Method of enhancing Anti-tumor response activity of t cell, method for treating tumor, t cell having enhanced Anti-tumor response activity, and pharmaceutical composition

Abstract

A method of enhancing anti-tumor response activity of a T cell is provided. The method includes overexpressing one or more AP-1 transcription factors in the T cell. Overexpressing the one or more AP-1 transcription factors leads to accumulation of the T cell in a tumor.

Description

METHOD OF ENHANCING ANTI-TUMOR RESPONSE ACTIVITY OF T CELL, METHOD FOR TREATING TUMOR, T CELL HAVING ENHANCED ANTI-TUMOR RESPONSE ACTIVITY, AND PHARMACEUTICAL COMPOSITION

[0001] The present disclosure relates to a method of enhancing anti-tumor response activity of a T cell, a method for treating a tumor in a subject, a T cell having enhanced anti-tumor response activity, and a pharmaceutical composition.Background

[0002] Recently, immunotherapy utilizing the immune response of T cell has been largely studied. For example, the CAR (Chimeric Antigen Receptor)-T cell is attracting attention and has been studied recently.

[0003] PTL 1, for example, suggests a method for treating mantle cell lymphoma (MCL) or B cell ALL in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of a T cell product comprising autologous T cells expressing an anti-CD 19 chimeric antigen receptor.

[0004] PTL 1: WO2021092290A1Summary

[0005] However, the efficacy of current T cell-based cancer immunotherapies, such as immune checkpoint blockage and CAR-T therapy, is limited in treating tumors, especially solid tumors. Recent studies have indicated that abundance of intra-tumoral T cells is positively associated with cancer prognosis and effects of PD1 blockage therapy. Therefore, the inventors focused on improving the efficacy of T cell-based cancer immunotherapy by inducing a sufficient number of cytotoxic T cells in a tumor.

[0006] The inventors conducted diligent investigation and discovered that the overexpression of a specific AP-1 transcription factor leads to the accumulation of a T cell in a tumor, thereby enhancing anti-tumor response activity of the T cell.

[0007] The gist of the present disclosure is as follows: [1] A method of enhancing anti-tumor response activity of a T cell comprising: overexpressing one or more AP-1 transcription factors in the T cell, wherein overexpressing the one or more AP-1 transcription factors leads to accumulation of the T cell in a tumor. [2] The method according to [1], wherein the T cell is selected from the group consisting of a CD8+T cell, a CD4+T cell and an NKT (natural killer T) cell. [3] The method according to [1] or [2], wherein the T cell is a CD8+T cell. [4] The method according to any one of [1] to [3], wherein the one or more AP-1 transcription factors include a member of a Jun family. [5] The method according to any one of [1] to [4], wherein the one or more AP-1 transcription factors include JunB. [6] The method according to any one of [1] to [5], wherein overexpressing the one or more AP-1 transcription factors promotes expression of Myb in the T cell. [7] The method according to any one of [1] to [6], further comprising: modifying the T cell to express a T cell receptor (TCR) and / or a chimeric antigen receptor (CAR). [8] The method according to any one of [1] to [7], wherein overexpressing the one or more AP-1 transcription factors is performed by retroviral transduction or lentiviral transduction. [9] A method for treating a tumor in a subject comprising: producing a T cell having enhanced anti-tumor response activity using the method according to any one of [1] to [8]; and administering a therapeutically effective amount of the T cell into the subject.

[0010] The method according to [9], wherein the tumor is a solid tumor.

[0011] The method according to

[0010] , wherein the solid tumor is melanoma.

[0012] The method of according to any one of [9] to

[0011] , further comprising: administering a therapeutically effective amount of an immune checkpoint inhibitor to the subject.

[0013] A T cell having enhanced anti-tumor response activity, wherein the T cell has overexpression of one or more AP-1 transcription factors, and the overexpression of the one or more AP-1 transcription factors leads to accumulation of the T cell in a tumor.

[0014] The T cell according to

[0013] , wherein the T cell is selected from the group consisting of a CD8+T cell, a CD4+T cell and an NKT (natural killer T) cell.

[0015] The T cell according to

[0013] or

[0014] , wherein the T cell is a CD8+T cell.

[0016] The T cell according to any one of

[0013] to

[0015] , wherein the one or more AP-1 transcription factors include a member of a Jun family.

[0017] The T cell according to any one of

[0013] to

[0016] , wherein the one or more AP-1 transcription factors include JunB.

[0018] The T cell according to any one of

[0013] to

[0017] , wherein the T cell further expresses a T cell receptor (TCR) and / or a chimeric antigen receptor (CAR).

[0019] A pharmaceutical composition comprising the T cell according to any one of

[0013] to

[0018] and at least one of a pharmaceutically acceptable carrier, a diluent and an excipient.

[0008] In the accompanying drawings: FIG. 1a is a diagram showing flow cytometry analysis of JunB expression in intra-tumoral PD1+ OT-I T cells in mice inoculated with B16-OVA and in splenic CD44+ OT-I T cells in mice inoculated with LM-OVA. FIG. 1b is a diagram showing flow cytometry analysis of JunB expression in OT-I T cells in TdLNs or tumors in mice inoculated with B16-OVA on day 14 after inoculation FIG. 2a is a diagram showing experimental design. FIG. 2b is a diagram showing the result of tumor growth analysis. FIG. 2c is a diagram showing frequencies of total CD4+and CD8+T cells in tumors. FIG. 2d is a diagram showing frequency of CD8+T cells stained with H2kb-restricted OVA tetramer in tumors. FIG. 2e is a diagram showing frequency of CD8+T cells stained with H2kb-restricted OVA tetramer in TdLNs. FIG. 2f is a diagram showing frequencies of PD-1 expressing CD4+and CD8+T cells in tumors. FIG. 2g is a diagram showing frequencies of cells expressing GzmB, IFN-γ, TNF-α or IL-2 upon restimulation in intra-tumoral CD8+T cells. FIG. 3a is a diagram showing experimental design. FIG. 3b is a diagram showing the result of tumor growth analysis. FIG. 3c is a diagram showing frequencies of OT-I T cells (CD45.1-, CD45.2+) in tumors. FIG. 3d is a diagram showing frequencies of OT-I T cells (CD45.1-, CD45.2+) in TdLNs. FIG. 3e is a diagram showing frequencies of PD-1 expressing cells in intra-tumoral OT-I T cells. FIG. 3f is a diagram showing frequencies of cells distinguished by expression of LY108 and TIM-3 in intra-tumoral OT-I T cells. FIG. 3g is a diagram showing experimental design. FIG. 3h is a diagram showing frequencies of JunB cKO or control OT-I T cells in tumors. FIG. 4a is a diagram showing frequencies of cells expressing Ki-67 or active caspase 3 in OT-I T cells in TdLNs. FIG. 4b is a diagram showing frequencies of cells expressing TCF1 and TOX in OT-I T cells in TdLNs. FIG. 4c is a diagram showing expression of LY108, CD62L, CD122, and CD127 in OT-I T cells in TdLNs. FIG. 4d is a diagram showing expression of GzmB, IFN-γ, TNF-α, and IL-2 in OT-I T cells in TdLNs. FIG. 4e is a scatter plot showing expression levels of genes in JunB cKO and control cells. FIG. 5a is a diagram showing experimental design. FIG. 5b is a diagram showing cell growth of OT-I T cells. FIG. 5c is a diagram showing frequencies of OT-I T cells expressing Ki-67 or active caspase 3. FIG. 5d is a diagram showing frequencies of OT-I T cells distinguished by expression of LY108 and TIM-3. FIG. 5e is a diagram showing expression of TCF1 and TOX in OT-I T cells. FIG. 5f is a diagram showing a diagram showing expression of PD-1 in OT-I T cells. FIG. 5g is a diagram showing frequencies of cells expressing GzmB, IFN-γ, Perforin, or TNF-α upon restimulation in OT-I T cells. FIG. 6a is a diagram showing expression of JunB in dTAG-JunB CD8 T cells. FIG. 6b is a bar graphs showing expression of Ki-67, active caspase 3, and PD-1, as well as restimulation-induced IFN-γ and GzmB in dTAG-JunB CD8 T cells. FIG. 6c is a bar graphs showing expression of Ki-67, active caspase 3, and PD-1, as well as restimulation-induced IFN-γ and GzmB in wild-type cells. FIG. 7 is a bar graph showing relative MFI of JunB expression. FIG. 8a is a diagram showing experimental design. FIG. 8b is a diagram showing JunB expression in dTAG-JunB OT-I T cells after co-culture. FIG. 8c is a diagram showing numbers of living dTAG-JunB OT-I T cells after co-culture. FIG. 8d is a diagram showing frequencies of cells expressing Ki-67, active caspase 3, or BIM in dTAG-JunB OT-I T cells after co-culture. FIG. 9a is a diagram showing frequencies of cells distinguished by expression of LY108 and TIM-3 in dTAG-JunB OT-I T cells after co-culture. FIG. 9b is a diagram showing expression of PD-1, LAG3, and TIGIT in dTAG-JunB OT-I T cells after co-culture. FIG. 9c is a diagram showing expression of TOX in dTAG-JunB OT-I T cells after co-culture. FIG. 9d is a diagram showing expression of GzmB, IFN-γ, perforin, and TNF-α upon restimulation in dTAG-JunB OT-I T cells after co-culture. FIG. 10a is a diagram showing numbers of living dTAG-JunB OT-I T cells after co-culture. FIG. 10b is a diagram showing frequencies of cells expressing Ki-67, active caspase 3, or BIM in dTAG-JunB OT-I T cells after co-culture. FIG. 10c is a diagram showing frequencies of cells distinguished by expression of LY108 and TIM-3 in dTAG-JunB OT-I after co-culture. FIG. 10d is a diagram showing expression of LAG3 and TIGIT in dTAG-JunB OT-I after co-culture. FIG. 10e is a diagram showing expression of GzmB, IFN-γ, Perforin, and TNF-α upon restimulation in dTAG-JunB OT-I after co-culture. FIG. 11a is a scatter plot showing expression levels of genes in Tpex cells with or without dTAGv-1 treatment. FIG. 11b is a scatter plot showing expression levels of genes in Ttex cells with or without dTAGV-1 treatment. FIG. 12a is a scatter plot showing levels of chromatin accessibility in ATAC-seq peaks in Tpex cells with or without dTAGV-1 treatment. FIG. 12b is a stacked bar plots showing the distance of accessible chromatin regions from transcription start sites (TSS). FIG. 12c is a bar graph showing accessible chromatin regions annotated to intron, intergenic, promoter-TSS, exon, 5’- or 3’-UTR (untranslated region), or other regions. FIG. 12d is a genomic browser image showing the Myb and Il7r loci. FIG. 13a is a diagram showing experimental design. FIG. 13b is a diagram showing the result of tumor growth analysis. FIG. 13c is a diagram showing JunB expression in intra-tumoral OT-I T cells. FIG. 13d is a diagram showing frequencies of OT-I T cells transduced with retroviral vectors (CD8+GFP+) in TdLNs and tumors. FIG. 13e is a diagram showing frequencies of cells distinguished by expression of LY108 and TIM-3 in OT-I T cells transduced with retroviral vectors (CD8+GFP+) in tumors. FIG. 13f is a diagram showing expression of GzmB, IFN-γ, perforin, and TNF-α in OT-I T cells transduced with retroviral vectors (CD8+GFP+) in tumors, in the presence or absence of restimulation.DETAILED DESCRIPTION

