing cells.

[0021] As is well known in the art, TCR is a receptor present on the surface of T cells that is used by T cells to recognize antigens and is composed of a dimer of an α chain and a β chain, or a dimer of a γ chain and a δ chain. Most T cells are αβ T cells that have a TCR (αβ TCR) composed of a dimer of an α chain and a β chain, and in the present invention, αβ TCR is usually used as the TCR to be expressed in immune cells.

[0022] As is well known in the art, CARs are artificially engineered receptors capable of specifically recognizing antigens by combining an antigen-recognition domain derived from an antibody that specifically recognizes the antigen with a transmembrane domain derived from a cell surface molecule such as CD8 or CD28, one or more costimulatory domains derived from the intracellular signaling domain of a costimulatory receptor molecule such as CD28, 4-IBB, OX40, or 2B4, and an activation domain derived from the intracellular signaling domain of a CD3ζ molecule. The antigen-recognition domain is preferably a short-chain variable fragment (scFv), which is constructed by linking the heavy-chain variable region (VH) and light-chain variable region (VL) of an antibody that specifically recognizes the desired antigen via an appropriate linker. Target antigens are not limited to tumor antigens; various antigens can be used as long as they are characteristically or specifically expressed in cells that are expected to have a therapeutic effect by reducing or eliminating specific cells. For example, CARs directed against antigens specifically expressed on B cells, such as CD19, are useful for treating not only hematological cancers but also autoimmune diseases. A suitable costimulatory domain can be selected depending on the type of immune cells to be used to express the CAR. For example, Non-Patent Document 1 reports that a domain derived from 2B4 can be used as a costimulatory domain for CARs to be expressed in NK cells.

[0023] A chimeric auto-antibody receptor (CAAR) is a well-known chimeric receptor similar to CAR (see WO2022 / 196719, WO2016 / 07006, Ellebrecht et al., Science, 08 Jul 2016: Vol. 353, Issue 6295, pp. 179-184, etc.). CAARs differ from CARs in that the antigen-recognition domain is derived from an autoantigen, but otherwise share the same molecular structure as CARs. The antigen-recognition domain is typically composed of an oligopeptide or polypeptide containing part or all of the amino acid sequence of an autoantigen that is the cause of autoimmune disease. CAAR-expressing immune cells have been reported to bind to the B cell receptor (BCR) on the surface of pathogenic B cells that recognize autoantigens, thereby reducing the pathogenic B cells that cause autoimmune disease and thereby demonstrating therapeutic efficacy against autoimmune diseases. Examples of such autoimmune diseases and autoantigens include DSG3 in pemphigus vulgaris and the nicotinic acetylcholine receptor α subunit in myasthenia gravis.

[0024] In the present invention, "culturing in the presence of a Notch ligand" refers to culture conditions under which the cells to be cultured can be cultured under conditions that allow contact between the Notch ligand and the cells, thereby stimulating and enhancing Notch signaling in the cells. The Notch ligand may be used in the form of a recombinant protein, cells expressing the protein (e.g., feeder cells), or a culture of the cells containing the Notch ligand protein (e.g., culture supernatant, cell lysate, etc.). Preferred Notch ligands include Delta-like ligands such as Delta-like 1 (DLL1; human gene: Gene ID: 28514, NCBI Reference Sequence: NM_005618.4, SEQ ID NOs: 19 and 20) and Delta-like 4 (DLL4; human gene: Gene ID: 54567, NCBI Reference Sequence: NM_019074.4, SEQ ID NOs: 21 and 22). The amino acid sequences of DLL1 and DLL4 usable as Notch ligands in the present application are not limited to those shown in SEQ ID NOs: 20 and 22; proteins having amino acid sequences with 90% or more identity, e.g., 95% or more, or 98% or more identity to these sequences can also be used as DLL1 and DLL4. Methods for culturing the cell population in the presence of a Notch ligand include, for example, culturing the target cells on feeder cells expressing a Notch ligand, or culturing the cell population in a culture vessel (plate, dish, etc.) whose inner surface is coated with a Notch ligand protein (or a fragment thereof containing at least the extracellular domain) or a culture of feeder cells expressing a Notch ligand. Various feeder cells expressing Notch ligands are known and can be easily obtained from cell banks, etc. Specific examples include OP9 cells (e.g., OP9 / DLL1 cells), Tst-4 cells (e.g., Tst-4 / DLL1 cells), S17 cells (e.g., S17 / DLL1 cells), and MS-5 cells (e.g., MS-5 / DLL1 cells) overexpressing a Notch ligand.

[0025] In the present invention, "culture in the absence of a Notch ligand" refers to culture in a culture system that does not substantially contain a Notch ligand. Co-culture with feeder cells that do not substantially express a Notch ligand corresponds to culture in the absence of a Notch ligand. In the "culture in the absence of a Notch ligand" of the present invention, feeder cells that do not substantially express a Notch ligand may be used, but basically do not need to be used.

[0026] Human T / NK precursor cells, immature NK cells, and mature NK cells are known to be cells defined by the following marker expression patterns. Immature NK cells are known to have a phenotype similar to that of NK cells isolated from umbilical cord blood (cord blood NK cells), while mature NK cells are known to display the phenotype of NK cells isolated from peripheral blood (peripheral blood NK cells). Human T / NK precursor cells: CD3-negative, CD4-negative, CD8-negative, CD56-negative, CD161-negative, and CD7-positive (CD3 - CD4 - CD8 - CD56 - CD161 - CD7 + Human immature NK cells: CD3 negative, CD4 negative, CD8 negative, CD56 positive, CD161 positive, CD16 negative, and KIR negative (CD3 - CD4 - CD8 - CD56 + CD161 + CD16 - KIR - Human mature NK cells: CD3 negative, CD4 negative, CD8 negative, CD56 positive, CD161 positive, CD16 positive, and KIR positive (CD3 - CD4 - CD8 - CD56 + CD161 + CD16 + KIR + )

[0027] CD16 and KIR (killer cell immunoglobulin-like receptor) are known as maturation markers that indicate the maturity of human NK cells. As NK cells mature, the expression levels of CD16 and KIR increase. Among NK cells that are positive for CD16 and KIR, the lower the expression levels of these markers, the more immature the cells are evaluated to be.

[0028] The cultured human T / NK progenitor cells, one of the inventions provided by the present invention, are human T / NK progenitor cells isolated from a living body and cultured, or induced to differentiate in vitro from pluripotent cells such as pluripotent blood progenitor cells, and cultured. These cells are characterized by being CD122-positive. The inventors of the present invention were the first to discover that CD122 serves as a specific marker for human T / NK progenitor cells in the Examples below. The cultured human T / NK progenitor cells of the present invention may further satisfy the expression condition of one, two, three, four, five, or six surface antigens selected from the group consisting of CD3-negative, CD4-negative, CD8-negative, CD56-negative, CD161-negative, and CD7-positive. In one embodiment, the cultured human T / NK progenitor cells of the present invention are CD122-positive and CD3-negative. In another embodiment, the cultured human T / NK progenitor cells of the present invention are CD122-positive, CD3-negative, and CD4-negative. In yet another embodiment, the cultured human T / NK progenitor cells of the present invention are CD122-positive, CD3-negative, CD4-negative, and CD8-negative. In yet another embodiment, the cultured human T / NK progenitor cells of the present invention are CD122-positive, CD3-negative, CD4-negative, CD8-negative, and CD56-negative. In yet another embodiment, the cultured human T / NK progenitor cells of the present invention are CD122-positive, CD3-negative, CD4-negative, CD8-negative, CD56-negative, and CD161-negative. In yet another embodiment, the cultured human T / NK progenitor cells of the present invention are CD122-positive, CD3-negative, CD4-negative, CD8-negative, CD56-negative, CD161-negative, and CD7-positive. The expression of these surface antigen markers can be examined by well-known techniques, such as a flow cytometer using antibodies against each surface antigen marker, or real-time PCR using primers and probes that detect the expression of each surface antigen marker.

[0029] I. Method for Producing Immune Cells Expressing an Exogenous Receptor There are two embodiments of the method for producing immune cells expressing an exogenous receptor, which is a TCR, a CAR, or a CAAR, according to the present invention, which differ in the step of introducing the exogenous receptor gene.

[0030] Aspect A is a method for producing TCR / CAR / CAAR-expressing immune cells by introducing an exogenous receptor gene into multipotent hematopoietic progenitor cells and then inducing their differentiation into immune cells, and includes the steps of: preparing multipotent hematopoietic progenitor cells from a cell population containing hematopoietic stem cells; preparing multipotent hematopoietic progenitor cells that express the exogenous receptor by introducing a gene encoding the exogenous receptor into the multipotent hematopoietic progenitor cells; and inducing the differentiation of the multipotent hematopoietic receptor-expressing multipotent hematopoietic progenitor cells into immune cells.

[0031] Aspect B is a method for producing TCR / CAR / CAAR-expressing immune cells by inducing the differentiation of multipotent blood progenitor cells into immune cells and then introducing an exogenous receptor gene, and includes the steps of: preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells; inducing the differentiation of the multipotent blood progenitor cells into immune cells; and introducing a gene encoding the exogenous receptor into immune cells.

[0032] Immune cells include T cells, NK cells, macrophages, and B cells.

[0033] When the exogenous receptor is a TCR, introduction of the TCR alone does not function in NK cells, macrophages, and B cells, so it is preferable to induce differentiation of multipotent blood progenitor cells into T cells. However, a system has been developed in which CD3, which forms a complex with the TCR and transmits the TCR signal intracellularly, is introduced into NK cells together with the TCR to induce antigen-specific signal transduction via the TCR (Mensali et al., eBioMed. 40, 106-117 (2019)). Using this technology, TCR can also be made to function in NK cells, macrophages, and B cells. Therefore, the present invention also encompasses the production of NK cells, macrophages, or B cells that express a TCR. In this case, the CD3 gene is also introduced into the cells in combination with the TCR gene.

[0034] When the exogenous receptor is a CAR, the CAR can function in various immune cells such as NK cells, macrophages, and B cells, and can therefore preferably induce differentiation into the various immune cells described above.

[0035] CAAR is a chimeric receptor that utilizes CAR, and like CAR, CAAR can function in various immune cells such as NK cells, macrophages, and B cells. Therefore, even when the exogenous receptor is CAAR, it can be preferably induced to differentiate into the various immune cells mentioned above.

[0036] I-1. Preparation of Multipotent Hematopoietic Progenitor Cells (Aspects A and B) Multipotent hematopoietic progenitor cells are cells that have a more restricted differentiation direction than hematopoietic stem cells, but have the pluripotency to differentiate into multiple cell lineages and the ability to self-replicate. In the present invention, induced leukocyte stem cells (iLS) or T / NK progenitor cells can be preferably used as multipotent hematopoietic progenitor cells.

[0037] A cell population containing hematopoietic stem cells can be obtained from umbilical cord blood, bone marrow aspirate, or peripheral blood, and typically contains hematopoietic stem cells and hematopoietic progenitor cells. Human hematopoietic stem cells are CD34-positive, CD59-positive, CD90 / Thy1-positive, CD38-weakly positive or negative, c-Kit-negative or weakly positive, and Lineage marker-negative (CD34 + CD59 + CD90 / Thy1 + CD38 low / - c-Kit - / low Lin - For example, a cell population containing hematopoietic stem cells (a cell population in which hematopoietic stem cells account for approximately 3% or more, for example 7% or more) can be obtained by separating and collecting CD34+ cells using cell sorting or magnetic beads.

[0038] As will be described later, when TCR / CAR / CAAR-expressing immune cells produced according to the present invention are used to treat tumors or autoimmune diseases, it is also possible to obtain a cell population containing hematopoietic stem cells from umbilical cord blood, which is generally not derived from the patient himself, or from bone marrow fluid or peripheral blood collected from the patient, and prepare multipotent blood progenitor cells derived from the patient's own cells.

[0039] When the multipotent blood progenitor cells are iLS cells, their preparation methods are known as described in Non-Patent Documents 2 and 3 and Patent Document 1. iLS cells can be prepared by culturing a cell population containing hematopoietic stem cells under conditions that induce differentiation into B cells, while inhibiting the function of the intracellular transcription factor E2A (also known as transcription factor 3 (TCF3)).

[0040] Methods for inhibiting E2A function include, for example, enhancing a factor that binds to E2A and inhibits its action on target genes, introducing a dominant-negative mutant of E2A that inactivates its functional domain, and inhibiting the expression or activity of other transcription factors that are under the control of E2A or function cooperatively with E2A. However, a preferred method involves forced expression of the differentiation inhibitor Id (Inhibitor of DNA binding) in hematopoietic stem cells. Id protein is known to inhibit E proteins such as E2A in a dominant-negative manner (Norton, JD et al., Trends Cell Biol. 8, 58-65 (1998); and Sayegh, CE et al., Nat. Immunol. 4, 586-593 (2003)). Forced expression of Id protein in hematopoietic stem cells inhibits E2A's action on target genes through its binding to E2A.

[0041] Any of the four Id factors, Id1 to Id4, which exist in mammals, may be used as the Id factor. Gene sequence information for Id1 to Id4 is registered in databases such as GenBank and is readily available. For example, the sequence information for the human Id1 gene is registered in GenBank under the accession numbers NM_002165 (transcript variant 1) and NM_181353 (transcript variant 2), the sequence information for the human Id2 gene is registered under the accession number NM_002166, the sequence information for the human Id3 gene is registered under the accession number NM_002167, and the sequence information for the human Id4 gene is registered under the accession number NM_001546. Of these, the sequences of the human Id3 gene and protein of NM_002167 used in the Examples below are shown in SEQ ID NOs: 5 and 6 in the Sequence Listing, respectively. DNA encoding Id can be amplified by designing appropriate primers based on this known sequence information and performing RT-PCR using RNA extracted from cells or tissues expressing Id as a template. The resulting DNA can be incorporated into an appropriate expression vector under the control of a suitable promoter (e.g., CMV promoter, SV40 promoter, LTR promoter) that constitutively exerts strong promoter activity in mammalian cells, and then introduced into a cell population containing hematopoietic stem cells to forcibly express the Id protein in the hematopoietic stem cells. Animal viral vectors, such as retroviral, adenoviral, and lentiviral vectors, are preferred expression vectors, but plasmid vectors for mammalian cells can also be used. To sustain the expression of Id factors in cells, the Id-encoding DNA is preferably introduced so that it is integrated into the chromosomes of hematopoietic stem cells. Retroviral and lentiviral vectors, which can integrate transgenes into chromosomes, are preferred. By infecting cells with these viral vectors carrying Id-encoding DNA using methods such as spin infection, the Id-encoding DNA can be integrated into the chromosomes by random integration.However, it is also possible to introduce the gene using a viral vector that can exist stably outside the chromosome, such as a persistent expression Sendai virus vector (e.g., J. Biol. Chem. 282, 27383-27391, 2007).

[0042] Modes of E2A inhibition by forced expression of Id factors include constitutive E2A inhibition, in which E2A is constitutively inhibited in cells, and controlled E2A inhibition, in which E2A is inhibited only under certain conditions. When, in a later step, pluripotent blood progenitor cells after introduction of a TCR gene or a CAR gene are induced to differentiate into B cells, controlled E2A inhibition is preferably performed so that the E2A inhibition can be released. When inducing differentiation into immune cells other than B cells, constitutive E2A inhibition may be used, but controlled E2A inhibition is also preferably used.

[0043] Constitutive E2A inhibition can be achieved by expressing DNA encoding the Id factor under the control of an appropriate promoter that constitutively exerts strong promoter activity in mammalian cells.

[0044] One example of a method for controlled E2A inhibition is the 4-hydroxytamoxifen (4-OHT) system, which utilizes the ligand-binding domain of the human estrogen receptor (HER), as demonstrated in the Examples below. In this system, the ID-ER gene, in which the human estrogen receptor (hER) ligand-binding domain sequence is ligated downstream of DNA encoding Id factor, is transduced into cells, resulting in intracellular expression of the ID-ER protein, in which the hER ligand-binding domain is fused to the C-terminus of Id factor. In the absence of 4-OHT, the expressed ID-ER protein remains in the cytoplasm, whereas in the presence of 4-OHT, it translocates to the nucleus and inhibits the transcription factor E2A (Non-Patent Document 3). Therefore, culturing iLS cells with the addition of 4-OHT to the culture medium allows for maintenance of iLS cells without differentiation. Since 4-OHT is not required for differentiation into immune cells, differentiation can be induced in a 4-OHT-free medium.

