Long-lived t cells, pharmaceutical composition, and method of use

Abstract

The present invention provides T cells that express a recombinant receptor and satisfying conditions (I) and (II).　(I) T cells having reduced or eliminated expression and / or function of the PRDM1 gene (II) T cells having reduced or eliminated expression and / or function of the RCOR1 gene, and / or increased or activated expression of the c-Jun gene

Description

Long-term viable T cells, pharmaceutical compositions, and methods of use

[0001] The present invention relates to long-lived T cells, pharmaceutical compositions containing the T cells, and methods of using the T cells.

[0002] Chimeric antigen receptor (CAR)-transduced T cell therapy is a promising treatment that can lead to a cure for intractable cancers, but the infused T cells gradually age, losing their viability and cell-killing ability (i.e., exhaustion).Several genetic modification techniques for T cells have been reported to prevent this functional decline.

[0003] For example, Patent Document 1 discloses that overexpression of the cJUN gene, an AP-1 transcription factor, can confer exhaustion resistance to T cells, but does not disclose whether the T cells acquire long-term viability.

[0004] Non-Patent Document 1 discloses that deletion of the PRDM1 gene conferred long-term survival to T cells. However, deletion of the PRDM1 gene alone inhibited the effector function of T cells, leading to exhaustion. This document uses a method to introduce Cas9 protein and guide RNA by electroporation using CRISPR / Cas9 technology.

[0005] Non-Patent Document 2 shows that simultaneous knockout of the PRDM1 gene and the NR4A3 gene enables CART cells to acquire long-term viability and fatigue resistance. In this document, electroporation was used for gene modification.

[0006] U.S. Patent No. 11,400,117

[0007] Yoshikawa et al. Blood. 2022;139(14):2156-2172.Jung In-Young et al. Sci Transl Med 2022;14(670):eabn7336.

[0008] T cell aging and exhaustion are controlled by distinct transcriptional networks, yet few strategies have been developed to simultaneously improve these properties.

[0009] The problem to be solved by the present invention is to provide T cells with long-term viability and exhaustion resistance, a pharmaceutical composition containing the T cells, and a method for using the T cells.

[0010] The present invention encompasses the following embodiments. Item 1. A T cell expressing a recombinant receptor, wherein the T cell satisfies the following (I) and (II): (I) has reduced or eliminated expression and / or function of the PRDM1 gene; (II) has reduced or eliminated expression and / or function of the RCOR1 gene, and / or has increased or activated expression of the c-Jun gene. Item 2. The T cell according to Item 1, wherein the recombinant receptor is a chimeric antigen receptor (CAR). Item 3. The T cell according to Item 1, wherein the recombinant receptor is a chimeric antigen receptor (CAR) that specifically recognizes a tumor antigen. Item 4. The T cell according to Item 3, wherein the tumor antigen is CD19. Item 5. Item 6. The T cell of any of Items 1 to 4, wherein the T cell is selected from the group consisting of CD3+ T cells, CD8+ T cells, CD4+ T cells, immune effector cells, natural killer (NK) T cells, γδ T cells, a combination of CD4+ cells and CD8T+ cells, memory cells, cytokine-induced killer cells, tumor-infiltrating lymphocytes, and combinations thereof. Item 7. The T cell of Items 1, wherein the cell further comprises an inhibitor of the PRDM1 gene and at least one of an inhibitor of the RCOR1 gene and an activator of the c-Jun gene. Item 7. The T cell of Items 6, wherein the inhibitor of the PRDM1 gene is either (1) or (2) below: (1) a gene editing system that targets one or more sites within the PRDM1 gene or its regulatory elements, or (2) a nucleic acid encoding one or more components of the gene editing system. Item 8. Item 6. The T cell of Item 6, wherein the inhibitor of the RCOR1 gene is the following (1) or (2): (1) a gene editing system that targets one or more sites in the RCOR1 gene or its regulatory elements, or (2) a nucleic acid encoding one or more components of the gene editing system. Item 9. The T cell of Item 6, wherein the activator of the c-Jun gene is integration of the c-Jun gene into cellular DNA via a virus or vector that expresses c-Jun. Item 10. A pharmaceutical composition for treating a disease in a subject, comprising the T cell of Item 1. Item 11. The pharmaceutical composition of Item 10, wherein the disease is a tumor or cancer.Item 12. The pharmaceutical composition according to Item 10, wherein the T cells maintain an early memory phenotype, enhance or maintain cytokine production, reduce expression of exhaustion markers, and / or suppress decline in effector function, compared to control T cells that do not satisfy either (I) or (II). Item 13. A method for enhancing the therapeutic efficacy of cells expressing a recombinant receptor that specifically recognizes a tumor antigen, comprising the steps of reducing or eliminating the expression and / or function of the PRDM1 gene in the T cells, and reducing or eliminating the expression and / or function of the RCOR1 gene in the T cells and / or increasing or activating the expression of the c-Jun gene in the T cells. Item 14. A method for enhancing the therapeutic efficacy of T cells expressing a recombinant receptor that specifically recognizes a tumor antigen, comprising the steps of contacting the T cells with an inhibitor of the PRDM1 gene, and contacting the T cells with an inhibitor of the RCOR1 gene and / or with an activator of the c-Jun gene. Item 15. Use of a PRDM1 gene inhibitor and an RCOR1 gene inhibitor and / or a c-Jun gene activator in the manufacture of a medicament for CAR-expressing cell therapy.

[0011] The present invention provides T cells that maintain high cytotoxicity while maintaining long-term viability. Pharmaceutical compositions containing such T cells and therapeutic methods using such T cells are useful for improving T cell-based therapies.

[0012] Searching for H3K9 methylation-associated epigenetic factors that influence memory T cell differentiation. Data are representative flow cytometry plots. Peripheral blood T cells were stimulated with K562 / mOKT3 / CD80 (day 0), transduced with a CD19-targeting CAR, and the indicated genes were individually disrupted using the CRISPR / Cas9 system. Memory marker profiles were analyzed on day 15. Searching for H3K9 methylation-associated epigenetic factors that influence memory T cell differentiation. Memory marker profiles were analyzed on day 15. (A) Mean fluorescence intensity of CD62L, (B) mean fluorescence intensity of CCR7, (C) Frequency (%) of CD62L+CCR7+ cells within the CD8+CAR-T cell population (C, n=3, one-way ANOVA with multiple comparison test compared to control CAR-T cells). *P<0.05. KDM1A knockout inhibits T cell proliferation. CD19-targeting CAR-T cells were knocked out of PRDM1 and KDM1A individually (PRDM1 KO, KDM1A KO) or simultaneously (DKO). Memory marker profiles were analyzed on days 11-13. (A) Frequency (%) of CD62L+CCR7+ cells, (B) Frequency (%) of CD62L+CCR7+CD27+CD28+ cells (n=5 each, one-way ANOVA with multiple comparison test compared to control T cells). (C) Fold expansion of CAR-T cells during 5 days of culture (days 8-13) (n=5, one-way ANOVA with multiple comparison test). Effect of KDM1A-related factors on memory T cell differentiation. CD19-targeting CAR-T cells were knocked out of PRDM1 alone or PRDM1 and RCOR1, and each memory marker was analyzed. Effect of KDM1A-related factors on memory T cell differentiation. CD19-targeted CAR-T cells were knocked out at the indicated epigenetic genes, and the frequencies of (A) CD62L+CCR7+ cells and (B) CD62L+CCR7+CD27+CD28+ cells were calculated. n=6 for each, one-way ANOVA with multiple comparison test. *p<0.05, **p<0.01. CD19-targeted CAR-T cells were knocked out at the indicated epigenetic genes, and the effect on CD19-targeted CAR-T cell proliferation was examined. Fold expansion during 5 days of culture (days 8-13) was calculated.n=5, one-way ANOVA with multiple comparison test. Cytotoxic activity of CAR-T cells. CD19-targeted CAR-T cells were co-cultured overnight with CD19-expressing K562 (K562-CD19), and viable K562-CD19 cells were quantified by flow cytometry (n=3, one-way ANOVA with multiple comparison test). Gene expression profile of knockout CAR-T cells. CD19-targeted CAR-T cells were disrupted with PRDM1, RCOR1, or both genes and expanded. Purified CD8. +Total RNA was isolated from CAR-T cell populations (n ​​= 3 different donors). (A) Hierarchical clustering (FDR < 0.05) of differentially expressed genes among the four groups. (B) Gene set enrichment analysis comparing PRDM1 knockout versus PRDM1 and RCOR knockout CAR-T cells using memory T cell-related genes retrieved from GSE83978. Comparison of the expression levels of effector transcription factors (TBX21 and RUNX3) and exhaustion molecules (TOX and TOX2) in control, PRDM1-knockout CAR-T cells, RCOR1-knockout CAR-T cells, and double-knockout CAR-T cells. n = 3 per group, paired t-test. The effect of epigenetic modifications on the antitumor efficacy of CAR-T cells. NSG mice were intravenously injected with NALM-6 expressing EGFP and luciferase (NALM6-GL) and treated with CD19-targeted CAR-T cells harboring knockouts of PRDM1, RCOR1, or both. Representative images of mice from each group. Mice were continuously monitored for leukemia progression and persistence of infused CAR-T cells. Quantitative total flux within mice at the indicated time points during treatment (n = 8 per group) in Figure 10. One-way analysis of variance (ANOVA) with multiple comparisons test on log-transformed values. Quantitative total flux within mice at the indicated time points during treatment (n = 8 per group) in Figure 10. Analysis of the frequency of human cells in peripheral blood in each group at weeks 2, 3, 4, and 6. One-way ANOVA with multiple comparisons test. Overall survival of treated mice (log-rank test). Role of AP1 transcription factors in PRDM1 knockout CAR-T cells. T cells were individually transduced with GD2-targeted CAR and AP1 transcription factors (BATF, BATF3, c-JUN, or JUNB). CAR-T cells were PRDM1-deleted, restimulated with NAL6-GD2, and expanded for 1 week. (A) Intracellular flow cytometry was used to analyze IL-2 production by CAR-T cells upon restimulation with NALM6-GD2 (n=3, one-way ANOVA with multiple comparisons compared to control CAR-T cells).(B) Granzyme B production by CAR-T cells upon restimulation with NALM6-GD2 was analyzed using intracellular flow cytometry (n = 3, one-way ANOVA with multiple comparisons test compared to control CAR-T cells). Analysis of exhaustion markers in expanded CAR-T cells. Representative flow cytometry plots. Analysis of exhaustion markers in expanded CAR-T cells. Mean fluorescence intensity of PD1, LAG3, and TIM3. Analysis of memory markers in expanded CAR-T cells. Representative flow cytometry plots. Analysis of memory markers in expanded CAR-T cells. CD8. +Frequency of CD62L+ CCR7+ cells (left) or CD62L+ CCR7+ CD27+ CD28+ cells (right) in CAR-T cell populations (n=3, one-way ANOVA with multiple comparisons compared to control CAR-T cells). *P<0.05, **P<0.01, NS, unbiased. PRDM1 knockout and c-JUN overexpression in mesothelin-targeted CAR-T cells. T cells were transduced with a mesothelin-targeted CAR and c-JUN. CAR-T cells were PRDM1-deleted and then restimulated with K562-mesothelin once or daily for a total of seven times. Granzyme B production by CAR-T cells upon restimulation with K562-mesothelin was analyzed (n=3, Student's t-test between CAR-T cells stimulated once and 7 times). Daily restimulated CAR-T cells were analyzed for cytokine production using flow cytometry (n=3, one-way ANOVA with multiple comparisons compared to control CAR-cells). Memory markers were analyzed after daily restimulation (n=3, one-way ANOVA with multiple comparisons compared to control CAR-cells). **P<0.01; NS, not significant. PRDM1 knockout and c-JUN overexpression in GD2-targeted CAR-T cells. GD2-targeted CAR-T cells were ectopically expressed with c-JUN and gene-disrupted with PRDM1. CAR-T cells were restimulated three times with NALM6-GD2 and analyzed for granzyme B production. n=3, one-way ANOVA with multiple comparisons. **P<0.01; NS, not significant. Analysis of exhaustion markers. n=3, one-way ANOVA with multiple comparisons. **P<0.01; NS, not significant. Analysis of cytokine production. n=3, one-way ANOVA with multiple comparison test. **P<0.01; NS, not significant. Analysis of memory marker profile. n=3, one-way ANOVA with multiple comparison test. **P<0.01; NS, not significant. Gene expression profile of GD2-targeted CAR-T cells. Hierarchical clustering (logFC >1, FDR < 0.01) of differentially expressed genes between PRDM1 knockout CAR-T cells with and without c-JUN overexpression. Results showed significant changes in the expression of 959 genes.Importantly, sample classification in the heatmap is based on gene modification, not donor origin. Furthermore, the PRDM1 KO + c-JUN group exhibits distinct gene expression profiles. Log-transformed counts per million reads for the indicated genes (n=3, one-way ANOVA with multiple comparisons). *P<0.05, **P<0.01. Frequency (%) of CD62L+CCR7+ cells in PRDM1 / RCOR1 double-knockdown CAR-T cells and control knockdown CAR-T cells before (left) and after (right) antigen stimulation. Quantitative total flux within mice after injection of control knockdown CAR-T cells (left) and PRDM1 / RCOR1 double-knockdown CAR-T cells (right) (n=4 and n=3, respectively). Frequency (%) of human CD45+ cells in peripheral blood 2 weeks (left bar of each graph) and 6 weeks (right bar of each graph) after infusion of non-transfected cells (NGMC), control knockdown CAR-T cells, and PRDM1 / RCOR1 double knockdown CAR-T cells, respectively.