[0009] The following provides a detailed description of embodiments of the present disclosure.

[0010] The term “anti-tumor activity” as used herein means a reduction in the rate of proliferation, viability, or metastatic activity of tumor cells. For example, anti-tumor activity can be shown by a decline in growth rate of tumor cells or tumor size stability or reduction, or longer survival due to therapy as compared to control without therapy. Such activity can be assessed using in vitro or in vivo tumor models, including but not limited to xenograft models, allograft models, and other known models known in the art to investigate anti-tumor activity.

[0011] The term “enhancing anti-tumor response activity of a T cell” as used herein means that the anti-tumor response by the T cells is enhanced as compared to control cell (e.g., a untreated cell, a cell before treatment, or cell received mock treatment).

[0012] The term “T cell” as used herein encompasses any known T cell. The term T cell encompasses, for example, CD8+T cell, CD4+T cell, and NKT cell.

[0013] The term “overexpression” of gene(s) as used herein encompasses excessive expression of the gene(s) as compared to control cell (e.g., a untreated cell, a cell before treatment, or cell received mock treatment). In one example, the level of gene overexpression can be 101% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, and 170% or more, compared to the normal level of gene expression. The level of gene overexpression can be evaluated through reverse transcription quantitative PCR (RT-qPCR), immunoblot, or flow cytometry analyses. Among these, flow cytometry is optimal in the light of feasibility.

[0014] The term “treatment (treating)” or “therapy” of a subject as used herein refers to any type of intervention or process performed on, or the administration of an active agent or the presently disclosed cells to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease.

[0015] The term “subject” as used herein may encompass any human or nonhuman animal.

[0016] The term “tumor” as used herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer”, “cancerous”, “cell proliferative disorder”, “proliferative disorder”, and “tumor” are not mutually exclusive as referred to herein.

[0017] The term “solid tumor” as used herein is a general term for cancers that form masses in organs and tissues, other than blood cancer. Examples of solid cancers include, but are not specifically limited to, sarcoma, breast cancer, prostate cancer, head and neck cancer, brain tumor, colorectal cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, melanoma, gastric cancer, renal cell cancer, endometrial cancer, and hepatocellular carcinoma.

[0018] The term “therapeutically effective amount” as used herein refers to an amount of a compound that results in prevention, delay of onset of symptoms, or amelioration of symptoms of a condition.

[0019] In the specification, the technical features described in different embodiments can be combined with each other unless otherwise stated.

[0020] The articles “a” and “an,” as used herein, should be understood to mean “at least one,” unless clearly indicated to the contrary.

[0021] The phrase “and / or,” when used between elements in a list, is intended to mean either (1) that only a single listed element is present, or (2) that more than one element of the list is present. For example, “A and / or B” indicates that the selection may be A alone; B alone; or A and B. The phrase “and / or” may be used interchangeably with “at least one of” or “one or more of” the elements in a list.

[0022] (Method of enhancing anti-tumor response activity of a T cell) The presently disclosed method of enhancing anti-tumor response activity of a T cell at least includes overexpressing one or more AP-1 transcription factors in the T cell (step (1)), and optionally further includes modifying the T cell to express a T cell receptor (TCR) and / or a chimeric antigen receptor (CAR) (step (2)). One feature of the presently disclosed method of enhancing anti-tumor response activity of a T cell is that the overexpression of the one or more AP-1 transcription factors in a T cell leads to accumulation of the T cell in a tumor. Note that the presently disclosed method of enhancing anti-tumor response activity of a T cell may include steps other than the steps (1) and (2) set forth above.

[0023] <Step (1)> In the step (1), one or more AP-1 transcription factors are overexpressed in a T cell.

[0024] The AP-1 (activator protein 1) transcription factor consists of the Jun family (c-Jun, JunB, and JunD), the FOS family (c-FOS, Fra-1, Fra-2, and FosB), the MAF family (c-Maf, MafB, MafA, Mafg / f / k, and Nrl), and the ATF family (ATF2, LRF1 / ATF3, BATF, BATF2, BATF3, JDP1, and JDP2).

[0025] AP-1 transcription factors form homodimers or heterodimers by interacting through their basic leucine zipper (bZIP) domains to exert transcriptional regulatory functions. AP-1 transcription factors bind to the 12-O-tetradecanoylphorbol-13-acetate (TPA) response element (TRE; TGAnTCA) or cAMP response element (CRE: TGACGTCA).

[0026] The AP-1 transcription factor that can be used in the presently disclosed method of enhancing anti-tumor response activity of a T cell is not limited, so long as its overexpression in a T cell leads to the accumulation of the T cell in a tumor. Suitable examples of such AP-1 transcription factors, for example, include JunB. In some embodiments, the one or more AP-1 transcription factors include a member of the Jun family. Preferably, the one or more AP-1 transcription factors include JunB.

[0027] Recent studies have reported that JunB / BATF heterodimer binds to a response element in a closed chromatin as a so-called pioneer factor, which promotes chromatin accessibility and the subsequent recruitment of other transcription factors to regulate gene expression (Reference 15). This raises the possibility that the overexpression of JunB may control expression of target genes, including Myb, by regulating chromatin accessibility. Consistently, experimental data described in the EXAMPLES section of the present specification suggests that the overexpression of JunB promotes chromatin accessibility and expression of Myb.

[0028] In some embodiments, the overexpression of the one or more AP-1 transcription factors promotes expression of Myb.

[0029] Overexpression of AP-1 transcription factors in a T cell can be performed by a commonly known method such as retroviral or lentiviral transduction. The overexpression is usually performed by retroviral transduction in mouse T cells and by lentiviral transduction in human T cells.