[0045] The sequence of positions 370 to 1311 in the nucleotide sequence shown in SEQ ID NO: 1 can be preferably used as the hER ligand-binding domain. This sequence is a mutant hER ligand-binding domain (ERT2) sequence obtained by adding several base substitutions to the ligand-binding domain sequence (positions 1078 to 2019 in SEQ ID NO: 7; the sequence of this 942-nucleotide region is shown in SEQ ID NO: 9) of hER isoform 1 (GenBank Accession No. NM_000125). SEQ ID NO: 1 is the nucleotide sequence of the ID3-ERT2 gene, a preferred example of an ID-ER gene, in which the ERT2 sequence is fused to the full-length cDNA of the human ID3 gene. However, the sequence encoding the hER ligand-binding domain used in the ID-ER gene is not limited to this mutant sequence; a sequence in which 10 or fewer bases are substituted or added in the nucleotide sequence of the ligand-binding domain of hER isoform 1 shown in SEQ ID NO: 9 can also be used as the hER ligand-binding domain sequence. A DNA having a structure in which a hER ligand-binding domain sequence is linked to the 3' end of the DNA encoding the Id factor via an appropriate spacer sequence can be used as the ID-ER gene.

[0046] The ID gene (DNA encoding the Id factor) or the ID-ER gene may be linked to DNA encoding any protein that serves as a marker for gene transfer. After gene transfer, cells expressing the marker protein are separated and collected using a cell sorter from a cell population containing hematopoietic stem cells, thereby obtaining hematopoietic stem cells into which the ID gene or ID-ER gene has been introduced and which express the Id factor.

[0047] After obtaining hematopoietic stem cells in which E2A is inhibited by the expression of Id factors, the cells are cultured under conditions that induce differentiation into B cells. When controlled E2A inhibition is performed, the cells are cultured under E2A-inhibiting conditions (in the presence of 4-OHT in the case of a system that utilizes the ligand-binding domain of the human estrogen receptor) to induce differentiation into B cells.

[0048] Culture under conditions that induce differentiation into B cells refers to culture on feeder cells capable of supporting B cell differentiation, with an appropriate amount of cytokines added to the medium. Examples of feeder cells capable of supporting B cell differentiation include OP9 cells, TSt-4 cells, S17 cells, and MS-5 cells. Examples of cytokines that can be used include IL-7, Flt3-L, and SCF, each at a concentration of approximately 0.1 to 20 ng / mL.

[0049] When the multipotent blood progenitor cells are T / NK progenitor cells (pT / NK cells), their preparation methods are known, as described in Non-Patent Documents 2, 4, and 5. pT / NK cells with the ability to differentiate into T cells and NK cells and the ability to self-renew can be generated by culturing a cell population containing CD34-positive hematopoietic stem cells in the presence of Notch signaling with the addition of an appropriate amount of cytokines. SCF, IL-7, and Flt3-L are preferred cytokines. In the case of mouse cells, pT / NK cells are vigorously expanded by adding SCF, IL-7, and Flt3-L to the medium at concentrations of 10 to 50 ng / ml, particularly a high concentration of 25 to 50 ng / ml, and culturing in the presence of Notch signaling. On the other hand, when mass-producing pT / NK cells from human hematopoietic stem cells, it is preferable to use SCF, IL-7, and Flt3-L at lower concentrations than those mentioned above. Specifically, as shown in Examples A-2-10, B-1, and B-2 below, pT / NK cells can be expanded in large quantities by adding the same cytokines to the medium at low concentrations of 1 ng / mL or more but less than 10 ng / mL and culturing them in the presence of Notch signaling. Furthermore, in the case of human cells, pT / NK cells can be expanded several-fold by further adding TPO or M-CSF to the medium (see Examples B-8 to B-10, etc.). The production of pT / NK cells from human hematopoietic stem cells is described in detail in "II. Method for producing human T / NK progenitor cells."

[0050] Culturing a cell population containing CD34-positive hematopoietic stem cells in the presence of a Notch ligand can stimulate and enhance Notch signaling in hematopoietic stem cells. The definition of the term "culture in the presence of a Notch ligand," the form of the Notch ligand used, the Notch ligand that can be preferably used, and the method of culturing in the presence of a Notch ligand are as described above.

[0051] The human hematopoietic stem cells used in the method may be subjected to a pre-expansion procedure. An example of such an expansion culture method is a method in which a cell population containing hematopoietic stem cells is cultured in a medium containing SCF, TPO, and G-CSF at high concentrations of 100 ng / mL or more, respectively, in the absence of a Notch ligand. By culturing hematopoietic stem cells in the presence of the above-mentioned high concentrations of cytokines in the absence of a Notch ligand, CD34-positive hematopoietic stem cells are expanded. Such expansion culture can be applied to all methods of the present invention that use a cell population containing hematopoietic stem cells. That is, the cell population containing hematopoietic stem cells used in the present invention may be a cell population obtained after CD34-positive hematopoietic stem cells have been expanded by the above-mentioned expansion culture.

[0052] I-2. Introduction of an Exogenous Receptor Gene into Multipotent Hematopoietic Progenitor Cells (Aspect A) In Aspect A, a gene encoding an exogenous receptor (TCR gene, CAR gene, or CAAR gene) is introduced into multipotent hematopoietic progenitor cells such as iLS cells or pT / NK cells to prepare multipotent hematopoietic progenitor cells expressing the receptor. If desired, other genes (e.g., CD3 gene) may be introduced into the multipotent hematopoietic progenitor cells in addition to the exogenous receptor gene.

[0053] When the exogenous receptor is an antigen-specific receptor, the target antigen can be selected depending on the intended use of the TCR / CAR-expressing multipotent hematopoietic progenitor cells or immune cells. When these cells are used for tumor therapy, tumor antigens are selected. Specific examples of tumor antigens include GD2 (a cell surface glycolipid specifically expressed in neuroblastoma), WT1 (a Wilms tumor gene expressed in leukemia and various solid cancers), survivin (a member of the inhibitor of apoptosis (IAP) family expressed in many solid and hematopoietic cancers), MAGE-A3 and MAGE-A4 (expressed in many cancer types, including melanoma, esophageal cancer, non-small cell lung cancer, head and neck cancer, and bladder cancer), tyrosinase (melanoma antigen), gp100 (melanoma antigen), CEA (expressed in colorectal cancer), Melan-A (melanoma antigen), TRP-2 (melanoma antigen), and CDK4 (cyclin-dependent kinase 4). Examples include NY-ESO-1 (expressed frequently in synovial cell sarcoma and also in other tumors), HER2 (expressed in breast cancer, ovarian cancer, etc.), MUC-1 (highly expressed in many cancers including breast cancer, lung cancer, pancreatic cancer, and colorectal cancer), and p53 (expressed in various types of cancer).

[0054] For each tumor antigen, sequence information such as gene sequence and amino acid sequence is publicly known, and antibodies that specifically recognize the tumor antigen are publicly known or can be produced by conventional methods. A CAR gene can be designed and produced using the variable region sequence of an antibody that specifically recognizes the tumor antigen. Regardless of the type of antigen, when expressing a CAR in human-derived cells, it is preferable to use domains derived from human proteins for the intracellular domains of the CAR (costimulatory domain and activation domain).

[0055] TCR genes that recognize a desired antigen can be obtained from antigen-specific killer T cell clones. A desired antigen protein or its CTL epitope peptide is incorporated into antigen-presenting cells such as dendritic cells to prepare antigen-presenting cells that present the CTL epitope peptide on their surface, and these antigen-presenting cells are co-cultured with T cells to bring them into contact with each other, thereby obtaining killer T cell clones specific to the desired antigen.

[0056] Furthermore, when an expression vector carrying a TCR gene that recognizes a desired antigen is already known, the TCR gene can be prepared by PCR amplification from such an expression vector.

[0057] Examples of antigens other than tumor antigens include viral antigens and parasitic antigens. Specific examples of viral antigens include constitutive proteins of viruses such as hepatitis viruses (HBV, HCV, etc.), human papillomavirus, and human immunodeficiency virus, while specific examples of parasitic antigens include malaria parasite proteins. Information on the gene sequences and amino acid sequences of these antigens is also publicly known, so TCR and CAR genes that specifically recognize these antigens can be obtained in the same manner as described above.

[0058] When the exogenous receptor is a CAAR, multipotent hematopoietic progenitor cells or immune cells expressing the CAAR can be used to treat autoimmune diseases. The autoantigen used in the antigen-derived portion of the CAAR can be selected from various autoantigens known to cause autoimmune diseases. Representative examples of autoimmune diseases and autoantigens are listed above, but are not limited to these. For example, the antigen-derived portion of the CAAR can be designed based on DSG3 for the treatment of pemphigus vulgaris, or based on the nicotinic acetylcholine receptor α subunit for the treatment of myasthenia gravis.

[0059] Introduction of exogenous receptor genes or CD3 genes into multipotent hematopoietic progenitor cells can be performed essentially in the same manner as introduction of ID or ID-ER genes into hematopoietic stem cells. When introducing genes into human pT / NK cells using a viral vector, the efficiency of gene transfer can be increased by using the Gibbon-ape leukemia virus (GALV) envelope as the viral envelope. Specifically, a TCR / CAR / CAAR gene-expressing virus carrying a GALV envelope is constructed and then used to infect pT / NK cells by spin infection or other methods. In the present invention, a protein having an amino acid sequence with 90% or more, e.g., 95% or more, or 98% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 16 or 24 can be used as the GAVL envelope. The amino acid sequence shown in SEQ ID NO: 24 is the amino acid sequence encoded by the envelope gene (SEQ ID NO: 23) of the GALV SEATO strain, registered in GenBank under AF055060.1. The amino acid sequence shown in SEQ ID NO: 16 is the amino acid sequence encoded by the synthetic gene GALV-MTR (SEQ ID NO: 15) of the GALV envelope, in which the membrane domain and cytoplasmic domain of the GALV envelope (residues 627-685 in the amino acid sequence shown in SEQ ID NO: 24) are replaced with the membrane domain and cytoplasmic domain of the mouse leukemia virus (MuLV) envelope, thereby increasing the efficiency of insertion of the GALV envelope protein into virus particles (Caeser et al. Nat Commun. 2019 Oct 4;10(1)4543).

[0060] Cells expressing a marker protein linked to a transgene can be separated and recovered using a cell sorter to isolate cells expressing the receptor via the introduction of an exogenous receptor gene. The isolated receptor-expressing cells are cultured and expanded while maintaining their pluripotency, with appropriate changes in medium and feeder cells. When introducing an exogenous receptor gene into iLS cells generated by controlled E2A inhibition, gene introduction, isolation, and expansion are carried out while culturing the iLS cells under E2A inhibition conditions. When using a system in which E2A inhibition is controlled by 4-OHT, receptor gene introduction, isolation, and expansion are carried out using a medium containing 4-OHT.

[0061] I-3. Induction of Differentiation of Exogenous Receptor-Expressing Multipotent Progenitor Cells into Immune Cells (Aspect A) In Aspect A, expanded TCR / CAR / CAAR-expressing multipotent hematopoietic progenitor cells are induced to differentiate into immune cells such as T cells, NK cells, macrophages, or B cells. When iLS cells generated by controlled E2A inhibition are used, it is not necessary to continue E2A inhibition conditions in this step. For example, when using a system in which E2A inhibition is controlled by 4-OHT, a 4-OHT-containing medium may be used at the start of the differentiation induction treatment, but the differentiation induction treatment may then be carried out using a 4-OHT-free medium.

[0062] When the multipotent blood progenitor cells are iLS cells, they can be induced to differentiate into various immune cells such as T cells, NK cells, macrophages, and B cells. On the other hand, pT / NK cells have the ability to differentiate into T cells, NK cells, and myeloid cells (macrophages, etc.), but not into B cells. Therefore, when the multipotent blood progenitor cells are pT / NK cells, they are induced to differentiate into T cells, NK cells, or macrophages, preferably NK cells or macrophages, and particularly preferably NK cells.

[0063] In general, iLS cells are preferably used when producing TCR-expressing T cells (TCR-T cells) and CAR-expressing T cells (CAR-T cells), and pT / NK cells and iLS cells, particularly pT / NK cells, are preferably used when producing NK cells expressing CAR or CAAR (CAR-NK cells, CAAR-NK cells) and macrophages expressing CAR or CAAR (CAR-macrophages, CAAR-macrophages).

[0064] In one embodiment, the TCR / CAR / CAAR-expressing multipotent blood progenitor cells are TCR-expressing iLS cells, which are induced to differentiate into T cells to obtain TCR-T cells.

[0065] In another embodiment, the TCR / CAR / CAAR-expressing multipotent hematopoietic progenitor cells are CAR-expressing iLS cells and are induced to differentiate into T cells, NK cells, macrophages, or B cells to obtain CAR-T cells, CAR-NK cells, CAR-macrophages, or CAR-expressing B cells (CAR-B cells), or into T cells, NK cells, or macrophages to obtain CAR-T cells, CAR-NK cells, or CAR-macrophages. In yet another embodiment, the TCR / CAR / CAAR-expressing multipotent hematopoietic progenitor cells are CAAR-expressing iLS cells and are induced to differentiate into T cells, NK cells, macrophages, or B cells to obtain CAAR-T cells, CAAR-NK cells, CAAR-macrophages, or CAAR-B cells, or into T cells, NK cells, or macrophages to obtain CAAR-T cells, CAAR-NK cells, or CAAR-macrophages.

[0066] In yet another embodiment, the TCR / CAR / CAAR-expressing multipotent blood progenitor cells are TCR / CAR / CAAR-expressing pT / NK cells, and are induced to differentiate into T cells to obtain TCR / CAR / CAAR-T cells. In yet another embodiment, the TCR / CAR / CAAR-expressing multipotent blood progenitor cells are CAR-expressing pT / NK cells, and are induced to differentiate into NK cells to obtain CAR-NK cells. In yet another embodiment, the TCR / CAR / CAAR-expressing multipotent blood progenitor cells are CAAR-expressing pT / NK cells, and are induced to differentiate into NK cells to obtain CAAR-NK cells. In yet another embodiment, the TCR / CAR / CAAR-expressing multipotent blood progenitor cells are CAR-expressing pT / NK cells, and are induced to differentiate into macrophages to obtain CAR-macrophages. In yet another embodiment, the TCR / CAR / CAAR-expressing multipotent blood progenitor cells are CAAR-expressing pT / NK cells, and are induced to differentiate into macrophages to obtain CAAR-macrophages.

[0067] In yet another embodiment, the TCR / CAR / CAAR-expressing multipotent hematopoietic progenitor cells are TCR-expressing iLS cells, which further express CD3. The cells are induced to differentiate into NK cells, macrophages, or B cells to obtain TCR-expressing NK cells, TCR-expressing macrophages, or TCR-expressing B cells (all of which also express CD3), or are induced to differentiate into NK cells or macrophages to obtain TCR-expressing NK cells or TCR-expressing macrophages (all of which also express CD3).

[0068] In yet another embodiment, the TCR / CAR / CAAR-expressing multipotent blood progenitor cells are TCR-expressing pT / NK cells, which further express CD3. The cells are induced to differentiate into NK cells or macrophages to obtain TCR-expressing NK cells or TCR-expressing macrophages.

[0069] Induction of TCR / CAR / CAAR-Expressing Multipotent Hematopoietic Progenitor Cells into T Cells. To induce differentiation of TCR, CAR, or CAAR-expressing multipotent hematopoietic progenitor cells into T cells, first, the cells are cultured in the presence of a Notch ligand, i.e., in a state where Notch signaling is stimulated, by adding an appropriate amount of cytokines to the culture medium. Methods for stimulating Notch signaling are as described above, including culturing on feeder cells expressing Notch ligands (specific examples of such feeder cells are described above) and culturing in a culture vessel whose inner surface is coated with a Notch ligand protein (or a fragment thereof containing at least the extracellular domain). Examples of cytokines that can be used include IL-7 and Flt3-L, each at a concentration of approximately 0.1–10 ng / mL. Culture is continued while appropriately changing the culture medium and, if using feeder cells, replacing them. Approximately three weeks after the start of induction, immature CD4 / CD8 double-positive (DP) T cells are generated.