[0013] DEFINITIONS As used herein, the singular forms (a, an, the) include both the singular and the plural unless otherwise expressly stated herein or clearly contradicted by context.

[0014] As used herein, the term "comprise" encompasses the concepts of "consist essentially of" and "consist only of."

[0015] The terms "host," "subject," or "patient" are used interchangeably herein to refer to an individual treated (e.g., administered) with the T cells, pharmaceutical compositions, and methods of the invention. Subjects include, but are not limited to, mammals (e.g., humans, mice, rats, monkeys, horses, cows, pigs, dogs, cats, etc.).

[0016] The term "T cell exhaustion" refers to the loss of T cell function that can occur as a result of disease. T cell exhaustion is associated with increased expression of PD-1, TIM-3, and LAG-3, increased apoptosis, and decreased cytokine secretion. Thus, the terms "ameliorating T cell exhaustion," "inhibiting T cell exhaustion," "reducing T cell exhaustion," and the like refer to a state of restored T cell function characterized by one or more of the following: decreased expression and / or levels of one or more of PD-1, TIM-3, and LAG-3; increased memory cell formation and / or maintenance of memory markers (e.g., CD62L); prevention of apoptosis; increased antigen-induced cytokine (e.g., IL-2) production and / or secretion; enhanced killing capacity; increased recognition of tumor targets with low surface antigens; and enhanced proliferation in response to antigen.

[0017] The terms "cancer" and "tumor" refer to a tissue or development containing cells that have lost the ability to control growth and proliferation. Cancer and tumor cells are generally characterized by a loss of contact inhibition and may be invasive and exhibit the ability to metastasize. The present invention is not limited by the type of cancer or type of treatment, but a variety of cancers may be treated with the T cells, pharmaceutical compositions, and methods described herein, including brain cancer or other cancers of the central nervous system, melanoma, lymphoma, bone cancer, epithelial cancer, breast cancer, ovarian cancer, endometrial cancer, colorectal cancer, lung cancer, renal cancer, melanoma, kidney cancer, prostate cancer, sarcoma, carcinoma, and / or combinations thereof.

[0018] The term "effective amount" refers to an amount of T cells, pharmaceutical composition, anti-cancer agent, or other drug effective, at dosages and time intervals necessary, to achieve the desired therapeutic or prophylactic result (e.g., alleviation of some or all symptoms of the disease being treated).

[0019] The term "therapeutically effective amount," as used herein, refers to an amount of a therapeutic agent sufficient to result in the improvement of one or more symptoms of a disorder, or to prevent the progression of a disorder, or to cause regression of a disorder. For example, with respect to the treatment of cancer, in one embodiment, a therapeutically effective amount refers to an amount of a therapeutic agent that reduces the rate of tumor growth (e.g., reduces and / or eliminates tumor burden in a patient), reduces tumor mass, reduces the number of metastases, reduces tumor progression, or increases survival time by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

[0020] The term "purified" refers to the removal of contaminants or undesired compounds from a sample or composition. As used herein, the term "substantially purified" refers to the removal of about 70-90%, up to 100%, of contaminants or undesired compounds from a sample or composition.

[0021] The terms "administration" and "administering" refer to the act of providing T cells or a pharmaceutical composition to a subject. Exemplary routes of administration to the human body include, but are not limited to, by eye (ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lung (inhalation), oral mucosa (buccal), ear, rectum, injection (e.g., intravenous, subcutaneous, intraperitoneal, intratumoral, etc.), topical, etc. In one embodiment, administration of the T cells of the invention is by intravenous infusion.

[0022] The terms "co-administration" and "co-administering" refer to the administration of at least two agents (e.g., genetically modified immune cells and one or more other agents—e.g., anti-cancer agents) or therapeutic agents to a subject. In some embodiments, co-administration of two or more agents or therapeutic agents is simultaneous. In another embodiment, a first agent / therapeutic agent is administered before a second agent / therapeutic agent. In some embodiments, co-administration can be via the same or different routes of administration. One of skill in the art will understand that the formulations and / or routes of administration of the various agents or therapeutic agents used may vary. Appropriate dosages for co-administration can be readily determined by one of skill in the art. In some embodiments, when agents or therapeutic agents are co-administered, each agent or therapeutic agent is administered at a lower dosage than would be appropriate for its administration alone. Thus, co-administration is particularly desirable in embodiments where co-administration of agents or therapeutic agents reduces the required dosage of a potentially harmful (e.g., toxic) agent(s) and / or where co-administration of two or more agents results in sensitization of the subject to the beneficial effects of one of the agents due to co-administration of the other agent.

[0023] The term "pharmaceutically acceptable," as used herein, refers to a composition that does not produce substantially any adverse reaction (e.g., toxic, allergic, or other immunological reaction) when administered to a subject.

[0024] As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, including, but not limited to, phosphate buffered saline solution, water, and various types of wetting agents (e.g., sodium lauryl sulfate), any and all solvents, dispersion media, coating agents, sodium lauryl sulfate, isotonic and absorption delaying agents, disintegrants (e.g., potato starch or sodium starch glycolate), polyethylene glycol, etc. Pharmaceutical compositions may also include stabilizers and preservatives. Examples of carriers, stabilizers, and adjuvants have been described and are known in the art (see, e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa. (1975) which is incorporated herein by reference).

[0025] The term "pharmaceutically acceptable salt" refers to any salt of the pharmaceutical composition of the present invention (e.g., obtained by reaction with an acid or base) that is physiologically acceptable in the target subject. The "salt" of the pharmaceutical composition of the present invention can be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, sulfonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Examples of bases include, but are not limited to, alkali metal (e.g., sodium) hydroxide, alkaline earth metal (e.g., magnesium) hydroxide, ammonia, and compounds of the formula NW4+ (wherein W is C1-4 alkyl).

[0026] Examples of salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, chloride, bromide, iodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include the anions of compounds of the invention combined with suitable cations, such as Na, NH, and NW (wherein W is a C alkyl group). For therapeutic use, it is contemplated that salts of compounds of the invention will be pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

[0027] For therapeutic use, salts included in pharmaceutical compositions of the invention are intended to be pharmaceutically acceptable salts. However, salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable pharmaceutical composition.

[0028] As used herein, the term "kit" refers to any delivery system for delivering materials. In the context of immunotherapeutics, such delivery systems include systems that allow for the storage, transport, or delivery of immunogenic agents and / or support materials (e.g., written instructions for using the materials) from one location to another. For example, a kit may include one or more enclosures (e.g., boxes) containing the relevant immunotherapeutics (e.g., engineered T cells and / or support materials). As used herein, the term "fragmented kit" refers to a delivery system that includes two or more separate containers, each containing a subportion of the components of the overall kit. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain a pharmaceutical composition, including an immunotherapeutic composition for a specific use, while a second container contains a second agent (e.g., a chemotherapeutic agent). Indeed, any delivery system that includes two or more separate containers, each containing a subportion of the components of the overall kit, is encompassed by the term "fragmented kit." In contrast, a "combined kit" refers to a delivery system that contains all of the components needed for a particular application in one container (e.g., in one box housing each of the desired components). The term "kit" includes both fragmented and combined kits.