[0030] <Step (2)> In the step (2), the T cell is modified to express a T cell receptor (TCR) and / or a chimeric antigen receptor (CAR) that recognizes and responds to a tumor antigen. By using a T cell whose anti-tumor response activity is enhanced by undergoing the steps (1) and (2), it is possible to further reduce tumor size in a subject.

[0031] CAR is a modular protein which grafts the specificity of a monoclonal antibody to the effector function of an immune cell (e.g., T cell). Typically, the CAR consists of a type I transmembrane domain protein with an antigen recognizing amino terminus, a spacer, and a transmembrane domain which are connected to a signaling endodomain which transmits survival and activation signals. The CAR may be a fusion of single-chain variable fragments (scFv) derived from monoclonal antibodies which recognize an antigen present on the tumor cell surface (tumor antigen), fused via a spacer and a trans-membrane domain to the signaling endodomain.

[0032] Expression of TCR and / or CAR in a T cell can be performed by any known method in the art. Typically, an engineered T cell having expression of TCR and / or CAR is obtained by synthesizing a new gene encoding the TCR and / or CAR and then incorporating the synthesized gene into a T cell isolated from a subject (e.g. subject’s blood). The synthesis of a new gene encoding the TCR and / or CAR as well as the incorporation of the synthesized gene into a T cell can be performed by commonly known methods in the art.

[0033] (Method of treating tumor in a subject) The presently disclosed method for treating a tumor at least includes producing a T cell having enhanced anti-tumor response activity according to the presently disclosed method of enhancing anti-tumor response activity of a T cell (production step) and administering a therapeutically effective amount of the T cell into the subject (administration step). Note that the presently disclosed method of treating a tumor may include steps other than the production step and the administration step described above (i.e., may include other steps).

[0034] Tumors for which the presently disclosed method for treating a tumor is useful include all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0035] In some embodiments, the tumor to be treated by the presently disclosed method of treating a tumor is a solid tumor. In some embodiments, the solid tumor is melanoma.

[0036] Specifically, experimental data described in the EXAMPLES section of the present specification indicates that T cells having enhanced anti-tumor activity caused by JunB overexpression are effective for melanoma treatment. Hence, the presently disclosed method for treating a tumor is also effective for solid tumor treatment.

[0037] <Production step> In the production step, T cells as a material can be taken out from a subject. The T cells taken out from the subject are subjected to several manipulations under the above-mentioned method so as to produce T cells having anti-tumor response activity.

[0038] The T cell may be extracted from blood (e.g., peripheral blood mononuclear cells (PBMC)). Alternatively, the T cell may be derived from a stem cell, e.g. by in vitro differentiation. The stem cell can be an adult stem cell, an embryonic stem cell, a cord blood stem cell, a progenitor cell, a bone marrow stem cell, a lymphoid stem cell, an induced pluripotent stem cell, or a hematopoietic stem cell. The T cell may be obtained from the subject, or from any source, such as a donor, a blood bank, or a cell bank (e.g., a stem cell bank).

[0039] <Administration step> In the administration step, the T cells having enhanced anti-tumor response activity obtained through the above-described production step is administered to the subject with a therapeutically effective amount of dose. The therapeutically effective amount of the T cell having enhanced anti-tumor activity can be determined appropriately based on the result of medical diagnosis.

[0040] The subject to which a therapeutically effective amount of the T cell having enhanced anti-tumor response activity is administered may be the same as or different from the subject from which the T cells as a material were taken out. In other words, administration of the T cell having enhanced anti-tumor response activity to the subject may be autologous or allogeneic administration.

[0041] <Other steps> Examples of other steps include, but are not specifically limited to, a step of administering a therapeutically effective amount of an immune checkpoint inhibitor to the subject and a step of expanding the T cell having enhanced anti-tumor response activity ex vivo prior to the administration step.

[0042] From a viewpoint of further reducing tumor size in a subject, the presently disclosed method of treating a tumor preferably further includes administering a therapeutically effective amount of an immune checkpoint inhibitor to the subject.

[0043] Examples of the immune checkpoint inhibitors include, but are not specifically limited to, PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, CD47 inhibitors, SIRPα inhibitors, BTLA inhibitors, TIM-3 inhibitors, TIGIT inhibitors, LAG-3 inhibitors, Siglec-15 inhibitors, and galectin-9 inhibitors. The therapeutically effective amount of the immune checkpoint inhibitor can be determined, for example, in the same way as the therapeutically effective amount of the T cell having enhanced anti-tumor response activity.

[0044] The presently disclosed method for treating a tumor may be used in combination with other methods of treating tumors for example by chemotherapy, irradiation therapy, tumor-targeted therapy, adjuvant therapy, immunotherapy, hormonal therapy, gene therapy or surgery. In particular, the presently disclosed method for treating a tumor is preferably used in combination with immunotherapy.

[0045] (T cell having enhanced anti-tumor response activity) The presently disclosed T cell having enhanced anti-tumor response activity has overexpression of one or more AP-1 transcription factors, and the overexpression of the one or more AP-1 transcription factors leads to accumulation of the T cell in a tumor.

[0046] The preferable attribute and preparation method for the T cell is as described above. In one example, the T cell is selected from the group consisting of a CD8+T cell, a CD4+T cell and an NKT cell. Preferably, the T cell is a CD8+T cell.

[0047] As described above, the T cell has overexpression of one or more AP-1 transcription factors whose overexpression in the T cell leads to the accumulation of the T cell in a tumor. The candidates for such AP-1 transcription factors are as explained above. In one example, the one or more AP-1 transcription factors include a member of the Jun family. Preferably, the one or more AP-1 transcription factors include JunB.

[0048] From a viewpoint of further reducing tumor size in a subject, the presently disclosed T cell having enhanced anti-tumor response activity preferably further expresses a T cell receptor (TCR) and / or a chimeric antigen receptor (CAR).

[0049] (Pharmaceutical composition) The presently disclosed pharmaceutical composition comprises the presently disclosed T cell set forth above and at least one of a pharmaceutically acceptable carrier, a diluent and an excipient. As a result of the presently disclosed pharmaceutical composition containing the T cell having enhanced anti-tumor response activity set forth above, it is possible to reduce tumor size in a subject by using the pharmaceutical composition.

[0050] Suitable pharmaceutically acceptable carriers include, but are not specifically limited to, magnesium carbonate, magnesium stearate, talc, sucrose, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, and sodium carboxymethylcellulose.

[0051] Suitable pharmaceutically acceptable diluents include, but are not specifically limited to, distilled water for injection, physiological saline, aqueous glucose solution, vegetable oil for injection, propylene glycol, and polyethylene glycol.

[0052] Suitable pharmaceutically acceptable excipients include, but are not specifically limited to, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavouring agents, flavour masking agents, colouring agents, anticaking agents, humectants, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents.

[0053] One of the pharmaceutically acceptable carriers, diluents and excipients described above may be used individually, or two or more of the pharmaceutically acceptable carriers, diluents and excipients described above may be used in combination.

[0054] The pharmaceutical composition may be used in combination with one or more additional pharmaceutical agents such as, for example, chemotherapeutics, anti-inflammatory agents, steroids, immunosuppressants, immune checkpoint inhibitors, therapeutic antibody, metabolic enzyme inhibitors, chemokine receptor inhibitors, and phosphatase inhibitors, as well as targeted therapies.Examples

[0055] Materials and methods The materials and methods used in the following examples are explained as below.

[0056] Mice Junbfl / flmice were previously generated in our laboratory (Reference 1). dTAG-JunB mice, which have FKBP12F36V(degradation tag [dTAG]) knock-in at the N-terminus of JunB, were also recently generated in our laboratory. CD4-Cre (stock #017336), B6SJL (stock# 002014), and OT-I TCR transgenic mice (stock #003831) were obtained from the Jackson Laboratory. All mice were maintained on a C57BL / 6 background under specific pathogen-free conditions. Litter-mate controls were used in most experiments. Sex-matched mice aged 6 to14-week-old were used for experiments. The Animal Care and Use Committee of the Okinawa Institute of Science and Technology Graduate School approved all animal protocols for this study.

[0057] Isolation of naive CD8+T cells Splenocytes were isolated by disrupting spleens through 70 μl cell strainers (BD; 352340) and were then treated with ACK lysis buffer (NH4Cl 150mM, KHCO310mM Na2EDTA 0.1mM, pH7.2-7.4) to remove red blood cells. CD8+T cells were isolated from single cell suspension using MojosortTMMouse naive CD8+T cell isolation Kit (Biolegend; 480008) following manufacturers instruction. Fluorescence-activated cell sorting (FACS) analysis confirmed that the purity of isolated naive CD8+T cells (CD8+CD62LloCD44hi) were above 95%.