[0070] TCR stimulation of DP cells can induce differentiation into CD8+ killer T cells (cytotoxic T cells). Methods for TCR stimulation of DP cells include treating DP cells with anti-CD3 and anti-CD28 antibodies, or, if the exogenous receptor is an antigen-specific receptor, co-culturing DP cells with antigen-presenting cells (e.g., dendritic cells) that present the antigen recognized by the introduced antigen-specific receptor. If the introduced exogenous receptor is a CAR or CAAR that utilizes the intracellular signaling domain of the costimulatory receptor molecule derived from 4-IBB, treatment of DP cells with anti-CD3 and anti-4-IBB antibodies can also be used. CD8+ CAR-T cells or CD8+ CAAR-T cells can be generated and expanded by TCR stimulation of DP cells using any of the above methods in a medium containing appropriate cytokines (e.g., IL-7, IL-15, IL-2).

[0071] Induction of TCR / CAR / CAAR-Expressing Multipotent Hematopoietic Progenitor Cells into NK Cells The method for inducing the differentiation of CAR- or CAAR-expressing multipotent hematopoietic progenitor cells into NK cells varies depending on the breadth of differentiation potential of the multipotent hematopoietic progenitor cells. CAR- or CAAR-expressing iLS cells can be generated by culturing them in a medium containing appropriate cytokines (e.g., IL-15 and IL-2) on feeder cells capable of supporting differentiation into B cells or under feeder-free conditions. CAR- or CAAR-expressing pT / NK cells can be generated by culturing them in a medium containing appropriate cytokines (e.g., IL-7, Flt3-L, SCF, IL-15). This culture may or may not contain a Notch ligand. Culture in the absence of a Notch ligand is preferred.

[0072] The induction of differentiation of multipotent blood progenitor cells expressing TCR and CD3 into NK cells can be carried out in the same manner as the induction of differentiation of multipotent blood progenitor cells expressing CAR or CAAR into NK cells.

[0073] The resulting CAR / TCR / CAAR-NK cells have sufficient proliferation potential, but if desired, receptor stimulation can be performed to further enhance their proliferation potential. CAR / TCR / CAAR-NK cells can be receptor-stimulated by, for example, co-culturing the CAR / TCR-NK cells with the K562 leukemia cell line, which expresses membrane-bound IL-21 (memIL-21) and 4-1BB ligand (41BBL), or by culturing the CAR / TCR / CAAR-NK cells in the presence of cytokines such as IL-12, IL-15, IL-18, IL-21, and IL-2.

[0074] Induction of TCR / CAR / CAAR-Expressing Multipotent Hematopoietic Progenitor Cells into Macrophages When inducing the differentiation of CAR- or CAAR-expressing multipotent hematopoietic progenitor cells into macrophages, macrophages expressing CAR or CAAR (CAR / CAAR-macrophages) can be generated by culturing them on OP9 cells in a medium containing appropriate cytokines (e.g., SCF, M-CSF, IL-3, and IL-6). Induction of the differentiation of TCR- and CD3-expressing multipotent hematopoietic progenitor cells into macrophages can also be carried out in the same manner as inducing the differentiation of CAR- or CAAR-expressing multipotent hematopoietic progenitor cells into macrophages.

[0075] The generated CAR / TCR / CAAR-macrophages have sufficient proliferation ability, but if desired, their proliferation ability can be further enhanced by receptor stimulation. CAR / TCR / CAAR-macrophages can be receptor-stimulated by, for example, culturing them in the presence of M-CSF or GM-CSF.

[0076] Induction of TCR / CAR / CAAR-Expressing Multipotent Hematopoietic Progenitor Cells into B Cells Induction of differentiation of CAR- or CAAR-expressing multipotent hematopoietic progenitor cells into B cells can be achieved by culturing them on OP9 cells in a medium containing appropriate cytokines (e.g., IL-7 and SCF) to generate CAR / CAAR-B cells. Induction of differentiation of TCR- and CD3-expressing multipotent hematopoietic progenitor cells into B cells can also be achieved in the same manner as induction of differentiation of CAR- or CAAR-expressing multipotent hematopoietic progenitor cells into B cells.

[0077] I-4. Induction of Differentiation of Multipotent Hematopoietic Progenitor Cells into Immune Cells (Aspect B) In Aspect B, the prepared multipotent hematopoietic progenitor cells are induced to differentiate into immune cells. This differentiation can be performed essentially by the same method as in I-3. Similarly, in Aspect B, iLS cells can be induced to differentiate into various immune cells such as T cells, NK cells, macrophages, and B cells; pT / NK cells are induced to differentiate into T cells, NK cells, or macrophages, preferably NK cells or macrophages, and particularly preferably NK cells; iLS cells are preferably used to produce TCR-expressing T cells (TCR-T cells), CAR-expressing T cells (CAR-T cells), and CAAR-expressing T cells (CAAR-T cells); and pT / NK and iLS cells, particularly pT / NK cells, are preferably used to produce CAR- or CAAR-expressing NK cells (CAR-NK cells, CAAR-NK cells) and CAR- or CAAR-expressing macrophages (CAR-macrophages, CAAR-macrophages).

[0078] In one embodiment, the multipotent blood progenitor cells are iLS cells and are induced to differentiate into T cells. In the next step, a TCR gene is introduced. In another embodiment, the multipotent blood progenitor cells are iLS cells and are induced to differentiate into T cells, NK cells, macrophages, or B cells, or are induced to differentiate into T cells, NK cells, or macrophages. In the next step, a CAR gene is introduced. In yet another embodiment, the multipotent blood progenitor cells are iLS cells and are induced to differentiate into T cells, NK cells, macrophages, or B cells, or are induced to differentiate into T cells, NK cells, or macrophages. In the next step, a CAAR gene is introduced.

[0079] In yet another embodiment, the multipotent blood progenitor cells are pT / NK cells and are induced to differentiate into T cells. In the next step, a TCR gene is introduced. In yet another embodiment, the multipotent blood progenitor cells are pT / NK cells and are induced to differentiate into NK cells. In the next step, a CAR gene is introduced. In yet another embodiment, the multipotent blood progenitor cells are pT / NK cells and are induced to differentiate into NK cells. In the next step, a CAAR gene is introduced. In yet another embodiment, the multipotent blood progenitor cells are pT / NK cells and are induced to differentiate into macrophages. In the next step, a CAR gene is introduced. In yet another embodiment, the multipotent blood progenitor cells are pT / NK cells and are induced to differentiate into macrophages. In the next step, a CAAR gene is introduced.

[0080] In yet another embodiment, the multipotent blood progenitor cells are iLS cells and are induced to differentiate into NK cells, macrophages, or B cells, or are induced to differentiate into NK cells or macrophages. In the next step, a TCR gene is introduced, and a CD3 gene is also introduced.

[0081] In yet another embodiment, the multipotent blood progenitor cells are pT / NK cells, and are induced to differentiate into NK cells or macrophages. In the next step, a TCR gene is introduced, and a CD3 gene is also introduced.

[0082] I-5. Introduction of an Exogenous Receptor Gene into Immune Cells (Aspect B) When introducing a TCR, CAR, or CAAR into immune cells, receptor stimulation appropriate to the type of immune cell (referred to as TCR stimulation in the case of T cells) is required, and the exogenous receptor gene is introduced into the immune cells after receptor stimulation. Below, a general method for stimulating cells before gene introduction is described, but stimulation methods appropriate to the type of cell are well known in the art, and other methods can also be used. For the introduction of an exogenous receptor gene into cells, an animal virus vector such as a retrovirus vector or a lentivirus vector can be preferably used, as explained in the method for preparing iLS cells in I-1. If desired, the CD3 gene may also be introduced simultaneously with the exogenous receptor gene.

[0083] When introducing a TCR / CAR / CAAR gene into T cells, T cells are stimulated with TCR using an anti-CD3 antibody (anti-CD28 antibody may also be added) and IL-2 to proliferate, and then the TCR / CAR / CAAR gene is introduced using a viral vector by spin-infection or other methods.

[0084] When introducing CAR / CAAR genes or TCR genes and CD3 genes into NK cells, NK cells are stimulated by receptor stimulation and proliferation by co-culturing them with K562 leukemia cells expressing membrane-bound IL-21 (memIL-21) and 4-1BB ligand (41BBL) in the presence of appropriate cytokines (IL-2 alone or IL-2, IL-15, and IL-21). Genes are then introduced into the NK cells by spin-infection or other methods using a viral vector.

[0085] When introducing the CAR / CAAR gene, or the TCR gene and the CD3 gene into macrophages, the macrophages are receptor stimulated with M-CSF or GM-CSF to proliferate, and then the genes are introduced using a viral vector by spin-infection or other methods.

[0086] When introducing the CAR / CAAR gene, or the TCR gene and the CD3 gene into B cells, the cells are receptor-stimulated with cytokines such as IL-7 and SCF, or CD40 ligand, depending on the differentiation stage, to allow proliferation, and then the genes are introduced using a viral vector by spin-infection or other methods.

[0087] After the exogenous receptor gene is introduced, receptor stimulation may be continued for a certain period (several days to several weeks).

[0088] When the exogenous receptor is an antigen-specific receptor, preferred examples of the antigen recognized by the receptor, specific examples of autoantigens used in the antigen-derived portion when the exogenous receptor is a CAAR, methods for designing a CAR gene or CAAR gene, and methods for preparing a TCR gene are as described in I-2.

[0089] II. Method for Producing Human T / NK Precursor Cells The present invention provides a novel method for producing human T / NK progenitor cells that achieves improved expansion efficiency in the expansion of human T / NK progenitor cells compared to the method described in Non-Patent Document 2, which was applied to mouse cells; a method for producing T cells or NK cells that includes a step of inducing differentiation of T / NK precursor cells produced by the novel production method into T cells or NK cells; and a method for producing T cells or NK cells that express an exogenous receptor, which combines the novel production method with the step of expressing the exogenous receptor described above.

[0090] The method for producing human T / NK progenitor cells (human pT / NK cell production method) of the present invention comprises culturing a cell population containing human hematopoietic stem cells in a medium containing SCF, Flt3-L, and IL-7 at concentrations of 1 ng / mL or more and less than 10 ng / mL, respectively, in the presence of a Notch ligand (hereinafter, the medium used in this step is referred to as the "human pT / NK cell induction medium"). Unlike in mice, the human pT / NK cell induction medium employs the aforementioned low concentrations of SCF, Flt3-L, and IL-7, thereby enabling the generation and expansion of large numbers of human pT / NK cells. Furthermore, the addition of at least one of TPO and M-CSF at a predetermined concentration to the human pT / NK cell induction medium can further increase the production of human pT / NK cells several-fold.

[0091] The human cell population containing human hematopoietic stem cells used in the method for producing human pT / NK cells can be as described in I-1. The human cell population may be a cell population after the above-mentioned expansion culture. The culture in the presence of a Notch ligand is also as described above, and the Notch ligand and culture conditions described in I-1 can be appropriately used. As the Notch ligand, a recombinant protein may be used, or feeder cells expressing a Notch ligand may be used.

[0092] The concentrations of SCF, Flt3-L, and IL-7 contained in the human pT / NK cell induction medium can be appropriately selected from the range of 1 ng / mL or more and less than 10 ng / mL. All three may be at the same concentration, or some or all of the three may have different concentrations within the above range. Preferably, each concentration is appropriately selected from the range of 3 to 7 ng / mL, more preferably from the range of 4 to 6 ng / mL, and even more preferably about 5 ng / mL for each.

[0093] The concentration of TPO contained in the human pT / NK cell induction medium can be appropriately selected from the range of 1 to 100 ng / mL, and may be, for example, 1 to 80 ng / mL, 1 to 70 ng / mL, 1 to 60 ng / mL, 1 to 55 ng / mL, 1 to 50 ng / mL, 5 to 80 ng / mL, 5 to 70 ng / mL, 5 to 60 ng / mL, 5 to 55 ng / mL, 5 to 50 ng / mL, 10 to 80 ng / mL, 10 to 70 ng / mL, 10 to 60 ng / mL, 10 to 55 ng / mL, or 10 to 50 ng / mL.

[0094] The concentration of M-CSF contained in the human pT / NK cell induction medium can be appropriately selected from the range of 1 to 100 ng / mL, and may be, for example, 1 to 50 ng / mL, 1 to 40 ng / mL, 1 to 30 ng / mL, 5 to 50 ng / mL, 5 to 40 ng / mL, 5 to 30 ng / mL, 5 to 25 ng / mL, 7 to 50 ng / mL, 7 to 40 ng / mL, 7 to 30 ng / mL, or 7 to 25 ng / mL.

[0095] The human pT / NK cell induction medium may contain either TPO or M-CSF alone or both. Particularly preferred examples of the medium include a medium containing SCF, Flt3-L, and IL-7 at concentrations of 4 to 6 ng / mL or 5 ng / mL, respectively, and TPO at concentrations of 10 to 55 ng / mL or 10 to 50 ng / mL.

[0096] The medium used in the method of the present invention is not particularly limited and may be any medium used for ordinary cell culture or a medium prepared for the culture of hematopoietic cells. Representative media used in ordinary cell culture include Iscove's Modified Dulbecco's Medium (IMDM), alpha Modified Eagle's Minimum Essential Medium (αMEM), Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, McCoy's 5A Medium, Eagle's Minimum Essential Medium (EMEM), and RPMI 1640 medium. Representative serum-free media suitable for culturing hematopoietic cells include StemPro34 (Invitrogen), Stemline (Sigma-Aldrich), Stemline II (Sigma-Aldrich), X-VIVO 10 (Lonza), X-VIVO 15 (Lonza), X-VIVO 20 (Lonza), HPGM (Lonza), StemSpan H3000 (Stem Cell Technology), StemSpanSFEM (Stem Cell Technology), and QBSF-60 (Quality Biologicals). Depending on the purpose, culture may be performed under serum-free conditions, or may be performed under conditions supplemented with serum such as FBS or a serum substitute such as Knockout Serum Replacement (Gibco).

[0097] The culture period is not particularly limited, but is, for example, 7 to 56 days (preferably 28 to 42 days), and the medium is preferably replaced with a fresh medium at intervals of 1 to 7 days (preferably 3 to 4 days).

[0098] Other culture conditions can be appropriately selected from conditions employed in ordinary cell culture. The culture temperature is, for example, 35 to 39°C, and the culture can be performed in the presence of 0 to 10% CO2. In the method for producing human pT / NK cells, the culture in the presence of a Notch ligand may be performed using feeder cells that express a Notch ligand, or may be performed using a culture vessel whose inner surface is coated with a Notch ligand protein or the like.

[0099] The method for producing human NK cells according to the present invention comprises the steps of producing human T / NK precursor cells by the human pT / NK cell production method, and inducing the differentiation of human NK cells by culturing the produced human T / NK precursor cells in a medium containing IL-7, Flt3-L, SCF, and IL-15 in the absence of a Notch ligand. As described in Example B-2 below, in the differentiation induction of human NK cells from human T / NK precursor cells, culturing in a medium containing the above four cytokines in the absence of a Notch ligand without using feeder cells expressing a Notch ligand allows the production of approximately 12 to 30 times more NK cells than when cultured in the presence of a Notch ligand.

[0100] IL-7, Flt3-L, SCF, and IL-15 can each be used at about 1 to 50 ng / ml, for example, about 5 to 30 ng / ml. For example, IL-15 and Flt3-L can each be used at about 1 to 30 ng / ml or about 5 to 20 ng / ml, and SCF and IL-15 can each be used at about 5 to 50 ng / ml or about 10 to 40 ng / ml. By performing the above feeder-free culture for about 2 to 4 weeks, with appropriate medium changes, the production of NK cells can be significantly improved as described above.

[0101] Human NK cells obtained by the above-mentioned method for producing human pT / NK cells maintain a juvenile state and exhibit a characteristic surface antigen profile. The present invention also provides cultured human NK cells characterized by such surface antigens.