[0029] The terms "immunoglobulin" or "antibody" refer to a protein that binds to one or more epitopes on a specific antigen. Immunoglobulins include, but are not limited to, polyclonal, monoclonal, chimeric, and humanized antibodies, as well as Fab and F(ab')2 fragments of the following classes: IgG, IgA, IgM, IgD, IgE, and secretory immunoglobulin (sIg). Immunoglobulins generally contain two identical heavy chains and two light chains. However, the terms "antibody" and "immunoglobulin" also include single-chain and two-chain antibodies.

[0030] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of a heavy chain may be referred to as "VH." The variable domain of a light chain may be referred to as "VL." These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

[0031] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further contains a polypeptide linker between the VH and VL domains which enables the scFv to form a structure for antigen binding.

[0032] The term "antigen-binding protein" refers to a protein that binds to a specific antigen. "Antigen-binding proteins" include, but are not limited to, immunoglobulins, including polyclonal, monoclonal, chimeric, and humanized antibodies; Fab fragments, F(ab')2 fragments, and Fab expression libraries; and single-chain antibodies.

[0033] The term "epitope," as used herein, refers to the portion of an antigen that makes contact with a specific immunoglobulin.

[0034] The terms "specific binding" or "specifically binding," when used in reference to the interaction of an antibody or portion thereof (e.g., an scFv) with a protein or peptide, mean that the interaction is dependent on the presence of a particular sequence or structure (e.g., an antigenic determinant or epitope) on the protein; i.e., the antibody or portion thereof (e.g., an scFv) recognizes and binds to a particular protein sequence or structure, rather than the protein in general. For example, if an antibody is specific for epitope "A," the presence of a protein containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A that binds to the antibody.

[0035] As used herein, the term "subject suspected of having cancer" refers to a subject who exhibits one or more symptoms indicative of cancer (e.g., a noticeable lump or mass) or who is being screened for cancer (e.g., during a routine physical exam). A subject suspected of having cancer may also have one or more risk factors for developing cancer. A subject suspected of having cancer generally has not been tested for cancer. However, "subject suspected of having cancer" includes individuals who have received a preliminary diagnosis (e.g., a CT scan showing a mass) but have not had a confirmatory test (e.g., biopsy and / or histology) or for whom the type and / or stage of the cancer is unknown. The term also includes people who previously had cancer (e.g., individuals in remission). A "subject suspected of having cancer" is sometimes diagnosed with cancer and sometimes found to be free of cancer.

[0036] The term "subject diagnosed with cancer" refers to a subject who has been tested and found to have cancerous cells. Cancer can be diagnosed using any suitable method, including but not limited to, biopsy, x-ray, blood test, etc.

[0037] The terms "treatment," "therapeutic use," or "medical use" refer to any and all uses of the T cells, pharmaceutical compositions, and methods of the invention to treat a disease or condition, or otherwise prevent, hinder, slow, or reverse the progression of any disease or other undesirable condition in any way. For example, the term "treatment of cancer" or "treatment of tumor," or grammatical equivalents herein, means the inhibition, regression, or partial or complete disappearance of a pre-existing cancer or tumor. The definition includes any decrease in size, aggressiveness, or growth rate of a pre-existing cancer or tumor.

[0038] The terms "improved therapeutic outcome" and "enhanced therapeutic efficacy" with respect to cancer refer to a slowing or reduction in the growth of cancer cells or solid tumors, or a reduction in the total cancer cell number or total tumor burden.

[0039] The term "gene transfer system" refers to any means of delivering a pharmaceutical composition containing a nucleic acid sequence to a cell or tissue. For example, gene transfer systems include, but are not limited to, vectors (e.g., retroviruses, adenoviruses, lentiviruses, adeno-associated viruses, and other nucleic acid-based delivery systems), microinjection of naked nucleic acids, polymer-based delivery systems (e.g., liposome-based and metal particle-based systems), biolistic injection, transduction using transposase-based systems for gene integration, Crispr / Cas9-mediated gene integration, non-integrating vectors such as RNA or adeno-associated viruses, etc.

[0040] The term "viral gene transfer system" refers to a gene transfer system that includes viral elements (e.g., intact viruses, modified viruses, and viral components such as nucleic acids or proteins) to facilitate delivery of a sample to a desired cell or tissue. Non-limiting examples of viral gene transfer systems useful in the T cells, pharmaceutical compositions, and methods of the invention are lentiviral and retroviral gene transfer systems.

[0041] The term "nucleic acid molecule" refers to any nucleic acid-containing molecule, including but not limited to DNA or RNA.

[0042] The term "heterologous gene" refers to a gene that is not in its natural environment. For example, a heterologous gene includes a gene from one species that has been introduced into another species. A heterologous gene also includes a gene native to an organism that has been modified in some way (e.g., mutated, added in multiple copies, linked to non-native regulatory sequences, etc.). Heterologous genes are distinguished from endogenous genes in that heterologous gene sequences are typically linked to DNA sequences not found naturally associated with gene sequences in a chromosome or associated with a part of a chromosome not found in nature (e.g., a gene expressed at a locus where the gene is not normally expressed). Cells containing heterologous genes are described herein as "modified" or "engineered" cells. For example, T cells containing a heterologous AP-1 transcription factor gene (e.g., a heterologous AP-1 transcription factor gene expression construct) and / or a heterologous receptor gene (e.g., a heterologous T cell receptor gene expression construct or a heterologous chimeric antigen receptor gene expression construct) are described herein as modified and / or engineered T cells.

[0043] The term "gene expression" refers to the process by which the genetic information encoded in a gene is converted into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) by "transcription" of the gene (i.e., by the enzymatic action of RNA polymerase), and, for protein-coding genes, by "translation" of the mRNA into protein. Gene expression can be controlled at many stages during the process. "Upregulation" or "activation" refers to regulation that increases production of the gene expression product (i.e., RNA or protein), while "downregulation" or "repression" refers to regulation that decreases production. Molecules (e.g., transcription factors) involved in upregulation or downregulation are sometimes called "activators" and "repressors," respectively.

[0044] The terms "nucleic acid molecule encoding," "DNA sequence encoding," and "DNA encoding" refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. Thus, a DNA sequence encodes a sequence of amino acids.

[0045] The term "native protein" includes only those amino acids found in the protein when it occurs in nature. A native protein can be produced by recombinant means or isolated from a naturally occurring source.

[0046] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Some vectors (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) can autonomously replicate in a host cell into which they are introduced. Lentiviral or retroviral vectors can be used (e.g., to introduce DNA encoding one or more AP-1 transcription factors and / or a CAR construct into cells (e.g., T cells)). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, thereby replicating along with the host genome. Furthermore, some vectors can direct the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors." In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector.

[0047] The term "expression vector" refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, sometimes in addition to other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

[0048] The term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell cultures. The term "in vivo" refers to a natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment.

[0049] The term "cell culture" refers to any in vitro culture of cells. This term includes continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, transformed cell lines, finite cell lines (e.g., non-transformed cells), and any other cell populations maintained in vitro.

[0050] The term "sample" is used in its broadest sense. In one sense, it is meant to include specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from animals (including humans) and can encompass fluids, solids, tissues, and gases. Biological samples include blood products such as plasma and serum. However, such examples are not to be construed as limiting the types of samples applicable to the present invention.

[0051] The term "knockout" (of a gene) refers to an alteration in the genetic code, which completely eliminates the expression and / or function of a gene.

[0052] The term "knockdown (of a gene)" refers to reducing the amount of transcription of a gene. Gene knockdown reduces, but does not completely eliminate, the expression and / or function of a gene.

[0053] The term "gene editing system" refers to one or more molecules that perform modifications, e.g., deletions, of one or more nucleic acids at or near a site in DNA that is a targeted gene. Gene editing systems are known in the art.

[0054] Detailed Description of Embodiments of the Invention The present invention is based on the finding that knockout or knockdown of the PRDM1 gene extensively alters the gene expression and epigenomic profile of T cells, resulting in the acquisition of a profile associated with memory T cells with long-term viability, and that knockout or knockdown of the RCOR1 gene or overexpression of the c-Jun gene inhibits the induction of a gene expression profile associated with exhaustion, a state of functional decline that occurs with continuous antigen stimulation. By combining modification of the PRDM1 gene with modification of the RCOR1 gene or the cJUN gene, a profile combining the characteristics of both can be obtained.

[0055] According to one aspect of the present invention, there is provided a T cell that expresses a recombinant receptor and satisfies the following (I) and (II): (I) has reduced or eliminated expression and / or function of the PRDM1 gene; (II) has reduced or eliminated expression and / or function of the RCOR1 gene and / or has increased or activated expression of the c-Jun gene.

[0056] In some embodiments, reduced expression and / or function of the PRDM1 gene in T cells expressing a recombinant receptor refers to a reduction in the expression and / or function of the PRDM1 gene, its transcription product, or its translation product due to PRDM1 gene knockdown, compared to control T cells in which the PRDM1 gene is not knocked down. The control T cells in which the PRDM1 gene is not knocked down may be T cells expressing the recombinant receptor before knockdown, or may be T cells different from the T cells in which the PRDM1 gene is knocked down, differing only in that the PRDM1 gene has not been knocked down. For example, reduced expression and / or function of the PRDM1 gene in T cells expressing a recombinant receptor refers to a reduction in the expression level of the PRDM1 gene or the activity of the PRDM1 protein, for example, by 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less, when compared to the expression and / or function of the PRDM1 gene in control T cells in which the PRDM1 gene is not knocked down.

[0057] In some embodiments, eliminated expression and / or function of the PRDM1 gene in T cells expressing a recombinant receptor refers to the elimination of expression and / or function of the PRDM1 gene, its transcription product, or its translation product by knocking out the PRDM1 gene.