[0058] Mouse tumor model B16-OVA cells (Sigma, SCC420) in the exponential phase were subcutaneously injected into right back of mice (5 x 105per mouse) either one day after adoptive transfer of naive OT-I T cells or 7 days before adoptive transfer of activated OT-I T cells. Tumor growth was monitored every two days, and tumor volume was calculated using the formula V = (L x W2) / 2, where V represents tumor volume, L represents the longest dimension (mm), and W represents the widest dimension (mm). For flow cytometry or RNA-seq analysis, tumor-draining lymph node (TdLN) cells were isolated by disrupting TdLNs through 70 μl cell strainers. Additionally, tumor-infiltrating cells were isolated using mouse tumor dissociation kit (Miltenyi Biotec, 5220305140). Briefly, tumors placed in gentle MACS C tubes were treated with the kit’s enzymes with shaking for 45 min at 37°C, followed by further dissociation with gentle MACS dissociator for 1 min. After lysing red blood cells with ACK buffer, cell suspensions were layered on a Percoll gradient between 40% and 80% (Cytiva, 17544501) and centrifuged at 800 g for 25 min. Lymphocytes were then collected from a cloudy interface layer.

[0059] LM-OVA infection The recombinant L. monocytogenes expressing ovalbumin (LM-OVA) (DMX; 09-082) were cultured on a brain heart infusion (BHI) agar plate (Sigma, 53286) containing erythromycin (10 μg / mL) at 37°C overnight. A single colony of bacteria was cultured overnight in BHI media at 37°C with agitation. The culture was then diluted 1:25 in fresh BHI media and incubated until the optical density at 600 nm (OD600) reached 0.1. An OD600 of 0.1 was confirmed to correspond to 2 x 107colony-forming units (CFU) / ml. A dose of 5 x 106CFU of LM-OVA in 100 μL of PBS was intravenously injected into mice.

[0060] Adoptive transfer of OT-I T cells Naive OT-I T cells isolated from OT-I (CD45.1+CD45.2+), Junbfl / flOT-I (CD45.2+), Cd4CreJunbfl / flOT-I (CD45.2+), or dTAG-JunB OT-I (CD45.2+) mice were used for adoptive transfer to congenic recipient (CD45.1+) mice. Adoptive transfer was performed by intravenous injection of 0.25 or 1 x 106OT-I T cells for experiments with B16-OVA inoculation or 1 x 104cells for experiments with LM-OVA infection.

[0061] Flow cytometry analysis Prior to antibody staining, cells were treated with anti-Fc receptor-blocking antibody (anti-CD16 / CD32; Biolegend; 101320) and NIR-Zombie cell viability dye (Biolegend; 423106). Cell surface molecules were stained with fluorochrome-conjugated antibodies in PBS containing 2% fetal calf serum (Biosera; FB-1061) for 30 min to 1 h on ice. Cells expressing GFP were fixed with cold 4% PFA immediately after surface marker staining. OVA-specific CD8+T cells were stained with H-2kb OVA tetramer (MBL). Subsequent staining of intracellular molecules was performed with fluorochrome-conjugated antibodies (1:200 dilution for transcription factors and 1:400 dilution for other molecules including cytokines) using a Foxp3 Staining Buffer set (eBioscience; 005253-00) following the manufacturer’s instructions. Intracellular cytokine staining was performed using cells re-stimulated with phorbol 12-myristate 13-acetate (PMA, Sigma; P8139; 50 ng / mL) and ionomycin (Sigma; I0634; 500 ng / mL) in the presence of brefeldin A (BFA, Biolegend; 420601; 5 μg / mL). Samples were analyzed using BD FACS Aria II, Aria III, or Fortessa, and data were analyzed using FlowJo software.

[0062] Antibodies The following antibodies were used in flow cytometry analysis. Anti-CD44 (clone:IM7), anti-CD62L (clone:MEL-14), anti-CD8 (clone:TK15 and 53-6.7), anti-LY108 (clone:330-AJ), anti-Tim3 (clone:RMP3-23), anti-PD1 (clone:RMP1-30), anti-CX3CR1 (clone:SA011f11), anti-CD45.1 (clone:A20), anti-CD45.2 (clone:104), anti-CD122 (clone: TM-β1), anti-CD127 (clone:SB / 199), anti-JunB (clone:C-11), anti-TCF1 (clone:S33-966), anti-TOX (clone:TXRX10), anti-Bim (clone:C34C5), anti-Ki67 (clone:16A8), anti-Active-Caspase-3 (clone:C92-605), anti-Granzyme B (clone:GB11), anti-IFN-γ (clone:XMG1.2), anti-TNF-α (clone:MP6-XT22), anti-Perforin (clone:S16009A), anti-IL-2 (clone:JES6-5H4), anti-MHC-II (clone:M5 / 114.15.2), anti-CD19 (clone:6D5), anti-CD11b (clone:M1 / 70), anti-CD11c (clone:N418). Antibodies were diluted as follows: 1:800 for targeting cell surface molecules, 1:400 for targeting intracellular molecules other than transcription factors and 1:200 for targeting transcription factors.

[0063] Culture of tumor-specific T cells isolated from TdLNs or tumors OT-I T cells in TdLNs or tumors as well as dendric cells (DCs) in tumors were isolated from B16-OVA-bearing mice for ex vivo co-culture experiments. Specifically, OT-I T cells in TdLNs (Zombie-CD8+CD45.1+CD45.2+CD44+LY-108+), as well as tumor-infiltrating OT-I T cells with Tpex phenotype (Zombie-CD8+CD45.1+CD45.2+PD-1+LY-108+TIM-3-) or Ttex phenotype (Zombie-CD8+CD45.1+CD45.2+PD-1+LY-108-TIM-3+), were sorted using FACS. Additionally, tumor-infiltrating dendritic cells (MHC-II+CD19-CD11b+CD11c+) were sorted. Dendric cells (DCs) were pulsed with 10 μg / mL of the OVA257-264(SIINFEKL) peptide and subsequently irradiated with 10 Gy using an X-ray irradiator (Softx co. LTD, M-150WE). Each OT-I T cell subset was cocultured with irradiated OVA-pulsed DCs in a 1:1 ratio using RPMI complete medium supplemented with IL-2 (10 ng / mL) for 5 days.

[0064] dTAG-mediated JunB degradation dTAGV-1 (R&D systems, 6914), which is a specific compound degrading FKBP12F36V-fused proteins, was used to induce dTAG-JunB degradation in T cells. In ex vivo experiments, cells were treated with dTAGV-1 (10-1000 nM) or DMSO vehicle in RPMI complete medium. In in vivo experiments, B16-OVA-bearing mice adoptively transferred with dTAG-JunB OT-I T cells were intratumorally injected with dTAG dissolved in 0.5 x HBSS at a dose of 35 μg / 0.5 cm3of tumor volume.

[0065] Retroviral transduction to overexpress JunB pMIGR-Flag-JunB vector was constructed by inserting cDNA encoding N-terminally Flag-tagged JunB into the pMIGR1 plasmid (Addgene, 27490), a bicistronic retroviral vector containing GFP reporter. To produce retroviral particles, Platinum E (Plat-E) cells (Cell Biolabs, RV-101) cultured with Dulbecco’s modified eagle medium (DMEM) (Invitrogen, 11995-073) supplemented with 10% FBS in 100 mm culture dishes were transfected with 10 μg of pMIGR1 plasmids and 4.5 μg pCL-Eco retrovirus packaging vector (Addgene, 12371) using polyethylenimine (PEI: Polysciences, lnc, 24765) at a ratio of DNA:PEI=1:5. One day later, transfection media were replaced with complete DMEM. Two days later, cell culture media were collected, and viral particles were purified by ultracentrifugation for 2-2.5 h at 24,000 g at 4°C using Beckman Coulter Optima XPN-100. Viral particles were then coated onto a plate using RetroNectin (Takara Bio). Specifically, plates were first coated with RetroNectin at a concentration of 5 μg / cm2at 4°C overnight, followed by blocking with 2% BSA for 30 minutes and washing with PBS. Retroviral particles resuspended in complete RPMI medium were placed on the RetroNectin-coated plates and centrifuged at 2,000g for 2 hours for retroviral coating. For retroviral transduction, naive CD8+T cells were activated with anti-CD3 antibody (5 μg / mL), anti-CD28 antibody (5 μg / mL) and IL-2 (10 ng / mL) for 24 h. Cells were then transferred to plates coated with retroviral particles and cultured in the presence of IL-2 for 48 h, followed by culture in the presence of IL-7 (10 ng / ml) and IL-15 (10 ng / ml). Flow cytometry analysis consistently showed that about 85% of cells expressed GFP four days after transduction.

