Adoption Immunotherapy

Abstract

The present invention provides methods and compositions for treating EBV-related disorders. [Solution] A method is provided for treating or preventing an EBV-related disease, disorder, or condition in a subject, comprising the steps of (a) administering to the subject a first population of allogeneic T cells that bind to or recognize a first epitope of an EBV antigen; and (b) administering to the subject a second population of allogeneic T cells that bind to or recognize a second epitope of the EBV antigen or a further EBV antigen, thereby treating or preventing the EBV-related disease, disorder, or condition in the subject.

Description

[Technical Field]

[0001] [Related applications]

[0001] This application claims priority to Australian Provisional Application No. 2019903995, entitled “Adoptive Immunotherapy,” filed on 23 October 2019, the entire contents of which are incorporated herein by reference.

[0002] [Technical field]

[0002] The present invention generally relates to the field of therapeutic compositions and methods of adoptive immunotherapy. More specifically, the present invention relates to methods of adoptive immunotherapy in subjects having Epstein-Barr virus (EBV) related diseases, disorders or conditions, such as cancer. Background of the Invention

[0003]

[0003] Adoptive immunotherapy, or cellular immunotherapy, has emerged as a powerful tool for treating cancer, infectious complications, and autoimmune diseases.[1] The first success of T-cell therapy in a clinic was demonstrated by Steven Rosenberg's group, who pioneered the in vitro expansion of patient-derived tumor-infiltrating cells (TILs) by injecting and reinjecting them into patients with advanced melanoma.[2] Since then, T-cell effector functions have been shown to demonstrate clinical success in treating drug-resistant bacterial and fungal infections[3], viral infections including HIV[4], CMV[5] and BKV[6], as well as hematological malignancies and EBV-associated post-transplant lymphoproliferative disorder (PTLD)[7] in hematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT) patients.[7] However, the impact of these therapies on achieving effective and sustained clinical responses against solid tumors remains a significant challenge.

[0004]

[0004] The mechanisms of action associated with effective adoptive cell transfer (ACT) responses against cancer revolve around the ability of T cells to recognize tumor-associated antigens (TAAs) presented by HLA molecules expressed in malignant cells [1]. TAAs include molecular factors that play a crucial role in cell proliferation, neoantigens arising from somatic mutations, and cancer testis / germline antigens (CTAs) located in immune privileged sites. In vitro expanded T cells derived from tumor-infiltrating lymphocytes or peripheral blood mononuclear cells have been widely used. This approach has attracted considerable attention for the treatment of virus-associated cancers and diseases in transplant patients. In particular, adoptive T cell therapy has shown remarkable clinical responses against Epstein-Barr virus (EBV)-associated post-transplant lymphoma (PTLD) [7]. EBV is a potent human ubiquitous B lymphotropic oncogenic herpesvirus known to be associated with a wide range of human malignancies.

[0005]

[0005] In healthy individuals, EBV infection is mainly recognized by functional CD8 that recognize the EBNA3-6 antigen expressed on virus-infected B cells. + Cytotoxic T lymphocytes (CTL) and CD4 + It is immunologically regulated via T lymphocytes [8, 9]. However, due to its ubiquitous nature and compelling cell-transforming ability, EBV infection is associated with a number of malignancies of both B-cell and epithelial origin, including Burkitt lymphoma (BL), Hodgkin lymphoma (HL), natural killer or T (NK / T) cell lymphoma, post-transplant lymphoproliferative disorders (PTLD), nasopharyngeal carcinoma (NPC), and gastric cancer (GC)

[10] . To date, radiotherapy and / or chemotherapy remain the central therapeutic treatment for EBV-related malignancies. Numerous clinical trials are currently underway using EBV-specific autologous T-cell immunotherapy [7]. However, the time required to produce autologous CTLs and test their safety before administration to patients is one of the major constraints on the generation of EBV-specific T cells for ACT.

[0006] [Overview of the prefecture] [

[0006] ]The present invention broadly relates to a method of treating or preventing an EBV-related disease, disorder or condition, such as an EBV-related cancer, in a subject by administering to the subject allogeneic EBV-specific T cells that bind or recognize an epitope of an EBV antigen.