[0102] The cultured human NK cells provided by the present invention are characterized by their immaturity and lower level of exhaustion than human cord blood NK cells isolated from human umbilical cord blood, due to lower expression of CD16 and KIR (killer cell immunoglobulin-like receptor), which are indicators of maturity, and lower expression of the exhaustion marker TIGIT (T cell immunoreceptor with Ig and ITIM domains; NCBI Gene ID: 201633, NCBI Reference Sequence: NM_173799.4). Human cord blood NK cells are known in the art to be immature compared to other human NK cells, such as human peripheral blood NK cells. However, as shown in Example B-11 below, human NK cells induced from human T / NK precursor cells produced by the method for producing human pT / NK cells of the present invention are even immature than human cord blood NK cells. Furthermore, the cultured human NK cells of the present invention are characterized by lower expression of the exhaustion marker TIGIT, which is an indicator of NK cell exhaustion, compared to human cord blood NK cells, resulting in a lower level of exhaustion. Thus, the cultured human NK cells of the present invention have characteristics different from those of human NK cells naturally occurring in the human body. The expression levels of CD16, KIR, and TIGIT in the cultured human NK cells of the present invention may be independently 1 / 2 or less, 1 / 3 or less, 1 / 4 or less, 1 / 5 or less, 1 / 6 or less, 1 / 7 or less, or 1 / 8 or less of the expression levels in human cord blood NK cells.

[0103] KIRs include KIR2DL2 (killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2; NCBI Gene ID: 3803, NCBI Reference Sequence: NM_014219.3) and KIR2DL3 (killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 3; NCBI Gene ID: 3804, NCBI Reference Sequence: NM_015868.3). Expression of KIR in the cultured human NK cells of the present invention may be either KIR2DL2 expression, KIR2DL3 expression, or both.

[0104] The cultured human NK cells of the present invention may further be characterized by higher expression of one, two, three, four, or five genes selected from CCR3, CCR6, CCR7, CCR8, and CCR9 compared to human cord blood NK cells and human peripheral blood NK cells. The expression of surface antigen markers CD3, CD4, CD8, CD56, and CD161 on the cultured human NK cells of the present invention is similar to that of other human NK cells, and the cells are CD3-negative, CD4-negative, CD8-negative, CD56-positive, and CD161-positive.

[0105] The expression of these surface antigen markers can be examined by well-known techniques such as a flow cytometer using antibodies against each surface antigen marker, or real-time PCR using primers and probes that detect the expression of each surface antigen marker.

[0106] III. Production of Immune Cell Therapy Agents for Tumors or Autoimmune Diseases The methods for producing immune cell therapy agents for tumors or autoimmune diseases according to the present invention include Production Method 1, which produces CD8-positive cytotoxic TCR-T, CAR-T, or CAAR-T cells as immune cell therapy agents, and Production Method 2, which produces CAR-NK cells, CAAR-NK cells, CAR-macrophages, or CAAR-macrophages as immune cell therapy agents. Production Method 1 and Production Method 2 each include two methods: a method in which cells expressing each receptor are produced by the method of Aspect A above, and a method in which cells expressing each receptor are produced by the method of Aspect B above.

[0107] Production Method 1A Production Method 1A is a method for producing CD8-positive cytotoxic TCR-T, CAR-T, or CAAR-T cells by the method for producing TCR / CAR / CAAR-expressing immune cells of Aspect A, which involves introducing an exogenous receptor gene into multipotent hematopoietic progenitor cells and then inducing their differentiation into immune cells, and using the produced CD8-positive cytotoxic TCR-T, CAR-T, or CAAR-T cells as an immune cell therapy for tumors or autoimmune diseases. The method comprises the following steps 1A-1 to 1A-5: Step 1A-1: preparing multipotent hematopoietic progenitor cells from a cell population containing hematopoietic stem cells; Step 1A-2: preparing multipotent hematopoietic progenitor cells expressing the exogenous receptor by introducing a gene encoding an exogenous receptor, which is a TCR or CAR that specifically recognizes a CAAR or a tumor antigen, into the multipotent hematopoietic progenitor cells; and Step 1A-3: preparing T cells expressing the exogenous receptor (TCR-T, CAR-T, or CAAR-T cells) by inducing the differentiation of the multipotent hematopoietic receptor-expressing multipotent hematopoietic progenitor cells into T cells. Step 1A-4 is to stimulate TCR in TCR-T, CAR-T, or CAAR-T cells and induce their differentiation into CD8-positive cytotoxic TCR-T, CAR-T, or CAAR-T cells. Step 1A-5 is to prepare CD8-positive cytotoxic TCR-T or CAR-T cells as an immune cell therapy agent for tumors expressing the tumor antigen, or prepare CD8-positive cytotoxic CAAR-T cells as an immune cell therapy agent for autoimmune diseases.

[0108] Production Method 1B Production Method 1B is a method for producing CD8-positive cytotoxic TCR-T, CAR-T, or CAAR-T cells by the method for producing TCR / CAR / CAAR-expressing immune cells of Aspect B, in which multipotent hematopoietic progenitor cells are induced to differentiate into immune cells and then an exogenous receptor gene is introduced, and using the produced CD8-positive cytotoxic TCR-T, CAR-T, or CAAR-T cells as an immune cell therapy agent for tumors or autoimmune diseases. It comprises the following steps 1B-1 to 1B-4: step 1B-1 of preparing multipotent hematopoietic progenitor cells from a cell population containing hematopoietic stem cells; step 1B-2 of inducing the differentiation of the multipotent hematopoietic progenitor cells into T cells; and step 1B-3 of preparing CD8-positive cytotoxic TCR-T, CAR-T, or CAAR-T cells by TCR-stimulating T cells and then introducing a gene encoding an exogenous receptor, which is a TCR or CAR that specifically recognizes a CAAR or tumor antigen. Step 1B-4, in which CD8-positive cytotoxic TCR-T or CAR-T cells are prepared as an immune cell therapy agent for tumors expressing the tumor antigen, or CD8-positive cytotoxic CAAR-T cells are prepared as an immune cell therapy agent for autoimmune diseases.

[0109] Production Method 2A Production Method 2A is a method for producing CAR-NK cells, CAAR-NK cells, CAR-macrophages, or CAAR-macrophages by the method for producing TCR / CAR / CAAR-expressing immune cells of Aspect A, which involves introducing an exogenous receptor gene into multipotent hematopoietic progenitor cells and then inducing their differentiation into immune cells, and using the cells as an immune cell therapy agent for tumors or autoimmune diseases, and includes the following steps 2A-1 to 2A-4: step 2A-1 of preparing multipotent hematopoietic progenitor cells from a cell population containing hematopoietic stem cells; and step 2A-2 of preparing multipotent hematopoietic progenitor cells that express a chimeric receptor by introducing a gene encoding a chimeric receptor that is a CAR that specifically recognizes a CAAR or a tumor antigen into the multipotent hematopoietic progenitor cells. Step 2A-3 is to prepare NK cells (CAR-NK or CAAR-NK cells) or macrophages (CAR-macrophages or CAAR-macrophages) expressing the chimeric receptor by inducing differentiation of multipotent blood progenitor cells expressing the chimeric receptor into NK cells or macrophages. Step 2A-4 is to prepare CAR-NK cells or CAR-macrophages as an immune cell therapy agent for tumors expressing the tumor antigen, or prepare CAAR-NK cells or CAAR-macrophages as an immune cell therapy agent for autoimmune diseases.

[0110] Because CAR / CAAR-NK cells and CAR / CAAR-macrophages have sufficient proliferation ability even before receptor stimulation, they can be prepared as immune cell therapy agents without receptor stimulation and administered to patients. To further activate them and enhance their proliferation ability, a step of receptor-stimulating the prepared CAR / CAAR-NK cells or CAR / CAAR-macrophages may be carried out after step 2A-3. In this case, in step 2A-4, the CAR / CAAR-NK cells or CAR / CAAR-macrophages after receptor stimulation are prepared as immune cell therapy agents.

[0111] Production Method 2B Production Method 2B is a method for producing CAR-NK cells, CAAR-NK cells, CAR-macrophages, or CAAR-macrophages by the method for producing TCR / CAR / CAAR-expressing immune cells of Aspect B, in which multipotent hematopoietic progenitor cells are induced to differentiate into immune cells and then an exogenous receptor gene is introduced, and using the produced CAR-NK cells, CAAR-NK cells, CAR-macrophages, or CAAR-macrophages as an immune cell therapy for tumors or autoimmune diseases. The method comprises the following steps 2B-1 to 2B-4: step 2B-1 of preparing multipotent hematopoietic progenitor cells from a cell population containing hematopoietic stem cells; step 2B-2 of inducing the differentiation of multipotent hematopoietic progenitor cells into NK cells or macrophages; and step 2B-3 of receptor-stimulating NK cells or macrophages and then introducing a gene encoding a chimeric receptor that is a CAR that specifically recognizes a CAAR or a tumor antigen, thereby preparing NK cells (CAR-NK or CAAR-NK cells) or macrophages (CAR-macrophages or CAAR-macrophages) that express the chimeric receptor. Step 2B-4, in which CAR-NK cells or CAR-macrophages are prepared as an immune cell therapy agent for tumors expressing the tumor antigen, or CAAR-NK cells or CAAR-macrophages are prepared as an immune cell therapy agent for autoimmune diseases.

[0112] In production method 2B, cells undergo receptor stimulation during CAR gene introduction, and therefore, the CAR / CAAR-NK cells or CAR / CAAR-macrophages obtained in step 2B-3 already have high proliferation potential and can be preferably prepared into an immune cell therapy agent without further receptor stimulation.

[0113] Steps 1A-1 to 1A-4 of Production Method 1A, and steps 2A-1 to 2A-3 of Production Method 2A, and the step of receptor-stimulating CAR / CAAR-NK cells or CAR / CAAR-macrophages can be carried out as described in I-1 to I-3.

[0114] Steps 1B-1 to 1B-3 of Production Method 1B and steps 2B-1 to 2B-3 of Production Method 2B can be carried out as described in I-1, I-4, and I-5.

[0115] The final steps of each production method, steps 1A-5, 1B-4, 2A-4, and 2B-4, involve preparing (formulating) cells expressing an exogenous receptor or chimeric receptor as an immune cell therapy agent for tumors expressing a tumor antigen specifically recognized by the receptor (when the receptor is a TCR or CAR) or as an immune cell therapy agent for autoimmune diseases (when the receptor is a CAAR). Receptor-expressing cells are generally administered to patients with tumors or autoimmune diseases intravenously, intraperitoneally, or intratumorally (in the case of tumor patients). Therefore, they can be formulated as intravenous infusions, drip infusions, injections, or the like, containing the cells in a medium suitable for these administration routes. Examples of the medium that can be used include an aqueous medium (e.g., highly purified sterile water) containing approximately 2.5-5% human serum albumin, approximately 0.2-0.5% sodium chloride, and approximately 3-8% dimethyl sulfoxide as a cryoprotectant. The number of receptor-expressing cells in a single dose of formulation (e.g., one bag of intravenous infusion) can be appropriately determined depending on the type of cells, the type of tumor, etc., but for example, the number of viable cells is 1 x 10 5 ~1 x 10 9 It can be about one.

[0116] The above-described production method of the present invention can produce immune cell therapies for tumors containing TCR-T cells, for tumors containing CAR-T cells, for tumors containing CAR-NK cells, for tumors containing CAR-macrophages, for autoimmune diseases containing CAAR-NK cells, and for autoimmune diseases containing CAAR-macrophages. Each of these cellular immunotherapies can be used alone to treat tumors or autoimmune diseases. Alternatively, an immune cell therapy for tumors containing CAR-macrophages can be administered to a tumor patient in combination with one or more immune cell therapies including TCR-T cells, CAR-T cells, or CAR-NK cells. When multiple immune cell therapies are combined, the exogenous receptors expressed by the immune cells of each immune cell therapy (in this case, the exogenous receptors are antigen-specific receptors) may all recognize the same tumor antigen, or some or all of the receptors may recognize different tumor antigens. The term "administered in combination" refers to administering multiple agents to a patient simultaneously, sequentially, or separately. "Administering sequentially" refers to administering the next agent immediately after the completion of administration of one agent. "Administering separately" refers to administering multiple agents at intervals, for example, at intervals of several hours or more on the same day, or on different days during a course of treatment. When administering multiple immune cell therapy agents in combination, it is preferable to administer them to the patient sequentially or separately.

[0117] According to the present invention, it is possible to produce immune cell therapy agents effective against various tumors expressing tumor antigens, as well as immune cell therapy agents effective against autoimmune diseases for which autoantigens have been identified. Examples of tumors expressing tumor antigens are as described above. Immune cell therapy agents can be produced for various solid cancers, including neuroblastoma, melanoma, esophageal cancer, lung cancer (e.g., non-small cell lung cancer), head and neck cancer, bladder cancer, colorectal cancer, synovial cell sarcoma, breast cancer, ovarian cancer, and pancreatic cancer, as well as various blood cancers, including leukemia. Examples of autoimmune diseases and autoantigens are also as described above. Immune cell therapy agents can be produced for various autoimmune diseases, including pemphigus vulgaris and myasthenia gravis. Furthermore, as described above, the present invention also allows the production of immune cell therapy agents effective against infectious diseases.

[0118] Patients to be treated with immune cell therapy agents are primarily humans, but various animals bearing tumors, including other mammals, can also be treated.

[0119] Treatment of tumors or autoimmune diseases using these immune cell therapy agents (various TCR / CAR / CAAR-expressing immune cells or multipotent blood progenitor cells) is further described below in "III. Treatment of tumors or autoimmune diseases."

[0120] IV. Treatment of Tumors or Autoimmune Diseases The method for treating tumors or autoimmune diseases according to the present invention is a method for treating tumors or autoimmune diseases using the immune cell therapy agent produced as described above, and has the following aspects.

[0121] Treatment Method 1a Treatment Method 1a is a method for treating tumors or autoimmune diseases by producing CD8-positive cytotoxic TCR-T, CAR-T, or CAAR-T cells by the method for producing TCR / CAR / CAAR-expressing immune cells of Aspect A, which involves introducing an exogenous receptor gene into multipotent hematopoietic progenitor cells and then inducing their differentiation into immune cells, and administering the produced CD8-positive cytotoxic TCR-T, CAR-T, or CAAR-T cells to a patient. The method comprises the following steps 1a-1 to 1a-5: Step 1a-1: preparing multipotent hematopoietic progenitor cells from a cell population containing hematopoietic stem cells; Step 1a-2: preparing multipotent hematopoietic progenitor cells expressing an exogenous receptor by introducing a gene encoding an exogenous receptor that is a TCR or CAR that specifically recognizes a CAAR or a tumor antigen into the multipotent hematopoietic progenitor cells; and Step 1a-3: preparing TCR-T, CAR-T, or CAAR-T cells, which are T cells expressing the exogenous receptor, by inducing the differentiation of the multipotent hematopoietic receptor-expressing multipotent hematopoietic progenitor cells into T cells. Step 1a-4 is TCR stimulation of TCR-T, CAR-T, or CAAR-T cells to induce their differentiation into CD8-positive cytotoxic TCR-T, CAR-T, or CAAR-T cells. Step 1a-5 is administration of CD8-positive cytotoxic TCR-T or CAR-T cells to a patient having a tumor expressing the tumor antigen, or administration of CD8-positive cytotoxic CAAR-T cells to a patient with an autoimmune disease.

[0122] Treatment Method 1b Treatment Method 1b is a method for treating tumors or autoimmune diseases by producing CD8-positive cytotoxic TCR-T, CAR-T, or CAAR-T cells by the method for producing TCR / CAR / CAAR-expressing immune cells of Aspect B, in which multipotent hematopoietic progenitor cells are induced to differentiate into immune cells and then an exogenous receptor gene is introduced, and administering the produced CD8-positive cytotoxic TCR-T, CAR-T, or CAAR-T cells to a patient, and comprises the following steps 1b-1 to 1b-4: step 1b-1 of preparing multipotent hematopoietic progenitor cells from a cell population containing hematopoietic stem cells; step 1b-2 of inducing the differentiation of the multipotent hematopoietic progenitor cells into T cells; and step 1b-3 of preparing CD8-positive cytotoxic TCR-T, CAR-T, or CAAR-T cells by TCR-stimulating T cells and then introducing a gene encoding an exogenous receptor, which is a TCR or CAR that specifically recognizes a CAAR or tumor antigen. Step 1b-4 is administering CD8-positive cytotoxic TCR-T or CAR-T cells to a patient having a tumor expressing the tumor antigen, or administering CD8-positive cytotoxic CAAR-T cells to a patient with an autoimmune disease.

