Method for activating and expanding antigen-exposed CD8 T cells, CD8 T cells with enhanced anti-cancer activity produced thereby, and uses thereof

Abstract

The present invention relates to a method for cell activation, cells activated thereby, and uses thereof. More specifically, the present invention provides a method for improving, restoring, and proliferating exhausted CD8 T cells under in vitro conditions, which includes a step of inducing overexpression of Klf4 protein in CD8 T cells isolated from an individual, a cell population containing CD8 T cells, or CAR-CD8 T cells obtained by genetically transforming CD8 T cells, and CD8 T cells in which Klf4 protein is overexpressed by the method, thereby enhancing anti-cancer activity, and uses thereof.

Description

Detailed Description of the Invention

[0001] [Technical field] The present invention relates to a method for cell proliferation and activation, cells activated thereby, and uses thereof, and more particularly to a method for activating and proliferating CD8 T cells chronically exposed to an antigen, CD8 T cells with enhanced anti-cancer activity produced thereby, and uses thereof. [Background technology] CD8 T cells present in tumor tissue recognize antigens on cancer cells, become activated, and then secrete effector cytokines such as granzyme B, interferon-gamma (IFN-γ), and perforin to eliminate cancer cells. Therefore, increasing the activity of CD8 T cells in tumor tissue can effectively control cancer. However, unlike a general immune response, in the special environment of cancer, these immune cells are continuously exposed to cancer antigens, causing their characteristics to change, making it much more difficult to control their activity.

[0002] In the tumor environment, chronic stimulation by persistent cancer cell antigens leads to exhaustion of CD8 T cells. Exhausted CD8 T cells are unable to perform normal immune responses due to impaired cytokine secretion and cell division, making them unable to effectively eliminate cancer cells. In particular, exhausted CD8 T cells are unable to easily return to normal function over time. Therefore, preventing CD8 T cell exhaustion in tumor tissue and activating the function of exhausted cells are key factors in cancer control. Recent research has shown that CD8 T cells persistently exposed to antigens are composed of subsets that undergo several stages of exhaustion: the chronic progenitor cell subset (exhausted progenitor cell 1, exhausted progenitor cell 2), the chronic effector cell subset (intermediate exhausted cells), and the terminally exhausted cell subset (terminally exhausted cells). Among these, chronic effector cells have the effector function to recognize and eliminate cancer cells, but terminally exhausted cells are known to have lost a significant portion of this function. Generally, CD8 T cells present in cancer tissues are known to have insufficient immune function when in a terminally exhausted state. Therefore, if a method can be developed to promote the development and function of cancer-specific CD8 T cells into chronic effector cells, it can be effectively used to control cancer cells.

[0003] In relation to anti-cancer treatments using CD8 T cells, U.S. Patent US8106092 discloses a method for treating secondary cancers in which ingenol-3-angelate is administered locally and / or intratumorally to an individual with secondary cancer, thereby inducing primary necrosis of cancer cells and promoting the production of anti-cancer-specific T cells such as CD8 T cells. U.S. Patent Publication US20190218515A discloses a method for producing activated T cells by reacting T cells isolated from a cancer patient with cancer cells together with a bispecific antibody against CD123 / CD3, a bispecific antibody against CD19 / CD3, or a bispecific antibody against EpCAM / CD3.

[0004] Meanwhile, three generations of T cell therapy drugs have been developed to date. First-generation T cell therapy drugs involved expanding all T cells (bulk T cells) present in the blood or cancer tissue and administering them to patients, resulting in low specificity for cancer cells and poor efficacy. Second-generation T cell therapy drugs showed improved therapeutic effects by isolating and mass-cultivating only tumor antigen-specific T cells (Ag-specific T cells) and administering them to cancer patients, but problems arose due to long culture times and complicated processes. Third-generation T cell therapy drugs not only improved antigen specificity and shortened manufacturing times by either 1) directly introducing a TCR gene that recognizes a specific cancer antigen into T cells, or 2) binding a T cell activation domain to the antigen recognition site (scFv) of a monoclonal antibody that recognizes a specific antigen and introducing it into T cells, but also showed excellent therapeutic efficacy, achieving nearly 100% therapeutic efficacy in some leukemias and lymphomas. The second type of third-generation T cell therapy is characterized by being genetically engineered to express a so-called chimeric antigen receptor (hereinafter abbreviated as "CAR").

[0005] The leading companies based on CAR-transduced T cell technology are Novartis, Juno Therapeutics, and Kite Pharma, all of which have developed CAR-transduced T cells that target the B cell-specific antigen CD19. These have demonstrated high cure rates of nearly 80-90% for resistant / relapsed acute lymphocytic leukemia (ALL) and non-Hodgkin's lymphoma (NHL), positioning them as leaders in the field of targeted immune cell therapy (Hartmann et al., EMBO Mol. Med. 9(9):1183-1197, 2017). Recently, Novartis Korea's application for the world's first chimeric antigen receptor T cell (CAR-T) therapeutic agent, Kymriah (registered trademark) (tisagenlecleucel), was approved as the first cutting-edge biopharmaceutical product developed using the "Advanced Regenerative Biotechnology Method." [Summary of the Invention] [Problem to be solved by the invention] However, the above prior art also has limitations in that it does not take into account the phenomenon of CD8 T cell exhaustion in the body and does not aim to utilize these specialized cells or overcome this condition.

[0006] The present invention aims to solve various problems, including those mentioned above, and aims to provide a method and composition capable of controlling cancer by regulating the in vivo exhaustion process of CD8 T cells, but the scope of the present invention is not limited to the above object. [Means for solving the problem] According to one aspect of the present invention, there is provided a method for suppressing CD8 T cell exhaustion in vitro, the method comprising inducing overexpression of Klf4 protein in cells selected from the group consisting of: a) CD8 T cells isolated from an individual; b) a cell population containing the CD8 T cells; and c) CAR-CD8 T cells obtained by transducing the CD8 T cells with a gene encoding a chimeric antigen receptor (CAR).

[0007] According to one aspect of the present invention, there is provided a method for enhancing the immune response of CD8 T cells in vitro, the method comprising the step of inducing overexpression of Klf4 protein in cells selected from the group consisting of: a) CD8 T cells isolated from an individual; b) a cell population containing the CD8 T cells; and c) CAR-CD8 T cells obtained by transducing the CD8 T cells with a gene encoding a chimeric antigen receptor (CAR).

[0008] According to one aspect of the present invention, there is provided a method for expanding CD8 T cells in vitro, the method comprising the step of inducing overexpression of Klf4 protein in cells selected from the group consisting of: a) CD8 T cells isolated from an individual; b) a cell population containing the CD8 T cells; and c) CAR-CD8 T cells obtained by transducing the CD8 T cells with a gene encoding a chimeric antigen receptor (CAR).

[0009] Another aspect of the present invention provides a pharmaceutical composition for cancer treatment comprising, as an active ingredient, transformed CD8 T cells transformed to overexpress the Klf4 protein or transformed CAR-CD8 T cells transformed to overexpress the Klf4 protein and a chimeric antigen receptor.

[0010] Another aspect of the present invention provides transformed CD8 T cells that have been transformed so that Klf4 protein is overexpressed.

[0011] Another aspect of the present invention provides transformed CAR-CD8 T cells that have been transformed to express an exogenous Klf4 protein and a chimeric antigen receptor (CAR).

[0012] Another aspect of the present invention provides a composition comprising the transformed CD8 T cells or the transformed CAR-CD8 T cells.