[0007] [

[0007] ]In a first aspect, the present invention is a method of treating or preventing an EBV-related disease, disorder or condition in a subject comprising:

[0008] (a) administering to the subject a first population of allogeneic T cells that bind or recognize a first epitope of an EBV antigen; and

[0009] (b) administering to the subject a second population of allogeneic T cells that bind or recognize a second epitope of an EBV antigen or a further EBV antigen thereby treating or preventing an EBV-related disease, disorder or condition in the subject.

[0010] [

[0008] ]

[0011] In some embodiments, the method of this aspect further comprises an initial step of generating the first and / or second population of allogeneic T cells in vitro. [

[0009] ]

[0012] Suitably, the method comprises a further step of administering a therapeutic agent to the subject. In one embodiment, the therapeutic agent is selected from the group consisting of an immunotherapeutic agent, a MAPK pathway inhibitor such as a MEK1 / 2 inhibitor, a BET inhibitor, and any combination thereof. In this regard, the immunotherapeutic agent is suitably an immune checkpoint inhibitor such as a PD1 inhibitor, a PDL1 inhibitor, a CTLA4 inhibitor, a LAG3 inhibitor, a TIM3 inhibitor or a CD96 inhibitor, or comprises the same. In some particular embodiments, the immune checkpoint inhibitor is a PD1 antibody or comprises an anti-PD1 antibody. [[00,10]]

[0013] [

[0011] ]In a second aspect, the present invention is a pharmaceutical composition for treating or preventing an EBV-related disease, disorder or condition in a subject comprising:

[0014] a first population of allogeneic T cells that binds or recognizes a first epitope of an EBV antigen;

[0015] a second population of allogeneic T cells that binds or recognizes a second epitope of an EBV antigen or a further EBV antigen;

[0016] optionally, a pharmaceutically acceptable carrier, diluent and / or excipient; in a composition.

[0011]

[0017] With respect to the above aspect, both the first population of allogeneic T cells and the cells of an EBV-related disease, disorder or condition suitably either contain a first human leukocyte antigen (HLA) allele encoding a first MHC protein or are restricted by a first human leukocyte antigen (HLA) allele encoding a first MHC protein. In this regard, the first MHC protein may present the first epitope of the EBV antigen to cells of an EBV-related disease, disorder or condition.

[0012]

[0018] With respect to the first and second aspects, both the second population of allogeneic T cells and the cells of an EBV-related disease, disorder or condition contain a second HLA allele encoding a second MHC protein or are restricted by a second HLA allele encoding a second MHC protein. For this purpose, the second MHC protein suitably presents the second epitope of the EBV antigen or a further EBV antigen to cells of an EBV-related disease, disorder or condition.

[0013]

[0019] In some embodiments of the above aspect, the second population of allogeneic T cells is administered before, simultaneously with and / or after administration of the first population of allogeneic T cells.

[0014]

[0020] In a third aspect, the invention is a method of treating or preventing an EBV-related disease, disorder or condition in a subject comprising:

[0021] (a) administering to the subject a population of allogeneic T cells that binds or recognizes an epitope of an EBV antigen; and

[0022] (b) A step of administering a therapeutic agent selected from the group consisting of immunotherapies, MAPK pathway inhibitors, BET inhibitors, and any combination thereof. Includes,

[0023] The present invention relates to a method for treating or preventing EBV-related diseases, disorders, or conditions in the subject.

[0015]

[0024] Ideally, the allogeneic T cell population should be administered before, concurrently with, and / or after the administration of the therapeutic agent.

[0016]

[0025] In some embodiments, the method further includes a first step of generating a population of allogeneic T cells in vitro.

[0017]

[0026] In a fourth aspect, the present invention relates to a pharmaceutical composition for treating or preventing EBV-related diseases, disorders, or conditions in a subject:

[0027] A population of allogeneic T cells that bind to or recognize the EBV antigen epitope;

[0028] A therapeutic agent selected from the group consisting of immunotherapeutic agents, MAPK pathway inhibitors, BET inhibitors, and any combination thereof;

[0029] Optionally, a pharmaceutically acceptable carrier, diluent and / or excipient. The present invention provides a composition containing [a certain substance].

[0018]

[0030] In the third and fourth embodiments, both the population of allogeneic T cells and the cells of EBV-related disease, disorder, or condition appropriately contain a first human leukocyte antigen (HLA) allele encoding a first MHC protein, or are constrained by a first human leukocyte antigen (HLA) allele encoding a first MHC protein. In some embodiments, the MHC protein presents an EBV antigen epitope to the cells of EBV-related disease, disorder, or condition.