[0058] In some embodiments, reduced expression and / or function of the RCOR1 gene in T cells expressing a recombinant receptor refers to reduced expression and / or function of the RCOR1 gene, its transcription product, or its translation product due to knockdown of the RCOR1 gene, compared to control T cells in which the RCOR1 gene has not been knocked down. The control T cells in which the RCOR1 gene has not been knocked down may be T cells expressing the recombinant receptor before knockdown, or may be T cells different from the T cells in which the RCOR1 gene has been knocked down, except that the RCOR1 gene has not been knocked down. For example, the reduced expression and / or function of the RCOR1 gene in T cells expressing a recombinant receptor is, for example, a reduction of 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less, when compared to the expression and / or function of the RCOR1 gene in control T cells that have not been subjected to knockdown of the RCOR1 gene, in terms of the expression level of the RCOR1 gene or the activity of the RCOR1 protein.

[0059] In some embodiments, eliminated expression and / or function of the RCOR1 gene in T cells expressing a recombinant receptor refers to the elimination of expression and / or function of the RCOR1 gene, its transcription product, or its translation product by knockout of the RCOR1 gene.

[0060] In some embodiments, increased or activated expression of the c-Jun gene in T cells expressing a recombinant receptor refers to increased or activated expression of the c-Jun gene due to overexpression of the c-Jun gene, compared to control T cells not subjected to overexpression of the c-Jun gene. The control T cells not subjected to overexpression of the c-Jun gene may be T cells expressing a recombinant receptor before being subjected to overexpression of the c-Jun gene, or may be T cells different from the T cells subjected to overexpression of the c-Jun gene, except that they are not subjected to overexpression of the c-Jun gene. For example, increased or activated expression of the c-Jun gene in T cells expressing a recombinant receptor refers to an increase in the amount of c-Jun gene expression, for example, by 120% or more, 150% or more, or 200% or more, compared to the expression level of the c-Jun gene in control T cells not subjected to overexpression of the c-Jun gene.

[0061] "Reduction of the RCOR1 gene" can be used interchangeably with "knockdown of the RCOR1 gene." "Elimination of the RCOR1 gene" can be used interchangeably with "knockout of the RCOR1 gene." Knockdown of the RCOR1 gene reduces the expression and / or function of the RCOR1 gene. Knockout of the RCOR1 gene eliminates the expression and / or function of the RCOR1 gene.

[0062] T cells may be natural T cells or engineered T cells (e.g., tumor-infiltrating lymphocytes (TILs)). T cells expressing a recombinant receptor may be T cells expressing a chimeric antigen receptor (CAR) or a T cell receptor (TCR). The type of T cell is not limited.

[0063] In some embodiments, the T cells are CD3+ T cells (e.g., a combination of CD4+ and CD8+ T cells). In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are natural killer (NK) T cells. In some embodiments, the T cells are γδ T cells. In some embodiments, the T cells are a combination of CD4+ and CD8 T+ cells (e.g., CD3+). In some embodiments, the T cells are memory T cells. In some embodiments, the T cells are a combination of CD8+ T cells, CD4+ T cells, NK T cells, memory T cells, and / or γδ T cells. In some embodiments, the T cells are immune effector cells. In some embodiments, the T cells are cytokine-induced killer cells.

[0064] In some embodiments, the T cells are selected from the group consisting of CD3+ T cells, CD8+ T cells, CD4+ T cells, immune effector cells, natural killer (NK) T cells, γδ T cells, a combination of CD4+ and CD8+ T cells, memory cells, cytokine-induced killer cells, tumor-infiltrating lymphocytes, and combinations thereof.

[0065] The means of genetically expressing a CAR or TCR in a T cell is not limited, and any means known in the art and / or described herein may be used. Non-limiting examples of methods for genetically engineering T cells include, but are not limited to, retroviral or lentiviral mediated transduction, transduction using transposase-based systems for gene integration, Crispr / Cas9 mediated gene integration, non-integrative vectors such as RNA or adeno-associated virus, or other methods described herein.

[0066] The T cells of the present invention may have a recombinant receptor comprising an antigen-binding domain. In some embodiments, the T cells of the present invention may have a chimeric antigen receptor (CAR) comprising the antigen-binding domain. The selection of the antigen-binding domain depends on the type and number of ligands defining the surface of the target cell. For example, the antigen-binding domain of the CAR can be selected to recognize a ligand that acts as a cell surface marker on the target cell associated with a particular disease. Examples of cell surface markers that can act as ligands for the antigen moiety domain in the CAR include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells. For example, a CAR can target a tumor antigen of interest by having a desired antigen-binding domain that specifically binds to an antigen on tumor cells.

[0067] In some embodiments, the T cells are T cells that express a recombinant receptor that specifically recognizes a tumor antigen. In another embodiment, the T cells are T cells that express, for example, a chimeric antigen receptor that specifically recognizes a tumor antigen. In another embodiment, the T cells are peripheral blood-derived T cells that have been genetically modified to express a chimeric antigen receptor that specifically recognizes a tumor antigen.

[0068] Tumor antigens are proteins produced by tumor cells that elicit an immune response, particularly a T cell-mediated immune response. Tumor antigens are well known in the art. Tumor antigens can include one or more antigenic cancer antigens / epitopes associated with malignant tumors.

[0069] Tumor antigens can also be tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are unique to tumor cells and do not occur on other cells in the body. TAAs are not unique to tumor cells, but instead are also expressed on some normal cells under conditions that do not induce a state of immunological tolerance to the antigen. Expression of an antigen on a tumor can occur under conditions that allow the immune system to respond to the antigen.

[0070] Examples of TSAs or TAAs include, but are not limited to, differentiation antigens such as MART-1 / MelanA (MART-1), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, and HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-A BL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO-1, p185erb B2, p180erb B-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791 Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA125, CA 15-3 / CA 27.291 / BCAA, CA195, CA242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, PSMA, TA-90 / Mac-2 binding protein / cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0071] In some embodiments, the tumor antigen is selected from the group consisting of CD5, CD19, CD20, CD22, CD33, CD38, CD44v6, CD47, CD70, CD116, CD123, CD138, CD269, ROR1, GD2, EBV protein or antigen, folate receptor, mesothelin, GM2, GPC3, human carcinoembryonic antigen, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, NY-ESO-1, MAGE-A3, MART-1, gp100, Her2, p53, and alpha-fetoprotein (AFP), and combinations thereof.

[0072] In some embodiments, the chimeric antigen receptor (CAR) expressed in the T cell comprises, as an antigen-binding domain, a fusion protein (e.g., a single-chain variable fragment (scFv)) of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin that specifically binds to a tumor antigen (e.g., GD2).

[0073] Any antibody or antibody fragment thereof that specifically binds to a tumor antigen (e.g., GD2) can be used to construct the antigen-binding site or antigen-binding domain of a CAR for expression in immune cells used in the therapeutic methods of the present invention. Examples of such antibodies / immunoglobulins include, but are not limited to, for GD2: 14 G2a, ch14.18, hu14.18K322A, m3F8, hu3 F8-IgG1, hu3 F8-IgG4, HM3F8, UNITUXIN, DMAb-20, or any other antibody that specifically binds to GD2.

[0074] The T cells of the present invention may have a recombinant receptor comprising a transmembrane domain. In some embodiments, the T cells of the present invention may have a chimeric antigen receptor (CAR) comprising a transmembrane domain. In some embodiments, the tumor antigen CAR may incorporate a variable length hinge region and / or transmembrane domain between the antigen binding domain and the signaling domain. The transmembrane domain may include any transmembrane domain, including, but not limited to, all or part of the transmembrane domain of the TCR ζ chain (CD3ζ), CD28, OX40 / CD134, 4-1BB / CD137 / TN FRSF9, FcERIγ, I COS / CD278, I LRB / CD122, I L-2RG / CD132, or CD40.

[0075] T cells of the present invention can have a recombinant receptor comprising an intracellular signaling domain that transduces a ligand binding event into an intracellular signal that activates the T cell. In some embodiments, T cells of the present invention can have a chimeric antigen receptor (CAR) comprising an intracellular signaling domain. The intracellular signaling domain can be, for example, CD3ζ and / or other signaling domains of the native T cell receptor complex (e.g., the MyD88 signaling domain).

[0076] In the absence of a costimulatory signal, receptor-ligand binding may be insufficient for sufficient activation and proliferation of T cells. Thus, recombinant receptors (e.g., CARs) expressed in T cells can include one or more costimulatory domains (e.g., to provide additional signals to stimulate sufficient T cell activation). In one embodiment, a costimulatory domain is used that increases CAR immune T cell cytokine production. In another embodiment, a costimulatory domain is used that facilitates T cell replication. In yet another embodiment, a costimulatory domain is used that prevents CAR T cell exhaustion. In another embodiment, a costimulatory domain is used that increases T cell anti-tumor activity. In yet a further embodiment, a costimulatory domain is used that enhances CAR T cell survival (e.g., after infusion into a patient).Examples of proteins or domains or portions thereof that can be used to provide costimulatory signals include, but are not limited to, B7-1 / CD80; CD28; B7-2 / CD86; CTLA-4; B7-H1 / PD-L1; ICOS / CD278; ILRB / CD122; IL-2RG / CD132; B7-H2; PD-1; B7-H3; PD-L2; B7-H4; PDCD6; BTLA; 4-1BB / TNFRSF9 / CD137; FcERIγ; CD40 ligand / TNFSF5; 4-1BB ligand / TNFSF9; GITR / TNFRSF18; BAFF / BLyS / TNFSF13B; GITR ligand / TNFSF18; BAFFR / TNFRS F13C; HVEM / TNFRSF14; CD27 / TNFRSF7; LIGHT / TNFSF14; CD27 ligand / TNFSF7; OX40 / TNFR SF4; CD30 / TNFRSF8; OX40 ligand / TNFSF4; CD30 ligand / TNFSF8; TACl / TNFRSF13B; CD40 / TNFRSF5; 2B4 / CD244 / SLAMF4; CD84 / SLAMF5; BLAME / SLAMF8; CD229 / SLAMF3; CD2CRACC / SLAMF7; CD2F-10 / SLAMF9; NTB-A / SLAMF6; CD48 / SLAMF2; SLAM / CD150; CD58 / LFA-3; CD2; Ikaros; CD53; integrin α4 / CD49d; CD82 / Kai-1; integrin α4β1; CD90 / Thy1; integrin α4β7 / LPAM-1; CD96; LAG-3; CD160; These include LMIR1 / CD300A; CRTAM; TCL1A; DAP12; TIM-1 / KIM-1 / HAVCR; Dectin-1 / CLEC7A; TIM-4; DPPIV / CD26; TSLP; EphB6; TSLP R and HLA-DR.