[0066] Bulk RNA-Seq analysis Total RNA was isolated from freeze-thawed cells using RNAdvance Cell V2 kit (Beckman Coulter; A47942), and its concentration was measured on a Qubit Flex Fluorometer using an RNA HS assay kit (ThermoFisher; 10034622). Sequencing libraries were then prepared by using the 3’mRNA-Seq Library Prep Kit FWD with Unique Dual Indices (Lexogen; 015.96) following either the standard or low-input RNA sample preparation protocol depending on the concentrations of each sample. Libraries were quantified using Qubit 1x dsDNA HS assay kit and Qubit Flex Fluorometer and their quality was assessed using High Sensitivity D5000 ScreenTape with Tapestation 2200 (Agilent; 50675592). Libraries were pooled and subsequently sequenced on a NovaSeq 6000 (Illumina) with 1x100-bp reads.

[0067] RNA-seq data analysis Adapters and low-quality sequences were removed from the sequence data using Cutadapt 2.10. Following alignment to the UCSC mouse genome (mm10), transcripts were quantified using Salmon 1.3.0 with default settings. Counts of each transcript were normalized both within and between samples to obtain transcript per kilobase million. DeSeq2 was used for differential gene expression analysis.

[0068] ATAC-seq analysis ATAC-seq analysis was performed using Tpex cells that had been co-cultured with dendric cells (DCs) for 5 days. FACS-sorted Zombie-cells (5 x 104) were lysed in lysis buffer (10mM Tris-HCl, 10mM NaCl, 3mM MgCl2, 0.1% NP40, 0.1% Tween20, and 0.01% Digitonin) to release nuclei. DNA was then tagmented using the Tagment DNA Kit (Illumina; 20034210) and purified with the Zymo DNA Clean and Concentrator 5 kit (Zymo; D4014). Tagmented DNA was then PCR amplified with index primers using the NEBNext High-Fidelity 2X PCR Master Mix (NEB; M0541S) and subsequently purified twice using Solid Phase Reversible Immobilization (SPRI) beads (Beckman Coulter; B23318). DNA concentration and size distribution of samples were then analyzed by dsDNA HS Assay Kit (Thermo Fisher) and High sensitivity DNA Kit (Agilent; 5067-4626), respectively. The pooled samples were sequenced on a Novaseq 6000 sequencer (Illumina) with paired-end reads of 150 bp in length.

[0069] ATAC-seq data analysis ATAC-seq fastq reads were trimmed using Trimmomatic v0.39. with arguments SLIDINGWINDOW420MINLEN:35 LEADING:20 TRAILING:20 183. Data quality was evaluated by FASTQC v0.11.9, and duplicates were removed using the MarkDuplicates function from Picard v2.7.0. Reads were then mapped to genomic regions listed in the ENCODE mm10 blacklist. Reads positions were then corrected by a constant offset to the read start (plus strands: +4 bp, - strands: -5 bp) with deepTools v.3.5.1 using “alignmentSieve-ATACshift” option. Peaks were called using MACS2 v2.2.7.1 to obtain narrow peaks with “-fBAMP-nomode--shift 75--extsize 150” option. BigWig files were created using bamCoverage from deepTools and were uploaded to the UCSC genome browser. Peaks with differential accessibility between assay conditions were identified with the Diffbind package. ChIPseeker was used for peak annotation.

[0070] Statistical analysis Unpaired two-tailed Student’s tests or one-way ANOVA followed by Tukey’s post-hoc tests were performed using Prism (GraphPad). P values < 0.05 were considered to be statistically significant.

[0071] (Experiment 1) JunB expression is induced in tumor-specific CD8 T cells.

[0072] Differentiation of exhausted CD8+T cell proceeds in a step-wise manner, in which progenitor exhausted T (Tpex) are induced and then differentiate into terminally exhausted T (Ttex) cells [89, 90]. Both Tpex and Ttex cells express PD-1, but they are distinguished by expression of TCF1, LY108, and TIM-3; Tpex cells are TCF1+LY108+TIM-3-, whereas Ttex cells are TCF1-LY108-TIM-3+. Tpex cells, like stem cells, have capacity to self-renew as well as differentiate into Ttex cells, while Ttex cells lack the ability to proliferate or differentiate. Accordingly, Tpex cells are critical for persistence of exhausted T cell populations in chronic infections and tumors. In the spleen with chronic infection, Tpex cells are maintained in white pulp, and migrate to red pulp to differentiate into Ttex cells. A similar niche enriched with Tpex cells has been observed in tertiary lymphoid structures in tumors. In anti-tumor CD8+T cell responses, prior to accumulation of intra-tumoral Tpex and Ttex cells, tumor-specific naive CD8+T cells need to be primed in the tumor-draining lymph nodes (TdLNs) (References 4, 8). This priming results in differentiation of naive CD8 T cells into tumor-specific memory T (Ttsm) cells, and subsequently into Tpex cells (Reference 4). Both Ttsm cells and Tpex cells express TCF1 and PD-1 and have self-renewal capacity, but a homing receptor, CD62L is expressed specifically in Ttsm cells, whereas Tox is expressed specifically in Tpex cells (References 4, 14). Likely due to differential expression of CD62L, Ttsm cells mainly accumulate in TdLNs, whereas Tpex cells accumulate in tumors (References 4, 14). Importantly, in response to PD-1 blockade, Ttsm cells, but not other exhausted CD8+T cell subsets, undergo proliferation burst, leading to increase in their progeny, Tpex and Ttex cells, which contributes to reinvigoration of anti-tumor effector responses (References 4, 14).

[0073] JunB expression is induced upon activation of naive CD4+and CD8+T cells in vitro and in vivo (References 1-3). Additionally, a previous transcriptomic study detected high levels of JunB mRNA expression in tumor-infiltrating exhausted CD8 T cells (Reference 4), but JunB expression during T cell exhaustion has not been fully investigated. To address this, we adoptively transferred naive CD8+T cells that express transgenic T cell receptor (TCR) specific to a model antigen, chicken ovalbumin (OVA) into congenic recipient mice. Subsequently, mice were subcutaneously injected with B16 melanoma expressing OVA (B16-OVA), followed by flow cytometry analysis of JunB expressing in OT-I T cells expressing an exhaustion marker, PD-1, in tumors on day 20. For comparison, mice were also infected with LM-OVA, and JunB expression in OT-I T cells with an activated phenotype (CD44+) in the spleen on day 5, which corresponds to a reported peak time point for JunB expression (Reference 5), was analyzed. This showed that JunB expression in exhausted OT-I T cells in tumors of mice transplanted with B16-OVA was comparable to that in activated OT-I T cells in mice infected with LM-OVA (FIG. 1a).

[0074] In responses to tumors, antigen-specific naive CD8+T cells are activated in the tumor-draining lymph nodes (TdLNs), migrate to tumor tissues, and differentiate to terminally exhausted cells due to chronic TCR stimulation with tumor antigens. To assess JunB expression kinetics in the process of anti-tumor CD8+T cell responses, 20 days after B16-OVA injection into mice that had adoptive transfer of naive OT-I T cells, we analyzed the expression of JunB in OT-I T cells in both the TdLNs and tumor microenvironment. In the TdLNs, JunB expression in activated OT-I T cells expressing a marker of progenitor exhausted cells LY108 (CD44+LY108+) was at undetectable levels (FIG. 1b). In contrast, in tumors, both progenitor exhausted T (Tpex) cells (LY108+TIM-3-) and terminal exhausted T (Ttex) cells (LY108-TIM-3+) expressed high levels of JunB (FIG. 1b). These observations indicate that JunB expression in CD8+T cells is induced in tumors but not in TdLNs.

[0075] (Experiment 2) JunB is necessary for accumulation of tumor-specific CD8 T cells in tumors.

[0076] To understand the role of JunB in anti-tumor CD8 T cell responses, we first assessed whether T-cell-specific conditional knockout of Junb affects growth of transplanted B16-OVA melanoma and polyclonal anti-tumor CD8 T cell responses. We found that tumor growth in Junbfl / flCD4Cre(JunB cKO) mice was accelerated compared to that in control mice (FIG. 2a). Strikingly, flow cytometry analysis revealed that a significant decrease in both CD4+and CD8+T cells in both the percentages in tumor-infiltrating lymphocytes (TILs) and in the absolute numbers per tumor volume in JunB cKO mice on Day 21 after tumor injection (FIG. 2b). Moreover, JunB deficiency reduced numbers of OVA-specific CD8+T cells, which were stained with an H-2Kb-restricted tetramer containing an OVA peptide, to the barely detectable levels in tumors, but not in tumor-draining lymph nodes (TdLNs) (FIGs 2c-d). We also found that JunB deficiency diminished expression of PD-1 in CD8+T cells, but not CD4+T cells, in tumors (FIG. 2e). Furthermore, JunB deficiency decreased expression of granzyme B (GzmB), but not interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and IL-2 in CD8+T cells in tumor microenvironment (FIG. 2f). Thus, JunB deficiency impairs intra-tumoral accumulation and functions of CD8+T cells responding tumors.