[0019]

[0031] Appropriately with respect to third and fourth embodiments, the immunotherapy agent is or comprises an immune checkpoint inhibitor such as a PD1 inhibitor, a PDL1 inhibitor, a CTLA4 inhibitor, a LAG3 inhibitor, a TIM3 inhibitor, or a CD96 inhibitor. In certain embodiments, the immune checkpoint inhibitor is or comprises an anti-PD1 antibody. In some embodiments, the MAPK pathway inhibitor is or comprises a MEK1 / 2 inhibitor.

[0020]

[0032] In a fifth aspect, the present invention relates to the use of a first population of allogeneic T cells that bind to or recognize a first epitope of EBV antigen in the manufacture of a pharmaceutical product for the treatment or prevention of EBV-related disease, disorder or condition in a subject; the first population of allogeneic T cells is administered in combination with: (a) a second population of allogeneic T cells that bind to or recognize a second epitope of EBV antigen or further EBV antigen; and / or (b) a therapeutic agent selected from the group consisting of immunotherapeutic agents, MAPK pathway inhibitors, BET inhibitors and any combination thereof.

[0021]

[0033] In a sixth aspect, the present invention provides a first population of allogeneic T cells that bind to or recognize a first epitope of EBV antigen for use in the treatment or prevention of EBV-related diseases, disorders or conditions in a subject; (a) a second population of allogeneic T cells that bind to or recognize a second epitope of EBV antigen or further EBV antigen; and / or (b) a first population of allogeneic T cells to be administered in combination with a therapeutic agent selected from the group consisting of immunotherapeutic agents, MAPK pathway inhibitors, BET inhibitors and any combination thereof.

[0022]

[0034] In the embodiments described above, the EBV antigen and / or further EBV antigens are appropriately selected from the group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2 and any combination thereof. In one embodiment, the EBV antigen and / or further EBV antigens are EBNA1, LMP1 and / or LMP2, or include EBNA1, LMP1 and / or LMP2.

[0023]

[0035] Appropriately with respect to the above embodiments, EBV-related disease, disorder, or condition is EBV-related cancer or includes EBV-related cancer. In certain embodiments, EBV-related cancer is selected from the group consisting of nasopharyngeal carcinoma, NKT cell lymphoma, Hodgkin lymphoma, post-transplant lymphoproliferative disorder, Burkitt lymphoma, diffuse large B-cell lymphoma, gastric cancer, and any combination thereof.

[0024]

[0036] More appropriately, the above-described embodiments of the present invention are intended for mammals.

[0025]

[0037] Preferably, the subject is a human.

[0026]

[0038] Throughout this specification, unless otherwise indicated, “comprise,” “comprises,” and “comprising” are used in an inclusive rather than exclusive sense, so that the integer or group of integers described may include one or more other integers or groups of integers not described.

[0027]

[0039] Furthermore, it should be understood that the indefinite articles "a" and "an" should not be interpreted as singular indefinite articles, or as excluding more than one or more than one object that the indefinite article refers to. For example, "a" protein includes one protein, one or more proteins, or more proteins. [Brief explanation of the drawing]