[0013] Another aspect of the present invention provides a method for treating cancer in an individual, comprising the steps of inducing overexpression of Klf4 protein in CD8 T cells isolated from the individual with cancer or in a cell population containing the CD8 T cells; and administering to the individual the CD8 T cells in which Klf4 protein is overexpressed or the cell population containing the CD8 T cells. [Effects of the invention] The method according to one embodiment of the present invention is useful for developing more effective anti-cancer cell therapeutic agents by preventing immune cell exhaustion. [Brief description of the drawing] FIG. 1A is a schematic diagram illustrating the design of a repeated antigen stimulation test for CD8 T cells according to one embodiment of the present invention. FIG. 1B is a series of graphs showing the results of an experiment conducted according to the experimental design of FIG. 1A, measuring the expression levels at the mRNA level of Tox (left) and Klf4 (right), which are markers related to the exhaustion state of CD8 T cells depending on the level of antigen stimulation. [FIG. 1C] A schematic diagram showing the marker phenotypes of various stages of CD8 T cell subsets present in the spleen and cancer tissue induced by injection of MC38 colon cancer cells into mice. [FIG. 1D] A graph showing the results of measuring the level of Klf4 protein expression in each of these CD8 T cell subsets at the mRNA level. [FIGS. 2A to 2E] According to one embodiment of the present invention, the characteristics of CD8 T cells transformed with a control retroviral vector (MigRI) or CD8 T cells transformed with a retroviral vector (Klf4) containing the Klf4 gene are analyzed. According to one aspect of the present invention, there is provided a method for suppressing exhaustion of CD8 T cells in vitro, comprising inducing overexpression of Klf4 protein in CD8 T cells isolated from an individual, a cell population containing the CD8 T cells, or CAR-CD8 T cells obtained by transfecting the CD8 T cells with a gene encoding a chimeric antigen receptor (CAR). FIG. 2A shows the results of FACS analysis of electron-transformed CD8 T cells using the above-mentioned subset markers. FIG. 2B is a graph showing the results of FACS analysis of the ratio of dead cancer cells when control (MigRI) and Klf4 gene-transformed CD8 T cells were co-cultured with target cancer cells. FIG. 2C is a graph showing the results of measuring the gene expression levels of Klf4 (left) and Tox (right) at the mRNA level in control (MigRI) and Klf4 gene-transformed CD8 T cells. FIG. 2D is a graph showing the results of FACS analysis of the expression level of granzyme B in control (MigRI) and Klf4 gene-transformed CD8 T cells (Klf4). - , MigRI GFP + , Klf4 GFP - , Klf4 GFP +) shows a graph (left) and a two-dimensional histogram (right) of the results of measuring the ratio of cells expressing granzyme B, which is essential for the target cell killing function of CD8 T cells. Figure 2E shows the results of FACS analysis of the expression level of IFN-γ, which is essential for the function of control (MigRI) and Klf4 gene-transfected CD8 T cells, and the results of the ... - , MigRI GFP + , Klf4 GFP - , Klf4 GFP + ) shows a graph (left) and a two-dimensional histogram (right) relating to the results of measuring the ratio of cells expressing IFN-γ. FIG. 3A is a schematic diagram illustrating the administration schedule of an animal experiment using CD8 T cells transduced with the Klf4 gene according to one embodiment of the present invention. [FIG. 3B] A graph showing the results of measuring the expression level of the Klf4 gene at the mRNA level in control and Klf4 gene-transduced CD8 T cells. FIG. 3C is a graph showing the change in tumor tissue volume over time in a control group (MigRI) and in a cancer model animal administered with CD8 T cells transduced with the Klf4 gene according to an embodiment of the present invention. [Figure 3D] The expression level of Ki-67, which indicates the degree of cell division, was analyzed by FACS analysis in CD8 T cell subsets isolated from tumor tissues of animals sacrificed after the animal experiment. The graph (left) and 2D histogram (right) show the results of measuring the ratio of Ki-67 expressing cells among all CD8 T cells according to the marker phenotype of the isolated CD8 T cells. [Figure 3E] The level of granzyme B expression in CD8 T cells isolated from tumor tissues of animals sacrificed after the animal experiment was analyzed by FACS analysis, and the percentage of granzyme B-expressing cells among all CD8 T cells was measured according to the marker phenotype of the isolated CD8 T cells. This graph (left) and two-dimensional histogram (right) show the results. [Figure 3F] The TNF-α and INF-γ expression levels of CD8 T cells isolated from tumor tissues of animals sacrificed after the animal experiment were analyzed by FACS analysis, and the percentage of TNF-α- and INF-γ-expressing cells among total CD8 T cells was measured according to the marker phenotype of the isolated CD8 T cells. The graph (left) and two-dimensional histogram (right) show the results. FIG. 4A is a schematic diagram illustrating the administration schedule of an animal experiment using CD8 T cells treated with APTO-253 according to one embodiment of the present invention. [FIG. 4B] A graph showing the results of measuring the expression level of the Klf4 gene at the mRNA level in experimental animals administered with control (PBS) and APTO-253-treated CD8 T cells. [Figure 4C] A graph showing the change in tumor tissue volume over time in a control group (PBS) and a cancer model animal to which CD8 T cells treated with APTO-253 according to one embodiment of the present invention were administered. [Figure 5] A schematic diagram showing the structures of various CAR constructs (EpCAM CAR, Trop-2 CAR, CEACAM6 CAR, and CEACAM5 CAR) according to one embodiment of the present invention. [Mode for Carrying Out the Invention] According to one aspect of the present invention, there is provided a method for suppressing CD8 T cell exhaustion in vitro, the method comprising inducing overexpression of Klf4 protein in cells selected from the group consisting of: a) CD8 T cells isolated from an individual; b) a cell population containing the CD8 T cells; and c) CAR-CD8 T cells obtained by transducing the CD8 T cells with a gene encoding a chimeric antigen receptor (CAR).

[0014] According to one aspect of the present invention, there is provided a method for enhancing the immune response of CD8 T cells in vitro, the method comprising the step of inducing overexpression of Klf4 protein in cells selected from the group consisting of: a) CD8 T cells isolated from an individual; b) a cell population containing the CD8 T cells; and c) CAR-CD8 T cells obtained by transducing the CD8 T cells with a gene encoding a chimeric antigen receptor (CAR).

[0015] According to one aspect of the present invention, there is provided a method for expanding CD8 T cells in vitro, the method comprising the step of inducing overexpression of Klf4 protein in cells selected from the group consisting of: a) CD8 T cells isolated from an individual; b) a cell population containing the CD8 T cells; and c) CAR-CD8 T cells obtained by transducing the CD8 T cells with a gene encoding a chimeric antigen receptor (CAR).

[0016] In the above method, the Klf4 protein is overexpressed by transfecting the CD8 T cells with an expression vector containing a polynucleotide encoding the Klf4 protein, by introducing mRNA expressing the Klf4 protein into the CD8 T cells, or by treating the CD8 T cells with a Klf4 inducer. The Klf4 protein may comprise the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5, and the polynucleotide encoding the Klf4 protein may comprise the nucleic acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 6. However, the present invention is not limited to these specific sequences, and any Klf4 protein derived from other mammals may also be used. Preferably, Klf4 protein or a nucleic acid molecule encoding the same derived from primates such as chimpanzees, gorillas, orangutans, primates, crab-eating monkeys, and red monkeys is used.

[0017] In the method, the expression vector is a viral vector or a non-viral vector, the viral vector is an adeno-associated virus (AAV) vector, an adenovirus vector, an alphavirus vector, a herpes simplex virus vector, a vaccinia virus vector, a Sendai virus vector, a flavivirus vector, a radvovirus vector, a retrovirus vector, a herpes virus vector, a poxvirus vector, or a lentivirus vector, the non-viral vector is a DNA vector, a nanoparticle, a cationic polymer, an exosome, an extracellular vesicle, or a liposome, and the DNA vector is a plasmid vector, a cosmid vector, a phagemid vector, or an artificial human chromosome.

[0018] The expression vector contains an appropriate regulatory sequence linked to a polynucleotide encoding the Klf4 protein so that the Klf4 protein is overexpressed in the CD8 T cells, and the regulatory sequence is operably linked to the polynucleotide. In this case, the regulatory sequence and the polynucleotide operably linked thereto are collectively referred to as a "gene construct." The gene construct may contain appropriate restriction enzyme recognition sites at both ends for appropriate cloning into an expression vector.

[0019] As used herein, the term "operably linked to" means that a nucleic acid sequence of interest (e.g., in an in vitro transcription / translation system or in a host cell) is linked to the regulatory sequence in a manner that allows its expression. The term "regulatory sequence" is intended to include promoters, enhancers, and other regulatory elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleic acid of interest in many host cells, those that direct expression of a nucleic acid of interest only in specific tissues (e.g., tissue-specific regulatory sequences), and those that direct expression in response to specific signals (e.g., inducible regulatory sequences). Those skilled in the art will appreciate that the design of an expression vector will vary depending on factors such as the choice of host cell to be transformed and the desired level of protein expression. The expression vector of the present invention can be introduced into a host cell to express the fusion protein. Regulatory sequences that enable expression in eukaryotic and prokaryotic cells are well known to those skilled in the art. As mentioned above, these typically include regulatory sequences responsible for transcription initiation and, optionally, poly-A signals responsible for transcription termination and stabilization of the transcript. In addition to transcriptional regulatory elements, additional regulatory sequences may also include translational enhancers and / or native, combined, or heterologous promoter regions. For example, possible regulatory sequences enabling expression in mammalian host cells include the CMV-HSV thymidine kinase promoter, SV40, RSV promoter (Rous sarcoma virus), human kidney element 1α promoter, glucocorticoid-inducible MMTV promoter (Moloney murine tumor virus), metallothionein-inducible or tetracycline-inducible promoters, or amplifiers such as CMV or SV40 amplifiers. For expression in neuronal cells, it is contemplated that the neurofilament promoter, PGDF promoter, NSE promoter, PrP promoter, or thy-1 promoter may be used.Such promoters are known in the art and are described in the literature (Charron, J. Biol. Chem. 270:25739-25745, 1995). For expression in prokaryotic cells, a number of promoters have been disclosed, including the lac promoter, tac promoter, or trp promoter. In addition to elements capable of initiating transcription, the regulatory sequence may also include a transcription termination signal, such as an SV40 poly-A site or a TK poly-A site, downstream of the polynucleotide according to one embodiment of the present invention. Suitable expression vectors for use herein are known in the art, and examples thereof include the Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc / CMV, pcDNA1, pcDNA3 (Invitrogene), pSPORT1 (GIBCO BRL), pGX27 (Japanese Patent No. 1442254), pX (Pagano (1992) Science 255, 1144-1147), yeast two-hybrid vectors such as pEG202 and dpJG4-5 (Gyuris (1995) Cell 75, 791-803), or prokaryotic expression vectors such as lambda gt11 or pGEX (Amersham-Pharmacia). In addition to the nucleic acid molecule of the present invention, the vector may further comprise a polynucleotide encoding a secretion signal. Such secretion signals are well known to those skilled in the art. Depending on the expression system used, a leader sequence capable of directing the fusion protein to a cellular compartment is combined with the coding sequence of a polynucleotide according to one embodiment of the present invention, preferably a detoxified protein or a leader sequence capable of secreting the protein directly into the periplasm or extracellular medium.

[0020] Furthermore, the expression vectors used in the present invention can be prepared by standard recombinant DNA techniques, including, for example, blunt-end and sticky-end ligation, restriction enzyme treatment to provide suitable termini, removal of phosphate groups by alkaline postase treatment to prevent improper ligation, and enzymatic ligation using T4 DNA ligase. The vectors of the present invention can be prepared by recombining DNA encoding a signal peptide obtained by chemical synthesis or genetic engineering and DNA encoding a fusion protein according to one embodiment of the present invention into a vector containing appropriate regulatory sequences. Vectors containing the regulatory sequences can be commercially purchased or prepared.

