Chimeric antigen receptor-expressing immune cells

Abstract

The present invention addresses the problems of increasing the cytotoxic activity of immunocompetent cells and increasing the therapeutic effect thereof against diseases such as cancer, and provides immunocompetent cells that have reduced diacylglycerol kinase activity and express the chimeric antigen receptor and a fusion protein containing IL-15 and the IL-15 receptor α subunit.

Description

[Technical Field]

[0001] This invention relates to immune cells expressing chimeric antigen receptors and pharmaceuticals containing them. [Background technology]

[0002] In recent years, immunotherapy has attracted attention as a treatment for cancer. Immunotherapy is a treatment method in which immune cells that have been proliferated and activated outside the patient's body are administered to the patient, and these immune cells are used to attack cancer cells. Immunotherapy has the advantage of having almost no side effects compared to the three major therapies: surgery, radiation therapy, and chemotherapy. There are various types of immunotherapy, but among them, treatment using T cells (cytotoxic T cells: CTLs), which are responsible for innate immunity and have cytotoxic activity against cancer cells, is attracting attention. Furthermore, to enable cytotoxic T cells to recognize and attack specific antigens expressed on the surface of cancer cells, researchers are also expressing chimeric antigen receptors (CARs) on the surface of cytotoxic T cells.

[0003] Patent documents 1 and 2 disclose a method for inducing hematopoietic progenitor cells and CD4 / CD8-positive T cells from pluripotent stem cells, and Patent document 1 discloses that chimeric antigen receptors may be expressed in CD8-positive cytotoxic T cells. Patent Document 3 discloses a method for inducing cytotoxic T cells from pluripotent stem cells, stating that the cytotoxic T cells may express a chimeric antigen receptor, and furthermore, a fusion protein containing interleukin-15 (IL-15) and the interleukin-15 receptor α subunit (IL-15Rα) may be expressed. This fusion protein is also disclosed in Non-Patent Document 1.

[0004] On the other hand, Non-Patent Document 2 describes that knocking out the diacylglycerol kinase gene in cytotoxic T cells can enhance the anti-cancer activity of cytotoxic T cells. However, there are no known examples of combining the expression of an IL-15 and IL-15Rα fusion protein in immune cells such as T cells with the knockout of the diacylglycerol kinase gene. Given the various known modifications of immune cells, there was no motivation to combine these methods, and the effects of doing so were unknown. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] WO2017 / 221975 [Patent Document 2] WO2018 / 135646 [Patent Document 3] WO2020 / 013315 [Non-patent literature]

[0006] [Non-Patent Document 1] Proc. Natl. Acad. Sci. 113, E7788-e7797 (2016) [Non-Patent Document 2] Cancer Res. 2018 Aug 15;78(16):4692-4703. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The present invention aims to enhance the cytotoxic activity of immune cells and thereby improve the therapeutic effect against diseases such as cancer. [Means for solving the problem]

[0008] The inventors of this invention conducted diligent research to solve the above problems. As a result, they discovered that by reducing diacylglycerol kinase activity in immune cells such as cytotoxic T cells expressing chimeric antigen receptors, and by expressing a fusion protein (IL-15 / IL-15Rα) containing IL-15 and IL-15Rα, it is possible to significantly improve cytotoxicity against cancer cells and other immune cells, thereby improving therapeutic effects, and thus completed the present invention.

[0009] The gist of this invention is as follows: [1] Immune cells characterized by reduced diacylglycerol kinase activity and the expression of a fusion protein (IL-15 / IL-15Rα) containing IL-15 and the IL-15 receptor α subunit (IL-15Rα), as well as a chimeric antigen receptor. [2] The immune cells described in [1], which possess one or more diacylglycerol kinase genes having mutations that reduce intracellular diacylglycerol kinase activity. [3] The immune cell according to [1] or [2], wherein the fusion protein is a transmembrane protein. [4] Immune cells described in any of [1] to [3] that are CD5-positive and CD8β-positive. [5] A T cell, an immune cell as described in any of [1] to [4]. [6] Immune cells differentiated from pluripotent stem cells, as described in any of [1] to [5]. [7] Immune cells as described in [6], wherein the pluripotent stem cells are induced pluripotent stem cells. A pharmaceutical composition comprising immune cells as described in any of [8][1] to [7]. [9] The pharmaceutical composition described in [8] for the treatment of cancer.

[10] The pharmaceutical composition according to [9], wherein the cancer is a solid tumor. A method for treating cancer, comprising the step of administering a therapeutically effective amount of immune cells described in any of

[11] [1] to [7] to a subject in need of treatment. Use of immune cells in the manufacture of a cancer treatment pharmaceutical composition as described in any of

[12] [1] to [7].

[13] Immune cells described in any of [1] to [7] for the treatment of cancer.

[14] A method for producing chimeric antigen receptor-expressing immune cells, comprising the step of (a) preparing immune cells that express chimeric antigen receptors, The aforementioned immune cells have reduced diacylglycerol kinase activity, and It expresses a fusion protein (IL-15 / IL-15Rα) containing IL-15 and the IL-15 receptor α subunit (IL-15Rα). The aforementioned manufacturing method.

[15] The method according to

[14] , further comprising the step of (b) differentiating pluripotent stem cells into immune cells prior to step (a).

[16] The method according to

[15] , wherein the cells obtained in step (b) express chimeric antigen receptors.

[17] The method according to

[16] , wherein pluripotent stem cells expressing a chimeric antigen receptor are used in step (b), or the chimeric antigen receptor is expressed at any stage of step (b).

[18] The cells obtained in step (b) have reduced diacylglycerol kinase activity, the method according to any one of

[15] to

[17] .

[19] The method according to

[18] , wherein in step (b), pluripotent stem cells with reduced diacylglycerol kinase activity are used, or a modification is made at any stage of step (b) to reduce diacylglycerol kinase activity.

[20] The method according to any one of

[15] to

[19] , wherein the cells obtained in step (b) express a fusion protein (IL-15 / IL-15Rα) containing IL-15 and the IL-15 receptor α subunit (IL-15Rα).

[21] The method according to

[20] , wherein pluripotent stem cells expressing IL-15 / IL-15Rα are used in step (b), or IL-15 / IL-15Rα is expressed at any stage of step (b). [Effects of the Invention]

[0010] According to the present invention, remarkably superior cytotoxic effects can be achieved in cell immunotherapy for cancer and other conditions. Furthermore, by preparing immune cells such as cytotoxic T cells from pluripotent stem cells, a stable supply of cells necessary for treatment can be achieved. [Brief explanation of the drawing]