[0077] In some embodiments, a CAR receptor specific for a tumor antigen comprises an antigen-binding domain that specifically binds to the tumor antigen, a transmembrane domain, an intracellular signaling domain, and optionally at least one costimulatory domain.

[0078] The T cells of the present invention may further comprise an inhibitor of the PRDM1 gene and at least one of an inhibitor of the RCOR1 gene and an activator of the c-Jun gene.

[0079] In some embodiments, the inhibitor of the PRDM1 gene is any of the following (i) to (v): (i) (1) a gene editing system that targets one or more sites within the PRDM1 gene or its regulatory elements, (2) a nucleic acid encoding one or more components of the gene editing system, or (3) a combination of (1) and (2); (ii) a PRDM1-specific siRNA or shRNA or a nucleic acid encoding the siRNA or shRNA; (iii) a protein or a small molecule; (iv) a dominant-negative binding partner of a protein encoded by the PRDM1 gene or a nucleic acid encoding the dominant-negative binding partner; or (v) a dominant-negative mutant of a protein encoded by the PRDM1 gene or a nucleic acid encoding the dominant-negative mutant.

[0080] By providing such an inhibitor of the PRDM1 gene, T cells can have reduced or eliminated expression and / or function of the PRDM1 gene.

[0081] In some embodiments, the gene editing system is selected from the group consisting of a CRISPR / Cas9 system, a zinc finger nuclease system, a TALEN system, and a meganuclease system. The gene editing system can bind to any target sequence of the PRDM1 gene, such as an exon, an intron, a 5'UTR, or a 3'UTR. In some embodiments, the gene editing system is a CRISPR / Cas system comprising a gRNA molecule comprising a targeting sequence that hybridizes to the target sequence of the PRDM1 gene.

[0082] In some embodiments, the inhibitor of the RCOR1 gene is any of the following (i) to (v): (i) (1) a gene editing system targeting one or more sites within the RCOR1 gene or its regulatory elements, (2) a nucleic acid encoding one or more components of the gene editing system, or (3) a combination of (1) and (2); (ii) an siRNA or shRNA specific to RCOR1 or a nucleic acid encoding the siRNA or shRNA; (iii) a protein or a small molecule; (iv) a dominant-negative binding partner of a protein encoded by the RCOR1 gene or a nucleic acid encoding the dominant-negative binding partner; or (v) a dominant-negative mutant of a protein encoded by the RCOR1 gene or a nucleic acid encoding the dominant-negative mutant.

[0083] By providing such an inhibitor of the RCOR1 gene, T cells can have reduced or eliminated expression and / or function of the RCOR1 gene.

[0084] In some embodiments, the gene editing system is selected from the group consisting of a CRISPR / Cas9 system, a zinc finger nuclease system, a TALEN system, and a meganuclease system. In some embodiments, the gene editing system can bind to any target sequence of the RCOR1 gene, such as an exon, an intron, a 5'UTR, or a 3'UTR. In some embodiments, the gene editing system is a CRISPR / Cas system comprising a gRNA molecule comprising a targeting sequence that hybridizes to the target sequence of the RCOR1 gene.

[0085] In some embodiments, the activator of the c-Jun gene is any of the following (i) to (vi): (i) incorporation of the c-Jun gene into cellular DNA via a virus or vector that expresses c-Jun; (ii) a small molecule or protein that controls the expression of c-Jun; (iii) incorporation of the c-Jun gene into cellular DNA via a CRISPR / Cas9-based system; (iv) expression of the c-Jun gene via RNA or an oncolytic virus or a transient expression system; (v) delivery of c-Jun to T cells ex vivo for adoptive transfer; or (vi) delivery of c-Jun to T cells by in vivo gene transfer.

[0086] By providing such an activator of the c-Jun gene, T cells can have increased or activated expression of the c-Jun gene.

[0087] c-Jun is one of the AP-1 transcription factors induced after T cell activation and is involved in the production and secretion of cytokines (e.g., interleukin-2) by T cells. CAR-expressing T cells experience sustained antigen-independent signaling through the receptor rastalin and exhibit the fundamental biology of T cell exhaustion, as evidenced by high levels of PD-1, TIM-3, and LAG-3 expression, reduced antigen-induced cytokine production, and excessive programmed cell death. c-Jun has been identified as being reduced in exhausted T cells, and overexpression of the c-Jun gene inhibits T cell exhaustion and prevents or suppresses T cell dysfunction associated with T cell exhaustion.

[0088] In some embodiments, the activator of the c-Jun gene is (i) incorporation of the c-Jun gene into cellular DNA via a virus or vector that expresses c-Jun. c-Jun can be expressed from an expression construct, such as a virus or vector, by well-known genetic engineering methods. In some embodiments, the chimeric antigen receptor and c-Jun are expressed from separate expression constructs, at least one of which is a viral vector. In some embodiments, the chimeric antigen receptor and c-Jun are co-expressed from a single expression construct, which is a viral vector.

[0089] In some embodiments, the c-Jun gene may be a mutated c-Jun gene or a truncated c-Jun gene (a portion of the c-Jun gene). c-Jun can be mutated and / or truncated without affecting the ability of the mutated / truncated c-Jun to mediate the rescue of dysfunctional T cells. T cells can be engineered to have increased or activated expression of such mutated or truncated c-Jun genes. For example, a c-Jun polypeptide with an N-terminal deletion and mutation maintains its ability to rescue the function of HA-28z exhausted CAR T cells and maintains a comparable increase in cytokine production compared to native c-Jun. In some embodiments, the c-Jun polypeptide contains mutations or deletions that render the transactivation and / or δ domains inactive or absent. In some embodiments, the mutant / truncated c-Jun polypeptide contains 70% or more sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any range therebetween) to the C-terminal amino acid residues (e.g., 50, 75, 100, 150, 200, 250, or any range therebetween), C-terminal portion (e.g., one-quarter, one-third, one-half of the full-length amino acids), or C-terminal domain (e.g., the ε, bZIP, and amino acids C-terminus thereof) of wild-type c-Jun. In some embodiments, the N-terminal amino acid residues (e.g., 50, 75, 100, 150, or any range therebetween), N-terminal portion (e.g., one-quarter, one-third, one-half of the full-length amino acids), or N-terminal domain (e.g., the δ, transactivation domain, and amino acids N-terminus thereof) of wild-type c-Jun are deleted, mutated, or inactivated.

[0090] The T cells of the present invention may be further modified to reduce and / or eliminate the expression and / or activity of one or more AP-1 inhibitory complex members, such as Jun B, BATF family members (e.g., BATF3), IRF4, and ATF family members. Such modifications may be achieved using techniques well known in the art, such as CRISPR / Cas9 systems, zinc finger nuclease targeting, TALEN, si RNA, sh RNA, protein inhibitors (e.g., chemical inhibitors, small molecule inhibitors, antibodies, etc.), and expression of dominant negatives, as described above for inhibitors of the PRDM1 gene and inhibitors of the RCOR1 gene.

[0091] The T cells of the present invention may be further modified to inhibit the activity of an agent that inhibits T cell activity (such as PD1, PD-L1, CTLA4, TIM3, CEACAM, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGFβ). For example, the T cells may be modified to carry the intracellular domain of the agent.

[0092] In some embodiments, the T cells of any of the above-described embodiments of the present invention maintain an early memory phenotype, enhance or maintain cytokine production, reduce expression of exhaustion markers, and / or suppress decline in effector function, compared to control T cells that do not satisfy either (I) or (II) below.

[0093] (I) having reduced or eliminated expression and / or function of the PRDM1 gene; (II) having reduced or eliminated expression and / or function of the RCOR1 gene and / or having increased or activated expression of the c-Jun gene.

[0094] In some embodiments, the T cells of any of the above embodiments of the present invention maintain the early memory phenotype and exhibit a suppressed decline in effector function compared to PRDM1 single knockout cells.

[0095] In some embodiments, maintenance of the initial memory phenotype is assessed based on the frequency of initial memory T cells. In some embodiments, maintenance of the initial memory phenotype is assessed based on the expression of genes characteristic of memory T cells (e.g., IL7R, TCF7, CCR7, MYB). For example, if the proportion of initial memory T cells among T cells after expansion of recombinant receptor-expressing T cells (particularly CAR-T cells) is at least twice the proportion among initial memory T cells before expansion, it can be said that the initial memory phenotype is maintained.

[0096] In some embodiments, the cytokine production is the production of IL-2, IFN-γ, and / or TNF-α.

[0097] In some embodiments, the exhaustion marker is PD1 and / or TIM3, hi some embodiments, the exhaustion marker is TOX and / or TOX2.

[0098] In some embodiments, suppression of the decline in effector function is assessed based on the expression of effector transcription factors (e.g., TBX21 and RUNX3). In some embodiments, suppression of the decline in effector function is assessed based on the expression of genes characteristic of non-exhausted effector T cells (PDCD1, granzyme B).

[0099] According to another aspect of the present invention, there is provided a pharmaceutical composition for treating a disease in a subject, comprising a T cell according to any of the above embodiments of the present invention.

[0100] In some embodiments, the disease is a tumor or cancer.

[0101] The pharmaceutical composition may include a pharmaceutically acceptable carrier. The pharmaceutical composition may further include one or more other drugs (e.g., chemotherapeutic drugs). The pharmaceutical composition may optionally include one or more additional drugs, such as other drugs for treating T cell exhaustion (e.g., anti-PD-1 checkpoint inhibitors such as nivolumab) or other drugs used to treat a subject for a disease associated with T cell exhaustion (e.g., antiviral agents, antibiotics, antibacterial agents, or anticancer drugs).

[0102] Unlike monoclonal antibodies, which do not efficiently cross the blood-brain barrier, activated CAR-T cells efficiently enter the central nervous system after adoptive transfer.