[0021] In addition, in the above method, the gene construct may further include a polynucleotide encoding one or more immune enhancing peptides, in which case the immune enhancing peptides may be included in an expression vector in a form linked to a separate regulatory sequence, i.e., in the form of a bicistrone, or may be linked to a single regulatory sequence, but with an IRES (internal ribosome entry site) inserted between the polynucleotides encoding the two proteins, so that they are transcribed into a single mRNA and then translated into their respective proteins. The immune-enhancing peptides include CD28, ICOS (inducible costimulator), CTLA4 (cytotoxic T lymphocyte associated protein 4), PD1 (programmed cell death protein 1), BTLA (B and T lymphocyte associated protein), DR3 (death receptor 3), 4-1BB, CD2, CD40, CD40L, CD30, CD27, and SLAM (signaling lymphocyte activation). molecule), 2B4(CD244), NKG2D(natural-killer group 2, member D) / DAP12(DNAX-activating protein 12), TIM1(T-Cell immunoglobulin and mucin domain containing protein 1), TIM2, TIM3, TIGIT, CD226, CD160, LAG3(lymphocyte activation gene 3), B7-1, B7-H1, GITR (glucocorticoid-induced TNFR family related) The cytoplasmic domain of Flt3 ligand (fms-like tyrosine kinase 3 ligand), flagellin, HVEM (herpesvirus entry mediator), or OX40L [ligand for CD134 (OX40), CD252], or a combination of two or more of these.

[0022] In the method, the Klf4 inducer is APTO-253 {2-(5-fluoro-2-methyl-1H-indol-3-yl)-1H-imidazo[4,5-f][1,10]phenanthroline}.

[0023] Another aspect of the present invention provides transformed CD8 T cells that have been transformed so that the Klf4 gene is overexpressed.

[0024] The transformed CD8+ T cells can be produced by transducing CD8+ T cells isolated from an individual or a cell population containing such CD8+ T cells with an expression vector containing a polynucleotide encoding the Klf4 protein. The expression vector can be a viral or non-viral vector. The viral vector can be an adeno-associated virus (AAV) vector, adenovirus vector, alphavirus vector, herpes simplex virus vector, vaccinia virus vector, Sendai virus vector, flavivirus vector, radovvirus vector, retrovirus vector, herpes virus vector, poxvirus vector, or lentivirus vector. The non-viral vector can be an mRNA, DNA vector, nanoparticle, cationic polymer, exosome, extracellular vesicle, or liposome. The mRNA can be an mRNA encoding the Klf4 protein alone or in combination with a non-viral vector other than the mRNA. The DNA vector can be a plasmid vector, cosmid vector, phagemid vector, or artificial human chromosome.

[0025] Another aspect of the present invention provides transformed CAR-CD8 T cells that have been transformed to express an exogenous Klf4 protein and a chimeric antigen receptor (CAR).

[0026] The transformed CAR-CD8 T cells can be prepared by transfecting CD8 T cells, preferably CD8 T cells isolated from a patient in need of treatment or a cell population containing the CD8 T cells, with a genetic construct containing a polynucleotide encoding the Klf4 protein and a genetic construct containing a polynucleotide encoding the CAR. The two genetic constructs can be cloned into separate vectors and co-transfected, or cloned into a single vector and transduced into host CD8 T cells. In this case, each gene can be operably linked to separate regulatory sites, such as a promoter and / or enhancer, to be expressed as separate mRNAs, or can be linked via an IRES under a single transcriptional regulator to be expressed as a single mRNA, with translation and modification processes occurring separately. Various techniques, such as vectors and promoters, for transducing foreign genes are described above.

[0027] The term "chimeric antigen receptor" as used herein refers to a synthetic protein composed of a single-chain antibody analog such as scFv or sdAb as an antigen recognition site, a cell membrane transduction domain, a costimulatory domain, and an intracellular signaling domain. It is well known that when transduced into immune cells such as T cells, it recognizes cancer cell-specific antigens and improves the anti-cancer activity of these immune cells against cancer cells.

[0028] In the transformed CAR-CD8 T cells, the CAR is a fusion protein comprising an antibody analog, a cell membrane-spanning domain, a costimulatory factor, and an intracellular signaling domain. The single-chain-based antibody analogs can specifically bind to cancer antigens or pathogen-derived antigens, and the cancer antigens include epithelial cell adhesion molecule (EpCAM), trophoblast cell surface antigen 2 (Trop-2), CEA cell adhesion molecule 5 (CEACAM5), CEA cell adhesion molecule 6 (CEACAM6), carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), prostate-specific membrane antigen (PSMA), Her2 / neu, MUC-1, BCR / ABL, α-fetoprotein (AFP), antigens derived from Epstein-Barr virus (EBV) such as LMP2a, antigens derived from human hepatitis B virus (HBV), antigens derived from human hepatitis C virus (HCV), proteinase 3, WT-1, PAP (prostatic acid phosphatase), and the like. phosphatase), G250, melanoma antigen gene (MAGE), BAGE, GAGE, NY-ESO-1, tyrosinase, tyrosinase-related protein-1 (TRP-1), TRP-2, gp100, MART-1, Ig idiotype, CDK4, caspase-8, β-catenin, BCR / ABL, human papillomavirus (HPV E6 / E7), HHV-8, 5T4, p53, cancer antigen 125 (CA-125), cancer antigen-72-4 (CA-72-4), cancer antigen-15-3 (CA-15-3), or cancer antigen-19-9 (CA-19-9).The pathogen-derived antigen is an antigen derived from a pathogenic microorganism, a virus, or a parasite, and the pathogenic microorganism is a pathogenic bacterium or a pathogenic fungus, and the pathogenic bacterium is Bordetella pertussis, Clostridium tetanus, Corynebacterium diphtheriae, Helicobacter pylori, Streptococcus pneumoniae, Mycobacterium tuberoculosis, Cholera sp., Staphylococcus sp., Shigella sp., Borrelia sp., or Salmonella sp., and the pathogenic fungus is Candida sp., Trichophyton sp., sp.), Aspergillus sp., Fonsecaea sp., Epidermophyton sp., Piedraia sp., Malassezia sp., Pseudallescheria sp., Basidiobolus sp., Conidiobolus sp., Rhinosporidium sp., Paracoccidioides sp., Cryptococcus sp., Blastomyces sp., Sporothrix sp., Mucor sp., Absidia sp., Rhizopus sp., Pneumocystis sp., Wangiella sp., Phialophora sp., Schizophyllum sp.The virus may be influenza virus, human papilloma virus (HPV), vesicular stomatitis virus, cytomegalovirus (CMV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C (HCV), hepatitis D virus (HDV), hepatitis G virus (HGV), respiratory syncytial virus (RSV), herpes simplex virus antigen, or human immunodeficiency virus (HIV). The parasite is a roundworm, pinworm, pork tapeworm, liver fluke, lung fluke, schistosome, liver fluke, lung fluke, Plasmodium falciparum, or Toxoplasma.

[0029] As used herein, "antibody mimetic" refers to a protein that has a similar function, i.e., antigen-binding ability, to an antibody, but is derived from an antibody, but is not composed of two chains, but is instead a single-chain antigen-binding fragment or a protein scaffold derived from a non-antibody. This antibody mimetic is different from a conventional full-length antibody, which functions by forming a quaternary structure of two heavy chains and two light chains. Specifically, such antibody mimetics include antibody-based single-chain antibody fragments that contain the V of an antibody. H and V L scFv (Glockshuber et al., Biochem. 29(6):1362-1367, 1990), a single-chain antibody fragment produced by linking the fragments with a linker; sdAb (single domain antibody), an antibody fragment composed of a single variable region fragment of an antibody; and antigen-binding fragments of antibodies derived from camelids or cartilaginous fishes, which are antibodies composed only of heavy chains without light chains (V). H H, V NARAntibody-like proteins produced from non-antibody-derived protein scaffolds include Affibodies derived from the Z domain of protein A (Nygren, PA, FEBS J. 275(11):2668-2676, 2008), Affilins derived from Gamma-B crystallin or Ubiquitin (Ebersbach et al., J. Mol. Biol. 372(1):172-185, 2007), Affimers derived from Cystatin (Johnson et al., Anal. Chem. 84(15):6553-6560, 2012), Affitins derived from Sac7d (Krehenbrink et al., J. Mol. Biol. 383(5):1058-1068, 2008), and Alphabodies derived from triple helix-coiled coil proteins (Desmet et al., J. Mol. Biol. 383(5):1058-1068, 2008). al., Nat. Commun. 5:5237, 2014), Anticalin derived from lipocalin (Skerra et al., FEBS J. 275(11):2677-2683, 2008), Avimers derived from domains of various membrane receptors (Silverman et al., Nat. Biotechnol. 23(12):1556-1561, 2005), DARPins derived from the Ankyrin repeat motif (Stumpp et al., Drug Discov. Today. 13(15-16):695-701, 2008), Fynomers derived from the SH3 domain of the Fyn protein (Grabulovski et al., J. Biol. Chem. 282(5):3196-3204, 2007), and Kunitz domain peptides derived from the Kunitz domains of various protein inhibitors (Nixon and Wood, Curr. Opin. Drug. Discov. Dev. 9(2):261-268, 2006), a monobody derived from the 10th type 3 domain of fibronectin (Koide and Koide, Methods Mol. Biol. 352:95-109, 2007), and nanoCLAMP derived from carbohydrate-binding module 32-2 (Suderman et al., Protein Exp. Purif.134:114-124, 2017), hagfish-derived variable lymphocyte receptors (VLRs) (Boehm et al., Ann. Rev. Immunol. 30:203-220, 2012), and repebodies engineered to improve antigen affinity based on the VLRs (Lee et al., Proc. Natl. Acad. Sci. USA, 109:3299-3304, 2012).

[0030] The term "scFv" as used herein is an abbreviation for "single chain variable fragment" and refers to the heavy chain variable region (V) of an antibody, rather than a fragment of an actual antibody. H ) and the light chain variable region (V L ) with a linker peptide of approximately 25 a.a. in size, and is known to have antigen-binding ability despite not being an inherent antibody fragment (Glockshuber et al., Biochem. 29(6):1362-1367, 1990).