[0011] [Figure 1] Schematic diagram of the CAR introduction vector used in the example. [Figure 2A] This figure shows the detection results of phosphorylated ERK (pERK) in iCAR-TCTL, DGK-dKO iCAR-TCTL, and pCAR-TCTL after co-culturing irradiated SK-Hep-GPC3 for 10 minutes. [Figure 2B] This figure shows the gRNA (sgRNA) sequences of the DGKα (DGKa) or DGKζ (DGKz) gene, as well as the mutated DGKα and DGKζ gene sequences of both alleles in DGK-dKO iCAR. The bars indicate the mutated sequences. [Figure 2C] Graphs showing the proliferation of iCAR-TCTLs and DGK-dKO iCAR-TCTLs via target cells. Co-cultured with irradiated SK-Hep-GPC3 cells, measurements taken at 72 hours using a standard 3H-thymidine uptake assay (n=3, mean ± SEM). [Figure 2D] This photograph shows in vivo imaging results based on luciferase levels at the indicated time point for iCAR-TCTL or DGK-dKO iCAR-TCTL (n=8 / group) injected into JHH7 xenograft mice. [Figure 2E] This graph shows the time course of luciferase expression levels at the indicated time point for iCAR-TCTL or DGK-dKO iCAR-TCTL (n=8 / group) injected into JHH7 xenograft mice. [Figure 2F] This image shows in vivo bioluminescence imaging results of luciferase-labeled KOC7c in NSG mice treated with PBS, iCAR-TCTL, DGK-dKO iCAR-TCTL, or pCAR-TCTL (n=8 / group). [Figure 2G]Graph showing the total body flux per mouse as the total tumor volume of luciferase-labeled KOC7c in NSG mice treated with PBS, iCAR-TCTL, DGK-dKO iCAR-TCTL, or pCAR-TCTL (n=8 / group). [Figure 2H] Graphs showing the changes in survival rate in NSG mice treated with PBS, iCAR-TCTL, DGK-dKO iCAR-TCTL, or pCAR-TCTL (n=8 / group) at the indicated time after injection (***p<0.01, ****p<0.001). [Figure 3A] A diagram showing the treatment plan for a KOC7c peritoneal transplantation model of ovarian cancer cells. [Figure 3B] This image shows in vivo bioluminescence imaging results of xenograft tumor KOC7c labeled with sea urchin luciferase in NSG mice. 5 × 10⁵ luciferase-expressing KOC7c cells were injected intraperitoneally into NSG mice. From day 3, 1 × 10⁶ iCAR-TCTL, DGK-dKO iCAR-TCTL, pCAR-TCTL, or DGK-pKO pCAR-TCTL (all genetically modified with mbIL-15) cells were injected intraperitoneally once (n=5 / group). [Figure 3C] Graphs showing the results of evaluating the change in survival rate of treated mice at the indicated time point after injection when various TCTLs were administered to KOC7c-transplanted mice (***p<0.001, ****p<0.0001). [Figure 3D] Photographs showing in vivo bioluminescence imaging of injected iCAR-TCTL mbIL15, DGK-dKO iCAR-TCTL mbIL15, pCAR-TCTL mbIL15, and DGK-pKO pCAR-TCTL mbIL15 (all labeled with firefly luciferase) on days 13 and 20. [Figure 3E]Graphs showing quantitative results of in vivo bioluminescence imaging of injected iCAR-TCTL mbIL15, DGK-dKO iCAR-TCTL mbIL15, pCAR-TCTL mbIL15, and DGK-pKO pCAR-TCTL mbIL15 (all labeled with firefly luciferase) on days 13 and 20 (**p<0.01, ***p<0.001). [Figure 3F] A diagram showing the treatment plan for a xenograft-transplant liver cancer model. [Figure 3G] This graph shows the change in subcutaneous tumor volume in each individual when a xenograft liver cancer model was treated with various TCTL cells. NSG mice were subcutaneously inoculated with 2 × 10⁵ JHH7 cells, and on day 3, 1 × 10⁶ cells of iCAR-TCTL mbIL15, DGK-dKO iCAR-TCTL mbIL15, pCAR-TCTL mbIL15, or DGK-pKO pCAR-TCTL mbIL15 were administered intravenously (n=5 / group). [Figure 3H] Graphs showing changes in subcutaneous tumor volume in each group of xenograft liver cancer models treated with various TCTL cells (mean ± SEM). [Figure 3I] This graph shows the results of evaluating the change in survival rate when xenograft liver cancer models were treated with various TCTL cells (**p<0.01). [Figure 3J] This figure shows the results of flow cytometry (FCM) analysis of peripheral blood from a xenograft liver cancer model, performed 28 days after T cell injection. [Figure 4A] A diagram showing the treatment plan for a xenograft-transplant liver cancer model. [Figure 4B] Graphs showing changes in subcutaneous tumor volume in each individual when a xenograft liver cancer model was treated with various TCTL cells. NSG mice were subcutaneously inoculated with 5 × 10⁵ SK-Hep-GPC3 cells, and on day 10, 1 × 10⁷ cells of pCAR-TCTL, DGK-pKO pCAR-TCTL mbIL15, or DGK-dKO iCAR-TCTL mbIL15 were administered intravenously (n=6 / group). [Figure 4C]Graphs showing changes in subcutaneous tumor volume in each group when xenograft liver cancer models were treated with various TCTL cells (mean ± SEM, **p<0.01, ****p<0.0001). [Figure 5A] A diagram showing the treatment plan for a KOC7c peritoneal transplantation model of ovarian cancer cells. [Figure 5B] Graphs showing the changes in survival rate when KOC7c-transplanted mice were treated with various TCTL cells (*p<0.05, **p<0.01). [Figure 5C] Photographs showing in vivo bioluminescence imaging of injected DGKdKO iCAR-TCTL mbIL15tg and DGKpKO pCAR-TCTL mbIL15tg (both labeled with sea urchin luciferase) on days 7, 14, 21, 28, 42, and 74. [Figure 5D] This figure shows the results of analyzing effector memory phenotype-related factors using FCM in ascites fluid from KOC7c-transplanted mice in the DGK-dKO iCAR-TCTL mbIL15 group, performed 80 days after T cell injection. [Modes for carrying out the invention]

[0012] The immune cells of the present invention exhibit reduced diacylglycerol kinase activity and express a fusion protein (IL-15 / IL-15Rα) containing IL-15 and IL-15Rα, as well as a chimeric antigen receptor (CAR).

[0013] In this specification, "immune cells" refers to cells that perform immune functions in living organisms. Examples of immune cells include lymphocytes such as T cells, natural killer cells (NK cells), and B cells; antigen-presenting cells such as monocytes, macrophages, and dendritic cells; and granulocytes such as neutrophils, eosinophils, basophils, and mast cells. Specifically, T cells derived from mammals such as humans, dogs, cats, pigs, and mice are preferred, and human-derived T cells are preferred. Furthermore, T cells can be isolated and purified from immune cells infiltrating bodily fluids such as blood and bone marrow fluid, tissues such as the spleen, thymus, and lymph nodes, or cancerous tissues such as primary tumors, metastatic tumors, and malignant ascites. In addition, T cells produced from ES cells or iPS cells (iPSCs) may also be used. Examples of such T cells include αβT cells, γδT cells, and CD8 + T cells, CD4 + T cells (CD4 + Examples of immune cells include helper T cells, tumor-infiltrating T cells, memory T cells, naive T cells, and NKT cells. The origin of the immune cells and the recipient may be the same or different. Furthermore, when the recipient is a human, the immune cells may be autologous cells collected from the patient themselves or allogeneic cells collected from another person. In other words, the donor and recipient may be the same or different, but it is preferable that they be the same.

[0014] Suitable targets for the above-mentioned administration include mammals or mammalian cells, and among such mammals, humans, mice, dogs, rats, guinea pigs, rabbits, birds, sheep, pigs, cattle, horses, cattle, monkeys, and chimpanzees are more preferably selected, with humans being particularly preferred.

[0015] The immune cells used in this invention can be any immune cells capable of expressing CARs. Since CARs do not normally exist in nature, these are immune cells capable of expressing exogenous CARs, not endogenous ones. The immune cells used in this invention may express IL-7 and / or CCL19. If the immune cells are cells that do not express IL-7 and / or CCL19, for example, T cells, or if the immune cells are other than T cells and have low expression of IL-7 and / or CCL19, the immune cells used in this invention may express exogenous IL-7 and / or CCL19.

[0016] <Cytotoxic T cells> Cytotoxic T cells are particularly preferred among immune cells. Cytotoxic T cells (CTLs) are T cells that specifically exert cytotoxic activity against foreign cells such as cancer cells that present antigen peptides. Cytotoxic activity can be confirmed using indicators such as the secretion or production of granzymes or perforins. The origin of the cytotoxic T cells is not particularly limited; for example, they may be cytotoxic T cells isolated from living organisms, or cytotoxic T cells differentiated from pluripotent stem cells.

[0017] Cytotoxic T cells are preferably CD8β-positive, and more preferably both CD8β and CD5-positive, in order to enhance cytotoxicity and trafficking of cytotoxic T cells. Cytotoxic T cells may also be CD3-positive or CD8α-positive. They may also be positive for both α and β T cell receptors. The presence or absence of these molecules can be confirmed by detection or labeling using antibodies against them, or by sorting using flow cytometry.

[0018] <Chimera antigen receptor> A chimeric antigen receptor (CAR) refers to a fusion protein that contains an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The antigen-binding domain of a CAR may be a short-chain antibody (scFv) in which the light chain (VL) and heavy chain (VH) of the variable region of the antibody are linked in series via a spacer such as a peptide linker (e.g., a GS linker consisting of glycine and serine). T cells expressing a CAR recognize the antigen via the antigen-binding domain and then transmit the recognition signal to other T cells through the intracellular signal transduction domain. Introducing a CAR into T cells makes it possible to confer specificity to the target antigen. Furthermore, since a CAR can directly recognize antigen molecules without depending on HLA class I or class II, it can elicit a strong immune response even against cells with reduced expression of HLA class I or class II genes.