[0077] To focus on the role of JunB in antigen-specific CD8+T cell responses, we adoptively transferred JunB cKO or control OT-I T cells into congenic mice, followed by inoculation with B16-OVA melanoma cells (FIG. 3a). Consistent with observations in JunB cKO mice, JunB deficiency significantly reduced anti-tumor effects of OT-I T cells (FIG. 3b) as well as their accumulation in tumors, but not in TdLNs (FIGs 3c-d). We also found that unlike in polyclonal CD8+T cells detected in tumors in JunB cKO mice (FIG. 2e), PD-1 expression in Junb-deficient OT-I T cells was comparable to controls (FIG. 3e). However, while PD-1-expressing control cells contained not only LY108+TIM-3-Tpex cells but also LY108-Tpex progeny cells including TIM-3+Ttex cells, almost all PD-1-expressing Junb-deficient OT-I T cells were LY108+TIM-3-Tpex cells (FIG. 3f). Analysis of Junb-deficient OT-I T cells co-transferred with congenic Junb-sufficient OT-I T cells revealed that Junb-deficient OT-I T cells did not accumulate in tumor tissues, even in the presence of Junb-sufficient OT-I T cells (FIGs 3g-h). Taken together, these results demonstrate that JunB is essential for the accumulation of tumor-specific CD8+T cells in tumors, but not in TdLNs.

[0078] (Experiment 3) JunB enhances stability of TdLN-Tpex cells under chronic antigen stimulation.

[0079] In response to acute infections, pathogen-specific CD8+T cells are activated and massively proliferate in the secondary lymphoid organs in a JunB-dependent manner (Reference 3). However, we observed that JunB is dispensable for clonal expansion of activated tumor-specific CD8+T cells in TdLNs, but it is indispensable for the subsequent responses. To understand how JunB promotes accumulation of tumor-specific CD8+T cells in tumors, we next assessed effects of JunB deficiency on phenotypes and activities of tumor-specific CD8+T cells in TdLNs. As expected from their normal cell numbers, Junb-deficient OT-I T cells showed no alterations in expression of active caspase 3, a marker for apoptosis, and Ki-67, a marker for cell proliferation, in the TdLNs (FIG. 4a). Naive CD8+T cells activated in the TdLNs differentiate into TCF1+TOX-tumor-specific memory T cells (Ttsm) cells, which further differentiate into TCF1+TOX+Tpex cells (Reference 4). We found that frequencies of Junb-deficient OT-I T cells exhibiting TCF1+TOX-Ttsm and TCF1+TOX+Tpex phenotypes in TdLNs were comparable to those of control OT-I T cells (FIG. 4b). Additionally, expression of LY108 as well as memory cell markers, CD62L, CD122, and CD127 were at the same levels between Junb-deficient OT-I T cells and controls in TdLNs (FIG. 4c). JunB deficiency also did not affect ability of OT-I T cells in TdLNs to express inflammatory cytokines, such as GzmB, IFN-γ, TNF-α, and IL-2 (FIG. 4d). Moreover, RNA-seq analysis of OT-I T cells in TdLNs showed that JunB deficiency affected expression of only 30 genes, including a slight upregulation of expression of Pdcd1 and Nr4a1 (FIG. 4d). These results indicate that JunB is largely dispensable for CD8+T cell responses in TdLNs, including generation of Ttsm and Tpex cells.

[0080] It has been suggested that Tpex cells migrate from TdLNs to the tumor tissues and generate a mixed population with Tpex and Ttex cells to adapt to tumor microenvironment (References 6-7). Considering this, we hypothesized that JunB deficiency might disturb adaptation of Tpex cells to tumor microenvironment, leading to loss of Tpex and Ttex cells in tumors. To test this possibility, we utilized an in vitro culture model of exhausted T cells with continuous antigen stimulation. Intra-tumoral dendric cells (DCs) are known to facilitate differentiation of Tpex to Ttex cells more efficiently than DCs in the lymphoid organs, likely due to their higher expression of co-stimulatory molecules (Reference 8). Therefore, we isolated intra-tumoral DCs (MHC-II+CD19-CD11b+CD11c+) from tumor-bearing mice. Subsequently, DCs were loaded with OVA peptides, irradiated, and co-cultured with control or Junb-deficient OT-I T cells isolated from TdLNs in tumor-bearing mice (FIG. 5a). In a five-day coculture, JunB-deficient OT-I T cells showed reduced cell growth compared to controls (FIG. 5b), along with reduced Ki67 expression and increased active caspase-3 expression (FIG. 5c). Notably, there was a significant decrease in the proportion of LY108+TIM-3-Tpex cells and an increase in the proportion of LY108-TIM-3+Ttex cells among Junb-deficient OT-I T cells compared to controls (FIG. 5d). Consistently, JunB deficiency also decreased expression of Tpex-related TCF1 expression in OT-I T cells (FIG. 5e). In contrast, JunB deficiency did not affect expression of T cell exhaustion-related molecules, PD-1 and TOX in OT-I T cells (FIG. 5e). Additionally, Junb-deficient OT-I T cells tended to exhibit decreased ability to express perforin and TNF-α, but not GzmB and IFN-γ (FIG. 5f). Taken together, these results suggest that JunB is required for adaptation of tumor-specific progenitor exhausted CD8+T cells to the tumor microenvironment.

[0081] (Experiment 4) Degradation of endogenous JunB protein using the degrading tag (dTAG) system

[0082] We next sought to understand JunB functions in Tpex and Tex cells in tumor microenvironment. The complete loss of Junb-deficient OT-I T cells in the tumor microenvironment did not allow further characterization of JunB functions. Furthermore, any Cre-expressing mouse lines for gene deletion specifically in CD8+T cell subsets in tumor microenvironment have not been reported. Under these circumstances, we decided to use a dTAG system for rapid degradation of endogenous JunB protein in Tpex and Tex cells in tumor microenvironment. We recently generated mice expressing JunB protein N-terminally fused with FKBP12F36Vdegradation tag (dTAG) in a manner dependent on the endogenous Junb promoter.

[0083] dTAG-fused proteins are selectively degraded upon treatment with membrane-permeable dTAG ligands, such as dTAGV-1, which bind to dTAG and promote ubiquitin-proteasome reactions (References 10-11). Indeed, while dTAG-JunB expression in activated dTAG-JunB CD8+T cells was comparable to JunB expression in control cells, treatment with dTAGV-1 reduced JunB-dTAG expression to almost undetectable levels (FIG. 6a). Importantly, as previously observed in Junb-deficient CD8+T cells (Reference 3), dTAGV-1 treatment resulted in decrease in expression of Ki-67 and GzmB and increase in expression of active caspase-3, IFN-γ, and PD-1 in dTAG-JunB CD8+T cells activated for 4 days, but not in wild-type CD8+T cells (FIG. 6b). In contrast, dTAG treatment did not affect expression of these molecules in CD8+T cells derived from wild-type C57BL / 6 mice (FIG. 6c).

[0084] Recent studies demonstrated that administration of dTAGV-1 to mice can lead to effective knockdown of dTAG-fused target proteins in various cells (References 11-13). To investigate the efficacy of dTAG-mediated JunB knockdown in effector CD8+T cells in tumors, we adoptively transferred dTAG-JunB naive OT-I T cells into mice, followed by injection of B16-OVA cells. Day 14, we intratumorally injected dTAGV-1 into mice and analyzed JunB expression in intra-tumoral OT-I T cells at 12 h after the injection. This showed that administration of dTAGV-1 did not significantly reduce JunB-dTAG expression in intra-tumoral OT-I T cells in vivo (FIG. 7).

[0085] (Experiment 5) JunB supports stability of Tpex cells and survival of Ttex cells.