[0031] In the transformed CAR-CD8 T cells, the cell membrane transduction domain is selected from the group consisting of 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a , CD11b , CD11c , CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, LFA-1, ligand specifically binding to CD83, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; transmembrane domains derived from CD1-la / CD18, MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocytic activation molecules (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6.

[0032] As used herein, the term "costimulatory domain" refers to the cytoplasmic domain of a costimulatory factor, an immune-related protein that assists T / NK activation, that is responsible for the T cell costimulatory function.

[0033] In the transformed CAR-CD8 T cells, the costimulatory domain is the cytoplasmic domain of CD28, ICOS, CTLA4, PD1, BTLA, DR3, 4-1BB, CD2, CD40, CD30, CD27, SLAM, 2B4 (CD244), NKG2D / DAP12, TIM1, TIM2, TIM3, TIGIT, CD226, CD160, LAG3, B7-1, B7-H1, GITR, HVEM, or OX40L (CD252), or a combination of two or more of these.

[0034] As used herein, the term "intracellular signaling domain" refers to a cytoplasmic domain that is responsible for transmitting an intracellular signal that mediates an immune response when a ligand binds to a receptor in an immune cell.

[0035] In the transformed CAR-CD8 T cells, the intracellular signaling domain comprises part or all of the cytoplasmic domain of one or more of CD3ζ, CD28, CD27, OX40 / CD134, 4-1BB / CD137, FcεRIγ, ICOS / CD278, IL-2Rβ / CD122, IL-2Rα / CD132, DAP10, DAP12, and CD40.

[0036] According to one embodiment of the present invention, transformed CAR-CD8 T cells can exhibit additive or synergistic effects in anti-cancer therapy by maximally suppressing the exhaustion phenomenon and increasing the recognition of cancer antigens and the immune response to the recognized cancer antigens.

[0037] Another aspect of the present invention provides a composition comprising the transformed CD8 T cells or the transformed CAR-CD8 T cells.

[0038] In the composition, the CD8 T cells overexpressing the Klf4 gene may be autologous CD8 T cells isolated from an individual in need of treatment or allogeneic CD8 T cells isolated from another person, preferably autologous CD8 T cells. The CD8 T cells overexpressing the Klf4 gene can be prepared by transfecting CD8 T cells with an expression vector containing the Klf4 gene and / or treating the CD8 T cells with a Klf4 inducer.

[0039] The transduction and Klf4 inducer are as described above.

[0040] The composition is used to treat a disease requiring an innate immune response, and the disease requiring an innate immune response is cancer, a bacterial infection, a fungal infection, a viral infection, or a parasitic infection.

[0041] According to another aspect of the present invention, there is provided a pharmaceutical composition for cancer treatment comprising the transformed CD8 T cells or the transformed CAR-CD8 T cells as an active ingredient.

[0042] The composition may further contain a pharmaceutically acceptable adjuvant, excipient or diluent in addition to the carrier.

[0043] As used herein, the term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not cause typical gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions when administered to humans. Examples of carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition may further contain fillers, anti-agglomerating agents, lubricants, wetting agents, flavorings, emulsifiers, preservatives, etc.

[0044] Furthermore, the pharmaceutical composition according to one embodiment of the present invention can be formulated using methods known to those skilled in the art to enable rapid, sustained, or delayed release of the active ingredient upon administration to a mammal, including powder, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injection solutions, and sterile powders.

[0045] The composition according to one embodiment of the present invention may be administered by various routes, including, but not limited to, general systemic or local administration, for example, subcutaneous injection, intrasynovial injection, intraperitoneal injection, intramuscular injection, or intravenous injection.

[0046] The composition according to one embodiment of the present invention may be formulated into a suitable form together with a commonly used pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, parenteral carriers such as water, suitable oils, saline, aqueous glucose, and glycols, and may further contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Furthermore, the composition according to the present invention may appropriately contain suspending agents, solubilizers, stabilizers, isotonicity adjusting agents, preservatives, anti-adsorption agents, surfactants, diluents, excipients, pH adjusters, soothing agents, buffers, antioxidants, and the like, as needed depending on the administration method and dosage form. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, latest edition.

[0047] Additionally, the compositions of the present invention are administered in therapeutically effective amounts.

[0048] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment. The effective dose level can be determined based on factors including the type and severity of the disease, age, sex, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, concomitant medications, and other factors well known in the medical field. The vaccine composition or pharmaceutical composition of the present invention is administered at a dose of 0.1 to 1 g / kg, more preferably at a dose of 1 to 500 mg / kg. The dosage may be appropriately adjusted depending on the age, sex, and condition of the patient.

[0049] Another aspect of the present invention provides a method for treating cancer in an individual, comprising the steps of inducing overexpression of the Klf4 gene in CD8 T cells isolated from the individual with cancer or in a cell population containing the CD8 T cells; and administering to the individual the CD8 T cells in which the Klf4 gene is overexpressed or the cell population containing the CD8 T cells.

[0050] In the method of treating cancer, the CD8 T cells or a cell population comprising the CD8 T cells are further transduced with a polynucleotide encoding a chimeric antigen receptor targeted to a cancer antigen.

[0051] The present invention will be described in more detail below through examples and experimental examples. However, the present invention is not limited to the examples and experimental examples disclosed below, and may be embodied in various different forms. The following examples and experimental examples are provided to fully disclose the present invention and to fully convey the scope of the invention to those skilled in the art.

[0052] Example General method Construction of retrovirus for Klf4 gene transduction The platinum E cell line (platE, CELL BIOLABS, USA) was cultured in DMEM medium (WELGENE) supplemented with 10% FBS (Gibco, USA) and the antibiotics streptomycin and penicillin (100 U / ml, WELGENE), blastomycin (10 μg / ml, Gibco), and puromycin (1 μg / ml, Gibco) in a 37°C, 5% CO2 incubator, while paying close attention to cell saturation and avoiding overgrowth. When cell saturation reached approximately 90%, the medium was removed and washed with 5 ml of PBS (Sigma), then removed. This washing process was repeated once more. Subsequently, 2–3 ml of trypsin-EDTA solution (WELGENE) was added dropwise and incubated for 3 minutes in a 37°C incubator. The isolated cells were then pipetted thoroughly into 10 ml of DMEM medium, transferred to a 15 ml tube, and centrifuged at 1,500 rpm at 4°C for 5 minutes. The supernatant was removed, and the pellet was resuspended in DMEM medium supplemented with 10% FBS only (antibiotic-free medium), after which the cells were counted. 2.5 x 10 cells were plated onto a 60 mm culture dish. 6100 platE cells were resuspended in 3 ml of antibiotic-free DMEM medium, uniformly seeded, and cultured for 6–8 hours at 37°C in a 5% CO2 cell incubator. After 6–8 hours, the cells were transfected with 2X HBS, 2 M CaCl2, and triple-distilled water. The MigRI-Klf4 vector (Addgene, USA) was then inserted with the CDS sequence (SEQ ID NO: 1) of the mouse klf4 gene to prepare the MigRI-Klf4 vector. The mouse klf42 gene was cloned into the MigRI vector by PCR amplification using a primer set (SEQ ID NO: 2 and SEQ ID NO: 3) containing restriction enzyme (BglII and EcoRI) recognition sites. The forward primer contained an ACC sequence with a Kozak sequence between the BglII recognition site and the start codon (ATG). Next, 312.5 μl of 2X HBS was dispensed into one 1.5 ml centrifuge tube, and 38.75 μl of 2M CaCl2 and a volume equivalent to 10 μg of DNA vector were added to the other centrifuge tube. The remaining volume was then filled to 312.5 μl with triple-distilled water. The centrifuge tube containing the 2X HBS was then vortexed at low intensity, and the solution from the centrifuge tube containing the DNA vector was transferred dropwise using a pipette to the centrifuge tube containing the 2X HBS and incubated at room temperature for 30 minutes. The mixture was then added dropwise using a pipette to the pre-seeded plateE culture dishes. The dishes were then cultured for approximately 16 hours in a 37°C, 5% CO2 cell incubator. After incubation, the culture medium was removed and the dishes were washed twice with warm PBS. 3 ml of antibiotic-free DMEM medium was dispensed into each 60 mm culture dish and incubated for an additional 48 hours in a 37°C, 5% CO2 cell incubator. After incubation, the culture supernatant was transferred and filtered using a 0.45 μm filter (ADVANTEC). The filtered virus-containing supernatant was either used immediately for experiments or flash-frozen using liquid nitrogen and stored at -80°C in a deep freezer.

[0053] Isolation and activation of uncontacted CD8 T cells Mice were sacrificed and their spleens were removed. The spleens in PBS were then finely ground and filtered through a mesh. The spleens were then transferred to a 15 ml centrifuge tube and centrifuged at 1,500 rpm at 4°C for 5 minutes. The supernatant was removed, and the pellet was resuspended in 1 ml of Ack lysis buffer and incubated at room temperature for 3 minutes. Subsequently, 10 ml of PBS was added and centrifuged at 1,500 rpm at 4°C for 5 minutes. Fluorescently labeled anti-CD8 and anti-CD44 antibodies were mixed with PBS at a volume ratio of 100:1 to prepare an antibody mixture. This mixture was then added to the cell pellet, resuspended, and incubated at 4°C for 30 minutes. Then, 10 ml of PBS was added and centrifuged at 1,500 rpm at 4°C for 5 minutes. The supernatant was removed, and the pellet was resuspended in 1 ml of PBS and filtered through a cell strainer. The cell density was adjusted by adding an appropriate amount of PBS or RPMI medium (WELGENE). Subsequently, CD8 cells were separated using a cell separator (SH800, Sony Biotechnology, USA). + CD44 low Cells (naive CD8 T cells) were isolated.