[0019] Antigens include tumor antigens and pathogen antigens. Examples of tumor antigens include glypican 3 (GPC3), CD19, MUC16, MUC1, CAlX, CEA, CDS, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49F, CD56, CD74, CD133, CD138, cytomegalovirus (CMV) infected cell antigen, EGP-2, EGP-40, EpCAM, and erb-B. 2,3,4, FBP, fetal acetylcholine receptor, folate receptor-α, GD2, GD3, HER-2, hTERT, IL-13R-A2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, NKG2D ligand, NY-ES0-1, tumor fetal antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, or one or more of WT-1.

[0020] The transmembrane domain of a CAR can be any peptide capable of transmembrane transmission across the cell membrane. Examples include, but are not limited to, transmembrane domains derived from one or more proteins selected from the group consisting of the α, β, or ζ chain of TCR, CD28, CD3ε chain, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, 4-1BB (CD137), CD154, ICOS, EGFR (epidermal growth factor receptor), and GITR. The transmembrane domain of the molecule from which the first intracellular signaling domain linked to the antigen-binding domain originates may also be used. For example, if the molecule from which the first intracellular signaling domain linked to the antigen-binding domain originates is CD28, the transmembrane domain may also be derived from CD28. Alternatively, an artificially designed transmembrane domain may be used.

[0021] The intracellular signaling domain of a CAR can be any region capable of signaling into immune cells when the antigen-binding domain binds to an antigen. Examples include, but are not limited to, intracellular domains derived from one or more proteins selected from the group consisting of CD3ζ chain (TCRζ chain), FcRγ chain, FcRβ chain, CD3γ chain, CD3δ chain, CD3ε chain, CD5, CD22, CD79a, CD79b, and CD66D. Among these, the intracellular signaling domain derived from the CD3ζ chain is preferred. Furthermore, the intracellular signaling domain may also include the intracellular domain of a co-stimulatory molecule. Examples of such co-stimulatory molecules include the intracellular domains of one or more proteins selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. By selecting the type and number of co-stimulatory molecules to bind, the strength and duration of CAR activity can be controlled (e.g., Mol Ther. 2009;17:1453-1464).

[0022] A spacer may be inserted between the antigen-binding domain and the transmembrane domain of the CAR, or between the intracellular signaling domain and the transmembrane domain of the CAR. The spacer can typically be a peptide consisting of 300 amino acids or less, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. Specifically, examples include, but are not limited to, a hinge region derived from IgG1 or IgG4, or a peptide containing the CH2CH3 region of immunoglobulin and a portion of CD3.

[0023] Specific examples of CARs include, but are not limited to, first-generation CARs in which scFV and CD3ζ chain are linked via a spacer; second-generation CARs in which a transmembrane domain and an intracellular domain derived from CD28 are incorporated between the scFV and CD3ζ chain of the first-generation CAR to enhance its ability to activate T cells; third-generation CARs in which the intracellular domain of a different co-stimulatory molecule (4-1BB or OX40) is incorporated between the intracellular domain of CD28 and the CD3ζ chain of the second-generation CAR; and fourth-generation CARs in which cytokines or molecules downstream of cytokine receptors are linked to the second-generation CAR to obtain more sustained T cell activation.

[0024] More specifically, examples include chimeric antigen receptors that include an scFv that recognizes GPC3 (e.g., an scFv derived from the antibody GC33) or an scFv that recognizes CD19 as an antigen-binding domain, a transmembrane domain of CD8 as a transmembrane domain, and intracellular domains derived from CD28, CD30, 4-1BB, and CD3ζ chain as intracellular signaling domains. The order of each of the above intracellular domains included in the intracellular signaling domain is not particularly restricted, but for example, they may be in the order of an intracellular domain derived from CD28, an intracellular domain derived from CD30 or an intracellular domain derived from 4-1BB, and an intracellular domain derived from CD3ζ chain.

[0025] As described below, CAR can be expressed by causing an immunocompetent cell to retain a polynucleotide encoding CAR.

[0026] <Fusion protein containing IL-15 and IL-15Rα> The fusion protein containing IL-15 and IL-15Rα (IL-15 / IL-15Rα) may be a transmembrane protein (sometimes called mbIL15) or a secreted protein. It is known that the IL-15 binding domain of 1-65 amino acids from the N-terminus of the mature protein is the responsible region for IL-15Rα to bind to IL-15 (Wei X. et al., J. Immunol., 167:277-282, 2001). Therefore, the transmembrane protein may be a fusion protein of an IL-15Rα protein retaining an IL-15 binding domain and a transmembrane domain and IL-15. On the other hand, as a secreted protein, it may be a fusion protein of an IL-15Rα protein retaining an IL-15 binding domain and lacking a transmembrane domain (for example, a protein consisting of 1-65 amino acid residues, 1-85 amino acid residues, or 1-185 amino acid residues of IL-15Rα, or a peptide containing an amino acid sequence having 90% or more identity with the amino acid sequence) and IL-15.

[0027] IL-15 / IL-15Rα may incorporate a spacer between IL-15 and IL-15Rα, and as the spacer, a peptide usually consisting of 300 amino acids or less, preferably 10-100 amino acids, and most preferably 20-50 amino acids can be used. Specifically, a GS linker etc. can be mentioned, but it is not limited thereto.

[0028] mbIL15 is not particularly limited as long as it is a transmembrane protein fused with IL-15 and IL-15Rα, but specifically, a polypeptide having the amino acid sequence of SEQ ID NO: 1 is an example. Alternatively, mbIL15 is not limited as long as it binds to the IL-15 receptor and transmits the IL-15 signal into the cell, but for example, a polypeptide containing an amino acid sequence having approximately 90% or more, preferably approximately 95% or more, more preferably approximately 97% or more, particularly preferably approximately 98% or more, and most preferably approximately 99% or more homology or identity with the amino acid sequence shown in SEQ ID NO: 1 is an example.

[0029] In amino acid sequences, "homology" or "identity" refers to the percentage of identical and similar amino acid residues (or identical amino acid residues in the case of identity) relative to all overlapping amino acid residues in the optimal alignment (preferably, the algorithm may consider introducing gaps into one or both sequences for optimal alignment) when two amino acid sequences are aligned using a mathematical algorithm known in the art. "Similar amino acids" refer to amino acids that are similar in physicochemical properties, and include, for example, amino acids classified into the same group such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), and amino acids with small side chains (Gly, Ala, Ser, Thr, Met). Substitutions by such similar amino acids are expected not to alter the protein phenotype (i.e., they are conservative amino acid substitutions). Specific examples of conservative amino acid substitutions are well known in the art and are described in various publications (see, for example, Bowie et al., Science, 247:1306-1310 (1990)). The homology or identity of amino acid sequences in this specification can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic LocalAlignment Search Tool) under the following conditions (expected value = 10; gaps allowed; matrix = BLOSUM62; filtering = OFF).

[0030] As described later, IL-15 / IL-15Rα can be expressed by having immune cells carry the polynucleotide encoding IL-15 / IL-15Rα.

[0031] <Decreased diacylglycerol kinase activity> Diacylglycerol kinase (DGK) is a lipid kinase that phosphorylates diacylglycerol and converts it to phosphatidic acid. DGK activity can be measured by known methods, and a decrease in DGK activity can be confirmed by a decrease in DGK expression levels using methods such as Western blotting or RT-PCR.

[0032] "Decreased DGK activity" in immune cells means that the DGK activity is reduced compared to control immune cells. For example, compared to control immune cells (immune cells with unmodified DGK genes), the DGK activity may be 50% or less, 20% or less, 10% or less, 5% or less, or 1% or less. DGK activity may also be below the detection limit. The immune cells used in this invention may be immune cells that originally have reduced DGK activity, or immune cells that have been modified to reduce DGK activity. Note that the reduction in DGK activity can also be achieved by adding a DGK activity inhibitor.

[0033] Although there are several subtypes of DGK, in T cells, subtypes α and ζ account for the majority of DGK activity. Therefore, it is preferable to reduce the activity of both subtype α and subtype ζ. Modifications that reduce DGK activity include introducing mutations that decrease DGK activity in the DGK gene on the chromosome, introducing mutations that decrease DGK stability in the DGK gene on the chromosome, introducing mutations that delete part or all of the DGK gene on the chromosome, and introducing mutations that reduce the expression level of the DGK gene on the chromosome, such as introducing mutations in the expression regulatory region or translation regulatory region. Such mutations can be introduced using, for example, genome editing technologies such as CRISPR-Cas9 and TALEN, RNA interference technologies and homologous recombination technologies using siRNA, shRNA, miRNA, etc., and site-directed recombination technologies such as the Cre / Lox P system. When using the Cre / Lox P system, for example, DGKα - / - DGKζ f / fBy introducing Cre into cells, it is possible to create cells in which both DGKα and DGKζ are knocked out.