[0103] Another aspect of the present invention provides a method for treating a disease, comprising administering an effective amount of any of the T cells of the present invention to a subject having the disease. The present invention is not limited by the type of disease being treated. Any disease treatable by administration of T cells can be treated more effectively by a pharmaceutical composition containing any of the T cells of the present invention and / or a method using any of the T cells of the present invention. In one embodiment, the disease is cancer or a tumor. The present invention is not limited by the type of cancer or tumor. In one embodiment, administering an effective amount of any of the T cells of the present invention to a subject having a disease enables long-term survival of T cells in the subject (e.g., compared to when the same amount of unmodified T cells is administered to the subject) and inhibits T cell exhaustion. For example, in one embodiment, the method includes a method for treating a subject having a disease responsive to treatment using adoptive cell therapy. In one embodiment, the method for treating a subject having a disease responsive to treatment using adoptive cell therapy includes administering an effective amount of a pharmaceutical composition containing any of the T cells of the present invention, enabling long-term survival of T cells in the subject (e.g., compared to when the same amount of unmodified T cells is administered to the subject) and inhibiting T cell exhaustion.

[0104] In one embodiment, a method is provided for treating (e.g., inhibiting the growth and / or killing) cancer and / or tumors using any of the above-described T cells of the present invention (e.g., CD3+ T cells) engineered to express a receptor that recognizes a tumor surface antigen (e.g., CD19, CD20, CD22, ROR1, GD2, EBV protein or antigen, folate receptor, mesothelin, human carcinoembryonic antigen, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, NY-ESO-1, MAGE-A3, MART-1, gp100, and / or p53) and deliver a signal that activates the T cell to induce T cell proliferation and / or tumor killing. A non-limiting example of a receptor is a chimeric antigen receptor (CAR) incorporating an antibody binding domain (e.g., scFv derived from a monoclonal antibody) that recognizes a tumor surface antigen (e.g., GD2), a transmembrane domain (e.g., the transmembrane domain of CD8), and one or more intracellular signaling domains.

[0105] In one embodiment, the present invention provides a method for treating or delaying the progression of cancer in a subject, comprising administering to the subject an effective amount of any of the above-described T cells of the present invention. In some embodiments, the treatment results in a durable response in the individual after cessation of treatment. In another embodiment, the present invention provides a method for enhancing immune function in a subject with cancer, comprising administering to the subject an effective amount of any of the above-described T cells of the present invention.

[0106] In some embodiments, the subject has a cancer that is resistant to one or more other forms of anti-cancer treatment (e.g., chemotherapy, immunotherapy, etc.). In some embodiments, resistance includes recurrence of cancer or refractory cancer. Recurrence can refer to the reappearance of cancer at the original site or at a new site after treatment. In some embodiments, resistance includes the progression of cancer during chemotherapy treatment. In some embodiments, resistance includes cancer that does not respond to conventional treatment with chemotherapeutic agents. The cancer can be resistant at the start of treatment or can become resistant during treatment. In some embodiments, the cancer is in an early stage or a late stage.

[0107] In one embodiment, immunotherapeutic compositions comprising T cells genetically modified to express a tumor-specific CAR of the present invention are used to treat, ameliorate, or prevent cancers characterized by resistance to one or more conventional cancer therapies (e.g., cancer cells that are chemoresistant, radioresistant, hormone resistant, etc.).

[0108] In certain embodiments, any of the above-described T cells of the present invention, immunotherapeutic compositions containing the same, and methods using the same can be used to induce cytotoxic activity against tumor cells and / or promote the survival and function of immune cells. For example, any of the above-described T cells of the present invention, immunotherapeutic compositions containing the same, and methods using the same can be used to induce interleukin-2 (IL-2) to promote T cell survival; induce Fas ligand and / or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL); and / or induce interferon-γ. In some embodiments, any of the above-described T cells of the present invention, immunotherapeutic compositions containing the same, and methods using the same are used to induce cell cycle arrest and / or apoptosis, and also to enhance the induction of cell cycle arrest and / or apoptosis, either alone or in response to additional apoptosis-inducing signals. In some embodiments, any of the above-described T cells of the present invention, immunotherapeutic compositions containing the same, and methods using the same sensitize cancer cells to the induction of cell cycle arrest and / or apoptosis, including cells that are normally resistant to such inductive stimuli.

[0109] In some embodiments, any of the above-described T cells of the present invention, pharmaceutical compositions containing same, and methods of using same are used to treat diseased cells, tissues, organs, or pathological conditions and / or diseases in animals (e.g., mammalian patients, including but not limited to humans and companion animals). In some embodiments, the cancer cells being treated are metastatic. In other aspects, the cancer cells being treated are resistant to anti-cancer drugs.

[0110] Any of the above-described T cells of the present invention or pharmaceutical compositions containing the same may be used in combination with at least one additional therapeutic agent (including but not limited to anti-cancer agents, apoptosis-regulating agents, antibacterial agents, antiviral agents, antifungal agents, and anti-inflammatory agents) in methods for treating a disease in a subject.

[0111] Any of the above-mentioned T cells of the present invention or pharmaceutical compositions containing the same may be used in combination with radiation therapy in a method for treating a disease in a subject.

[0112] The T cells of any embodiment of the invention are included in an amount effective to achieve its intended purpose. While individual needs vary, determining the optimal range of effective amounts of each component is within the skill of the art. In one non-limiting example, T cells of any embodiment of the invention are administered to a human at a dose of 1000-10 per day (e.g., to treat cancer). 10 In another embodiment, 1000 to 10 10 The engineered T cells are administered to treat, ameliorate, or prevent cancer (e.g., prevent metastasis, recurrence, and / or progression of cancer). The unit dose can be administered one or more times daily in one or more administrations (e.g., for 1, 2, 3, 4, 5, 6, or more days or weeks).

[0113] T cells can be administered as part of a pharmaceutical formulation comprising a suitable pharmaceutically acceptable carrier, including excipients and adjuvants that facilitate processing and / or administration of the modified cells into a pharmaceutically usable formulation. T cells and / or pharmaceutical formulations comprising the T cells can be administered intravenously, intramuscularly, subcutaneously, intratumorally, intraperitoneally, intrathecally, or intraventricularly. An effective amount of T cells and / or pharmaceutical formulations comprising the T cells can be administered for the prevention or treatment of disease. The appropriate dose can be determined based on the type of disease being treated, the type of modified T cells, the severity and course of the disease, the individual's clinical condition, the individual's clinical history and response to treatment, and the judgment of a treating physician.

[0114] The efficacy of any of the methods described herein (e.g., treatment with T cells of any embodiment of the invention alone, in combination with one or more chemotherapeutic agents described herein) can be tested in a variety of ways known in the art, such as clinical or preclinical models. Suitable preclinical models are exemplified herein. For any exemplary model, after tumors are allowed to develop, mice are randomly placed into treatment groups that receive treatment or control treatment. Tumor size (e.g., tumor volume) is measured over the course of treatment, and overall survival is also monitored.

[0115] According to another aspect of the present invention, there is provided a method for enhancing the therapeutic efficacy of T cells having a chimeric antigen receptor (CAR) or a T cell receptor (TCR), the method comprising the steps of: reducing or eliminating the expression and / or function of the PRDM1 gene in the T cells; and reducing or eliminating the expression and / or function of the RCOR1 gene in the T cells and / or increasing or activating the expression of the c-Jun gene in the T cells.

[0116] In some embodiments, the step of reducing or eliminating the expression and / or function of the PRDM1 gene in the T cells comprises administering an inhibitor of the PRDM1 gene to the T cells, thereby reducing or eliminating the expression and / or function of the PRDM1 gene.

[0117] In some embodiments, the step of reducing or eliminating the expression and / or function of the RCOR1 gene in the T cells and / or increasing or activating the expression of the c-Jun gene in the T cells comprises reducing or eliminating the expression and / or function of the RCOR1 gene by administering an inhibitor of the RCOR1 gene to the T cells.

[0118] In some embodiments, the step of reducing or eliminating the expression and / or function of the RCOR1 gene in the T cells and / or increasing or activating the expression of the c-Jun gene in the T cells comprises increasing or activating the expression of the c-Jun gene by administering to the T cells an activator of the c-Jun gene.

[0119] In some embodiments, the step of reducing or eliminating the expression and / or function of the RCOR1 gene in the T cells and / or increasing or activating the expression of the c-Jun gene in the T cells comprises administering an inhibitor of the RCOR1 gene to the T cells to reduce or eliminate the expression and / or function of the RCOR1 gene, and administering an activator of the c-Jun gene to the T cells to increase or activate the expression of the c-Jun gene.

[0120] According to another aspect of the present invention, there is provided a method for enhancing the therapeutic efficacy of T cells having a chimeric antigen receptor (CAR) or a T cell receptor (TCR), comprising the steps of: contacting the T cells with an inhibitor of the PRDM1 gene; and contacting the T cells with an inhibitor of the RCOR1 gene and / or with an activator of the c-Jun gene.

[0121] According to another aspect of the present invention, there is provided use of a PRDM1 gene inhibitor, and a COR1 gene inhibitor and / or a c-Jun gene activator in the manufacture of a medicament for CAR-expressing cell therapy.

[0122] Therefore, the T cells, pharmaceutical compositions containing T cells, and methods using T cells according to the above embodiments of the present invention may be useful for the treatment of any T cell-mediated disease because the T cells have long-term viability due to the knockout or knockdown of the PRDM1 gene, and the knockout or knockdown of the RCOR1 gene or the overexpression of the c-Jun gene inhibit the induction of exhaustion, a state of functional decline associated with continuous antigen stimulation, and thus have improved cytotoxicity. In particular, this technology can be applied to general adoptive immunotherapy for cancer. In genetically modified T cell therapy such as CAR-T cells and TCR-T cells, or TIL therapy using tumor-infiltrating T cells, genetic modification during ex vivo culture and proliferation can produce antitumor T cells that can induce sustained antitumor effects after infusion. This technology can be applied regardless of the target antigen or cancer type.

[0123] The disclosures of all patent applications and publications cited herein are hereby incorporated by reference in their entirety.

[0124] The following examples are intended for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Unless otherwise specified, reagents are commercially available or are obtained or prepared according to conventional techniques or literature procedures in the art.