[0028] [Figure 1AB]This figure shows the effectiveness of allogeneic "off-the-shelf" EBV-specific T cells in recognizing and eliminating multiple cancers in vitro. (A) Statistical representation of the relative transcriptional expression levels of the indicated EBV-related genes in each EBV-associated cancer cell line compared with NP43(EBV) cancer cells. Housekeeping genes, HPRT1 and 18s RNA were used as loading controls. (B) FACS plot showing IFN-γ expression in the presence of LMP1 / 2 and EBNA1-specific peptides, observed in a live CD8+ population of indicated allogeneic EBV-specific effector AdE1-LMPpoly-transfected T cells, as previously described [1]. (C) Statistical representation of cytotoxicity measured by LDH-releasing assays of the indicated EBV-associated cancer cell lines, highlighting the dose-dependent increase in T cell-derived cytotoxicity across various effector-to-target cell ratios (5:1 to 100:1). The cytotoxicity of HLA-matched T cells is expressed as a relative multiplier change in LDH release 24 hours after T cell therapy of cancer cells compared to a positive control (surfactant lysis control). (D) Statistical representation of cell viability measured by the MTS assay, highlighting the effect of HLA-matched T cells in dose-dependent suppression of cell proliferation in multiple EBV-associated cancer cell lines of different origins across various effector-to-target cell ratios (5:1 to 100:1). SNKT16(HLA) was treated with both TI_001 and TI_002, and its cell viability was compared to that of SNU719(HLA) and C17(HLA), respectively. The cell viability of each cancer cell is expressed as a relative multiplier change in viable cells 24 hours after T cell therapy compared to a mock (PBS) treatment control. (E) Statistical representation of cell death (50:1 effector-to-target cell ratio) measured by the annexin V binding assay of the indicated EBV-associated cancer cell lines in the presence of HLA-matched T cells. Cell death in each cancer cell type is expressed as the relative magnification change in annexin V binding in T-cell therapy samples compared to mock (PBS) treatment 48 hours after cell therapy. Error bars represent ±SEM from three independent experiments. [Figure 1CE]This figure shows the effectiveness of allogeneic "off-the-shelf" EBV-specific T cells in recognizing and eliminating multiple cancers in vitro. (A) Statistical representation of the relative transcriptional expression levels of the indicated EBV-related genes in each EBV-associated cancer cell line compared with NP43(EBV) cancer cells. Housekeeping genes, HPRT1 and 18s RNA were used as loading controls. (B) FACS plot showing IFN-γ expression in the presence of LMP1 / 2 and EBNA1-specific peptides, observed in a live CD8+ population of indicated allogeneic EBV-specific effector AdE1-LMPpoly-transfected T cells, as previously described [1]. (C) Statistical representation of cytotoxicity measured by LDH-releasing assays of the indicated EBV-associated cancer cell lines, highlighting the dose-dependent increase in T cell-derived cytotoxicity across various effector-to-target cell ratios (5:1 to 100:1). The cytotoxicity of HLA-matched T cells is expressed as a relative multiplier change in LDH release 24 hours after T cell therapy of cancer cells compared to a positive control (surfactant lysis control). (D) Statistical representation of cell viability measured by the MTS assay, highlighting the effect of HLA-matched T cells in dose-dependent suppression of cell proliferation in multiple EBV-associated cancer cell lines of different origins across various effector-to-target cell ratios (5:1 to 100:1). SNKT16(HLA) was treated with both TI_001 and TI_002, and its cell viability was compared to that of SNU719(HLA) and C17(HLA), respectively. The cell viability of each cancer cell is expressed as a relative multiplier change in viable cells 24 hours after T cell therapy compared to a mock (PBS) treatment control. (E) Statistical representation of cell death (50:1 effector-to-target cell ratio) measured by the annexin V binding assay of the indicated EBV-associated cancer cell lines in the presence of HLA-matched T cells. Cell death in each cancer cell type is expressed as the relative magnification change in annexin V binding in T-cell therapy samples compared to mock (PBS) treatment 48 hours after cell therapy. Error bars represent ±SEM from three independent experiments. [Figure 2A]This figure shows the phenotypic determination of EBV-associated cancer cell effector T cells in vitro. (A) Statistical representation comparing (i) the percentage of the Ki67+ population; (ii) the percentage of the active caspase 3+ population; and (iii) the percentage of the BCL2+ population of the shown live EBV-associated cancer cell lines 24 hours after mock (PBS) and HLA-matched T cell therapy. SNU719 and C17 were treated with TI_001 and TI_002 T cells, respectively, while SNKT16 was treated with both T cells. Both SNU719 and C17 were gated as CD45- populations, while SNKT16 was gated as a CD45+ CD3+ CD56+ population to distinguish it from the T cell population. (B)Statistical display comparing the percentage of (i) CD8+ population; (ii) Ki67+ population; (iii) GnzB+ (granzyme B) population; (iv) GnzK+ (granzyme K) population; and (iii) Perf+ (perforin) population of live T cells used to