[0034] <Method for producing immune cells> The present invention provides a method for producing immune cells in which DGK activity is reduced and immune cells expressing IL-15 / IL-15Rα and CAR are prepared.

[0035] The immune cells of the present invention can be obtained by modifying immune cells to express CAR, further reducing DGK activity, and expressing IL-15 / IL-15Rα. Furthermore, if DGK activity is already reduced in immune cells, the immune cells of the present invention can be obtained by expressing CAR and IL-15 / IL-15Rα in those immune cells. Furthermore, there are no particular restrictions on the order in which the following steps are performed: modification to reduce DGK activity (i), CAR expression (ii), and IL-15 / IL-15Rα expression (iii); these operations may be performed in any order.

[0036] As the immune cells used as material for the immune cells of the present invention, immune cells such as cytotoxic T cells isolated from living organisms, such as peripheral blood mononuclear cells (PBMCs), can be used. In this case, it is preferable to use immune cells such as cytotoxic T cells derived from the patient to whom the immune cells of the present invention will be administered.

[0037] Furthermore, the immune cells that serve as the material for the immune cells of the present invention may be immune cells obtained by differentiating pluripotent stem cells. Alternatively, the immune cells that serve as the material for the immune cells of the present invention may be derived from hematopoietic stem cells or hematopoietic progenitor cells after inducing them from pluripotent stem cells using a known method (e.g., WO2015 / 199127). In these cases, the modifications described in (i) to (iii) above may be applied after differentiating pluripotent stem cells into immune cells, or the modifications described in (i) to (iii) above may be applied at the pluripotent stem cell stage, or the modifications described in (i) to (iii) above may be applied at any stage during differentiation. In other words, these modifications can be applied in any order and at any stage before differentiation, during differentiation, or after differentiation.

[0038] Furthermore, CAR and IL-15 / IL-15Rα are preferably introduced into cells in the form of polynucleotides encoding them. The polynucleotide may be DNA, RNA, or a DNA / RNA chimera, but DNA is preferred. For example, the polynucleotide can be constructed by chemically synthesizing a DNA strand based on the amino acid sequence or base sequence of a known CAR, or by joining partially overlapping oligoDNA short chains using PCR or Gibson Assembly to construct DNA encoding the full length or part of the CAR. Polynucleotides encoding IL-15 / IL-15Rα can be constructed in a similar manner.

[0039] The polynucleotides described above can be incorporated into an expression vector. This vector may be one that is incorporated into the genome of the target cell, or it may not be one that is incorporated into the genome. The expression vector is not particularly limited as long as it can express CAR or mblL15 for a period sufficient to treat the disease when introduced into cells, but examples include viral vectors and plasmid vectors. Examples of viral vectors include retroviral vectors (including lentiviral vectors and pseudotype vectors), adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, Sendai virus, and episomal vectors. Transposon expression systems (PiggyBac systems) may also be used. Examples of plasmid vectors include animal cell expression plasmids (e.g., pa1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo).

[0040] Examples of promoters used in the above vectors include the EF1α promoter, CAG promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney mouse leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, TCR V α gene promoter, and TCR Vβ gene promoter.

[0041] In addition to the promoter described above, the vector may optionally include transcription and translation regulatory sequences, ribosome binding sites, enhancers, origins of replication, poly(A) addition signals, and selection marker genes. Examples of selection marker genes include dihydrofolate reductase genes, neomycin resistance genes, and puromycin resistance genes.

[0042] Vectors expressing CARs may also contain suicide genes. Such suicide genes, when co-expressed with CARs and activated by drugs such as medications or antibodies, can induce cell death. These suicide genes may be used to improve the safety of immune cells expressing CARs. Examples of such suicide genes include, but are not limited to, inducible caspase 9 (iC9), truncated EGFR (tEGFR), and herpes simplex virus thymidine kinase (HSV-TK). tEGFR consists of a truncated transmembrane domain and an extracellular domain of the EGFR protein. The suicide genes described above may be linked to the polynucleotide encoding the CAR via a linker (e.g., T2A or IRES).

[0043] There are no particular limitations on the method for introducing the above-mentioned polynucleotides or vectors into cells, and known methods can be used. Examples include electroporation (Cytotechnology, 3,133 (1990)), calcium phosphate method (Japanese Patent Publication No. 2-227075), lipofection method (Proc. Natl. Acad. Sci. USA, 84,7413 (1987)), and viral infection methods. As an example of such a viral infection method, a CAR expression vector (International Publication No. 2016 / 056228) and a packaging plasmid are transfected into packaging cells such as GP2-293 cells (Takara Bio Inc.), Plat-GP cells (Cosmo Bio Inc.), PG13 cells (ATCC CRL-10686), and PA317 cells (ATCC CRL-9078) to produce recombinant viruses, and these recombinant viruses are used to infect T cells.

[0044] Alternatively, CAR-coding nucleotides or IL-15 / IL-15Rα-coding nucleotides may be manufactured by incorporating them into the cell genome using known gene editing techniques so that they can be expressed under the control of an appropriate promoter. Known gene editing techniques include those using zinc finger nucleases, TALEN (transcriptional activation-like effector nucleases), and endonucleases such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas system.

[0045] On the other hand, examples of modification methods to reduce DGK activity include knocking out or knocking down one or more DGK genes on the chromosome.

[0046] Methods for knocking out the DGK gene include, for example, methods known in the art, such as methods that eliminate the expression of a functional protein by replacing all or part of a drug resistance gene with a target gene using homologous recombination, methods that utilize site-directed recombination reactions such as the Cre / Lox P system, and genome editing technologies using TALEN or CRISPR-Cas9. A method for knocking out the DGK gene by genome editing is disclosed, for example, in Non-Patent Document 2. Note that knocking out the DGK gene may also be carried out by deleting one or more exons of the gene.

[0047] Alternatively, the DGK gene may be knocked down. Methods for knocking down the DGK gene include, for example, antisense methods, which involve introducing RNA equivalent to the antisense strand of mRNA into cells, and RNAi methods, which use siRNA, shRNA, microRNA, etc., all of which are well-known in the art. The knockdown efficiency can be confirmed by measuring changes in mRNA levels or protein expression levels. Knockdown of the DGK gene may also be performed by targeting one or more exons of the gene.

[0048] <Method for inducing immune cells from pluripotent stem cells> Pluripotent stem cells are stem cells that possess pluripotency, meaning they can differentiate into many types of cells present in the body, and also possess proliferative capacity. They are not particularly limited as long as they are cells that can be induced by immune cells. Pluripotent stem cells are preferably of mammalian origin, and more preferably of human origin. Pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, spermatogonial stem cells ("GS cells"), embryonic germ cells ("EG cells"), induced pluripotent stem (iPS) cells, cultured fibroblasts, umbilical cord blood-derived pluripotent stem cells, and bone marrow stem cell-derived pluripotent cells (Muse cells). Preferred pluripotent stem cells are iPS cells, and more preferably human iPS cells, from the viewpoint that they can be obtained without destroying embryos, eggs, etc., in the manufacturing process.

[0049] Methods for producing iPS cells are well known in the field and can be produced by introducing reprogramming factors into any somatic cell. Examples of reprogramming factors include genes or gene products such as Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5A2, Tbx3, or Glis1. These reprogramming factors may be used individually or in combination. The combinations of initialization factors are WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, W O2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO 2010 / 056831, WO2010 / 068955, WO2010 / 098419, WO2010 / 102267, WO 2010 / 111409, WO2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al. (2008), Nat. Biotechnol., 26: 795-797, Shi Y, etal. (2008), Cell Stem Cell, 2: 525-528, EminliS, et al. (2008), Stem Cells. 26:2467-2474, Huangfu D, et al. al. (2008), Nat. Biotechnol. 26:1269-1275, Shi Y, et al.(2008), Cell Stem Cell,3, 568-574、Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479、Marson A, (2008), Cell Stem Cell, 3, 132-135、Feng B, et al. (2009), Nat. CellBiol. 11:197-203、RL Judson et al., (2009), Nat. Biotechnol., 27:459-461、Lyssiotis CA, etal. (2009), Proc Natl Acad Sci US A. 106:8912-8917、Kim JB, et al. (2009), Nature. 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell.5:491-503、Heng JC, et al. (2010), Cell Stem Cell. 6:167-74、Han J, et al. (2010), Nature. 463:1096-100、Mali P, et al. (2010), Stem Cells.28:713-720、Maekawa M, et al. (2011), Nature.474:225-9.