[0054] Anti-CD3 antibody was coated onto a 48-well plate (SPL Life Sciences) 1.5 to 2 hours before isolation was complete. Specifically, anti-CD3 antibody (Biolegend) was diluted with PBS to a concentration of 5 μg / ml and then dispensed in 150 μl portions into each well of the 48-well plate. The plate was then incubated in a 37°C cell incubator for 1.5 to 2 hours to coat the antibody. The antibody mixture was removed from each well of the plate after antibody coating was complete, and the plate was washed with 150 μl of PBS. The same washing process was repeated once more. The isolated, uncontacted CD8 T cells were centrifuged at 4°C and 1,500 rpm for 5 minutes, the supernatant was removed, and the pellet was resuspended in RPMI medium supplemented with 10% FBS, streptomycin and penicillin (100 U / ml), and 2-mercaptoethanol (Gibco). 1.5 x 10 cells were used per well of the prepared anti-CD3 antibody-coated plate. 6 The cells were then added to the cells. They were then treated with anti-CD28 antibody (2 μg / ml, BD Pharmigen) and mIL-2 (100 U / ml, R&D Systems). They were then cultured in a 37°C, 5% CO2 cell incubator for 18 to 24 hours.

[0055] Proliferation of activated CD8 cells and retroviral transduction of Klf4 gene To achieve high retroviral transduction efficiency, we first expanded activated CD8 T cells. The cells activated for 18–24 hours were transferred to a 15 ml centrifuge tube and centrifuged at 25°C and 1,500 rpm for 5 minutes. Subsequently, Percoll solutions were prepared by diluting 100% Percoll, RPMI medium, and PBS to 30% and 60%, respectively. The centrifuged pellet was resuspended in 4 ml of 30% Percoll solution, and 3 ml of 60% Percoll solution was carefully placed on the bottom layer. The tube was then centrifuged at 25°C and 2,000 rpm for 20 minutes, with minimum acceleration and deceleration. After centrifugation, the upper 3 ml of the supernatant was removed, and the cells at the interface were transferred to a new 15 ml centrifuge tube. The tube was then washed with 10 ml of PBS and centrifuged at 25°C and 1,500 rpm for 5 minutes. After centrifugation, the pellet was washed once more with 10 ml of PBS and centrifuged at 25°C and 1,500 rpm for 5 minutes. The cell pellet was resuspended in RPMI medium and then counted. Subsequently, 5 x 10 cells were plated per well in a 12-well non-coated plate (SPL Life Sciences). 5 The cells were dispensed into individual cells.

[0056] One to two milliliters of the prepared retroviral supernatant (MigRI-Klf4 and MigRI) was added to each well, followed by the addition of mIL-2 (100 U / ml) and polybrene (8 μg / ml, Sigma). The wells were then sealed with parafilm and centrifuged at 25°C and 1,800 x g for 1 hour. Acceleration and deceleration were also set to minimum. After centrifugation, the parafilm was removed and the cells were incubated in a 37°C, 5% CO2 incubator for 30 minutes. The cells were then transferred to a 15 ml centrifuge tube, washed with 10 ml of RPMI medium, and centrifuged at 25°C and 1,500 rpm for 5 minutes. After centrifugation, the cell pellet was resuspended in 10 ml of RPMI medium, washed once more, and centrifuged at 25°C and 1,500 rpm for 5 minutes. After centrifugation, the cell pellet was resuspended in PBS and immediately injected intravenously into mice for adoptive cell transfer or further cultured for use in cell experiments.

[0057] Induction of Klf4 overexpression using APTO-253 The present inventors predicted that overexpression of the endogenous Klf4 gene in naive CD8 T cells could be achieved by regulating the upstream signaling pathway of the transcription factor Klf4, in addition to retroviral-mediated Klf4 gene transduction. Therefore, we predicted that overexpression of the Klf4 gene by treating naive CD8 T cells with APO-253 (Cercek et al., Invest. New. Drug. 33(5):1086-1092, 2015), a known Klf4 expression inducer, would also produce effects similar to those achieved by retroviral vector-mediated transduction.

[0058] Uncontacted CD8 T cells were isolated as described above and then placed on anti-CD3 antibody-coated plates. During activation, the uncontacted CD8 T cells were treated with anti-CD28 antibody (2 μg / ml) and mIL-2 (100 U / ml), followed by 3 μM APTO-253 (MedChemExpress, LLC). The cells were then cultured for 72 hours in a 37°C, 5% CO2 incubator. After incubation, the cells were transferred to a 15ml centrifuge tube and adjusted to a volume of 3ml with additional RPMI medium. Then, 3ml of Histopaque (Sigma) was carefully placed on the bottom layer. The tube was then centrifuged at 25°C and 2,000 rpm for 30 minutes, with minimum acceleration and deceleration. After centrifugation, the upper layer of the culture medium was removed, and the cells at the interface were collected, transferred to a 15ml centrifuge tube, resuspended in 10ml of PBS, and centrifuged at 4°C and 1,500 rpm for 5 minutes. After centrifugation, the supernatant was removed, and the cell pellet was washed once more with 10 ml of PBS and centrifuged at 1,500 rpm at 4°C for 5 minutes. After centrifugation, the supernatant was removed, and the cell pellet was resuspended in an appropriate amount of PBS and then counted. Finally, 5 x 10 5 The cells were diluted in 200 μl of PBS, loaded into a 1 ml syringe, and then intravenously injected into mice for immune cell transplantation.

[0059] Example 1: Mechanism of CD8 T cell exhaustion As mentioned above, when CD8 T cells are chronically exposed to cancer antigens, they become exhausted. These exhausted CD8 T cells are unable to secrete normal cytokines and divide properly, and are therefore unable to mount a proper immune response. Furthermore, they do not recover to normal function even after time has passed.

[0060] Therefore, the present inventors conducted an experiment to mimic the exhaustion state under test tube conditions in order to investigate the cause of the phenomenon in which CD8 T cells become exhausted over time after activation.

[0061] 1-1: In vitro test of fatigue imitation To this end, we isolated CD8 T cells from OT-I transgenic mice bearing OVA (ovalbumin) peptide-specific T cell receptors (TCRs) and performed experiments in which the cells were repeatedly treated with OVA peptide for five days. In all experimental sets, the cells were treated with IL-15 (5 ng / ml) and IL-7 (5 ng / ml) to enhance CD8 T cell viability. After treatment with OVA peptide (10 ng / ml), the cells were washed the next day and then treated with the same cytokine and OVA peptide in the same composition for five days (repeated stimulation treatment group). A control group was treated with cytokines only, without OVA peptide treatment, and a single stimulation treatment group was treated with OVA peptide (10 ng / ml) for two days, washed, and then treated with cytokines only for resting. A group was also treated with IL-21, a known factor that promotes CD8 T cell activation (repeated stimulation + IL-21 treatment group) (Figure 1A). After 5 days, viable CD8 T cells were isolated using a cell sorter, RNA was extracted, and reverse transcribed into cDNA. The expression levels of various genes were then examined by qRT-PCR. The results showed that the expression of the Tox gene, a representative marker of exhaustion, increased in the repeated stimulation group, which mimicked the exhaustion state, while Tox expression decreased in the repeated stimulation + IL-21 treatment group. Under these conditions, we examined the expression of Klf4. We found that Klf4 expression increased in the repeated stimulation group, and, unlike Tox, Klf4 expression further increased in the repeated stimulation + IL-21 treatment group (Figure 1B). This suggests that Klf4 expression increased in response to the activating factor IL-21, suggesting that Klf4 is a factor involved in CD8 T cell activation in the exhausted state.

[0062] 1-2: In vivo experiment to mimic fatigue To confirm whether the results of the cell experiment in Example 1-1 can be reproduced in animal experiments, the present inventors performed fatigue imitation experiments under in vivo conditions using experimental animals.

[0063] Specifically, C57BL / 6 mice were inoculated with MC38 cancer cells (3 x 10 5 ) and then on the 18th day, the spleen and tumor tissues were excised. The animal experiments were conducted in accordance with the regulations of the Animal Experiment Ethics Committee of Seoul National University, Republic of Korea. In the spleen, CD44 low Naive (Na△ve) CD8 T cells and antigen-stimulated CD44 T cells with high CD44 expression high Effector CD8 T cells were isolated using a cell separator. In tumor tissue, Ly108 cells, known as chronic progenitor cells, were isolated based on information from previous studies. + CD8 T cells, also known as chronic effector cells, Ly108 - CD69 - Tim3 + CD8 T cells and Ly108, known as terminally exhausted CD8 T cells - CD69 + Tim3 + CD8 T cells were isolated using a cell separator (Figure 1C). RNA from the five isolated groups of cells was extracted and reverse transcribed into cDNA. The expression levels of various genes were then confirmed by qRT-PCR.

[0064] As a result, we confirmed that the expression level of Klf4 was generally higher in exhausted CD8 T cells (chronic precursor cells, chronic effector cells, and terminally exhausted cells) present in tumor tissue compared to unexposed CD8 T cells. In particular, the expression level of Ly108, known as a chronic effector cell, was significantly higher in exhausted CD8 T cells present in tumor tissue compared to unexposed CD8 T cells. - CD69 - Tim3 + We were able to confirm that Klf4 gene expression was highest in CD8 T cells (Figure 1D). Based on these findings, we were able to confirm that exhausted and highly activated CD8 T cells exhibited even higher levels of Klf4 gene expression both in vitro and in vivo.

[0065] Example 2: Generation of Klf4-overexpressing CD8 T cells through gene transduction Based on the results of Example 1, the inventors observed that Klf4 gene expression is elevated in exhausted cells, but that Klf4 expression is even higher in effector cells that retain anti-cancer activity even in exhausted cells. Based on this finding, they hypothesized that Klf4 may promote the development and function of effector cells.