[0123] Treatment Method 2a Treatment Method 2a is a method for treating tumors or autoimmune diseases by producing CAR-NK cells, CAAR-NK cells, CAR-macrophages, or CAAR-macrophages by the method for producing TCR / CAR / CAAR-expressing immune cells of Aspect A, which involves introducing an exogenous receptor gene into multipotent hematopoietic progenitor cells and then inducing their differentiation into immune cells, and administering the produced cells to a patient. The method comprises the following steps 2a-1 to 2a-4: step 2a-1 of preparing multipotent hematopoietic progenitor cells from a cell population containing hematopoietic stem cells; step 2a-2 of preparing multipotent hematopoietic progenitor cells expressing a chimeric receptor by introducing a gene encoding a chimeric receptor that is a CAR that specifically recognizes a CAAR or a tumor antigen into the multipotent hematopoietic progenitor cells; and step 2a-3 of preparing chimeric receptor-expressing immune cells that are CAR-NK cells, CAAR-NK cells, CAR-macrophages, or CAAR-macrophages by inducing the differentiation of the chimeric receptor-expressing multipotent hematopoietic progenitor cells into NK cells or macrophages. Step 2a-4 of administering CAR-NK cells or CAR-macrophages to a patient having a tumor that expresses the tumor antigen, or administering CAAR-NK cells or CAAR-macrophages to a patient with an autoimmune disease.

[0124] As explained in Production Method 2A in III., CAR-NK cells, CAAR-NK cells, CAR-macrophages, and CAAR-macrophages have sufficient proliferation ability even before receptor stimulation, and therefore, these chimeric receptor-expressing immune cells can be administered to patients without receptor stimulation. Alternatively, after step 2a-3, the prepared chimeric receptor-expressing immune cells may be receptor-stimulated to further enhance their proliferation ability before being administered to patients.

[0125] Treatment Method 2b Treatment Method 2b is a method for treating tumors or autoimmune diseases by producing CAR-NK cells, CAAR-NK cells, CAR-macrophages, or CAAR-macrophages by the method for producing TCR / CAR / CAAR-expressing immune cells of Aspect B, in which multipotent blood progenitor cells are induced to differentiate into immune cells and then an exogenous receptor gene is introduced, and administering the produced CAR-NK cells, CAAR-NK cells, CAR-macrophages, or CAAR-macrophages to a patient, and comprises the following steps 2b-1 to 2b-4: step 2b-1 of preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells; step 2b-2 of inducing the differentiation of the multipotent blood progenitor cells into NK cells or macrophages; and step 2b-3 of preparing CAR-NK cells, CAAR-NK cells, CAR-macrophages, or CAAR-macrophages by receptor stimulation of NK cells or macrophages and then introducing a gene encoding a chimeric receptor that is a CAR that specifically recognizes a CAAR or a tumor antigen. Step 2b-4 of administering CAR-NK cells or CAR-macrophages to a patient having a tumor that expresses the tumor antigen, or administering CAAR-NK cells or CAAR-macrophages to a patient with an autoimmune disease.

[0126] Treatment Method 3 Treatment Method 3 is a method for treating tumors by administering a combination of CAR-macrophages and CAR-T cells to a patient, and includes the following steps 3-1 to 3-3: Step 3-1: Preparing CAR-macrophages by the following (1) or (2): (1) preparing multipotent hematopoietic progenitor cells from a cell population containing hematopoietic stem cells, introducing a gene encoding a CAR that specifically recognizes a tumor antigen into the multipotent hematopoietic progenitor cells to prepare multipotent hematopoietic progenitor cells that express the CAR, and then inducing the differentiation of the CAR-expressing multipotent hematopoietic progenitor cells into macrophages; (2) preparing artificial leukocyte stem cells or T / NK progenitor cells from a cell population containing hematopoietic stem cells, inducing their differentiation into macrophages to stimulate receptors, and then introducing the gene encoding the CAR. Step 3-2 of preparing CAR-T cells by the following (3) or (4): (3) a step of preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells, introducing a gene encoding the CAR into the multipotent blood progenitor cells to prepare multipotent blood progenitor cells that express the CAR, inducing the differentiation of the CAR-expressing multipotent blood progenitor cells into T cells, and then stimulating the TCR to induce differentiation into CD8-positive cytotoxic TCR-T or CAR-T cells; (4) a step of preparing artificial leukocyte stem cells or T / NK progenitor cells from a cell population containing hematopoietic stem cells, inducing their differentiation into T cells, stimulating the TCR, and then introducing a gene encoding the CAR. Step 3-3 of combining CAR-macrophages and CAR-T cells and administering them to a patient with a tumor that expresses the tumor antigen.

[0127] Treatment Method 4 Treatment Method 4 is a method for treating tumors by administering a combination of CAR-macrophages and CAR-NK cells to a patient, and includes the following steps 4-1 to 4-3: Step 4-1: Preparing CAR-macrophages by the following steps (1) or (2): (1) preparing multipotent hematopoietic progenitor cells from a cell population containing hematopoietic stem cells, introducing a gene encoding a CAR that specifically recognizes a tumor antigen into the multipotent hematopoietic progenitor cells to prepare multipotent hematopoietic progenitor cells that express the CAR, and then inducing the differentiation of the CAR-expressing multipotent hematopoietic progenitor cells into macrophages; (2) preparing artificial leukocyte stem cells or T / NK progenitor cells from a cell population containing hematopoietic stem cells, inducing their differentiation into macrophages to stimulate their receptors, and then introducing the gene encoding the CAR. Step 4-2: preparing CAR-NK cells by the following step (5) or (6): (5) preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells, introducing a gene encoding the CAR into the multipotent blood progenitor cells to prepare multipotent blood progenitor cells that express the CAR, and then inducing the differentiation of the CAR-expressing multipotent blood progenitor cells into NK cells; (6) preparing artificial leukocyte stem cells or T / NK progenitor cells from a cell population containing hematopoietic stem cells, inducing their differentiation into NK cells to stimulate the receptor, and then introducing the gene encoding the CAR. Step 4-3: combining CAR-macrophages and CAR-NK cells, and administering them to a patient with a tumor that expresses the tumor antigen.

[0128] As explained in Production Method 2A of III., CAR-NK cells and CAR-macrophages have sufficient proliferation capacity even before receptor stimulation, and therefore receptor stimulation is not essential in steps 3-1(1), 4-1(1), and 4-2(5). Alternatively, in steps 3-1(1), 4-1(1), and 4-2(5), the prepared CAR-NK cells and CAR-macrophages may be receptor-stimulated to further enhance their proliferation capacity, and then administered to patients.

[0129] Steps 1a-1 to 1a-4 of therapeutic method 1a, steps 2a-1 to 2a-3 of therapeutic method 2a, steps 3-1(1) and 3-2(3) of therapeutic method 3, steps 4-1(1) and 4-2(5) of therapeutic method 4, and the step of receptor-stimulating CAR-NK cells, CAAR-NK cells, CAR-macrophages, or CAAR-macrophages, which is optionally performed in therapeutic method 2a, therapeutic method 3, and therapeutic method 4, can be performed as described in I-1 to I-3.

[0130] Steps 1b-1 to 1b-3 of Treatment Method 1b, steps 2b-1 to 2b-3 of Treatment Method 2b, steps 3-1(2) and 3-2(4) of Treatment Method 3, and steps 4-1(2) and 4-2(6) of Treatment Method 4 can be carried out as described in I-1, I-4, and I-5.

[0131] In each of the above-mentioned aspects of the treatment method, the multipotent blood progenitor cells, artificial leukocyte stem cells, or T / NK progenitor cells may be prepared from umbilical cord blood (which is generally not derived from the patient himself / herself), or a cell population containing hematopoietic stem cells may be obtained from bone marrow fluid or peripheral blood collected from the patient, and the multipotent blood progenitor cells, artificial leukocyte stem cells, or T / NK progenitor cells derived from the patient's own cells may be prepared.

[0132] In steps 1a-5, 1b-4, 2a-4, 2b-4, 3-3, and 4-3, immune cells expressing an exogenous receptor are prepared in an appropriate dosage form, such as an intravenous infusion, as described in III., and administered to a tumor patient or an autoimmune disease patient. The tumor patient to whom the immune cells are administered is a patient with a tumor expressing a tumor antigen recognized by the exogenous receptor (in this case, the receptor is an antigen-specific receptor) expressed on the immune cells. The autoimmune disease patient to whom the immune cells are administered is a patient with an autoimmune disease caused by an autoantigen incorporated in the antigen-derived portion of the CAAR expressed on the immune cells, or pathogenic B cells that recognize the autoantigen. The immune cells may be administered to a patient once, or two or more times at appropriate intervals. In steps 3-3 and 4-3, the CAR-macrophages and CAR-T / CAR-NK cells may each be administered once, or two or more times at appropriate intervals. The meaning of the term "administered in combination" is as explained in III. The dose of each immune cell may vary depending on the type of cell to be administered, the type of tumor, the patient's weight, etc., but the number of viable cells in one administration is 1 x 10 5 ~1 x 10 9 Approximately one dose should be administered.

[0133] In conventional TCR-T / CAR-T / CAR-NK therapy, TCR-T / CAR-T / CAR-NK cells are administered after reducing lymphocytes in the patient's body by anticancer drug treatment, etc. In the tumor treatment method of the present invention, a pretreatment to reduce lymphocytes in the patient's body may also be performed prior to the administration of immune cells expressing tumor antigen-specific TCR or CAR.

[0134] The tumor to be treated is a tumor expressing a tumor antigen recognized by an exogenous receptor (in this case, an antigen-specific receptor). The autoimmune disease to be treated is an autoimmune disease caused by an autoantigen incorporated into the antigen-derived portion of CAAR or pathogenic B cells that recognize the autoantigen. Specific examples of tumors and autoimmune diseases are listed in I-2., along with specific examples of tumor antigens and autoantigens.

[0135] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0136] A. Generation of TCR- or CAR-Expressing Immune Cells A-1. Experimental Materials Media The media used were Roswell Park Memorial Institute (RPMI)-1640 (Sigma-Aldrich), Isocove's Modified Dulbecco's Medium (IMDM; Sigma-Aldrich), Minimum Essential Medium alpha (MEMα; Invitrogen), and Stemline II (Sigma-Aldrich). All media were supplemented with 100 μg / mL penicillin and 100 U / mL streptomycin. RPMI-1640 and IMDM were supplemented with 10% fetal bovine serum (FBS; Gibco), and MEMα was supplemented with 20% FBS. RPMI-1640 supplemented with 1% MEM Non-Essential Amino Acid Solution (Gibco) and 1 mM Sodium Pyruvate (Gibco) was used as RPMI complete (RPMIcomp).

[0137] Cytokines: Recombinant human (rh) interleukin-2 (IL-2), IL-7, IL-15, FMS-related tyrosine kinase 3 ligand (Flt3-L), stem cell factor (SCF), thrombopoietin (TPO), and granulocyte-colony stimulating factor (G-CSF); recombinant mouse (rm) IL-3, IL-6, IL-7, IL-15, SCF, Flt3-L, TPO, and macrophage-stimulating factor (M-CSF) were purchased from R&D Systems.

[0138] Preparation of Retrovirus The ID3-ERT2 retroviral vector used was that previously reported (Non-Patent Document 3). This retroviral vector was constructed by cloning the ID3-ERT2 gene, which is a full-length human ID3 gene cDNA fused with a mutant human estrogen receptor ligand-binding domain (ERT2) sequence, into the publicly known pCSRet retroviral vector. In the nucleotide sequence of the ID3-ERT2 gene shown in SEQ ID NO: 1, positions 1 to 357 represent the human ID3 gene cDNA, and positions 370 to 1311 represent the mutant ERT2 ligand-binding domain sequence.

[0139] A plasmid (murine TCR OTI-2A.pMIG II) encoding the OTI-TCR gene (a gene encoding a T cell receptor that specifically reacts with OVA antigen; Fig. 2, upper panel, SEQ ID NO: 17) was purchased from Addgene. In the nucleotide sequence of the OTI-TCR gene shown in SEQ ID NO: 17, positions 1 to 828 (residues 1 to 276 in the amino acid sequence of SEQ ID NO: 18) represent the OTI TCR α chain sequence, positions 829 to 882 (residues 277 to 294) represent the T2A self-cleavage peptide sequence, and positions 883 to 1821 (residues 295 to 607) represent the OTI TCR β chain sequence.

[0140] The pMIGII-hCD19-m28z-CAR plasmid carrying the hCD19-m28z-CAR gene was constructed by fusing the 3xFlag gene, the human CD19-specific FMC63 antibody single-chain variable region fragment gene, the mouse CD28 plasma membrane domain and intracellular signaling domain, and the mouse CD3ε intracellular signaling domain gene downstream of the mouse IgG2aκ signal peptide gene (Figure 10B). In the nucleotide sequence of the hCD19-m28z-CAR gene shown in SEQ ID NO: 3, positions 1 to 60 are the mouse IgG2aκ signal peptide sequence, positions 61 to 126 are the 3xFlag gene, positions 127 to 870 are the human CD19-specific FMC63 antibody single-chain variable region fragment gene, positions 871 to 312 are the mouse CD28 plasma membrane domain and intracellular signaling domain gene, and positions 1183 to 1521 are the mouse CD3ε intracellular signaling domain gene sequence.

[0141] Retroviruses harboring these genes were produced by transfecting Plat E cells (Cell Biolabs Inc.) with the target plasmid and VSV-G plasmid using FuGENE 6 Transfection Reagent (Roche).

[0142] Lentivirus production: A CSII vector was used as the lentiviral vector. A plasmid encoding a gene of interest (e.g., a CAR gene), a Gag / Pol plasmid, a Rev plasmid, and a VSV-G or GALV envelope plasmid were transfected into 293T cells using FuGENE 6 Transfection Reagent (Roche) to generate lentiviruses expressing the gene of interest. The GALV envelope plasmid used was the commercially available phCMV-GALV-MTR, which contains the synthetic gene GALV-MTR (a gene encoding an envelope in which the membrane and cytoplasmic domains of the GALV envelope are replaced with those of Mouse Leukemia virus (MuLV) (SEQ ID NO: 15)).

[0143] A-2. Experiments and Results A-2-1. Generation of iLS Cells Bone marrow aspirates were collected from C57BL / 6 mice, and LSK cells, which were lineage (CD90, B220, Gr-1, CD11b, Ter119) negative and c-Kit and Sca-I positive, were isolated using a cell sorter (FACS Aria III). LSK cells were seeded on OP9 feeder cells and cultured at 37°C in 5% CO2 in RPMIcomp medium supplemented with 30 ng / mL rmIL-7, 30 ng / mL rmFlt3-L, 30 ng / mL rmSCF, and 30 ng / mL rmTPO. Cells were harvested after 4–6 days and transfected with the ID3-ERT2 gene using a retroviral spin-infection method. To identify and isolate ID3-ERT2 transfected cells, human CD25 was used as a transfection marker. The ID3-ERT2 gene was linked to the human CD25 gene and transfected into cells. Human CD25-positive ID3-ERT2 transfected cells were isolated using a cell sorter. Sorted iLS cells were cultured on OP9 cells in iLS cell medium (IMDM supplemented with 10 ng / mL rmIL-7, 10 ng / mL rmFlt3-L, 10 ng / mL rmSCF, and 40 nM 4-hydroxytamoxifen (4-OHT)). iLS cells were expanded by passage onto fresh OP9 cells every 3–4 days. After approximately one month, stable growth of iLS cells was confirmed by FCM analysis and used for subsequent experiments (Figure 1).

[0144] A-2-2. Transfection of Cancer-Specific Receptors into iLS Cells To generate iLS cells stably expressing cancer-specific receptors, the OTI-TCR gene or hCD19-m28z-CAR gene was introduced into iLS cells using retroviral spin-infection. After gene transfection, iLS cells positive for the marker protein GFP were isolated using a cell sorter. The isolated OTI-TCR transfected iLS (OTI TCR-iLS) cells and hCD19-m28z-CAR transfected iLS (CD19-CAR iLS) cells were cultured under iLS cell culture conditions for maintenance and expansion (Figures 2 and 10).