[0086] Since dTAG-JunB was efficiently knocked down by dTAGV-1 treatment in vitro but not in vivo, we decided to use the dTAG system to evaluate the impact of JunB knockdown on ex vivo culture of Tpex and Tex cells isolated from tumors. To this end, we used FACS to separately collect dTAG-JunB OT-I T cells with PD-1+Ly108+TIM-3-Tpex or PD-1+Ly108-TIM-3+Ttex phenotypes from tumors in mice injected with B16-OVA cells. Subsequently, these Tpex and Ttex cells were cocultured with intra-tumoral dendric cells (DCs) preloaded with OVA peptides in the presence or absence of dTAGV-1 treatment for five days (FIG. 8a). We confirmed that dTAGV-1 treatment reduced dTAG-JunB expression in both Tpex and Ttex cells in a dose dependent manner (FIG. 8b). We next examined how Tpex and Ttex cells respond to antigen-loaded DCs and how the responses are affected by dTAG-mediated JunB knockdown. We observed that upon co-coculture with antigen-loaded DCs, Tpex cells, but not Ttex cells, exhibited marked cell proliferation (FIG. 8c). Notably, dTAG-mediated JunB knockdown significantly reduced cell numbers in Ttex cells and slightly in Tpex cells (FIG. 8c). Consistently, dTAG-mediated JunB knockdown resulted in an increase in expression of active caspase 3 and BIM and a decrease in Ki67 expression in Tex cells but not in Tpex cells (FIG. 8d).

[0087] During coculture with antigen-loaded DCs, majority of control Tpex cells and Ttex cells retained their phonotypes (LY108+TIM-3-and LY108-TIM-3+, respectively) (FIG. 9a). However, dTAG-mediated JunB knockdown resulted in a phenotypic shift of Tpex cells to LY108-TIM-3-or Ttex-like Ly108-TIM-3+as well as a shift of Ttex cells to LY108-TIM-3-(FIG. 9a). Analysis of inhibitory receptors (IRs) revealed that dTAG-mediated JunB knockdown increased TIGIT expression in both Tpex and Ttex cells and reduced LAG3 expression in Ttex cells but not in Tpex cells (FIG. 9b) In contrast, dTAG-mediated JunB knockdown did not affect PD-1 expression in either cell type (FIG. 9b). Additionally, analysis of key transcription factors showed that dTAG-mediated JunB knockdown led to a slight increase in expression of TOX in both Tpex and Ttex cells (FIG. 9c). Furthermore, analysis of effector molecules revealed that dTAG-mediated JunB knockdown resulted in a significant decrease in expression of IFN-γ, perforin, and TNF-α in both Tpex and Ttex cells (FIG. 9d). Interestingly, JunB knockdown increased GzmB expression in Tpex cells while reducing it in Ttex cells (FIG. 9d).

[0088] Next, to investigate effects of re-expression of JunB on gene expression changed by JunB knockdown, we treated dTAG-JunB Tpex cells with dTAGV-1 for 5 days, washed out dTAGV-1, and cultured them for another 3 days in the presence or absence of dTAGV-1. dTAGV-1 removal significantly increased living cell numbers (FIG. 10a) accompanied by decreased expression of active caspase 3 and BIM without affecting expression of Ki67 (FIG. 10b). In contrast, frequency of LY108-TIM-3-or Ttex-like LY108-TIM-3+cells, which were induced by JunB knockdown, was not affected by dTAGV-1 removal (FIG. 10c). We also found that dTAGV-1 removal resulted in increased expression of LAG3, GzmB, and perforin and decreased expression of TIGIT (FIGs 10d-e), which is contrary to the effect of JunB knockdown. In contrast, expression of IFN-γ and TNF-α, which was decreased by JunB knockdown, was not influenced by dTAGV-1 removal (FIGs 10d-e). Thus, effects of dTAG-mediated JunB knockdown on cell viability and expression of specific cytokines, but not loss of Tpex phenotypic identity, in Tpex cells appear to be reversible by re-expression of JunB.

[0089] Taken together, these results suggest that JunB facilitates phenotypic stability of both Tpex and Ttex cells. Moreover, JunB promotes survival and expression of effector molecules in Ttex cells. Taken together, these findings imply that JunB plays a critical role in the adaptation of CD8+T cells in the tumor microenvironment.

[0090] (Experiment 6) JunB regulates transcriptional programs for Tpex identity and Ttex survival.

[0091] To understand roles of JunB in transcriptional program of intra-tumoral Tpex and Ttex cells, we performed bulk RNA-seq analysis on these cells with dTAG-mediated JunB knockdown during co-culture with antigen-loaded dendric cells (DCs). This analysis revealed that expression of 216 genes (123 downregulated genes and 93 upregulated genes) in Tpex cells and 1698 genes (716 downregulated genes and 982 upregulated genes) in Ttex cells were significantly altered (fold change > 2, adjusted p < 0.05) by JunB knockdown (FIGs 11a-b). Consistent with our observations in flow cytometry analysis, JunB knockdown altered expression of Slamf6 (encoding LY108), Havcr2 (encoding TIM-3), Tigit, Bcl2l11 (encoding BIM), prf1 (encoding perforin), and Gzmb in Tpex and / or Ttex cells (FIGs 11a-b). Notably, in addition to Slamf6, JunB knockdown reduced expression of other Tpex-related genes, such as Myb, an essential transcription factor for maintenance of Tpex cells (Reference 14), and Il7r (FIG. 11a). However, JunB knockdown did not affect expression of other transcription factors critical for Tpex cells, such as Tcf7 (encoding TCF1), Foxo1, Bach2, Bcl6, Id3, and Eomes (FIG. 11a). Moreover, JunB knockdown decreased expression of Myc, which encodes a critical transcription factor for glycolysis in both Tpex and Ttex cells, with a greater reduction in Ttex cells (FIGs 11a-b). Consistently, JunB knockdown reduced expression of other glycolytic genes, such as aldolase A (Aldoa), Aldob, and enolase 1 (Eno1) in Ttex cells (FIG. 11b). We also found that an inhibitory receptor (IR) gene, Cd160, was significantly upregulated by JunB knockdown in Tpex cells (FIGs 12a-b). These results suggest that JunB contributes to regulation of transcriptional programs for both Tpex and Ttex cells.

[0092] To investigate whether JunB is also involved in epigenetic regulation of exhausted T cells, we performed ATAC-seq analysis of Tpex cells with dTAG-mediated JunB knockdown during co-culture with antigen-loaded DCs. Here we focused on analysis of only Tpex cells because of severe reduction of living Ttex cells after JunB knockdown (FIG. 8c). ATAC-seq analysis of Tpex cells identified JunB knockdown-increased chromatin accessibility in 129 regions, while decreasing accessibility in 353 regions (FIG. 12a). Most of these chromatin regions were located in introns or intergenic regions more than 5 kb away from transcription start sties (FIGs 12b-c). We found that JunB deficiency affected both expression and chromatin accessibility of 23 genes, including Myb and Il7r (FIGs 12d-e). Taken together, these results suggest that JunB controls expression of a subset of target genes, including Myb, by regulating chromatin accessibility at their loci.

[0093] (Experiment 7) Overexpression of JunB enhances anti-tumor CD8 T cell responses.

[0094] Finally, we investigated whether overexpression of JunB improves anti-tumor CD8+T cell responses. We activated naive OT-I T cells in vitro and transduced them with MIGR / FLAG-JunB, which bicistronically express FLAG-tagged JunB and GFP reporter. We injected these cells into mice that had been transplanted with B16-OVA cells. Notably, OT-I T cells transduced with MIGR / FLAG-JunB suppressed tumor growth more efficiently than those transduced with control MIGR vector (FIG. 13a). We confirmed that JunB was overexpressed in intra-tumoral OT-I T cells transduced with the JunB-expressing retroviral vector (FIG. 13b). We found that JunB overexpression significantly increased numbers of intra-tumoral OT-I T cells (FIG. 13c) without altering ratios between LY108+TIM-3-Tpex cells and LY108-TIM-3+Ttex cells (FIG. 13d). However, JunB overexpression did not affect ability of OT-I T cells to express GzmB, IFN-γ, perforin, and TNF-α (FIG. 13e). These results suggest that JunB overexpression can improve anti-tumor effects of adoptive therapy with tumor-specific CD8+T cells through increasing frequency of both Tpex and Ttex cells in tumors.