[0066] Therefore, to confirm the accuracy of this hypothesis, the present inventors attempted to determine whether artificially increasing Klf4 expression in CD8 T cells would promote the generation of effector cells, and whether the exhaustion caused by chronic stimulation with antigens would be suppressed and effector cell function would be promoted.

[0067] To this end, the present inventors first conducted an experiment in which isolated CD8 T cells were transduced with the Klf4 gene to overexpress the Klf4 gene.

[0068] Specifically, CD8 T cells were treated with anti-CD3 antibody, anti-CD28 antibody, and mIL-2 for 24 hours, and then retrovirally overexpressed the MigRI vector as a control group, or a vector in which Klf4 was inserted into the MigRI vector (hereinafter referred to as "Klf4") as an experimental group. Retroviral transduction was performed using the general method described above. Then, resting CD8 T cells were induced by treatment with mIL-7 and mIL-15 for 3 days. Since the MigRI vector primarily expresses GFP, the overexpressed cells expressed GFP. + FACS analysis was performed using the untransfected control group, MigRI GFP. - , MigRI GFP + , Klf4 GFP - Compared to cells in which Klf4 is not overexpressed, Klf4 GFP +In CD8 T cells, we confirmed that the aforementioned chronic effector cell (Ly108-CD69-) subset significantly increased (Figure 2A), indicating that overexpression of Klf4 is an important factor in the generation of the chronic effector cell subset.

[0069] Next, to confirm whether Klf4 overexpression enhances effector cell function, CD8 T cells from Pmel mice, which possess a T cell receptor specifically recognizing the gp100 antigen, were isolated and co-cultured for 6 hours with control (MigRI) and target cancer cells (MC38-gp100) overexpressing the Klf4 gene using the same method as above, and the percentage of dead target cancer cells was measured. The percentage of dead target cancer cells increased when co-cultured with PmelI CD8 T cells overexpressing the Klf4 gene compared to the control (MigRI), indicating that Klf4-overexpressing CD8 T cells were able to kill cancer cells more effectively (Figure 2B).

[0070] Furthermore, to confirm whether Klf4 overexpression promotes chronic effector cell function in a direct exhaustion state, we applied the in vitro exhaustion model described in Example 1-1. After 24 hours of treatment with OVA peptide, CD8 T cells were retrovirally overexpressed with the control MigRI vector or the Klf4 vector. Subsequently, treatment with OVA peptide was repeated for the remaining four days to induce an in vitro exhaustion state, and MigRI GFP was expressed. + and Klf4 GFP +CD8 T cells were isolated using a cell separator and gene expression was analyzed by qRT-PCR using the primer pair listed in Table 1. The results confirmed that Klf4 gene expression was significantly higher in Klf4-overexpressing CD8 T cells than in the MigRI vector control group. Conversely, Tox gene expression, a marker of exhaustion, was significantly reduced (Figure 2C). Furthermore, the levels of granzyme B and interferon-gamma (IFN-γ) secreted by these CD8 T cells were measured using FACS analysis. Significant increases in cytokine secretion were confirmed in Klf4-overexpressing CD8 T cells (Figures 2D and 2E). These results suggest that higher Klf4 gene expression promotes the formation of chronic effector cells (Ly108-CD69-), enhancing their ability to kill target cancer cells, and suppressing further exhaustion in exhausted CD8 T cells by promoting increased secretion of active cytokines.

[0071] [Table 1]

[0072] Example 3: Analysis of fatigue suppression through animal experiments 3-1: Animal experiments using Klf4 gene transduction Based on these results, the present inventors used a mouse tumor model to confirm whether Klf4 overexpression in CD8 T cells can increase cell activity and suppress cancer development in vivo. Specifically, 3x10 MC38 cancer cells expressing gp100 (MC38-gp100) were inoculated into Rag2 KO mice lacking T cells. 5 One day after inoculation, CD8 T cells derived from PmelI transgenic mice (provided by the Jackson Laboratory, National Cancer Center) transformed to express MigRI and gp100-specific TCR were transfected with the expression vector, and 1 x 10 CD8 T cells transformed to overexpress Klf4 were injected. 6The tumors were administered intravenously. Tumor volume was monitored until day 15 after tumor cell inoculation, at which point the mice were sacrificed for the experiment (Figure 3A). Prior to analysis of the mice, the expression level of the Klf4 gene was measured in CD8 T cells overexpressing MigRI and Klf4, confirming successful overexpression of the Klf4 gene (Figure 3B). Mice administered with Klf4-overexpressing CD8 T cells showed significantly reduced tumor development compared to the control group administered only MigRI (Figure 3C). Furthermore, FACS analysis of Ki-67 expression, a measure of cell division, in infiltrating CD8 T cell subsets in tumor tissue revealed that CD8 T cell subsets overexpressing Klf4, particularly chronic effector cells, proliferated more efficiently than the control group (MigRI) (Figure 3D). Furthermore, we confirmed that activating cytokines such as granzyme B, IFN-γ, and TNF-α all increased (Figures 3E and 3F). These results demonstrate that Klf4-overexpressing CD8 T cells exhibit significantly superior anti-cancer immune responses.

[0073] 3-2: Analysis of fatigue suppression using Klf4 inducers Based on the results of Example 3-1, the present inventors believed that if a drug capable of overexpressing Klf4 were used instead of Klf4 gene transduction, the anti-cancer effect due to increased Klf4 gene expression could be confirmed, as described above. Therefore, animal experiments were performed using APTO-253, which has been known through previous research as a Klf4 inducer. To this end, specifically, 3x10 MC38 cancer cells were inoculated into lymphocyte-deficient Rag2 KO mice. 5 The next day, 5x10 CD8 T cells treated with PBS or APTO-253 for 3 days were injected. 5The tumors were administered intravenously. Tumor volume was monitored until day 15 after cancer cell inoculation, and the mice were sacrificed on day 15 for the experiment (Figure 4A). Prior to mouse analysis, Klf4 gene expression levels were measured in CD8 T cells treated with APTO-253 for 3 days. Klf4 gene expression levels were confirmed to be higher in APTO-253-treated CD8 T cells compared to PBS-treated CD8 T cells (Figure 4B). Furthermore, tumor growth was suppressed in mice treated with APTO-253-treated CD8 T cells compared to mice treated with PBS (Figure 4C). These results demonstrate that, in addition to Klf4 gene transduction, increased Klf4 gene expression in CD8 T cells via the Klf4 inducer APTO-253 also enhances the anti-cancer immune response of CD8 T cells.

[0074] This result demonstrates that CD8 T cells in which the Klf4 gene is overexpressed by gene transduction or drug treatment in accordance with one embodiment of the present invention can effectively kill cancer cells through innate immune responses by suppressing the immune exhaustion effect induced by repeated antigen stimulation in the body.

[0075] Example 4: Production of induced CAR-CD8 T cells 4-1: Construction of EpCAM-binding CAR construct A schematic diagram showing an exemplary CAR construct is provided in Figure 5. CAR constructs are generated through the following method. The nucleic acid sequence for the cDNA encoding a fusion protein containing the amino acid sequence of the CAR construct (EpCAM-CD28-CD3ζ, Figure 5) sequentially containing anti-EpCAM scFv (SEQ ID NO: 11), CD8α hinge (SEQ ID NO: 12), CD28 TM (SEQ ID NO: 13), CD28 ICD (SEQ ID NO: 14), and CD3ζ ICD (SEQ ID NO: 15) was synthesized by standard techniques, amplified by PCR, and inserted into pCLPS (Parry et al., J. Virol. 72:8463-8471, 1998), a third-generation self-inactivating lentiviral vector based on pRRL-SIN-CMV-eGFP-WPRE (Dull et al., J. Virol. 72:8463-8471, 1998) or pELNS (Carpenito et al., Proc. Natl. Acad. Sci. USA 106:3360-3365, 2009). (J. Immunol., 171:166-174, 2003). However, the vector used in the present invention differs from pCLPS in that the promoter for foreign gene expression, CMV, is replaced with EF-1α. The encoded CAR contains an scFv for binding to EpCAM (SEQ ID NO: 11).

[0076] 4-2: Construction of anti-Trop-2 CAR construct A lentiviral vector for expressing a CAR consisting of anti-Trop-2 scFv (SEQ ID NO: 16), CD8α hinge, CD28, and CD3ζ ICD linked in sequence (anti-Trop-2-CD28-CD3ζ, Figure 5) was prepared in the same manner as in Example 4-1, except that the nucleotide sequence encoding anti-EpCAM-scFv was substituted for the nucleic acid sequence of anti-Trop-2 scFv.

[0077] 4-3: Construction of anti-CEACAM6 CAR construct A lentiviral vector for expressing a CAR (anti-CEACM6-CD28-CD3ζ, Figure 5) in which anti-CEACAM6 scFv (SEQ ID NO: 17), CD8α hinge, CD28 TM, and CD3ζ ICD are linked sequentially is prepared in the same manner as in Example 4-1, except that the nucleotide sequence encoding anti-EpCAM-scFv replaces the nucleic acid sequence of anti-CEACAM6 scFv.

[0078] 4-4: Construction of anti-CEACAM5 CAR construct A lentiviral vector for expressing a CAR (anti-CEACAM5-CD28-CD3ζ, Figure 5) in which anti-CEACAM5 scFv (SEQ ID NO: 18), CD8α hinge, CD28 TM, and CD3ζ ICD are linked in sequence is prepared in the same manner as in Example 4-1, except that the nucleotide sequence encoding anti-EpCAM-scFv replaces the nucleic acid sequence of anti-CEACAM5 scFv.