[0145] A-2-3. T cell induction from cancer-specific receptor gene-transfected iLS cells. OTI TCR-iLS cells or CD19-CAR iLS cells were seeded on OP9 / Delta-like 1 (OP9 / DLL1) cells and cultured in MEMα supplemented with 5 ng / mL rmIL-7, 5 ng / mL rmFlt3-L, and 40 nM 4-OHT. Half of the medium was replaced with the same medium without 4-OHT every 3–4 days, and after 1 week, the iLS cells were seeded on fresh OP9 / DLL1 cells. After 2 weeks of culture, the cells were cultured in MEMα supplemented with 1 ng / mL rmIL-7 and 1 ng / mL rmFlt3-L. Three weeks after the start of induction, cells were harvested, and CD4 / CD8 double-positive (DP) cells were isolated using CD8a (Ly-2) MicroBeads (Miltenyi Biotech) (Figures 3, 4, and 6).

[0146] To stimulate DP cells generated from OTI TCR-iLS cells, spleen cells were collected from C57BL / 6 mice and treated with 10 μg / mL mitomycin C (MMC) for 2.5 hours. The DP cells derived from OTI TCR-iLS cells and the MMC-treated antigen-presenting cells were mixed at a 2:1 ratio and incubated with 5 ng / mL rmIL-7, 5 ng / mL rmIL-15, 50 IU / mL rhIL-2, and 10 μg / mL OVA peptide (OVA). 257-264 TCR stimulation was performed by co-culturing in RPMIcomp medium containing 1000kJ / ml of CD8+ T cells, resulting in the generation of CD8+ T cells (Figure 5).

[0147] Meanwhile, DP cells generated from CD19-CAR iLS cells were TCR-stimulated by culturing them on anti-CD3 and anti-CD28 antibody-coated plates in RPMIcomp medium supplemented with 5 ng / mL rmIL-7, 5 ng / mL rmIL-15, and 50 IU / mL rhIL-2. One week after TCR stimulation, the cells were harvested and cultured for an additional 4 days in RPMIcomp medium supplemented with 5 ng / mL rmIL-7, 5 ng / mL rmIL-15, and 50 IU / mL rhIL-2 without TCR stimulation. FCM analysis confirmed the generation of CD8+ CAR-T cells (Figure 6).

[0148] A-2-4. Evaluation of antigen-specific cytokine release from CD8+ T cells derived from OTI TCR-iLS cells. OVA antigen-presenting cells were prepared by incubating spleen cells collected from C57BL / 6 mice with 1 μg / mL OVA. 257-264 CD8+ T cells derived from OTI TCR-iLS cells were cultured for 1 hour with OVA antigen-presenting cells. CD8+ T cells derived from OTI TCR-iLS cells were mixed with 10x the amount of OVA antigen-presenting cells or treated with 10 ng / mL PMA and 1 μg / mL ionomycin, and cultured in RPMIcomp medium supplemented with 1 / 1000 of a PE-Cy7-conjugated CD107a antibody and 1x Protein transport inhibitor (eBioscience). After 6 hours, intracellular staining was performed and cytokine expression was assessed by FCM analysis. The iLS cell-derived OTI TCR-T cells were activated by stimulation and showed expression of CD107a, IFNγ, and TNFα (Figure 7).

[0149] A-2-5. Cytotoxic Activity of CD8+ T Cells Derived from OTI TCR-iLS Cells. B16 cells stably expressing OVA antigen (B16-OVA) or B16 cells expressing only the selectable marker neomycin (B16-Neo) were treated overnight with 500 ng / mL IFNγ and stained with Cell Tracer Far-Red (Invitrogen). They were then mixed at different ratios with CD8+ T cells derived from OTI TCR-iLS cells or CD8+ T cells from the spleen of OTI Tg mice (transgenic mice expressing the OTI-TCR gene) and co-cultured for 18 hours in RPMIcomp medium supplemented with 50 IU / mL rhIL-2. After co-culture, dead cells were stained with Live / Dead Near-IR (Invitrogen) and the percentage of dead cells was measured by FCM analysis. The iLS cell-derived OTI TCR-T cells exhibited high cytotoxic activity, similar to that of T cells derived from OTI Tg mice (Figure 8).

[0150] A-2-6. In vivo antitumor activity of OTI TCR-iLS cell-derived CD8+ T cells. C57BL / 6 mice were treated with 1x10 5 B16-OVA cells were subcutaneously transplanted to generate B16-OVA tumor-bearing mice. Five days after the transplantation, the tumor-bearing mice were irradiated with 4 Gy of X-rays, and then 3x10 6 OTI TCR T-iLS cells were administered via the tail vein. 50,000 IU rhIL-2 was administered intraperitoneally once daily for three days from the day of T cell transplantation. Tumor diameter was measured over time to evaluate the antitumor activity of TCR-T cells. Tumor volume (short side) 2 The calculation was made using x (long side) x 0.5. As a result, iLS-derived OTI TCR-T cells exhibited antitumor activity equivalent to that of T cells derived from OTI Tg mice (Figure 9).

[0151] CD19-CAR iLS cells were seeded onto OP9 cells and cultured for 10 days in RPMIcomp medium supplemented with 10 ng / mL rmIL-15 and 100 IU / mL rhIL-2, resulting in the generation of a high percentage of CAR-NK cells (Figure 10).

[0152] A-2-8. Cytotoxic activity of iLS cell-derived CD19-CAR-NK cells. CD19-CAR-NK cells were mixed with a B16 melanoma cell line expressing human CD19 at various cell ratios and co-cultured for 4 hours. These cells exhibited higher cytotoxic activity than the B16 melanoma cell line that did not express human CD19 (Figure 11).

[0153] A-2-9. Induction of differentiation of CD19-CAR iLS cells into macrophages. CD19-CAR iLS cells were seeded on OP9 cells and cultured in IMDM supplemented with 10 ng / mL rmSCF, 5 ng / mL rmM-CSF, 5 ng / mL rmIL-3, and 5 ng / mL rmIL-6. Half of the medium was replaced after 4 days. After 1 week of culture, FCM analysis confirmed the generation of CD11b-positive, F4 / 80-positive, MHC class II (IA)-positive macrophages (Figure 12). Therefore, CAR iLS cells were shown to have the ability to generate CAR-macrophages.

[0154] A-2-10. Generation of pT / NK cells from human umbilical cord blood CD34+ cells. hCB-CD34+ cells were obtained from the RIKEN BioResource Research Center. hCB-CD34+ cells were cultured and expanded for one week in Stemline II medium supplemented with 100 ng / mL rhSCF, 100 ng / mL rhTPO, and 100 ng / mL rhG-CSF. These cells were seeded on OP9 / DLL1 cells and cultured in MEMα supplemented with 5 ng / mL rhIL-7, 5 ng / mL rhFlt3-L, and 5 ng / mL rhSCF. On day 17 of culture, pT / NK cells (CD5 + CD7 + cells) were generated (FIG. 13, bottom left).

[0155] A-2-11. Induction of pT / NK Cell Differentiation into NK Cells. pT / NK cells were seeded on OP9 / DLL1 cells and differentiated into NK cells by culturing in MEMα supplemented with 20 ng / mL rhIL-7, 10 ng / mL rhFlt3-L, 20 ng / mL rhSCF, and 10 ng / mL rhIL-15. The efficiency of differentiation into NK cells was evaluated by FCM analysis. For comparison, hCB-HSPCs expanded in Stemline II medium were seeded on OP9 / DLL1 cells and cultured in the presence of 20 ng / mL rhIL-7, 10 ng / mL rhFlt3-L, 20 ng / mL rhSCF, and 10 ng / mL rhIL-15. NK cell differentiation was evaluated by FCM analysis. As a control, pT / NK cells generated 17-fold more NK cells than hCB-HSPCs (Figure 13, bottom right).

[0156] To evaluate the cytokine production of pT / NK cell-derived NK cells, NK cells were mixed with target cells, the leukemia cell line K562 (K562 cells), or treated with 10 ng / mL PMA and 1 μg / mL ionomycin, and cultured in MEMα medium containing 1 / 500 of an APC-conjugated CD107a antibody and 1x protein transport inhibitor. After 4 hours, intracellular staining was performed, and FCM analysis of cytokines revealed that they were activated by the target cells (Figure 14).

[0157] A-2-13. Cytotoxic activity of pT / NK cell-derived NK cells. pT / NK cell-derived NK cells or hCB-HSPC-derived NK cells were mixed with K562 cells at various cell ratios and co-cultured for 4 hours. pT / NK cell-derived NK cells exhibited high cytotoxic activity comparable to that of hCB-HSPC-derived NK cells (Figure 15).

[0158] A-2-14. Gene transfer into pT / NK cell-derived NK cells CAR-NK cells were generated by transferring a CAR gene into pT / NK cell-derived NK cells. The CAR genes used were the anti-CD19 CAR gene (SEQ ID NOs: 11 and 12), which specifically recognizes human CD19 and was constructed using a human protein in the intracellular domain, and the anti-GD2 CAR gene (SEQ ID NOs: 13 and 14), which encodes a CAR specifically recognizing GD2, a neuroblastoma antigen. In the anti-CD19 CAR gene sequence of SEQ ID NO: 11, positions 1-63 (AA 1-21 of SEQ ID NO: 12) represent the CD8α signal peptide sequence, positions 64-129 (AA 22-43) represent the 3xFlag gene, positions 130-864 (AA 44-288) represent the anti-CD19 scFv (antibody clone FMC63) gene, positions 874-1014 (AA 292-338) represent the human CD8α hinge region gene, positions 1024-1119 (AA 342-373) represent the human CD28 transmembrane domain gene, positions 1120-1245 (AA 374-415) represent the human 4-1BB intracellular signaling domain gene, and positions 1252-1593 (AA 418-531) represent the human CD3ζ intracellular signaling domain gene. In the anti-GD2 CAR gene sequence of SEQ ID NO: 13, positions 130-867 (44-289 AA of SEQ ID NO: 14) are the anti-GD2 scFv (antibody Clone 14g2a) gene, and its upstream and downstream sequences are identical to SEQ ID NO: 11 (positions 1-63 (1-21 AA) are the CD8α signal peptide sequence, positions 64-129 (22-43 AA) are the 3xFlag sequence, positions 877-1017 (293-339 AA) are the human CD8α hinge region, positions 1027-1122 (343-374 AA) are the human CD28 transmembrane domain, positions 1123-1248 (375-416 AA) are the human 4-1BB intracellular signaling domain, and positions 1255-1596 (419-532 AA) are the human CD3ζ intracellular signaling domain).

[0159] K562 cells stably expressing membrane-bound IL-21 (memIL-21) and 4-1BB ligand (41BBL) (K562 / memIL-21 / 41BBL) were treated with 10 μg / mL mitomycin C for 2.5 hours. NK cells were expanded by mixing mitomycin C-treated K562 / memIL-21 / 41BBL cells with pT / NK cell-derived NK cells at a 2:1 ratio and culturing them in MEMα medium supplemented with 200 IU / mL rhIL-2. After 1 week of culture, NK cells were cultured in MEMα medium containing 10 ng / mL rhIL-15, 10 ng / mL rhIL-21, and 100 IU / mL rhIL-2 for 2 days and then transfected with anti-CD19 or anti-GD2 CAR genes using lentivirus spin-infection. CAR-NK cells positive for the marker protein Venus were isolated using a cell sorter and further expanded by co-culture with K562 / memIL-21 / 41BBL cells. After expansion, FCM analysis of Venus expression confirmed that all cells maintained transgene expression (Figure 16A).

[0160] A-2-15. Cytotoxic Activity of pT / NK Cell-Derived Anti-CD19 CAR-NK Cells. NALM6 cells, a B-cell leukemia line expressing the CD19 antigen, were stained with Cell Tracer Far-Red (Invitrogen). Next, pT / NK cell-derived anti-CD19 CAR-NK cells were mixed with NALM6 cells at a 1:1 ratio and co-cultured for 4 hours. After co-culture, dead cells were stained with Live / Dead Near-IR (Invitrogen), and the percentage of dead cells was measured using FCM analysis. The CAR-NK cells demonstrated antigen-specific cytotoxic activity (Figure 16B).

[0161] A-2-16. CAR gene transduction into pT / NK cells The results of comparing the gene transduction efficiency into pT / NK cells using lentiviruses employing VSV-G or GALV envelopes (only the gene encoding Venus was used) are shown in Figure 17. The use of GALV envelopes increased the transduction efficiency.

[0162] The resulting pT / NK cells were transduced with a lentivirus encoding a CAR gene produced using a GALV envelope (SEQ ID NO: 16). The lentivirus was seeded onto a retronectin-coated plate and centrifuged at 2000 x g for 1.5 hours to allow the lentivirus to bind to retronectin, after which pT / NK cells were seeded. After centrifugation at 800 x g for 10 minutes, the cells were cultured in an incubator. The CAR-transduced cells were sorted using a cell sorter to obtain CAR-transduced pT / NK cells (CAR-pT / NK cells) (Figure 18A). The resulting CAR-pT / NK cells were cultured under NK cell-inducing conditions to obtain pT / NK cell-derived NK cells stably expressing the CAR gene (Figure 18B).

[0163] B. Examination of methods for expanding human pT / NK cells and inducing differentiation of human pT / NK cells into NK cells. B-1. Examination of cytokine concentrations for inducing human pT / NK cells. We investigated the optimal cytokine concentration for generating and expanding pT / NK cells from hCB-CD34+ cells. HSPCs (a cell population expanded from CD34+ cells) derived from hCB-CD34+ cells obtained from the RIKEN BioResource Research Center were cultured on OP9 / DLL1 feeder cells (in the presence of Notch signaling) with recombinant human (rh) IL-7, rhFlt3-L, and rhSCF at the cytokine concentrations shown in Table 1 for 6 weeks. After culture, the number of pT / NK cells produced was evaluated by FCM analysis.

[0164] Previous studies have confirmed that mouse pT / NK cell expansion efficiency is significantly improved by increasing the cytokine concentration to approximately 25–50 ng / ml, rather than the 10 ng / ml reported in Non-Patent Document 2. Figure 19 shows the results of counting pT / NK cells after culturing mouse HSPCs (Lineage-Sca1-ckit+: LSK) on OP9 / DLL1 feeder cells in medium supplemented with SCF, IL-7, and Flt3-L at concentrations of 10 ng / ml or 30 ng / ml, respectively, for two weeks. Even at 10 ng / ml, a large number of pT / NK cells (approximately 10,000) were generated from a single HSPC. However, increasing the concentration of each cytokine to 30 ng / ml further expanded the number of pT / NK cells generated from a single HSPC to approximately 60,000.

[0165] On the other hand, unlike mouse pT / NK cells, human pT / NK cells were not expanded when high concentrations (≥25 ng / ml) of cytokines were added, as shown in Figure 20. It was revealed that human pT / NK cells could be expanded most efficiently when rhIL-7, rhFlt3-L, and rhSCF were added at 5 ng / ml each. Based on these results, in the above-mentioned A-2-10, each cytokine was used at 5 ng / ml.