[0125] Materials and Methods Cell Lines The erythroleukemia cell line K562 was obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan). + The B-cell leukemia cell line NALM6 was obtained from the Tohoku University Medical Cell Resource Center (Sendai, Japan). K562-OKT3 / CD80 was generated by retroviral transfection of CD80 with an scFV derived from an anti-CD3 monoclonal antibody (clone OKT3) linking the transmembrane and cytoplasmic domains of CD8α (Yoshikawa, T. et al. Blood 139, 2156-2172 (2022)). NALM6-GL was prepared by retroviral transfection of NALM6 with EGFP linked to codon-optimized luciferase (Luc2) via the porcine teschovirus-1 (P2A)-derived 2A peptide sequence. GD2-expressing NALM6-GL was generated by transfecting NALM6-GL with GM2 / GD2 synthase and GD3 synthase. K562, NALM6, and their derivatives were cultured in RPMI-1640 (Nacalai Tesque, Kyoto, Japan) supplemented with 10% FBS, and PG13 cells were cultured in DMEM (Nacalai Tesque) supplemented with 10% FBS.

[0126] Human T cell culture. Peripheral blood mononuclear cells (PBMCs) from healthy donors (Cellular Technology Limited, Cleveland, OH) were stimulated with mitomycin C-treated K562-OKT3 / CD80 at an effector:target ratio of 7:1 as previously described (Yoshikawa, T. et al. Blood 139, 2156-2172 (2022)). Stimulated T cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 1% penicillin / streptomycin, and recombinant IL-2 (100 IU / mL, Nipro, Osaka, Japan).

[0127] To generate CAR-T cells, T cells were stimulated with RetroNectin (Takara Bio, Kusatsu, Japan) and then retrovirally transduced with CD19-, mesothelin-, or GD2-targeting CAR genes 2 days later. Retrovirus production was performed using PG13 packaging cells. The CAR constructs consist of single-chain variable fragments (scFv) derived from clones FMC63 (targeting CD19) (Nicholson, IC et al. Immunol 34, 1157-1165 (1997)), ss1 (targeting mesothelin) (Li, Q., Verschraegen et al., Anticancer Res 24, 1327-1335 (2004)), or 14g2a (targeting GD2) with the E101K high-affinity mutation (Richman, SA et al., Cancer Immunol Res 6, 36-46 (2018)) linked to CD28 and CD3z signaling domains. K562-CD19 or NALM-6, K562-mesothelin, and NALM6-GD2 cells were used to restimulate CD19-targeted CAR-T cells, mesothelin-targeted CAR-T cells, and GD2-targeted CAR-T cells, respectively.

[0128] CRISPR / Cas9-mediated gene knockout. CRISPR / Cas9-mediated knockout was performed using the Alt-R CRISPR / Cas9 system provided by Integrated DNA Technologies (IDT, Coralville, IA) (Yoshikawa, T. et al. Blood 139, 2156-2172 (2022)). Ribonucleoprotein (RNP) complexes were electroporated using a NEPA 21 electroporator (Nepa Gene, Ichikawa, Japan) after T cell stimulation. Chemically synthesized crRNA and tracrRNA (IDT) were mixed and annealed at 95°C for 5 minutes, then gradually cooled to room temperature. Next, Alt-R Cas9 Nuclease V3 (25 μg, IDT) was incubated with annealed guide RNA (300 pmol) at 37°C for 10–15 minutes to generate RNP complexes. Alt-R Cas9 Electroporation Enhancer was added to a final concentration of 2 μM before electroporation. The following parameters were used for electroporation: poling pulse: voltage 275 V, pulse length 1 msec, pulse interval 50 msec, number of pulses 2, decay rate 10%, polarity +; transfer pulse: voltage 20 V, pulse length 50 msec, pulse interval 50 msec, number of pulses ±5, decay rate 40%, polarity ±.

[0129] Genomic DNA was extracted 48-72 hours later using a NucleoSpin DNA Rapid Lyse kit (Macherey Nagel, Düren, Germany). The genomic region containing the target site was amplified by PCR, and the amplicons were subjected to Sanger sequencing. The gene editing efficiency of each gRNA was calculated using Inference of CRISPR Edits (ICE) analysis (Conant, D. et al. CRISPR J 5, 123-130 (2022)).

[0130] Flow cytometry analysis Flow cytometry analysis was performed using an LSRFortessa (BD Biosciences, Franklin Lakes, NJ) or a CytoFLEX-S (Beckman Coulter, Brea, CA). The following antibodies were used: PerCP / Cyanine5.5-anti human CD271 (ME20.4, BioLegend), APC-anti CD8 (RPA-T8, BioLegend), FITC-anti CD8 (RPA-T8, BioLegend), FITC-anti CD45RA (clone HI100, BD Biosciences), Pacific blue-anti CCR7 (G043H7, BioLegend), APC-Cy7-anti CD62L (DREG-56, Biolegend), PE-Cy7-anti CD27 (M-T271, Biolegend), PE-anti CD28 (CD28.2, BioLegend), FITC-anti IL2 (clone MQ1-17H12, BioLegend), PE-Cy7-anti IFNγ (clone 4S.B3, BioLegend), Brilliant Violet Antibodies used were 421-anti-TNFα (clone monoclonal antibody 11, BioLegend), Alexa Fluor 647-anti-CD45 (clone HI30, BioLegend), PE-Cy7-anti-CD279 (PD1) (clone EH12.1, BD Biosciences), BV421-anti-TIM3 (clone 7D3, BD Biosciences), APC-R700-anti-LAG-3 (clone T47-530, BD Biosciences), and PE-anti-GD2 (clone 14g2a, BioLegend, #357304). Data were analyzed using FlowJo v10 software (BD Biosciences).

[0131] To analyze cytokine production by CAR-T cells, T cells were co-cultured with the indicated target cells and incubated for 6 hours. Brefeldin A (BioLegend) was added 2 hours after stimulation. Cells were then fixed and permeabilized using the Cyto-Fas Fix / Perm kit (BioLegend) according to the manufacturer's protocol.

[0132] In vitro cytotoxicity test: To evaluate the cytotoxic activity of CAR-T cells, 1x10 5 CAR-T cells were cultured at the indicated ratios of EGFP + The cells were co-cultured with target cells. The absolute number of viable target tumor cells was measured by flow cytometry. The frequency of viable tumor cells was calculated as a ratio to the number of cells cultured without CAR-T cells. Dead cells were distinguished by staining with the LIVE / DEAD Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific).

[0133] RNA Sequencing Analysis. Paired-end RNA sequencing analysis was performed using NovaSeq 6000 (Illumina) from RIKEN GENESIS (Tokyo, Japan) or Rhelixa (Tokyo, Japan). FASTQ files were quality trimmed and mapped to the GRCh38 human reference genome using Hisat2 (version 2.1.0). Differential gene expression between groups was identified using edgeR. Unsupervised hierarchical clustering of differential gene expression between groups was performed using the heatmap.2 function in the gplots package. Log-transformed counts-per-million reads were used as gene expression levels. GSEA v2 (Broad Institute) was performed using a memory T cell-related gene set extracted from GSE83978 (FDR <0.05, log fold-change >2). All analysis of read count data was performed in the R environment.

[0134] Statistical Analysis: The significance of differences between two groups was assessed using unpaired or paired two-tailed t-tests. Differences between three or more groups were compared using one-way ANOVA. Where indicated, data were compared using nonparametric tests: the Mann-Whitney test for two groups and the Kruskal-Wallis test for three or more groups. Survival analysis in animal experiments was performed using the Kaplan-Meier method, and differences between two groups were evaluated using the log-rank test. Significant differences were considered statistically significant when p-values ​​were less than 0.05. Significant correlations were defined as p-values ​​less than 0.05. GraphPad Prism 8 software was used for statistical analysis and graphic design.

[0135] Results: We previously demonstrated that genetic disruption of PRDM1 promotes the maintenance of the initial memory phenotype, resulting in improved persistence of CAR-T cells and sustained antitumor efficacy (Yoshikawa, T. et al. Blood 139, 2156-2172 (2022)). Blimp-1, encoded by PRDM1, forms a transcriptional repression complex with multiple epigenetic factors and controls chromatin accessibility, in part, through modification of histone H3K9 trimethylation (Shin, HM et al. Immunity 39, 661-675 (2013)). We investigated whether other epigenetic factors associated with H3K9 methylation also affect the differentiation state of memory T cells in human CAR-T cells. When CD19-targeted CAR-T cells were individually knocked out at H3K9 methylation-related genes, we found that knockout of KDM1A significantly increased the expression levels of early memory markers such as CCR7 and CD62L (Figures 1, 2(A)-(C)).

[0136] We next considered whether simultaneous knockout of PRDM1 and KDM1A might further enhance the maintenance of the early memory phenotype. However, double knockout of PRDM1 and KDM1A significantly inhibited T cell proliferation without additively increasing the frequency of early memory T cells (Fig. 3(A)-(C)).

[0137] KDM1A is also involved in the formation of various transcriptional complexes, primarily including CoREST, NuRD, and CtBP (Lynch, JT, Expert Opin Ther Targets 16, 1239-1249 (2012)). We therefore investigated whether disrupting these complexes, instead of KDM1A, would promote memory T cell formation without inducing toxicity. As shown in Figures 4 and 5(A)-(B), PRDM1 and RCOR1 double knockout significantly increased the frequency of CD62L+CCR7+ or CD62L+CCR7+CD27+CD28+ CAR-T cells compared with PRDM1 single knockout. Importantly, RCOR1 knockout did not suppress CAR-T cell proliferation (Figure 6). Unexpectedly, PRDM1 / RCOR1 double knockout CAR-T cells also showed improved cytotoxic activity against CD19+ target cells compared with PRDM1 knockout CAR-T cells (Figure 7). These results suggest that genetic disruption of PRDM1 and RCOR1 may enhance the sustained effector function of CAR-T cells.