[0095] The following abbreviations are used in this specification: AHR - Aryl Hydrocarbon Receptor AICE - Antibody In Cell Extract ALDOA - Aldolase A ENO1 - Enolase 1 AMPK - AMP-Activated Protein Kinase AP-1 - Activator Protein 1 ATAC-seq - Assay for Transposase-Accessible Chromatin using sequencing BACH2 - BTB and CNC homology 2 BATF - Basic leucine zipper ATF-like transcription factor BCL6 - B-cell lymphoma 6 BCR - B cell receptor BHI - Brain Heart Infusion BIM - Bcl-2-interacting mediator of cell death BLIMP1 - B lymphocyte-induced maturation protein-1 bZIP - Basic leucine zipper CAR-T - Chimeric Antigen Receptor T cells CCR - C-C chemokine receptor CD - Cluster of Differentiation CFU - Colony-Forming Unit CTL - Cytotoxic T Lymphocyte CTLA4 - Cytotoxic T-Lymphocyte-Associated protein 4 CXCR - C-X-C chemokine receptor DC - Dendritic Cell DMSO - Dimethyl Sulfoxide EOMES - Eomesodermin E2A - E2A immunoglobulin enhancer-binding factors E12 / E47 FACS - Fluorescence-Activated Cell Sorting FBS - Fetal Bovine Serum FLI1 - Friend Leukemia Integration 1 FOX - Forkhead box GFP - Green Fluorescent Protein GzmB - Granzyme B HAVCR2 - Hepatitis A Virus Cellular Receptor 2 (also known as TIM-3) HIF - Hypoxia-Inducible Factor HIV - Human Immunodeficiency Virus ID - Inhibitor of DNA-binding IFN - Interferon IL - Interleukin IRF - Interferon Regulatory Factor ITAM - Immunoreceptor Tyrosine-based Activation Motif JNK - c-Jun N-terminal Kinase KLRG - Killer Cell Lectin-Like Receptor G1 LAG-3 - Lymphocyte Activation Gene-3 LCMV - Lymphocytic Choriomeningitis Virus LM - Listeria monocytogenes LPS - Lipopolysaccharide MACS - Magnetic-Activated Cell Sorting MHC - Major Histocompatibility Complex MPEC - Memory Precursor Effector Cell MYB - Myeloblastosis MYC - Myelocytomatosis mTOR - The mammalian Target Of Rapamycin NFAT - Nuclear Factor of Activated T-cells NK - Natural Killer NR4A - Nuclear Receptor Subfamily 4 Group A OVA - Ovalbumin PBS - Phosphate-Buffered Saline PD-1 - Programmed Death-1 PD-L1 - Programmed Death-Ligand 1 PGC1α - Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha PRF - Perforin PRR - Pattern Recognition Receptor RNA-seq - RNA sequencing RPMI - Roswell Park Memorial Institute medium RUNX - Runt-Related Transcription Factor SOX13 - SRY-Box Transcription Factor 13 SLEC - Short-Lived Effector Cell STAT - Signal Transducer and Activator of Transcription T-BET - T-box Transcription Factor TBX21 TCF7 - Transcription Factor 7 Tcm - Central Memory T cell TCR - T cell Receptor TdLN - Tumor-draining Lymph Node Teff - Effector T cell Tem - Effector Memory T cell TF - Transcription Factor TGF - Transforming Growth Factor Tfh - Follicular Helper T cell Th - T helper cell TIGIT - T-cell Immunoreceptor with Ig and ITIM domains TIL - Tumor-Infiltrating Lymphocyte TIM-3 - T-cell Immunoglobulin and Mucin-domain containing-3 TME - Tumor Microenvironment TNF - Tumor Necrosis Factor TOX - Thymocyte Selection-Associated High Mobility Group Box Tpex - Progenitor Exhausted T cell Treg - Regulatory T cell Trm - Tissue Resident Memory T cell Ttex - Terminally Exhausted T cell Ttsm - Tumor-specific memory T cell ZEB - Zinc Finger E-box-Binding Homeobox

[0096] An innovative method of enhancing anti-tumor response activity of a T cell is provided.REFERENCES

[0097] 1. Hasan, Z., et al., JunB is essential for IL-23-dependent pathogenicity of Th17 cells. Nat Commun, 2017. 8: p. 15628. 2. Hsieh, T., et al., JunB Is Critical for Survival of T Helper Cells. Front Immunol, 2022. 13: p. 901030. 3. Sarker, S., Analysis of the function of JunB in regulation of CD8+ T cell response. 2023, Okinawa Institute of Science and Technology. 4. Huang, Q., et al., The primordial differentiation of tumor-specific memory CD8(+) T cells as bona fide responders to PD-1 / PD-L1 blockade in draining lymph nodes. Cell, 2022. 185(22): p. 4049-4066 e25. 5. Betz, B.C., et al., Batf coordinates multiple aspects of B and T cell function required for normal antibody responses. J Exp Med, 2010. 207(5): p. 933-42. 6. Dahling, S., et al., Type 1 conventional dendritic cells maintain and guide the differentiation of precursors of exhausted T cells in distinct cellular niches. Immunity, 2022. 55(4): p. 656-670 e8. 7. Connolly, K.A., et al., A reservoir of stem-like CD8(+) T cells in the tumor-draining lymph node preserves the ongoing antitumor immune response. Sci Immunol, 2021. 6(64): p. eabg7836. 8. Prokhnevska, N., et al., CD8(+) T cell activation in cancer comprises an initial activation phase in lymph nodes followed by effector differentiation within the tumor. Immunity, 2023. 56(1): p. 107-124 e5. 9. Nabet, B., et al., The dTAG system for immediate and target-specific protein degradation. Nat Chem Biol, 2018. 14(5): p. 431-441. 10. Nabet, B., et al., Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules. Nat Commun, 2020. 11(1): p. 4687. 11. Zumer, K., et al., Two distinct mechanisms of RNA polymerase II elongation stimulation in vivo. Mol Cell, 2021. 81(15): p. 3096-3109 e8. 12. Ellegast, J.M., et al., Unleashing Cell-Intrinsic Inflammation as a Strategy to Kill AML Blasts. Cancer Discov, 2022. 12(7): p. 1760-1781. 13. Zhang, Y., et al., Collateral lethality between HDAC1 and HDAC2 exploits cancer-specific NuRD complex vulnerabilities. Nat Struct Mol Biol, 2023. 30(8): p. 1160-1171. 14. Tsui, C., et al., MYB orchestrates T cell exhaustion and response to checkpoint inhibition. Nature, 2022. 609(7926): p. 354-360. 15. Ciofani, M., et al., A validated regulatory network for Th17 cell specification. Cell, 2012. 151(2): p. 289-303.

Claims

1. A method of enhancing anti-tumor response activity of a T cell comprising: overexpressing one or more AP-1 transcription factors in the T cell, wherein overexpressing the one or more AP-1 transcription factors leads to accumulation of the T cell in a tumor.

2. The method according to claim 1, wherein the T cell is selected from the group consisting of a CD8+T cell, a CD4+T cell and an NKT (natural killer T) cell.

3. The method according to claim 1, wherein the T cell is a CD8+T cell.

4. The method according to claim 1, wherein the one or more AP-1 transcription factors include a member of a Jun family.

5. The method according to claim 1, wherein the one or more AP-1 transcription factors include JunB.

6. The method according to claim 1, wherein overexpressing the one or more AP-1 transcription factors promotes expression of Myb in the T cell.

7. The method according to claim 1, further comprising: modifying the T cell to express a T cell receptor (TCR) and / or a chimeric antigen receptor (CAR).

8. The method according to claim 1, wherein overexpressing the one or more AP-1 transcription factors is performed by retroviral transduction or lentiviral transduction.

9. A method for treating a tumor in a subject comprising: producing a T cell having enhanced anti-tumor response activity using the method according to any one of claims 1 to 8; and administering a therapeutically effective amount of the T cell into the subject.

10. The method according to claim 9, wherein the tumor is a solid tumor.

11. The method according to claim 10, wherein the solid tumor is melanoma.

12. The method according to claim 9, further comprising: administering a therapeutically effective amount of an immune checkpoint inhibitor to the subject.

13. A T cell having enhanced anti-tumor response activity, wherein the T cell has overexpression of one or more AP-1 transcription factors, and the overexpression of the one or more AP-1 transcription factors leads to accumulation of the T cell in a tumor.

14. The T cell according to claim 13, wherein the T cell is selected from the group consisting of a CD8+T cell, a CD4+T cell and an NKT (natural killer T) cell.

15. The T cell according to claim 13, wherein the T cell is a CD8+T cell.

16. The T cell according to claim 13, wherein the one or more AP-1 transcription factors include a member of a Jun family.

17. The T cell according to claim 13, wherein the one or more AP-1 transcription factors include JunB.

18. The T cell according to claim 13, wherein the T cell further expresses a T cell receptor (TCR) and / or a chimeric antigen receptor (CAR).

19. A pharmaceutical composition comprising the T cell according to any one of claim 13 to 18 and at least one of a pharmaceutically acceptable carrier, a diluent and an excipient.

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