[0166]

[0167] B-2. Mass Production of NK Cells Using the Human pT / NK Cell Expansion Method. The number of NK cells generated using pT / NK cells was evaluated. The culture protocol is shown in Figure 21A. First, to expand hCB-HSPCs, CD34+ cells were cultured for one week in Stemline II medium supplemented with 100 ng / ml rhTPO, 100 ng / ml rhSCF, and 100 ng / ml rhG-CSF. The expanded HSPCs were cultured on OP9 / DLL1 cells in MEMα medium containing 5 ng / ml rhIL-7, 5 ng / ml rhFlt3-L, and 5 ng / ml rhSCF to generate and expand pT / NK cells. After 4 weeks of culture, pT / NK cells were induced to differentiate into NK cells by culturing them in MEMα medium containing 10 ng / ml rhIL-15, 10 ng / ml rhFlt3-L, 20 ng / ml rhIL-7, and 20 ng / ml rhSCF in the absence of Notch signaling. As a control, NK cells were directly induced to differentiate using a previously reported culture method (Li et al., Cell Stem Cell, Vol. 23, Issue 2, pp. 181-192.e5, August 2, 2018) in which HSPCs were cultured in MEMα medium containing 10 ng / ml rhIL-15, 10 ng / ml rhFlt3-L, 20 ng / ml IL-7, and 20 ng / ml rhSCF. Figure 21B shows the proliferation rate of cells from CB-CD34+ cells. Figure 21C shows the results of FCM analysis after NK cell generation. Figure 21D shows the number of NK cells produced when NK cells were directly induced from HSPCs (conventional method) and when the pT / NK cell expansion method was used. These results demonstrate that this method can produce large amounts of CD56-positive NK cells from pT / NK cells. Compared to the conventional method, 200-500 times more NK cells were produced.Furthermore, according to the method described above in A-2-11, in which Notch ligand co-culture was performed using feeder cells expressing a Notch ligand when inducing differentiation of pT / NK cells into NK cells, the amount of NK cells produced from pT / NK cells was 17 times higher than that of the conventional method. Therefore, when comparing this method, in which culture is performed in the absence of a Notch signal when inducing differentiation of NK cells, with the method described in A-2-11, the amount of NK cells produced by this method is approximately 12 to 30 times higher than that of the method described in A-2-11, and this method can further significantly improve the amount of NK cells produced.

[0168] B-3. ​​Proliferative Potential of Human pT / NK Cell-Derived NK Cells The proliferative potential of pT / NK cell-derived NK cells was evaluated. NK cells collected from CB (human umbilical cord blood) or PB (human peripheral blood) (CB-NK and PB-NK, respectively) were used as controls. Figure 22A shows the cell proliferation rate after 2 weeks of culture in MEMα medium supplemented with 10 ng / ml rhIL-15. Figure 22B shows the cell counts obtained when NK cells were expanded in MEMα medium containing 200 IU / ml rhIL-2 after mixing with MMC-treated aAPCs (artificial antigen presenting cells, cells expressing a ligand that stimulates NK cells) at an E:T ratio of 1:2. Fresh aAPCs were added after 1 week of culture, and the cells were cultured for a total of 2 weeks. These results demonstrated that pT / NK-derived NK cells have a proliferative potential comparable to that of CB-NK and PB-NK cells.

[0169] B-4. Expression of Surface Antigen Markers on Human pT / NK Cell-Derived NK Cells (Figure 23). The expression levels of NK cell-associated receptors on pT / NK-derived NK cells were assessed by FCM analysis. Expression levels of NKp44, NKp46, and NKG2D on pT / NK-derived NK cells were comparable to those of CB-NK and PB-NK cells. However, expression levels of CD16 and KIR2DL2 / L3 were lower than those of PB-NK cells, and expression levels of NKG2A were lower than those of CB-NK cells. These findings suggest that pT / NK-derived NK cells are at an early stage of maturation. Furthermore, expression of TIGIT on pT / NK-derived NK cells was significantly lower than that of CB-NK and PB-NK cells, suggesting a lower degree of exhaustion.

[0170] B-5. Cytotoxic Activity of Human pT / NK Cell-Derived NK Cells The cytotoxic activity of pT / NK cell-derived NK cells against K562 cells was compared with that of PB- or CB-derived NK cells. NK cells and K562 cells were mixed at various E:T ratios and cocultured for 4 hours. After incubation, the percentage of K562 cell death was assessed by FCM analysis (Figure 24A). To assess the expression of effector molecules on activated NK cells, NK cells and K562 cells were mixed at a 1:2 E:T ratio and cultured in MEMα medium containing 1 / 500 of an APC-conjugated CD107a antibody and 1x protein transport inhibitor. After 4 hours, intracellular staining was performed, and cytokine expression was analyzed by FCM (Figure 24B). pT / NK-derived NK cells exhibited high cytotoxic activity comparable to that of CB-NK cells and PB-NK cells. Furthermore, the expression of effector molecules upon K562 cell stimulation was similar to that of CB-NK cells and PB-NK cells (Figure 24C).

[0171] B-6. Function of cryopreserved human pT / NK cell-derived NK cells. The effect of cryopreservation on pT / NK cell-derived NK cells was evaluated. The number of pT / NK cell-derived NK cells was measured after freeze-thawing, and the recovery rate (number of cells after freezing / 100 cells before freezing) was calculated. As a result, 60-100% of the pre-freezing NK cells were recovered (Figure 25A). Next, the cell proliferation ability was evaluated by culturing the frozen pT / NK cell-derived NK cells in medium supplemented with aAPC and 200 IU / ml rhIL-2. After 2 weeks of co-culture, the cells expanded 200-650-fold, confirming that cryopreservation did not impair the proliferation ability of the NK cells (Figure 25B). Next, the pT / NK cell-derived NK cells were co-cultured in medium supplemented with aAPC and 200 IU / ml rhIL-2, and their cytotoxic activity against K562 cells was evaluated. After culturing NK cells and K562 cells at various E:T ratios for 4 hours, the percentage of dead K562 cells was measured by FCM analysis. The results showed that the NK cells derived from frozen pT / NK cells maintained the same high cytotoxic activity as before freezing (Fig. 25C).

[0172] B-7. Identification of factors promoting the expansion of human pT / NK cells. To identify factors promoting the differentiation and proliferation of human pT / NK cells from hCB-HSPCs, we cultured hCB-HSPCs on OP9 / DLL1 cells in the presence of 5 ng / ml rhIL-7, 5 ng / ml rhFlt3-L, and 5 ng / ml rhSCF. We then added 10 ng / ml of each cytokine or 0.1 mM nicotinamide to the culture and calculated the expansion rate of pT / NK cells. Results indicated that M-CSF, TPO, IL-6, G-CSF, and nicotinamide promoted the differentiation and proliferation of pT / NK cells (Figure 26).

[0173] B-8. Expansion of human pT / NK cells by TPO and M-CSF We investigated the concentrations at which TPO or M-CSF promotes the generation of pT / NK cells. hCB-HSPCs were cultured on OP9 / DLL1 cells in the presence of 5 ng / ml rhIL-7, 5 ng / ml rhFlt3-L, and 5 ng / ml rhSCF, and the proliferation rate of pT / NK cells was examined by adding 0-50 ng / ml TPO or M-CSF. The results showed that the addition of 10-50 ng / ml TPO or 10-25 ng / ml M-CSF promoted the differentiation and proliferation of pT / NK cells (Figure 27).

[0174] B-9. NK cell-producing ability of human pT / NK cells expanded with TPO or M-CSF. The NK cell differentiation ability of pT / NK cells expanded with TPO or M-CSF was evaluated. Specifically, pT / NK cells were expanded by culturing hCB-HSPCs on OP9 / DLL1 cells in medium containing 5 ng / ml rhIL-7, 5 ng / ml rhFlt3-L, 5 ng / ml rhSCF, and 25 ng / ml TPO or M-CSF for 4 weeks. Next, pT / NK cells were induced to differentiate into NK cells by culturing them in MEMα medium containing 10 ng / ml rhIL-15, 10 ng / ml rhFlt3-L, 20 ng / ml rhIL-7, and 20 ng / ml rhSCF. Compared to the control group without the respective cytokines, TPO yielded 4.3-fold more NK cells, while M-CSF yielded 1.6-fold more NK cells (Figure 28).

[0175] B-10. TPO-Induced pT / NK and NK Cells from Bone Marrow HSPCs. To determine whether TPO is also effective in inducing pT / NK cells from bone marrow-derived HSPCs, human bone marrow-derived CD34+ cells were cultured for 1 week in Stemline II medium supplemented with 100 ng / ml rhTPO, 100 ng / ml rhSCF, and 100 ng / ml rhG-CSF. Then, they were cultured on OP9 / DLL1 cells for 8 weeks in MEMα medium containing 5 ng / ml rhIL-7, 5 ng / ml rhFlt3-L, 5 ng / ml rhSCF, and 25 ng / ml TPO to generate and expand pT / NK cells. The results demonstrated that the addition of TPO significantly expanded bone marrow-derived pT / NK cells several-fold (Figure 29, bottom left). Furthermore, NK cell induction from pT / NK cells after 5 weeks of culture yielded approximately 100 times more NK cells than without TPO (Figure 29, bottom right).

[0176] B-11. Expression of surface antigen markers on NK cells derived from human HSPC-derived pT / NK cells. NK cells derived from human bone marrow HSPCs (hBM-pT / NK-derived NK cells) were evaluated for NK cell-associated receptor expression by FCM analysis. Similar to hCB blood-derived NK cells, expression of NKp44, NKp46, and NKG2D was observed, but expression of CD16 and KIR (KIR2DL2 / L3) was low, suggesting that these NK cells are in an early stage of maturation (Figure 30A).

[0177] Figure 30B shows the results of comparing the expression of activating receptors, inhibitory receptors, and exhaustion markers among human T / NK progenitor-derived NK cells (pT / NK-NK cells), human CB-NK cells, and human PB-NK cells induced from human umbilical cord blood HSPCs. CB-NK cells are known to be immature NK cells with a low degree of maturity, but human pT / NK-NK cells were characterized as being even more immature than CB-NK cells. Furthermore, human pT / NK-NK cells also expressed lower levels of the exhaustion marker TIGIT than CB-NK cells, indicating a lower degree of exhaustion.

[0178] Figure 30C shows RNA-seq data comparing gene expression in human pT / NK-NK cells, human CB-NK cells, and human PB-NK cells induced from human umbilical cord blood HSPCs. Human pT / NK-NK cells (NK.pTNK_1-3) showed higher expression of CCR3, CCR6, CCR7, CCR8, and CCR9 than human CB-NK cells (NK.CB_1-3) and human PB-NK cells (NK.PB_1-3).

[0179] B-12. Cytotoxic Activity of NK Cells Differentiated from Bone Marrow HSPCs. The cytotoxic activity of hBM-pT / NK cell-derived NK cells against K562 cells was evaluated. NK cells and K562 cells were mixed at various E:T ratios and co-cultured for 4 hours. FCM analysis of the percentage of dead K562 cells after culture revealed that hBM-pT / NK cell-derived NK cells exhibited high anti-cancer activity against K562 cells (Figure 31).

[0180] B-13. Identification of specific markers for pT / NK cells. The expression of CD122 (IL-2 receptor β chain) on pT / NK cells was evaluated by FCM analysis (Fig. 32A). pT / NK cells were characterized by ProT1 (CD7 + CD5 - CD1a - ) cells, ProT2 (CD7 + CD5 + CD1a - ) cells, PreT (CD7 + CD5 + CD1a + ) cells. CD122 is a hematopoietic precursor cell that expresses CD34 before receiving Notch signaling. + CD7 - No expression was observed in ProT1 (CD7 + CD5 - CD1a - ) cells and ProT2 (CD7 + CD5 + CD1a - ) cells and PreT (CD7 + CD5 + CD1a +) cells. CD122 has been used as a marker for NK progenitor cells in mice (Non-Patent Document 4), but its expression has not been reported in humans. Thus, CD122 is considered to be a novel marker molecule for human T / NK progenitor cells. To evaluate the proliferation potential of these CD122+ cells, a CFSE assay was performed (Figure 32B). After staining in 0.1% BSA / PBS containing 10 μg / ml CFSE, CD122+ cells were isolated using a cell sorter and cultured on OP9 / DLL1 cells in the presence of 5 ng / ml rhIL-7, 5 ng / ml rhFlt3-L, and 5 ng / ml rhSCF for one week. CFSE fluorescence intensity was then analyzed by FCM. The results demonstrated that CD122+ cells have high proliferation potential. Next, the differentiation potential of CD122+ cells into T cells and NK cells was evaluated (Figure 32C). After culturing hCB-derived HSPCs on OP9 / DLL1 cells for 2 weeks, lineage marker-negative CD122-positive cells were isolated by cell sorting and cultured under T cell and NK cell-inducing conditions. The CD122-positive cells generated both CD4 / CD8 double-positive (DP) cells and CD56-positive NK cells, suggesting that they were pT / NK cells.

[0181] C. Generation and functional evaluation of human pT / NK cell-derived CAR-NK cells C-1. CAR retroviral vector The anti-CD19 CAR gene or anti-HER2 CAR gene was loaded into the pMYs-IG retroviral vector to generate the pMYs-CD19 CAR-BBz-IG (middle panel of Figure 33) and pMYs-HER2 CAR-BBz-IG (lower panel of Figure 33) vectors.

[0182] C-2. CAR-NK cells generated from pT / NK cell-derived NK cells. CAR genes were introduced into pT / NK cell-derived NK cells by retroviral spin-infection. CAR-NK cells positive for the marker protein GFP were isolated using a cell sorter and further expanded by co-culture with aAPCs. After culture, GFP expression was analyzed by FCM to confirm stable transgene expression (Figure 34A). Next, the antigen-specific cytotoxic activity of anti-CD19 CAR-NK cells or anti-HER2 CAR-NK cells was evaluated. Anti-CD19 CAR-NK cells derived from pT / NK cells were mixed with the CD19 antigen-positive B-cell leukemia cell line NALM6 (NALM6 cells), and anti-HER2 CAR-NK cells were mixed with the HER2 antigen-positive breast cancer cell line MDA-MB-453 (MDA-MB-453 cells) at various E:T ratios. After 4 hours of co-culture, the percentage of target cell death was measured by FCM analysis. As a result, both anti-CD19 CAR-NK cells and anti-HER2 CAR-NK cells exhibited higher cytotoxic activity against target cells compared to mock NK cells (Figure 34B).

[0183] C-3. CAR gene transduction into pT / NK cells. pT / NK cells were transduced with an anti-CD19 CAR gene or an anti-HER2 CAR gene. Cells expressing the marker protein GFP were isolated using a cell sorter and expanded by culturing on OP9 / DLL1 cells in the presence of 5 ng / ml rhIL-7, 5 ng / ml rhFlt3-L, and 5 ng / ml rhSCF for 2 weeks. After culturing, FCM analysis of GFP expression in pT / NK cells demonstrated stable expression of the transgene (Figure 35).

[0184] C-4. Induction of Differentiation from CAR-pT / NK Cells to CAR-NK Cells We attempted to generate CAR-NK cells from CAR gene-transduced pT / NK cells (CAR-pT / NK cells). Figure 36A shows the scheme for generating CAR-NK cells from CAR-pT / NK cells. CAR-pT / NK cells were expanded by culturing on OP9 / DLL1 cells in the presence of 5 ng / ml rhIL-7, 5 ng / ml rhFlt3-L, and 5 ng / ml rhSCF, and then induced into NK cells by culturing in MEMα medium containing 10 ng / ml rhIL-15, 10 ng / ml rhFlt3-L, 20 ng / ml IL-7, and 20 ng / ml rhSCF. The resulting CAR-NK cells were expanded for 2 weeks by culturing in medium containing aAPC and 200 IU / ml rhIL-2. Figure 36B shows the results of FCM analysis of CAR-NK cells derived from CAR-pT / NK cells on day 42 of culture, confirming that NK cells stably expressing the transgene were generated. CAR-NK cells were generated at a density of 1 x 10 per CAR-pT / NK cell. 4 It was revealed that approximately 1000 cells were produced (Figure 36C).

[0185] C-5. Cytotoxic Activity of CAR-pT / NK Cell-Derived CAR-NK Cells The cytotoxic activity of CAR-pT / NK cell-derived CAR-NK cells against target cells was evaluated. Anti-CD19 CAR-NK cells were co-cultured with NALM6 cells, or anti-HER2 CAR-NK cells were co-cultured with MDA-MB-453 cells for 4 hours, followed by FCM analysis. The results showed that both cells possessed antigen-specific cytotoxic activity (Figure 37).

[0186] C-6. Expression of Cell Surface Markers on CAR-pT / NK Cell-Derived CAR-NK Cells. The expression levels of representative NK cell-associated receptors on pT / NK-derived anti-HER2 CAR-NK cells, CB-derived anti-HER2 CAR-NK cells, and PB-derived anti-HER2 CAR-NK cells were analyzed by FCM. The results are shown in Figure 38. Compared with CB-derived anti-HER2 CAR-NK cells and PB-derived anti-HER2 CAR-NK cells, pT / NK cell-derived anti-HER2 CAR-NK cells exhibited a more mature NK cell phenotype. Furthermore, pT / NK cell-derived anti-HER2 CAR-NK cells expressed lower levels of TIGIT than CB-derived anti-HER2 CAR-NK cells and PB-derived anti-HER2 CAR-NK cells.