[0079] Example 5: Production of lentiviral particles containing a CAR construct High-titer, replication-defective lentiviral vectors were produced as described in the previous examples by the method described by Parry et al. (J. Immunol., 171:166-174, 2003). Briefly, HEK 293T cells (ATCC CRL-3216) were cultured in RPMI 1640, 10% heat-inactivated FCS, 2 mM glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin sulfate. Cells were cultured at 5 x 10 per T150 tissue culture flask 24 hours prior to transfection with 7 μg of pMDG.1 (VSV-G envelope), 18 μg of pRSV.rev (HIV-1 Rev-encoding plasmid), and 18 μg of pMDLg. 6Cells were seeded with p.RRE (packaging plasmid) and 15 μg of lentiviral vector transduced using Fugene 6 (Roche Molecular Biochemicals). The medium was replaced 6 hours after transfection, and viral supernatants were harvested at 24 and 48 hours post-transfection. Viral particles were concentrated 10-fold by ultracentrifugation at 28,000 rpm for 3 hours using a Beckman SW28 rotor.

[0080] Example 6: Transduction of T cells using CAR lentivirus For specific purposes, T cells from normal individuals are used with the target CAR construct for construct testing and design. Primary culture of human CD4 + and CD8 + T cells are isolated from PBMCs of healthy volunteers, donors, or cancer patients after collection of leukocyte components by negative selection using the RosetteSep kit (Stem Cell Technologies). T cells are cultured in complete medium (RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin sulfate, and 10 mM HEPES) and treated with beads coated with monoclonal anti-CD3 and anti-CD28 antibodies for 12–24 h. They are then transduced with the target lentiviral vector at an MOI (multiplicity of infection) of 5–10. Human recombinant IL-2 is injected every other day at a final concentration of 50 U / mL and 0.5–1.0 x 10 ng / mL. 6 The cell density is maintained at 1000 cells / mL. Transduction using the Klf4 construct can be performed by transducing a polynucleotide encoding Klf4 in a CAR construct, or can be performed together with transduction of a separate CAR construct, or can be performed sequentially.

[0081] Example 7: Generation and evaluation of autologous CAR-T cells from cancer patients The generation of autologous CAR-T cells from cancer patients was investigated using the method described by Brentjens et al. (Sci. Transl. Med. 5:177ra38, 2013). Briefly, PBMCs were obtained from cancer patients by leukapheresis, washed, and cryopreserved. T cells were isolated from the thawed leukapheresis population, activated with Dynabeads Human T-Activator CD3 / CD28 magnetic beads (Invitrogen), and transduced with the lentiviral vector of interest. The transduced T cells were further expanded in a WAVE bioreactor to achieve the desired transduced T cell volume.

[0082] Although the present invention has been described with reference to the above-described embodiments, these are merely illustrative, and those skilled in the art will recognize that various modifications and equivalent embodiments are possible. Therefore, the true technical scope of the present invention should be determined by the technical spirit of the claims. [Industrial Applicability] The methods and materials according to one embodiment of the present invention are utilized in the manufacture of pharmaceuticals, particularly anti-cancer drugs. [Brief explanation of the drawings]

[0083] [Figure 1A] FIG. 1 is a schematic diagram illustrating the design of a repeated antigen stimulation test for CD8 T cells according to one embodiment of the present invention. [Figure 1B] FIG. 1C is a series of graphs showing the results of an experiment conducted according to the experimental design of FIG. 1A, measuring the expression levels at the mRNA level of Tox (left) and Klf4 (right), which are markers related to the exhaustion state of CD8 T cells depending on the level of antigen stimulation. [Figure 1C] FIG. 1 is a schematic diagram showing the marker phenotypes of various stages of CD8 T cell subsets present in the spleen and cancer tissue induced by injection of MC38 colon cancer cells into mice. [Figure 1D] This is a graph showing the results of measuring the level of Klf4 protein expression in each of these CD8 T cell subsets at the mRNA level. [Figure 2A] According to one embodiment of the present invention, the characteristics of CD8 T cells transformed with a control retroviral vector (MigRI) or a retroviral vector (Klf4) containing the Klf4 gene were analyzed. According to one aspect of the present invention, there is provided a method for suppressing the exhaustion of CD8 T cells in vitro, comprising inducing overexpression of Klf4 protein in CD8 T cells isolated from an individual, a cell population containing the CD8 T cells, or CAR-CD8 T cells obtained by transducing the CD8 T cells with a gene encoding a chimeric antigen receptor (CAR). Figure 2A shows the results of FACS analysis of electron-transfected CD8 T cells using the above-mentioned subset markers. [Figure 2B] According to one embodiment of the present invention, the characteristics of CD8 T cells transformed with a control retroviral vector (MigRI) or a retroviral vector (Klf4) containing the Klf4 gene were analyzed. According to one aspect of the present invention, there is provided a method for suppressing the exhaustion of CD8 T cells in vitro, comprising inducing overexpression of Klf4 protein in CD8 T cells isolated from an individual, a cell population containing the CD8 T cells, or CAR-CD8 T cells obtained by transfecting the CD8 T cells with a gene encoding a chimeric antigen receptor (CAR). Figure 2B is a graph showing the results of FACS analysis of the proportion of dead cancer cells when control (MigRI) and CD8 T cells transformed with the Klf4 gene were co-cultured with target cancer cells. [Figure 2C]According to one embodiment of the present invention, the characteristics of CD8 T cells transformed with a control retroviral vector (MigRI) or a retroviral vector (Klf4) containing the Klf4 gene were analyzed. According to one aspect of the present invention, there is provided a method for suppressing the exhaustion of CD8 T cells in vitro, comprising inducing overexpression of Klf4 protein in CD8 T cells isolated from an individual, a cell population containing the CD8 T cells, or CAR-CD8 T cells obtained by transfecting the CD8 T cells with a gene encoding a chimeric antigen receptor (CAR). Figure 2C is a graph showing the results of measuring the gene expression levels of Klf4 (left) and Tox (right) at the mRNA level in control (MigRI) and CD8 T cells transformed with the Klf4 gene. [Figure 2D] According to one embodiment of the present invention, the characteristics of CD8 T cells transformed with a control retroviral vector (MigRI) or CD8 T cells transformed with a retroviral vector (Klf4) containing the Klf4 gene were analyzed. According to one aspect of the present invention, there is provided a method for suppressing exhaustion of CD8 T cells in vitro, comprising inducing overexpression of Klf4 protein in CD8 T cells isolated from an individual, a cell population containing the CD8 T cells, or CAR-CD8 T cells obtained by transfecting the CD8 T cells with a gene encoding a chimeric antigen receptor (CAR). Figure 2D shows a graph (left) and a 2D histogram (right) of the results of FACS analysis of the level of granzyme B expression in control (MigRI) and Klf4 gene-transfected CD8 T cells (Klf4), and measurement of the proportion of cells expressing granzyme B, which is essential for the target cell killing function of CD8 T cells, in each cell type (MigRI GFP-, MigRI GFP+, Klf4 GFP-, Klf4 GFP+). [Figure 2E]According to one embodiment of the present invention, the characteristics of CD8 T cells transformed with a control retroviral vector (MigRI) or CD8 T cells transformed with a retroviral vector (Klf4) containing the Klf4 gene were analyzed. According to one aspect of the present invention, there is provided a method for suppressing exhaustion of CD8 T cells in vitro, comprising inducing overexpression of Klf4 protein in CD8 T cells isolated from an individual, a cell population containing the CD8 T cells, or CAR-CD8 T cells obtained by transfecting the CD8 T cells with a gene encoding a chimeric antigen receptor (CAR). Figure 2E shows a graph (left) and a two-dimensional histogram (right) of the results of FACS analysis of the expression level of IFN-γ, which is crucial for the function of control (MigRI) and Klf4 gene-transfected CD8 T cells, and measurement of the proportion of IFN-γ-expressing cells in each cell type (MigRI GFP-, MigRI GFP+, Klf4 GFP-, Klf4 GFP+). [Figure 3A] FIG. 1 is a schematic diagram illustrating the administration schedule of an animal experiment using CD8 T cells transduced with the Klf4 gene according to one embodiment of the present invention. [Figure 3B] 10 is a graph showing the results of measuring the expression level of the Klf4 gene at the mRNA level in control and Klf4 gene-transduced CD8 T cells. [Figure 3C] 1 is a graph showing the change in tumor tissue volume over time in a control group (MigRI) and in a cancer model animal administered with CD8 T cells transduced with the Klf4 gene according to an example of the present invention. [Figure 3D] After the animal experiment, the CD8 T cell subsets isolated from the tumor tissues of the sacrificed animals were analyzed by FACS for Ki-67 expression, which indicates the degree of cell division. The graph (left) and two-dimensional histogram (right) show the results of measuring the ratio of Ki-67 expressing cells among all CD8 T cells according to the marker phenotype of the isolated CD8 T cells. [Figure 3E]After the animal experiment, the level of granzyme B expression in CD8 T cells isolated from tumor tissues of sacrificed animals was analyzed by FACS analysis. The percentage of granzyme B-expressing cells among all CD8 T cells was measured according to the marker phenotype of the isolated CD8 T cells. The graph (left) and two-dimensional histogram (right) show the results. [Figure 3F] After the animal experiment, the levels of TNF-α and INF-γ expression in CD8 T cells isolated from tumor tissues of sacrificed animals were analyzed by FACS analysis. The percentages of TNF-α- and INF-γ-expressing cells among total CD8 T cells were measured according to the marker phenotype of the isolated CD8 T cells. The graph (left) and two-dimensional histogram (right) show the results. [Figure 4A] FIG. 1 is a schematic diagram illustrating the administration schedule of an animal experiment using CD8 T cells treated with APTO-253 according to one embodiment of the present invention. [Figure 4B] 1 is a graph showing the results of measuring the expression level of the Klf4 gene at the mRNA level in experimental animals administered with control (PBS) and APTO-253-treated CD8 T cells. [Figure 4C] 1 is a graph showing the change in tumor tissue volume over time in a control group (PBS) and in a cancer model animal to which CD8 T cells treated with APTO-253 according to an embodiment of the present invention were administered. [Figure 5] FIG. 1 is a schematic diagram showing the structures of various CAR constructs (EpCAM CAR, Trop-2 CAR, CEACAM6 CAR, and CEACAM5 CAR) according to one embodiment of the present invention.