[0050] Somatic cells used to create iPS cells include fetal (offspring) somatic cells, neonatal (offspring) somatic cells, and mature healthy or diseased somatic cells, as well as primary cultured cells, passaged cells, and established cell lines. Specifically, examples of somatic cells include (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue progenitor cells, and (3) differentiated cells such as blood cells (peripheral blood cells, umbilical cord blood cells, etc.), lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes. It is preferable to use immune cells as somatic cells for inducing immune cells, but it is also possible to produce immune cells from iPS cells using non-immune cells.

[0051] In the present invention, the mammalian individual from which somatic cells are collected is not particularly limited, but is preferably human. When administering the immune cells of the present invention to a target, it is preferable that the somatic cells that will become iPS cells be isolated from the target, from the viewpoint of facilitating matching of the human leukocyte antigen (HLA) type with that of the target.

[0052] The method for inducing differentiation from pluripotent stem cells into cytotoxic T cells is not particularly limited, and known methods can be used. For example, one method involves inducing hematopoietic progenitor cells from pluripotent stem cells, then inducing CD4 and CD8-positive cells from the hematopoietic progenitor cells, and finally inducing CD8-positive cells from the CD4 and CD8-positive cells. Specifically, the methods described in Patent Documents 1 to 3 are examples of methods for inducing differentiation from pluripotent stem cells into cytotoxic T cells, but the method is not particularly limited as long as cytotoxic T cells can be obtained. Furthermore, immune cells such as cytotoxic T cells differentiated from pluripotent stem cells can be used as is, but they can also be selected using markers such as CD8β and CD5 before use.

[0053] Methods for inducing differentiation from pluripotent stem cells into various immune cells other than cytotoxic T cells include those described in the following literature. CD4 helper T cells...WO2017 / 065288, WO2018 / 168829 γδ T cells...WO2020 / 013315 NK cells...Blood(2016)128(22):1345, WO2013 / 163171, WO2010 / 099539 NKT cells Stem cell Rep. 2016 Feb 9;6(2):213-227. doi: 10.1016 / j.stemcr.2016.01.005., Stem Cells. 2016 Dec;34(12):2852-2860 Monocyte / Macrophage···WO2015 / 199127 Lymphocytes such as B cells...Japanese Patent Publication No. 2006-141356 Antigen-presenting cells such as dendritic cells...WO2012 / 115276, Clin CancerRes. 2008 Oct 1;14(19):6207-6217 Granulocytes such as neutrophils, eosinophils, basophils, and mast cells...WO2015 / 199127, JP 2006-141356

[0054] <Uses of immune cells> The present invention provides a pharmaceutical composition containing immune cells of the present invention. Since the immune cells of the present invention can exhibit cytotoxic activity against, for example, cancer cells and virus-infected cells, the pharmaceutical composition containing the cytotoxic T cells of the present invention can be used for the treatment of cancer, infectious diseases (e.g., chronic infections), autoimmune deficiency, and the like. The pharmaceutical compositions of the present invention can be administered, for example, to mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cattle, sheep, monkeys, and humans), preferably to humans. Accordingly, in one embodiment of the present invention, a pharmaceutical composition comprising the immune cells of the present invention for use in the treatment of cancer is provided. Furthermore, a method for treating cancer is provided, comprising administering an effective amount of the immune cells of the present invention or a pharmaceutical composition containing them to a subject in need of treatment.

[0055] Examples of cancers include, but are not limited to, liver cancer (e.g., hepatocellular carcinoma), ovarian cancer (e.g., clear cell adenocarcinoma of the ovary), childhood cancer, lung cancer (e.g., squamous cell carcinoma, small cell lung cancer), testicular cancer (e.g., non-seminoma germ cell tumor), soft tissue tumors (e.g., liposarcoma, malignant fibrous histiocytoma), uterine cancer (e.g., intraepithelial neoplasia of the cervix, squamous cell carcinoma of the cervix), melanoma, adrenal tumors (e.g., adenoma of the adrenal gland), neurological tumors (e.g., Schwannoma), stomach cancer (e.g., adenocarcinoma of the stomach), kidney cancer (e.g., Gravitz tumor), breast cancer (e.g., invasive lobular carcinoma, mucinous carcinoma), thyroid cancer (e.g., medullary carcinoma), laryngeal cancer (e.g., squamous cell carcinoma), and bladder cancer (e.g., invasive transitional cell carcinoma).

[0056] The immune cells of the present invention may be cultured and / or stimulated using a suitable medium and / or stimulating molecule before administration to a target. Examples of stimulating molecules include, but are not limited to, cytokines, suitable proteins, and other components. Examples of cytokines include IL-2, IL-7, IL-12, IL-15, and IFN-γ, with IL-2 being preferred. The concentration of IL-2 in the medium is not particularly limited, but is preferably, for example, 0.01 U / ml ~ 1 × 10⁻⁶. 5 U / ml, more preferably 1 U / ml to 1 × 10 4 The concentration is U / ml. Suitable proteins include, for example, CD3 ligand, CD28 ligand, and anti-IL-4 antibody. In addition, lymphocyte-stimulating factors such as lectins can also be added. Furthermore, serum or plasma may be added to the culture medium. There are no particular limitations on the amount of these added to the medium, but 0% to 20% by volume is an example, and the amount of serum or plasma used can be changed depending on the culture stage. For example, the serum or plasma concentration can be gradually reduced. The serum or plasma can be from either the patient's own body or a non-patient body, but from a safety standpoint, patient-derived serum or plasma is preferred.

[0057] The pharmaceutical composition of the present invention is preferably administered to a subject parenterally. Examples of parenteral administration methods include intravenous, intra-arterial, intramuscular, intraperitoneal, subcutaneous administration, and direct administration to the affected area. The dosage is appropriately selected according to the condition, body weight, age, etc. of the subject. Usually, as the number of cells, for a subject weighing 60 kg, per administration, usually 1×10 6 ~1×10 10 cells, preferably 1×10 7 ~1×10 9 cells, more preferably 5×10 7 ~5×10 8 cells are administered. Also, it may be administered once or multiple times. The pharmaceutical composition of the present invention can be in a known form suitable for parenteral administration, for example, an injection or an infusion. The pharmaceutical composition of the present invention may optionally contain pharmacologically acceptable excipients or carriers. The pharmaceutical composition of the present invention may contain physiological saline, phosphate-buffered saline (PBS), a medium, etc. to maintain the cells stably. The medium is not particularly limited, and examples include, but are not limited to, media such as RPMI, AIM-V, and X-VIVO10. Also, pharmaceutically acceptable carriers (e.g., human serum albumin), preservatives, etc. may be added to the pharmaceutical for the purpose of stabilization.

[0058] Furthermore, since the immune cells of the present invention can kill cells expressing target antigens such as the above-mentioned tumor antigens, they can be used as a killing agent for cells expressing the antigen (e.g., cancer cells, cancer stem cells, tumor cells, etc.). Such a killing agent can be prepared and used in the same manner as the above-mentioned pharmaceutical.

[0059] The immune cells of the present invention can be used in combination with other anticancer agents. Other anticancer agents include alkylating agents such as cyclophosphamide, bendamustine, eosfamide, and dacarbazine; antimetabolites such as pentostatin, fludarabine, cladribine, methotrexate, 5-fluorouracil, 6-mercaptopurine, and enocitabine; molecular targeted drugs such as rituximab, cetuximab, and trastuzumab; and kinase inhibitors such as imatinib, gefetinib, erlotinib, afatinib, dasatinib, sunitinib, and trametinib. Examples include proteasome inhibitors such as bortezomib, calcineurin inhibitors such as cyclosporine and tacrolimus, anticancer antibiotics such as idarubidine and doxorubicin mitomycin C, plant alkaloids such as irinotecan and etoposide, platinum-based drugs such as cisplatin, oxaliplatin, and carboplatin, hormone therapy drugs such as tamoxifen and bicardiamide, and immunosuppressants such as interferon, nivolumab, and pembrolizumab.

[0060] Methods for using the immune cells of the present invention in combination with other anticancer drugs include treating with other anticancer drugs and then using the immune cells of the present invention, using the immune cells of the present invention and other anticancer drugs simultaneously, and treating with the immune cells of the present invention and then using other anticancer drugs. Furthermore, when the immune cells of the present invention are used in combination with other anticancer drugs, the therapeutic effect on cancer is further improved, and the side effects of each drug can be reduced by reducing the number of administrations or the dosage of each drug.