[0138] We compared the gene expression profiles of PRDM1 knockout, RCOR1 knockout, and double-knockout CAR-T cells using RNA sequencing. Hierarchical clustering (FDR < 0.05) of differentially expressed genes among the four groups separated control, PRDM1 knockout, and RCOR1 knockout CAR-T cells into distinct clusters, suggesting that PRDM1 and RCOR1 regulate the expression of different sets of genes (Figure 8(A)). Gene set enrichment analysis showed that double-knockout CAR-T cells were significantly enriched for genes upregulated in memory T cells compared with exhausted T cells (FDE < 0.05, log-fold change > 2) (Figure 8(B)). Consistent with the increased cytotoxic activity, the double knockout CAR-T cells exhibited increased expression levels of effector transcription factors, such as TBX21 and RUNX3, and decreased expression levels of exhaustion molecules, such as TOX and TOX2, compared with PRDM1 knockout CAR-T cells (Figure 9).

[0139] Next, we investigated the in vivo therapeutic efficacy of PRDM1 / RCOR1 double-knockout CAR-T cells. NALM6-bearing NSG mice were injected with CD19-targeted CAR-T cells with or without PRDM1 or RCOR1 knockout. Surprisingly, the double-knockout CAR-T cells, in which both PRDM1 and RCOR1 were knocked out, demonstrated superior therapeutic efficacy compared with controls or with single-knockout CAR-T cells, in which either PRDM1 or RCOR1 was knocked out (Figures 10-12). Importantly, the double-knockout CAR-T cells exhibited the most significant persistence in peripheral blood (Figure 13), resulting in a better overall survival rate than the other groups (Figure 14).

[0140] We further explored other factors that enhance the sustained effector function of PRDM1-disrupted CAR-T cells. AP-1 transcription factors (AP1 TFs) play an important role in effector T cell function, but their role in inducing T cell exhaustion remains controversial. While some studies have shown that AP1 TFs promote T cell exhaustion (Lynch, J. et al. Expert Opin Ther Targets 16, 1239-1249 (2012); Man, K. et al. Immunity 47, 1129-1141.e1125 (2017)), other studies have shown that AP1 TFs counteract T cell exhaustion and maintain robust effector function (Chen, J. et al., Nature 567, 530-534 (2019); Chen, Y. et al., Nat Immunol 22, 996-1007 (2021)). To clarify how individual AP1 TFs affect the functional properties of PRDM1-knockout CAR-T cells, we ectopically expressed BATF, BATF3, c-JUN, and JUNB using retroviral vectors in PRDM1-deficient anti-GD2 28z CAR-T cells, which are known to undergo T cell exhaustion through tonic signaling (Long, AH et al., Nat Med 21, 581-590 (2015)). As shown in Figure 15(A), overexpression of c-JUN significantly inhibited CD8 + PRDM1 significantly improved IL-2 production by CAR-T cells, a hallmark of non-exhausted, polyfunctional T cells. PRDM1 knockout enhances memory T cell properties but impairs effector function. Importantly, expression of c-JUN in PRDM1-knockout CAR-T cells restored granzyme B production to the same level as control CAR-T cells (Figure 15(B)). In addition, c-JUN-overexpressing CAR-T cells showed reduced levels of immune inhibitory molecules such as PD1 and TIM3 (Figures 16 and 17). Neither AP-1 TF impaired the memory phenotype enhanced by PRDM1 knockout (Figures 18 and 19).

[0141] We also tested the strategy of PRDM1 knockout and c-JUN overexpression in different types of CAR-T cells. Mesothelin-targeted CAR-T cells were stimulated daily with K562-mesothelin and their effector function was analyzed. Repeated stimulation of CAR-T cells significantly attenuated granzyme B production by control CAR-T cells, confirming that this experimental protocol recapitulated the induction of T cell exhaustion (Figure 20). In contrast, ectopic expression of c-JUN in PRDM1-deficient CAR-T cells reversed the decline in effector function associated with repeated stimulation. Furthermore, PRDM1-deficient c-JUN-overexpressing CAR-T cells retained an early memory phenotype compared with control CAR-T cells, while exhibiting enhanced cytokine production (Figure 21).

[0142] Furthermore, we analyzed the effector function of GD2-targeting CAR-T cells after repeated antigen stimulation with NALM6-GD2. In contrast to the negative effect of PRDM1 knockout on granzyme B secretion, c-JUN expression significantly enhanced granzyme B production, independent of PRDM1 knockout (Fig. 23).

[0143] PRDM1 / c-JUN double-modified CAR-T cells showed reduced expression of exhaustion markers (PD1 and TIM3, Figure 24), increased cytokine production (Figure 25), and maintained the primary memory phenotype better than control CAR-T cells (Figure 26). Notably, IL-2 production exceeded the combined levels of PRDM1 knockout and c-JUN overexpression alone.

[0144] These results suggest that PRDM1 knockout and c-JUN overexpression conferred both long-life and potent effector functions. To confirm these findings, we analyzed the gene expression profiles of PRDM1 / c-JUN-modified anti-GD2 CAR-T cells. Hierarchical clustering of differentially expressed genes revealed four distinct clusters, regardless of donor sample type (Figure 27). PRDM1-deficient c-JUN-expressing CAR-T cells acquired gene expression profiles characteristic of both memory T cells (e.g., elevated levels of IL7R, TCF7, CCR7, and MYB) and non-exhausted effector T cells (e.g., decreased levels of PDCD1 and elevated levels of GZMB) (Figure 28). These results indicate that PRDM1 knockout and c-JUN-expressing CAR-T cells acquire a long-life and non-exhausted phenotype required for sustained antitumor efficacy.

[0145] Next, we investigated the in vivo therapeutic efficacy of PRDM1 / RCOR1 double knockout siRNA-coexpressing mesothelin-targeting CAR-T cells. We generated siRNA-coexpressing mesothelin-targeting CAR-T cells using lentiviral vectors in human peripheral blood mononuclear cells (PBMCs) (HemaCare) derived from healthy donors. The PRDM1 / RCOR1 double knockdown vector contained two siRNA sequences targeting PRDM1 and two siRNA sequences targeting RCOR1, while the control knockdown vector contained four siRNA sequences that did not target the human genome (Table 1).

[0146]

[0147] CAR-T cells were cultured on plates coated with mesothelin protein (MSN-H526x, ACRO Biosystems) for antigen stimulation. Before and after three rounds of stimulation, CAR-T cells were stained with Brilliant Violet 421 anti-CCR7 (Cat. No. 353208, BioLegend) and APC / cy7 anti-CD62L (Cat. No. 304814, BioLegend) and then analyzed by flow cytometry. As shown in Figure 29, PRDM1 / RCOR1 double knockdown increased the frequency of CD62L+CCR7+ cells before and after antigen stimulation.

[0148] The in vivo therapeutic efficacy of PRDM1 / RCOR1 double-knockdown CAR-T cells was investigated. AsPC-1 / CMV-Luc (JCRB1454)-bearing NOG mice (Invivo Science) were injected with PRDM1 / RCOR1 double-knockdown or control knockdown CAR-T cells, or non-transfected cells (NGMC). Mice injected with PRDM1 / RCOR1 double-knockdown CAR-T cells showed significantly reduced tumor growth after 6 weeks compared to the control knockout group (Figure 30), and the increase in total flux over time was suppressed (Figure 31). Thus, PRDM1 / RCOR1 double-knockdown CAR-T cells demonstrated superior therapeutic efficacy (Figures 30 and 31) and were significantly persistent in the peripheral blood (Figure 32).

Claims

1. T cells expressing a recombinant receptor, which satisfy the following (I) and (II): (I) have reduced or eliminated expression and / or function of the PRDM1 gene; (II) have reduced or eliminated expression and / or function of the RCOR1 gene, and / or have increased or activated expression of the c-Jun gene.

2. The T cell of claim 1, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

3. The T cell of claim 1, wherein the recombinant receptor is a chimeric antigen receptor (CAR) that specifically recognizes a tumor antigen.

4. The T cell of claim 3, wherein the tumor antigen is CD19.

5. The T cell of any one of claims 1 to 4, wherein the T cell is selected from the group consisting of CD3+ T cells, CD8+ T cells, CD4+ T cells, immune effector cells, natural killer (NK) T cells, gamma delta T cells, a combination of CD4+ and CD8 T+ cells, memory cells, cytokine-induced killer cells, tumor-infiltrating lymphocytes, and combinations thereof.

6. The T cell of claim 1, wherein the cell further comprises an inhibitor of the PRDM1 gene and at least one of an inhibitor of the RCOR1 gene and an activator of the c-Jun gene.

7. The T cell of claim 6, wherein the inhibitor of the PRDM1 gene is (1) or (2) below: (1) a gene editing system that targets one or more sites within the PRDM1 gene or its regulatory elements; (2) a nucleic acid encoding one or more components of the gene editing system.

8. The T cell of claim 6, wherein the inhibitor of the RCOR1 gene is (1) or (2) below: (1) a gene editing system that targets one or more sites within the RCOR1 gene or its regulatory elements; or (2) a nucleic acid encoding one or more components of the gene editing system.

9. The T cell according to claim 6, wherein the activator of the c-Jun gene is incorporation of the c-Jun gene into cellular DNA via a virus or vector expressing c-Jun.

10. A pharmaceutical composition for treating a disease in a subject, comprising the T cells described in claim 1.

11. The pharmaceutical composition according to claim 10, wherein the disease is a tumor or cancer.

12. The pharmaceutical composition of claim 10, wherein the T cells maintain an early memory phenotype, enhance or maintain cytokine production, reduce expression of exhaustion markers, and / or suppress decline in effector function, compared to control T cells that do not satisfy either (I) or (II).

13. A method for enhancing the therapeutic efficacy of T cells expressing a recombinant receptor that specifically recognizes a tumor antigen, comprising the steps of reducing or eliminating the expression and / or function of the PRDM1 gene in the T cells, and reducing or eliminating the expression and / or function of the RCOR1 gene in the T cells and / or increasing or activating the expression of the c-Jun gene in the T cells.

14. A method for enhancing the therapeutic efficacy of T cells expressing a recombinant receptor that specifically recognizes a tumor antigen, comprising the steps of: contacting the T cells with an inhibitor of the PRDM1 gene; and contacting the T cells with an inhibitor of the RCOR1 gene and / or with an activator of the c-Jun gene.

15. Use of a PRDM1 gene inhibitor, an RCOR1 gene inhibitor, and / or a c-Jun gene activator in the manufacture of a medicament for CAR-expressing cell therapy.