[0187] C-7. Cytotoxic activity of CAR-pT / NK cell-derived CAR-NK cells. Anti-HER2 CAR-NK cells derived from pT / NK, CB-NK, and PB-NK were co-cultured with MDA-MB-453 cells for 4 hours, and the percentage of dead MDA-MB-453 cells was measured by FCM analysis. The results showed that pT / NK cell-derived anti-HER2 CAR-NK cells exhibited superior anti-cancer activity to CB-derived and PB-derived anti-HER2 CAR-NK cells (Figure 39).

[0188] C-8. In vivo antitumor activity of CAR-pT / NK cell-derived CAR-NK cells. The in vivo antitumor activity of pT / NK cell-derived anti-HER2 CAR-NK cells was evaluated. First, 1 x 10 6 MDA-MB-453 cells stably expressing luciferase were cultured in NOD.Cg-Prkdc scid / IL2rg tm1Sug MDA-MB-453 cells were subcutaneously transplanted into / ShiJic (NOG) mice to generate tumor-bearing mice. 14, 17, 21, and 24 days after the transplantation, 5 x 10 6CAR-NK cells were administered via the tail vein. 50,000 IU rhIL-2 was administered intraperitoneally every 2–3 days for a total of 13 doses. Tumor growth was measured by bioluminescence imaging. Tumor growth was not suppressed in the CB-derived anti-HER2 CAR-NK cell and pT / NK-derived mock NK cell groups, whereas tumor growth was significantly suppressed in the pT / NK-derived anti-HER2 CAR-NK cell group (Figure 40B, Figure 40C, top). No weight loss was observed in either NK cell group (Figure 40C, bottom), and no serious side effects were observed.

Claims

1. A method for producing immune cells expressing an exogenous receptor, which is a T cell receptor (TCR), a chimeric antigen receptor (CAR), or a chimeric autoantibody receptor (CAAR), comprising the steps of: preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells; preparing multipotent blood progenitor cells expressing the exogenous receptor by introducing a gene encoding the exogenous receptor into the multipotent blood progenitor cells; and inducing differentiation of the multipotent blood progenitor cells expressing the exogenous receptor into immune cells.

2. A method for producing immune cells expressing an exogenous receptor which is a TCR, a CAR or a CAAR, comprising the steps of: preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells; inducing the differentiation of the multipotent blood progenitor cells into immune cells; and introducing a gene encoding the exogenous receptor into immune cells, wherein the multipotent blood progenitor cells are artificial white blood stem cells or T / NK progenitor cells.

3. The method according to claim 1, wherein the multipotent blood progenitor cells are induced leukocyte stem cells or T / NK progenitor cells.

4. The method according to claim 1 or 2, wherein the multipotent blood progenitor cells are artificial white blood stem cells, and in the process of preparing the multipotent blood progenitor cells, the artificial white blood stem cells are prepared by culturing a cell population containing hematopoietic stem cells under conditions that induce differentiation into B cells while inhibiting the function of the intracellular transcription factor E2A.

5. The method according to claim 4, wherein the inhibition of E2A function is achieved by forced expression of differentiation inhibitor Id in hematopoietic stem cells.

6. The method according to claim 1 or 2, wherein the multipotent blood progenitor cells are T / NK progenitor cells, and in the step of preparing the multipotent blood progenitor cells, the T / NK progenitor cells are prepared by culturing a cell population containing hematopoietic stem cells in the presence of a Notch ligand.

7. The method according to claim 6, wherein a cell population containing hematopoietic stem cells is cultured in a medium containing SCF, TPO and G-CSF at a concentration of 100 ng / mL or more each in the absence of a Notch ligand, and then cultured in the presence of a Notch ligand.

8. The method according to any one of claims 1 to 7, wherein the immune cell is a T cell, a NK cell, a macrophage, or a B cell.

9. The method of claim 8, wherein the exogenous receptor is a TCR and the immune cell is a T cell.

10. The method of claim 8, wherein the exogenous receptor is CAR or CAAR and the immune cell is a T cell, a NK cell, or a macrophage.

11. The method according to any one of claims 1 to 10, wherein the exogenous receptor is an antigen-specific receptor and the antigen is a tumor antigen.

12. A method for producing an immune cell therapy for tumors or autoimmune diseases, comprising the steps of: preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells; preparing multipotent blood progenitor cells expressing an exogenous receptor, which is a TCR or CAR that specifically recognizes a CAAR or a tumor antigen, by introducing a gene encoding the exogenous receptor into the multipotent blood progenitor cells; preparing T cells expressing the exogenous receptor (TCR-T, CAR-T or CAAR-T cells) by inducing differentiation of the multipotent blood progenitor cells expressing the exogenous receptor into T cells; stimulating the TCR-T, CAR-T or CAAR-T cells with TCR to induce differentiation into CD8-positive cytotoxic TCR-T, CAR-T or CAAR-T cells; and preparing the CD8-positive cytotoxic TCR-T or CAR-T cells as an immune cell therapy for tumors expressing the tumor antigen, or preparing the CD8-positive cytotoxic CAAR-T cells as an immune cell therapy for autoimmune diseases. A method comprising:

13. A method for producing an immune cell therapy for tumors or autoimmune diseases, comprising the steps of: preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells; inducing the differentiation of the multipotent blood progenitor cells into T cells; preparing CD8-positive cytotoxic TCR-T, CAR-T or CAAR-T cells by TCR-stimulating the T cells and then introducing a gene encoding an exogenous receptor which is a TCR or CAR that specifically recognizes a CAAR or a tumor antigen; and preparing the CD8-positive cytotoxic TCR-T or CAR-T cells as an immune cell therapy for tumors expressing the tumor antigen, or preparing the CD8-positive cytotoxic CAAR-T cells as an immune cell therapy for autoimmune diseases, wherein the multipotent blood progenitor cells are artificial leukocyte stem cells or T / NK progenitor cells.

14. A method for producing an immune cell therapy for tumors or autoimmune diseases, comprising the steps of: preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells; preparing multipotent blood progenitor cells expressing a chimeric receptor that is a CAR that specifically recognizes a CAAR or a tumor antigen by introducing a gene encoding the chimeric receptor into the multipotent blood progenitor cells; preparing NK cells (CAR-NK or CAAR-NK cells) or macrophages (CAR-macrophages or CAAR-macrophages) expressing the chimeric receptor by inducing differentiation of the multipotent blood progenitor cells expressing the chimeric receptor into NK cells or macrophages; and preparing the CAR-NK cells or CAR-macrophages as an immune cell therapy for tumors expressing the tumor antigen, or preparing the CAAR-NK cells or CAAR-macrophages as an immune cell therapy for autoimmune diseases.

15. The method according to claim 14, further comprising a step of receptor-stimulating the prepared NK cells or macrophages expressing the chimeric receptor, and preparing the chimeric receptor-expressing NK cells or macrophages after receptor stimulation as the immune cell therapy agent.

16. A method for producing an immune cell therapy for tumors or autoimmune diseases, comprising the steps of: preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells; inducing the differentiation of the multipotent blood progenitor cells into NK cells or macrophages; preparing NK cells (CAR-NK or CAAR-NK cells) or macrophages (CAR-macrophages or CAAR-macrophages) that express a chimeric receptor by stimulating the NK cells or macrophages with a receptor and then introducing a gene encoding a chimeric receptor that is a CAR that specifically recognizes a CAAR or a tumor antigen; and preparing the CAR-NK cells or CAR-macrophages as an immune cell therapy for tumors expressing the tumor antigen, or preparing the CAAR-NK cells or CAAR-macrophages as an immune cell therapy for autoimmune diseases, wherein the multipotent blood progenitor cells are artificial leukocyte stem cells or T / NK progenitor cells.

17. A method for treating a tumor or an autoimmune disease in a patient, comprising the steps of: preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells; preparing multipotent blood progenitor cells expressing an exogenous receptor, which is a TCR or CAR that specifically recognizes a CAAR or a tumor antigen, by introducing a gene encoding the exogenous receptor into multipotent blood progenitor cells; preparing TCR-T, CAR-T or CAAR-T cells, which are T cells expressing the exogenous receptor, by inducing differentiation of the multipotent blood progenitor cells expressing the exogenous receptor into T cells; stimulating the TCR-T, CAR-T or CAAR-T cells via TCR, and inducing differentiation into CD8-positive cytotoxic TCR-T, CAR-T or CAAR-T cells; and administering the CD8-positive cytotoxic TCR-T or CAR-T cells to a patient having a tumor expressing the tumor antigen, or administering the CD8-positive cytotoxic CAAR-T cells to a patient with an autoimmune disease.

18. A method for treating a tumor or an autoimmune disease in a patient, comprising the steps of: preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells; inducing differentiation of the multipotent blood progenitor cells into T cells; preparing CD8-positive cytotoxic TCR-T, CAR-T or CAAR-T cells by TCR-stimulating the T cells and then introducing a gene encoding an exogenous receptor which is a TCR or CAR that specifically recognizes a CAAR or a tumor antigen; and administering the CD8-positive cytotoxic TCR-T or CAR-T cells to a patient having a tumor expressing the tumor antigen, or administering the CD8-positive cytotoxic CAAR-T cells to a patient with an autoimmune disease, wherein the multipotent blood progenitor cells are artificial leukocyte stem cells or T / NK progenitor cells.

19. A method for treating a tumor or an autoimmune disease in a patient, comprising the steps of: preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells; preparing multipotent blood progenitor cells expressing a chimeric receptor that is a CAR that specifically recognizes a CAAR or a tumor antigen by introducing a gene encoding the chimeric receptor into multipotent blood progenitor cells; preparing chimeric receptor-expressing immune cells that are CAR-NK cells, CAAR-NK cells, CAR-macrophages or CAAR-macrophages by inducing differentiation of the multipotent blood progenitor cells expressing the chimeric receptor into NK cells or macrophages; administering the CAR-NK cells or CAR-macrophages to a patient having a tumor that expresses the tumor antigen, or administering the CAAR-NK cells or CAAR-macrophages to a patient with an autoimmune disease.

20. The method according to claim 19, further comprising a step of receptor-stimulating the prepared chimeric receptor-expressing immune cells, and administering the chimeric receptor-expressing immune cells after receptor stimulation to the patient.

21. A method for treating a tumor or an autoimmune disease in a patient, comprising the steps of: preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells; inducing the differentiation of the multipotent blood progenitor cells into NK cells or macrophages; preparing CAR-NK cells, CAAR-NK, CAR-macrophages or CAAR-macrophages by receptor-stimulating the NK cells or macrophages and then introducing a gene encoding a chimeric receptor that is a CAR that specifically recognizes a CAAR or a tumor antigen; and administering the CAR-NK cells or CAR-macrophages to a patient having a tumor that expresses the tumor antigen, or administering the CAAR-NK cells or CAAR-macrophages to a patient with an autoimmune disease, wherein the multipotent blood progenitor cells are artificial leukocyte stem cells or T / NK progenitor cells.

22. A method for treating a tumor in a patient, comprising the steps of: preparing CAR-macrophages by the following (1) or (2): (1) preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells, introducing a gene encoding a CAR that specifically recognizes a tumor antigen into the multipotent blood progenitor cells to prepare multipotent blood progenitor cells expressing the CAR, and then inducing the differentiation of the CAR-expressing multipotent blood progenitor cells into macrophages; (2) preparing artificial leukocyte stem cells or T / NK progenitor cells from a cell population containing hematopoietic stem cells, inducing their differentiation into macrophages to stimulate the receptor, and then introducing the gene encoding the CAR; preparing CAR-T cells by the following (3) or (4): (3) (3) preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells, introducing a gene encoding the CAR into the multipotent blood progenitor cells to prepare multipotent blood progenitor cells expressing the CAR, inducing the differentiation of the CAR-expressing multipotent blood progenitor cells into T cells and then stimulating the TCR to induce differentiation into CD8-positive cytotoxic TCR-T or CAR-T cells; (4) preparing artificial leukocyte stem cells or T / NK progenitor cells from a cell population containing hematopoietic stem cells, inducing their differentiation into T cells and stimulating the TCR, and then introducing a gene encoding the CAR; and (5) combining CAR-macrophages and CAR-T cells and administering them to a patient having a tumor expressing the tumor antigen.

23. A method for treating a tumor in a patient, comprising the steps of: preparing CAR-macrophages by the following step (1) or (2): (1) preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells, introducing a gene encoding a CAR that specifically recognizes a tumor antigen into the multipotent blood progenitor cells to prepare multipotent blood progenitor cells expressing the CAR, and then inducing the differentiation of the CAR-expressing multipotent blood progenitor cells into macrophages; (2) preparing artificial leukocyte stem cells or T / NK progenitor cells from a cell population containing hematopoietic stem cells, inducing their differentiation into macrophages to stimulate the receptor, and then introducing the gene encoding the CAR; preparing CAR-NK cells by the following step (5) or (6): (5) (5) preparing multipotent blood progenitor cells from a cell population containing hematopoietic stem cells, introducing a gene encoding the CAR into the multipotent blood progenitor cells to prepare multipotent blood progenitor cells expressing the CAR, and then inducing the differentiation of the CAR-expressing multipotent blood progenitor cells into NK cells; (6) preparing artificial white blood stem cells or T / NK progenitor cells from a cell population containing hematopoietic stem cells, inducing their differentiation into NK cells to stimulate the receptor, and then introducing the gene encoding the CAR; and (7) combining CAR-macrophages and CAR-NK cells, and administering the combined product to a patient having a tumor expressing the tumor antigen.

24. The method of claim 23, wherein step (5) further comprises inducing differentiation of the CAR-expressing multipotent blood progenitor cells into NK cells to prepare CAR-NK cells, and then receptor-stimulating the CAR-NK cells.

25. The method according to any one of claims 22 to 24, wherein the step (1) further comprises inducing differentiation of the CAR-expressing multipotent blood progenitor cells into macrophages to prepare CAR-macrophages, and then receptor-stimulating the CAR-macrophages.

26. A method for producing human T / NK progenitor cells, comprising the step of culturing a cell population containing human hematopoietic stem cells in a medium containing SCF, Flt3-L and IL-7 at concentrations of 1 ng / mL or more and less than 10 ng / mL, respectively, in the presence of a Notch ligand.

27. The method of claim 26, wherein the medium further comprises at least one of TPO and M-CSF.

28. The method according to any one of claims 26 to 27, wherein the cell population containing human hematopoietic stem cells is a cell population obtained by culturing a cell population containing human hematopoietic stem cells in a medium containing SCF, TPO and G-CSF at concentrations of 100 ng / mL or more each in the absence of a Notch ligand.

29. A method for producing human NK cells, comprising: producing human T / NK precursor cells by the method according to any one of claims 26 to 28; and inducing differentiation of the produced human T / NK precursor cells into human NK cells by culturing the produced human T / NK precursor cells in a medium containing IL-7, Flt3-L, SCF and IL-15 in the absence of a Notch ligand.

30. A method for producing the immune cells described in claim 6 or 7, wherein the T / NK precursor cells are human T / NK precursor cells, and in the step of preparing multipotent blood precursor cells, human T / NK precursor cells are produced from a cell population containing human hematopoietic stem cells by a method described in any one of claims 26 to 28.

31. The method for producing the immune cells described in claim 30, wherein the immune cells are human NK cells, and in the step of inducing differentiation of the multipotent blood progenitor cells into immune cells, human T / NK progenitor cells are cultured in a medium containing IL-7, Flt3-L, SCF and IL-15 in the absence of a Notch ligand, thereby inducing differentiation of the human NK cells.

32. Cultured human T / NK progenitor cells, characterized by being CD122 positive.

33. Cultured human NK cells, which are characterized by being younger and less exhausted than human cord blood NK cells isolated from human umbilical cord blood, because they have lower expression of CD16 and KIR, which are indicators of maturity, and lower expression of the exhaustion marker TIGIT.

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