[0061] Furthermore, the present invention also includes embodiments of the use of the immune cells of the present invention in the manufacture of cancer therapeutic agents, in accordance with the pharmaceutical composition containing the immune cells of the present invention. Cancer therapeutic agents can be manufactured by methods known to the extent of the invention. For example, they can be manufactured in known forms suitable for parenteral administration, such as injections or infusions, similar to the preparation method of the pharmaceutical of the present invention described above. [Examples]

[0062] The present invention will be specifically described below with reference to examples. However, the present invention is not limited to the embodiments described below.

[0063] <Experimental Procedure> Production of CAR-induced iPS cells As the CAR, GC33-CD28-41BB-T2A EGFR was designed by Professor Tamada of Yamaguchi University and manufactured by Genscript. This CAR has a CD8 transmembrane domain. The lentiviral vector CS-UbC-RfA-IRES-hKO1 was used to introduce the CAR gene into iPS cells. This vector expresses a humanized Kusabira-Orange 1 (hKO1) fluorescent protein gene bound to the IRES sequence under the control of the human ubiquitin C (UbC) promoter. As iPS cells, TKT3v strains 1-7 were established from human CD3-positive T cells isolated after notification and consent were obtained, using the method described in Nishimura T, et al., Cell Stem Cell. 12(1):114-126, 2013. The lentiviral vector CS-UbC-RfA-IRES-hKO1 (a schematic diagram of a portion of the vector is shown in Figure 1), incorporating the GC33-CD28-41BB-T2A EGFR gene, was introduced into the TKT3v1-7 cell line, and iPS cells expressing hKO1 (CAR-iPSCs) were selected.

[0064] CRISPR-Cas9 synthesis of iPSCs lacking DGK We designed single guide RNAs specific to exon 7 of DGKα (diacylglycerol kinase alpha) and exon 2 of DGKζ (diacylglycerol kinase zeta) (SEQ ID NOs: 2 and 3, respectively). To construct a custom sgRNA expression vector, we PCR-amplified two oligonucleotides containing the sgRNA target site and universal reverse primers (SEQ ID NOs: 4 and 5 for DGKα deficiency; SEQ ID NOs: 6 and 5 for DGKζ deficiency) and cloned them into the BamHI-EcoRI site of the pHL-H1-ccdB-mEFα-RiH vector (addgene #60601), which uses the H1 promoter for sgRNA expression. A plasmid expressing a nuclease (5 μg pHL-EF1α-SphcCas9-iP) and a 5 μg guide RNA expression vector (for DGK deficiency) were electroporated using a NEPA 21 electroporator (perforation pulse: pulse voltage 175 V; pulse width 2.5 ms; number of pulses 2; Negagene) to obtain CAR-iPSC 1 × 10⁶ 6 The mutations were introduced individually. iPSC clones in which nonsense mutations were introduced into both DGKα and DGKζ were selected (DGK-dKO CAR-iPSC).

[0065] T cell differentiation from CAR-iPSCs To differentiate iPSCs into T cells, we modified a previously reported protocol (Cell Stem Cell1-9 (2018). doi:10.1016 / j.stem.2018.10.005). First, CAR-iPSCs were differentiated into hematopoietic progenitor cells via SAC formation on feeder cells or via embryoid body formation under feeder-free conditions. In the method for SAC formation, iPSC aggregates were seeded onto C3H10T1 / 2 feeder cells and cultured in rhVEGF-supplemented EB medium (R&D). On day 7, rhSCF (Peprotech) and rhFlt-3L (Peprotech) were added to the culture medium. In the feeder-free embryoid body formation method, undifferentiated CAR-iPSC colonies were treated with TrypLE select (Gibco) for 8 minutes, then seeded on low-adhesion plates and allowed to form embryoid bodies in Stemfit AK03N supplemented with 10 μM Y-27632. After overnight incubation, embryoid body formation was promoted. The embryoid bodies were harvested and seeded on EB medium (StemPro-34 (Invitrogen), 2 mM l-glutamine, 400 μM monothioglycerol, 50 μg / ml ascorbic acid 2-phosphate, and insulin-transferrin-selenium medium additives) and cultured in the presence of 40 ng / ml hBMP-4, 10 ng / ml hbFGF, and 50 ng / ml VEGF. On day 4, embryoid bodies were cultured with a cocktail of hematopoietic cytokines (50 ng / ml hSCF, 20 ng / ml hFlt3L, 20 ng / ml hIL-3, and 30 ng / ml TPO). From days 8 to 14 of culture, differentiated cells were seeded on Delta-like 1-expressing OP9 (OP9-DL1) feeder cells or on plates coated with FcDLL4 and cultured in the presence of a cocktail of T-lineage cytokines (10 ng / ml hFlt3L and 5 ng / ml IL-7). From days 21 to 28 of culture, hematopoietic cells differentiated into CD3+ T-lineage cells.T-series cells were collected and stimulated with 500 ng / ml OKT3 (Miltenyi Biotec) in the presence of 10 ng / ml rhIL-7 and 10 nM dexamethasone (Dexart, Japan, Fuji Pharmaceutical Co., Ltd.) in a-MEM (SIGMA) supplemented with 15% fetal bovine serum (Thermo), 2 mM L-glutamine (Thermo), 100 U / ml penicillin (Thermo), and 1 G00 ng / ml streptomycin (Thermo). CD5 / CD8β double-positive T cells were selected from the obtained cell population and then treated as iCAR-T cells. CTL It was used in the following experiments. iCAR-T CTL The irradiated PBMCs were mixed and cultured on a large scale, and then co-cultured in alpha-MEM medium in the presence of 10 ng / ml IL-7, 5 ng / ml IL-15 (Peprotech), and 2 μg / ml PHA (SIGMA). DGK-dKO CAR-iPSCs were differentiated using a similar method, and CD5 / CD8β double-positive T cells were selected and designated as DGK-dKO iCAR-T cells. CTL It was used in the following experiments.

[0066] Flow cytometry analysis Stained cell samples were analyzed using LSR or FACS AriaII Flow Cytometer (BD Biosciences), and the data was processed using FlowJo (Tree Star). The following antibodies were used for phenotypic analysis of T cells: APC-cy7-CD3 (clone UCHT1, BioLegend), eFluor 450-CD3 (clone UCHT1, eBioscience), BV421-CD4 (clone OKT4, BioLegend), BV510-CD4 (clone OKT4, BioLegend), FITC-CD5 (clone UCHT2, eBioscience), PE-Cy7-CD5 (clone UCHT2, eBioscience), CD7-APC (clone CD7-6B7, BioLegend), PerCPcy5.5-CD8 (clone SK1, BioLegend), APC-CD8β (clone 2ST8.5H7, BD), PE-Cy7-CD8β (clone SIDI8BEE, eBioscience), BV510-CD45RA (clone HI100 (BioLegend), APC-cy7-CD45RO (clone UCHL1, BioLegend), FITC-CD62L (clone DREG-56, BioLegend), APC-CD197 (CCR7) (clone G043H7, BioLegend), BV421-CD28 (clone CD28.2, BioLegend), APC-CD27 (clone O323, BioLegend), BV421-CD279 (PD-1) (clone EH12.2H7, BioLegend), and PerCP-eFluor 710-TIGIT (clone MBSA43, eBioscience).

[0067] 3 H-thymidine uptake-mediated growth assay Proliferation of T cells 3 Measurement was performed using an H-thymidine uptake assay. 1 × 10 5 Irradiated 1 × 10⁶ effector cells 4 Individual target cells were co-cultured for 3 days. 3 Pulse-labeled with H, after 16 hours 3H-thymidine was measured using a β-counter.

[0068] CAR introduction into primary T cells and creation of DGK-deficient primary T cells Primary T cells were isolated from blood collected from healthy donors, and T cells were isolated and stimulated using Thermo Fisher Dynabeads Human T-Activator CD3 / CD28. CAR was introduced into primary T cells three days after stimulation using a lentivirus. CAR-introduced cells were sorted by flow cytometry based on CAR positivity and CD8 positivity (pCAR-T). CTL cell).