Claims

1. A method for suppressing exhaustion of CD8 T cells in vitro, the method comprising the step of transducing cells selected from the group consisting of: a) CD8 T cells isolated from an individual; b) a cell population containing the CD8 T cells; and c) CAR-CD8 T cells obtained by transducing the CD8 T cells with a gene encoding a chimeric antigen receptor (CAR), with an expression vector containing a polynucleotide encoding a Klf4 protein, or treating the cells with APTO-253, thereby inducing overexpression of the Klf4 protein in the cells.

2. A method for expanding CD8 T cells in vitro, the method comprising the step of transducing cells selected from the group consisting of: a) CD8 T cells isolated from an individual; b) a cell population containing the CD8 T cells; and c) CAR-CD8 T cells obtained by transducing the CD8 T cells with a gene encoding a chimeric antigen receptor (CAR), with an expression vector containing a polynucleotide encoding a Klf4 protein, or treating the cells with APTO-253, thereby inducing overexpression of the Klf4 protein in the cells.

3. A method for enhancing anti-cancer immune responses of CD8 T cells in vitro, the method comprising the step of transducing cells selected from the group consisting of: a) CD8 T cells isolated from an individual; b) a cell population containing the CD8 T cells; and c) CAR-CD8 T cells obtained by transducing the CD8 T cells with a gene encoding a chimeric antigen receptor (CAR), with an expression vector containing a polynucleotide encoding a Klf4 protein, or treating the cells with APTO-253, thereby inducing overexpression of the Klf4 protein in the cells.

4. The method of claim 3 , wherein the expression vector is a viral vector or a non-viral vector.

5. 5. The method of claim 4, wherein the viral vector is an adeno-associated virus (AAV) vector, an adenovirus vector, an alphavirus vector, a herpes simplex virus vector, a vaccinia virus vector, a Sendai virus vector, a flavivirus vector, a radvovirus vector, a retrovirus vector, a herpes virus vector, a poxvirus vector, or a lentivirus vector.

6. 5. The method of claim 4, wherein the non-viral vector is an mRNA, a DNA vector, a nanoparticle, a cationic polymer, an exosome, an extracellular vesicle, or a liposome.

7. The method of claim 6, wherein the mRNA is used alone or in combination with the non-viral vector other than the mRNA encoding the Klf4 protein.

8. A pharmaceutical composition for cancer treatment comprising CD8 T cells in which the Klf4 gene is overexpressed as an active ingredient, wherein the pharmaceutical composition for cancer treatment comprises: The composition, wherein the Klf4 protein is overexpressed by transforming the CD8 T cells with an expression vector containing a polynucleotide encoding it, or by treating the cells with APTO-253.

9. 9. The pharmaceutical composition for cancer treatment according to claim 8, wherein the CD8 T cells in which the Klf4 gene is overexpressed are autologous CD8 T cells isolated from an individual in need of treatment or allogeneic CD8 T cells isolated from another person.

10. 10. The pharmaceutical composition for cancer therapy according to claim 9, wherein the CD8 T cells overexpressing the Klf4 gene are prepared by transducing CD8 T cells with an expression vector containing the Klf4 gene and / or treating the CD8 T cells with a Klf4 inducer.

11. Transformed CD8 T cells that have been transformed with only an expression vector containing only a polynucleotide encoding the Klf4 protein as a foreign gene so that the Klf4 gene is overexpressed.

12. The transformed CD8 T cell of claim 11, wherein the expression vector is a viral vector or a non-viral vector.

13. 13. The transformed CD8+ T cell of claim 12, wherein the viral vector is an adeno-associated virus (AAV) vector, an adenovirus vector, an alphavirus vector, a herpes simplex virus vector, a vaccinia virus vector, a Sendai virus vector, a flavivirus vector, a radovvirus vector, a retrovirus vector, a herpes virus vector, a poxvirus vector, or a lentivirus vector.

14. The transformed CD8 T cell of claim 12, wherein the non-viral vector is an mRNA, a DNA vector, a nanoparticle, a cationic polymer, an exosome, an extracellular vesicle, or a liposome.

15. The transformed CD8 T cell of claim 14, wherein the mRNA is an mRNA encoding the Klf4 protein alone or in combination with a non-viral vector other than the mRNA.

16. A transformed CAR-CD8 T cell that has been transformed with only a single expression vector that contains, as exogenous genes, a gene construct containing only a polynucleotide encoding the Klf4 protein and a gene construct containing only a polynucleotide encoding the CAR, or a gene construct containing only a polynucleotide encoding the Klf4 protein and a gene construct containing only a polynucleotide encoding the CAR, so that an exogenous Klf4 protein and a chimeric antigen receptor (CAR) are expressed.

17. The transformed CAR-CD8 T cell of claim 16, wherein the CAR is a fusion protein comprising a single-chain-based antibody analog, a cell membrane-transducing domain, a costimulatory factor, and an intracellular signaling domain.

18. The transformed CAR-CD8 T cell of claim 17, wherein the single-chain-based antibody analog is an antibody analog that specifically binds to a cancer antigen or an antigen derived from a pathogen.

19. The cancer antigens include EpCAM, Trop-2, CEACAM5, CEACAM6, carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), prostate-specific membrane antigen (PSMA), Her2 / neu, MUC-1, BCR / ABL, α-fetoprotein (AFP), antigens derived from Epstein-Barr virus (EBV), antigens derived from human hepatitis B virus (HBV), antigens derived from human hepatitis C virus (HCV), Proteinase 3, WT-1, PAP, G250, melanoma antigen gene (MAGE), BAGE, GAGE, NY-ESO-1, tyrosinase, tyrosinase-related protein-1 (TRP-1), TRP-2, gp100, MART-1, and Ig The transformed CAR-CD8 T cell of claim 18, wherein the antigen is selected from the group consisting of idiotype, CDK4, caspase-8, β-catenin, BCR / ABL, human papillomavirus (HPV E6 / E7), HHV-8, 5T4, p53, cancer antigen 125 (CA-125), cancer antigen-72-4 (CA-72-4), cancer antigen-15-3 (CA-15-3), and cancer antigen-19-9 (CA-19-9).

20. The transformed CAR-CD8 T cell of claim 18, wherein the pathogen-derived antigen is an antigen derived from a pathogenic microorganism, virus, or parasite.

21. The transformed CAR-CD8 T cell of claim 20, wherein the pathogenic microorganism is a pathogenic bacterium or a pathogenic fungus.

22. The transformed CAR-CD8 T cell of claim 21, wherein the pathogenic bacterium is Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae, Helicobacter pylori, Streptococcus pneumoniae, Mycobacterium tuberculosis, Mycobacterium cholera, Staphylococcus aureus, Shigella, Borrelia, or Salmonella.

23. The pathogenic fungi include Candida, Trichophyton, Aspergillus, Fonsecaea sp., Epidermophyton sp., Piedraia sp., Malassezia sp., Pseudallescheria sp., Basidiobolus sp., Conidiobolus sp., Rhinosporidium sp., Paracoccidioides sp., Cryptococcus sp., Blastomyces sp., Sporothrix sp., Mucor sp., and Absidia sp. The transformed CAR-CD8 T cell according to claim 21, wherein the transformed CAR-CD8 T cell is selected from the group consisting of Rhizopus sp., Pneumocystis sp., Wangiella sp., Phialophora sp., and Schizophyllum sp.

24. The transformed CAR-CD8 T cell of claim 20, wherein the virus is influenza virus, human papillomavirus (HPV), vesicular stomatitis virus, cytomegalovirus (CMV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis G virus (HGV), respiratory syncytial virus (RSV), herpes simplex virus, or human immunodeficiency virus (HIV).

25. The single chain-based antibody analogs include scFv, sdAb, V Ｈ H, V ＮＡＲ , Affibody, Affilin, Affimer, Affitin, Alphabody, Anticalin, Avimer, DARPin, Fynomer, Kunitz domain peptide, monobody, nanoCLAMP, variable lymphocyte receptor (VLR), or repebody. T cells.

26. The cell membrane translocation domain may be selected from the group consisting of 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, and CRT. AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA 4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, a ligand that specifically binds to CD83, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CD1-1a / CD1 8), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocytic activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6. T cells.

27. The transformed CAR-CD8 T cell of claim 17, wherein the costimulatory factor is the cytoplasmic domain of CD28, ICOS, CTLA4, PD1, BTLA, DR3, 4-1BB, CD2, CD40, CD30, CD27, SLAM, 2B4 (CD244), NKG2D / DAP12, TIM1, TIM2, TIM3, TIGIT, CD226, CD160, LAG3, B7-1, B7-H1, GITR, HVEM, or OX40L (CD252), or a conjugate of two or more of these.

28. The transformed CAR-CD8 T cell according to claim 17, wherein the intracellular signaling domain comprises a part or the entirety of one or more cytoplasmic domains selected from CD3ζ, CD28, CD27, OX40 / CD134, 4-1BB / CD137, FcεRIγ, ICOS / CD278, IL-2Rβ / CD122, IL-2Rα / CD132, DAP10, DAP12, and CD40.

29. A composition comprising the transformed CD8 T cell according to claim 11 or the transformed CAR-CD8 T cell according to claim 16 as an active ingredient.

30. 30. The composition of claim 29, for use in treating a disease requiring an innate immune response.

31. 31. The composition of claim 30, wherein the disease requiring an innate immune response is cancer, a bacterial infection, a fungal infection, a viral infection, or a parasitic infection.

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