[0069] DGK-deficient primary T cells were generated using the following procedure (DGK-pKO pCAR-T CTL cell). Primary T cells were stimulated with Dynabeads Human T Activator anti-CD3 / 28 (Thermo Scientific). Four days after stimulation, electroporation was performed using the Amaxa P3 Primary Cell Kit and 4D-Nucleofecter (Lonza). Cas9-gRNA RNP complexes were constructed by incubating 10 μg of genetically modified S. pyogenes Cas9 (Thermo Fisher Scientific) and 1.25 μg of chemically synthesized DGKα and ζ-deficient sgRNA (synthesized using oligonucleotides of SEQ ID NOs. 7 and 8 for α-deficiency, and oligonucleotides of SEQ ID NOs. 9 and 8 for ζ-deficiency) for 20 minutes, followed by electroporation. A total of 5 × 10⁶ 5 Individual cells were resuspended, and P3 buffer was added to the pre-incubated Cas9-gRNA RNP complex. The complex was introduced into the cell nucleus (nucleofect) using program EO-115. Cells were harvested one week after electroporation, and genomic DNA was isolated. Indel frequencies were calculated using Tracking of Indels by Decomposition (TIDE) (NucleicAcids Res. 42, (2014)) analysis.

[0070] Creation of CAR-T cells with mbIL-15 gene transfer. We received RD18 cells capable of producing a retroviral vector encoding mbIL15-T2A-NGFR from Dr. Kazuhide Nakayama of Takeda Pharmaceutical Company Limited. This retroviral vector contains a nucleotide sequence encoding a polypeptide (SEQ ID NO: 1) that includes (i) the read sequence of human IL-2 (23 amino acids), (ii) the C-terminal sequence of human IL-15 (114 amino acids), (iii) the GS linker (24 amino acids), and (iv) the C-terminal sequence of human IL-15Rα (239 amino acids) in order from the N-terminus to the C-terminus. CTL Cells were stimulated with Dynabeads Human T Activator anti-CD3 / 28 (Thermo Scientific). Three days after stimulation, these cells were transfused using a retroviral vector encoding mbIL15-T2A-NGFR. NGFR-positive cells were purified by FACS. iCAR-T CTL Cells or DGK-dKO iCAR-T CTL Cells obtained by introducing mbIL-15 into cells were each classified as iCAR-T CTL mbIL cells (in the figure, "iCAR-T CTL mbIL15tg and iCAR-T CTL (Also known as mbIL15), DGK-dKO iCAR-T CTL mbIL cells (in the figure, "DGK-dKO iCAR-T CTL mbIL15tg”, “DGK-dKO iCAR-T CTL mbIL15" and "DGKdKO iCAR-T CTL It was named "mbIL15tg" (also known as "mbIL15tg"). pCAR-T CTL Cells or DGK-pKO pCAR-T CTL Cells obtained by introducing mbIL-15 into cells were each subjected to pCAR-T CTL mbIL cells (in the figure, "pCAR-T CTL mbIL15tg and pCAR-T CTL(Also known as mbIL15), DGK-pKO pCAR-T CTL mbIL cells (in the figure, "DGK-pKO pCAR-T CTL mbIL15tg”, “DGK-pKO pCAR-T CTL mbIL15" and "DGKpKO pCAR-T CTL It was named "mbIL15tg" (also known as "mbIL15tg").

[0071] Bioluminescence Imaging Bioluminescence was detected using the Xenogen IVIS Imaging System (Xenogen). VivoGlo® luciferin (3 mg / mouse; Promega) was injected intraperitoneally, and imaging was performed 10-15 minutes later.

[0072] in vivo tumor model Peritoneal dissemination animal model #1 A total of 5 × 10¹⁶ ovarian cancer cells KOC7c were transfected with luciferase. 5 After injecting the individual into the peritoneal cavity of NSG mice, various CAR-T cells were administered. CTL Cells 5 × 10 6 Inject the cells intraperitoneally on days 3, 7, 10, and 14 (Figure 2F~H), or administer CAR-T cells at a dose of 1 × 10⁶. 6 The drug was administered intraperitoneally on day 3 (Figures 3A-E). Tumor volume was measured using in vivo bioluminescence imaging (IVIS 100 Imaging System, Caliper).

[0073] Analysis of tumor-infiltrating CAR-T cell models A total of 5 × 10⁶ SK-Hep-1 cells that have been genetically modified with GPC3. 6 The individual was subcutaneously injected into the left flank of NSG mice. Various CAR-T cells were administered on day 0. CTLCells were injected intravenously. Mice were sacrificed on day 7, and subcutaneous tumors were collected. Each tumor was divided into two sections. One section was carefully finely chopped with a razor and gently disrupted using a MACS Dissociator. This was then filtered through a 70 mm nylon mesh cell strainer. The single-cell suspension was stained with a fluorescently labeled antibody as described. The other section was prepared for histopathological analysis.

[0074] Subcutaneous tumor animal model #1 On day 14, 5 × 10⁶ liver cancer cells (SK-Hep-1 cells) that had been genetically modified with GPC3 were used. 6 The individual cells were subcutaneously injected into the left flank of NSG mice, and a total of 5 × 10⁶ untransferred SK-Hep-1 cells were added. 6 The individual was subcutaneously injected into the right flank of NSG mice. Various CAR-T cells were administered on day 0. CTL Cells were injected intravenously. Various CAR-T cells were used. CTL The luciferase activity of cells was measured using in vivo bioluminescence imaging.

[0075] Subcutaneous tumor animal model #2 Total 2 x 10 5 Inject 1 x 10¹ JHH7 cells subcutaneously. 7 Individual various CAR-T CTL Inject the cells intravenously on days 3 and 10 (Figures 2D and E), or 1 × 10 6 Individual CAR-T cells were intravenously injected on day 3 (Figure 3F-J). Luciferase activity of the T cells was measured using in vivo bioluminescence imaging. Tumor dimensions were measured with calipers, and tumor volume was calculated using the formula: V = LW 2 The calculation was performed according to / 2 (where L represents the length (major axis) and W represents the width (minor axis)).

[0076] Subcutaneous tumor animal model #3 Total 5 x 10 5 Individual SK-Hep-GPC cells were subcutaneously injected, and 1 x 10⁶ cells were obtained on day 10. 7 Individual various CAR-T CTL Cells were injected intravenously (Figures 4A-C). The dimensions of the tumor were measured with calipers, and the tumor volume was calculated using the formula: V = LW. 2The calculation was performed according to / 2 (where L represents the length (major axis) and W represents the width (minor axis)).

[0077] Peritoneal dissemination animal model #2 A total of 5 × 10¹⁶ KOC7c cells that have been genetically modified with luciferase. 5 After injecting the individual into the peritoneal cavity of NSG mice, various CAR-T cells were administered on day 10. CTL Cells 1 × 10 7 The drug was injected intraperitoneally once (Figures 5A-D). Luciferase activity of T cells was measured using in vivo bioluminescence imaging.

[0078] statistics All data are expressed as mean ± SEM. All statistical analyses were performed using Prism (GraphPad software). When comparing multiple groups, ANOVA and Turkey's multiple comparison test were used, and for two-group comparisons of parameter data, Student's t-test was performed as a two-tailed test. A p-value of less than 0.05 was considered statistically significant.

[0079] <Result> When cytotoxic T cells obtained by differentiation induction from CAR-iPSCs were analyzed for markers and cell selection based on these markers, it was found that CD5-positive and CD8β-positive cells had gene expression profiles similar to those of cytotoxic T cells in vivo, and exhibited superior trafficking ability and cytotoxicity towards cancer cells. Therefore, cytotoxic T cells obtained by differentiation induction from CAR-iPSCs were selected using CD5 positivity and CD8β positivity as indicators, and these were classified as iCAR-T CTL We decided to use them as cells.

[0080] In vivo bioluminescence imaging results from subcutaneous tumor animal model #1 showed that iCAR-T was present at the transplantation site of liver cancer cells SK-Hep-1 (SK-Hep-GPC3) to which GPC3 had been gene-transformed. CTL Mice injected with pCAR-T CTLIn mice injected with the drug, luminescence intensity gradually and selectively increased from day 3 to day 7. No accumulation of these cells was observed at the transplantation site of non-transgenic SK-Hep-1 cells (SK-Hep).

[0081] However, in a xenotumor transplantation model with peritoneal dissemination, iCAR-T CTL The survival rate when treated with pCAR-T CTL It did not reach the same level as the other. Flow cytometry-based phosphorylation analysis (Phosflow) revealed that one possible reason is the attenuation of CAR-mediated MEK / ERK signaling (Figure 2A, left panel). When CAR binds to a target molecule, the CD3ζ of phosphorylated CAR produces diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), thereby activating the low molecular weight GTP-binding protein RAS and its downstream molecules ERK1, ERK2, and AP1. iCAR-T when co-cultured with SK-Hep-GPC3 CTL Phosphorylation of ERK1 / 2 in pCAR-T CTL It was lower than that (Figure 2A).

[0082] Therefore, we focused on regulating DAG metabolism to enhance T cell signaling intensity. When CD3ζ is phosphorylated via the TCR or CAR, DAG recruits and activates RasGRP1 (Ras guanylate-releasing protein 1), thereby activating the MEK / ERK pathway. DGKα and DGKζ are two major DGK isoforms found in T cells, and they are known to attenuate MEK / ERK signaling by degrading DAG (Sci. Signal. 8, re6-re6 (2015)). Furthermore, inhibiting DGK promotes the activation of AP-1 and NF-κB via the RAS / ERK pathway (Nat. Immunol. 7, 1166-1173 (2006)). Based on these findings regarding the enhancement of T cell signaling, we deleted both DGKα and DGKζ at the iPSC stage using CRISPR-Cas9 (DGK-dKO iCAR, Figure 2B), and then performed iCAR-T CTL The cells were differentiated into DGK-dKO iCAR-T cells. CTL It was named DGK-dKO iCAR-T. CTL Phosflow measurements showed that stimulating cells with the target antigen partially restored ERK1 / 2 phosphorylation (center panel in Figure 2A). This restoration of ERK phosphorylation led to a response to SK-Hep-GPC3-responsive DGK-dKO iCAR-T CTL The proliferation ability of iCAR-T cells that do not lack DGK CTL This represents a significant improvement compared to the previous version (Figure 2C).

[0083] Next, DGK deletion is iCAR-T CTL To determine whether or not it affects the in vivo function, we used a subcutaneous tumor xenograft model with DGK-dKO iCAR-T CTL The accumulation and survival persistence of DGK-dKO iCAR-T were evaluated based on the results of in vivo imaging assays. CTL iCAR-T CTL It was found that they accumulated in larger quantities and survived for longer periods (Figure 2D, E).

[0084] Next, a peritoneal seeding model of KOC7c was used for the purpose of evaluating the effector function in tumor-inoculated NSG mice. As a result, DGK-dKO iCAR-T CTL strongly suppressed tumor growth compared with iCAR-T CTL (Figs. 2F, G), and extended the survival period of mice to the same extent as the pCAR-T CTL treatment group (Fig. 2H).

[0085] Next, to design DGK dKO iCAR-T CTL showing a higher cancer treatment effect than pCAR-T CTL , attention was paid to the apoptosis inhibitory effect and proliferation effect of the transmembrane IL-15 / IL-15Rα (mbIL15) gene. The mbIL15 gene was introduced into pCAR-T[[ID=![

[18] ] CTL , DGK-pKO pCAR-T CTL , iCAR-T CTL and DGK-dKO iCAR-T CTL by a retroviral vector to examine the cell proliferation ability. As a result of introducing the mbIL15 gene, the target-mediated proliferation in vitro of iCAR-T CTL was significantly improved regardless of the presence or absence of DGK gene modification.

[0086] To investigate whether the in vivo antitumor effect can be improved by treating with DGK knockout CAR-T cells, various CAR-T CTL cells (1×10 6 cells) were injected into the peritoneal cavity of peritoneal seeding mice pre-inoculated with KOC7c (Fig. 3A). In this xenograft animal model, the treatment group with DGK-dKO iCAR-T CTL genetically modified with mbIL-15 had a significantly extended survival period compared with other groups (Figs. 3B, C), and the cell survival persistence was also significantly higher (Figs. 3D, E).

[0087] Furthermore, to test the cells under a more clinically relevant situation, various CAR-T were used in a subcutaneous tumor model pre-inoculated with JHH7CTL 1×10 cells 6 were intravenously injected (Figure 3F). As a result, similar to the results obtained in the intraperitoneal injection model, DGK-dKO iCAR-T genetically modified with mbIL-15 CTL showed significantly higher tumor suppression and survival compared to other groups (Figure 3G-I). In addition, in the FCM analysis of peripheral blood performed on the 28th day after injection, among all groups, only DGK-dKO iCAR-T genetically modified with mbIL-15 CTL was detected (Figure 3J).

[0088] In addition, to test the anti-tumor effect on established palpable tumors, various CAR-T CTL 1×10 cells 7 were intravenously injected into a subcutaneous tumor animal model 10 days after inoculation of SK-Hep-GPC3 (Figure 4A). As a result, DGK-dKO iCAR-T genetically modified with mbIL-15 CTL showed a significantly superior tumor growth inhibitory effect compared to other groups (Figure 4B, C).

[0089] Furthermore, various CAR-T CTL 1×10 cells 7 were intraperitoneally injected into a peritoneal dissemination animal model 10 days after inoculation of KOC7c (Figure 5A). The treatment group with DGK-dKO iCAR-T genetically modified with mbIL-15 CTL showed a significantly extended survival period (Figure 5B) and significantly higher cell survival persistence compared to other groups (Figure 5C). In addition, in the FCM analysis of ascites performed on the 80th day after injection in the treatment group with DGK-dKO iCAR-T genetically modified with mbIL-15 CTL it was shown that the effector memory phenotype was maintained (Figure 5D).

[0090] The above results strongly suggest the possibility that by making these modifications, iCAR-T CTL can be used as a platform for allogeneic therapy in immunotherapy using genetically modified regenerated T cells expressing CAR.

Claims

1. T cells differentiated from induced pluripotent stem cells, wherein the activity of diacylglycerol kinase encoded by the diacylglycerol kinase α gene and the diacylglycerol kinase ζ gene is deficient by knockout of these genes, and the T cells express a fusion protein (IL-15 / IL-15Rα) containing IL-15 and the IL-15 receptor α subunit (IL-15Rα), as well as a chimeric antigen receptor.

2. The T cell according to claim 1, wherein the fusion protein is a transmembrane protein.

3. The T cell according to claim 1 or 2, which is CD5-positive and CD8β-positive.

4. A pharmaceutical composition comprising T cells according to any one of claims 1 to 3.

5. A pharmaceutical composition according to claim 4 for treating cancer.

6. The pharmaceutical composition according to claim 5, wherein the cancer is a solid tumor.

7. Use in the manufacture of a T cell-based pharmaceutical composition for cancer treatment according to any one of claims 1 to 6.

8. (a) A step of preparing T cells expressing chimeric antigen receptors that have been differentiated from induced pluripotent stem cells, A method for producing chimeric antigen receptor-expressing T cells, including, The T cells described above lack the activity of diacylglycerol kinase encoded by the diacylglycerol kinase (DGK)α gene and the diacylglycerol kinase (DGK)ζ gene, as these genes are knocked out, and It expresses a fusion protein (IL-15 / IL-15Rα) containing IL-15 and the IL-15 receptor α subunit (IL-15Rα), The aforementioned manufacturing method.

9. A claim comprising, before step (a), a step of differentiating induced pluripotent stem cells into T cells. The method described in 8.

10. The method according to claim 9, wherein the cells obtained in step (b) express a chimeric antigen receptor.

11. The method according to claim 10, wherein in step (b), induced pluripotent stem cells expressing a chimeric antigen receptor are used, or the chimeric antigen receptor is expressed at any stage in step (b).

12. The method according to any one of claims 9 to 11, wherein the cells obtained in step (b) lack the activity of the diacylglycerol kinase encoded by the DGKα gene and the DGKζ gene, respectively, by knocking out these genes.

13. The method according to claim 12, wherein in step (b), pluripotent stem cells are used in which the activity of the diacylglycerol kinase encoded by the DGKα gene and the DGKζ gene is lost by knocking out these genes, or a modification is made in which the activity of the diacylglycerol kinase encoded by the DGKα gene and the DGKζ gene is lost by knocking out these genes at any stage of step (b).

14. The method according to any one of claims 9 to 13, wherein the cells obtained in step (b) express a fusion protein (IL-15 / IL-15Rα) containing IL-15 and the IL-15 receptor α subunit (IL-15Rα).

15. The method according to claim 14, wherein in step (b), induced pluripotent stem cells expressing IL-15 / IL-15Rα are used, or IL-15 / IL-15Rα is expressed at any stage of step (b).

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