treat each EBV-associated cancer cell line as described in (A). Error bars represent ±SEM from three independent experiments. P values ​​were calculated using one-way ANOVA: **p<0.01 and ***p<0.001. [Figure 2B]This figure shows the phenotypic determination of EBV-associated cancer cell effector T cells in vitro. (A) Statistical representation comparing (i) the percentage of the Ki67+ population; (ii) the percentage of the active caspase 3+ population; and (iii) the percentage of the BCL2+ population of the shown live EBV-associated cancer cell lines 24 hours after mock (PBS) and HLA-matched T cell therapy. SNU719 and C17 were treated with TI_001 and TI_002 T cells, respectively, while SNKT16 was treated with both T cells. Both SNU719 and C17 were gated as CD45- populations, while SNKT16 was gated as a CD45+ CD3+ CD56+ population to distinguish it from the T cell population. (B)Statistical display comparing the percentage of (i) CD8+ population; (ii) Ki67+ population; (iii) GnzB+ (granzyme B) population; (iv) GnzK+ (granzyme K) population; and (iii) Perf+ (perforin) population of live T cells used to treat each EBV-associated cancer cell line as described in (A). Error bars represent ±SEM from three independent experiments. P values ​​were calculated using one-way ANOVA: **p<0.01 and ***p<0.001. [Figure 3AD]This figure shows the evaluation of the therapeutic efficacy of allogeneic EBV-specific cytotoxic T cells against solid tumors in vivo. Statistical representation of tumor growth and survival percentage after T cell therapy, observed after a single T cell dose in (A) C17 and (B) C666.1 derived tumor xenografts; and after two T cell therapy doses (indicated by black arrows) at 96-hour intervals in (C) C17 and (D) C666.1 derived tumor xenografts. C17 was treated with TI_002, while C666.1 was treated with TI_004. Tumor size (area, mm2) was measured using digital calipers, and the mean tumor size for each cohort is shown. Tumor growth in xenografts from each cell line is expressed as mean tumor area ± SEM from n≧4 mice / group. Mouse survival was monitored over the indicated period, and the statistical significance of the data was analyzed by log-rank tests: *p<0.05, **p<0.01, and ***p<0.001. [Figure 4AB] This figure shows that the evaluation of "switch antigen" therapy leads to improved efficacy of EBV-specific cytotoxic T cells in vivo. (A) Statistical representation of SNU719-derived xenografts showing (i) tumor growth; (ii) tumor weight (gm) at the ethical limit of tumor growth; and (iii) survival percentage after T cell therapy (two doses, each at 96-hour intervals) compared to the mock (PBS) treatment control group. (B) Statistical representation of SNU719-derived xenografts showing (i) tumor growth; (ii) tumor weight (gm) at the ethical limit of tumor growth; and (iii) survival percentage after T cell therapy (three doses) compared to the mock (PBS) treatment control group. Red arrows indicate administration of three consecutive doses of TI_001 (each at 96-hour intervals), while green arrows indicate switching to TI_004 for the third dose administered after two doses of TI_001. Tumor growth of xenografts derived from each cell line is expressed as mean tumor area ± SEM from n≧5 mice / group. The statistical significance of tumor weight data was analyzed by the Mann-Whitney t-test. Mouse survival was monitored over the indicated period, and the statistical significance of the data was analyzed by the log-rank test:**p<0.01,***p<0.001, and****p<0.0001. [Figure 5AB] This figure shows the evaluation of the therapeutic efficacy of allogeneic EBV-specific cytotoxic T cells against lymphoid malignancies in vivo. (A) A schematic diagram showing a 12-week human immune system rearrangement schedule in NRG mice using irradiated CD34+ cells, and mouse monitoring for graft-versus-host disease (GVHD). The schematic also shows that EBV virus (QIMR-WIL strain) was administered after rearrangement and monitored for 2 weeks for EBV incubation. HLA-matched T cells were administered on the indicated days after EBV infection (highlighted by red arrows), and mice were sacrificed 2 weeks after T cell therapy. (B) A statistical display highlighting the 12-week human immune system rearrangement, indicated by the presence of percentages of live (i) CD45+; (ii) CD45+ CD3+; and (iii) CD45+ CD19+ populations in each group. (C) Macroscopic morphology of the pancreas showing the size and presence of lymphoid malignancies in the pancreas of n=3 mice reconstituted with CB33A CD34+ umbilical cord blood cells between each treatment group. G1 represents administration of three consecutive doses of TI_005 (each at 96-hour intervals), while G2 represents switching to TI_002 for the third dose after two doses of TI_005. (D) Statistical display comparing pancreatic weight (gm) between each treatment group. (E) Macroscopic morphology of the pancreas showing the size and presence of lymphoid malignancies in the pancreas of n=3 mice reconstituted with CB03 CD34+ umbilical cord blood cells between each treatment group. According to the strategy shown in (C), G1 was treated with TI_002, while G2 was treated with TI_003. (F) Statistical display comparing pancreatic weight (gm) between each treatment group. The statistical significance of tumor weight data was analyzed by one-way ANOVA: **p<0.01, and ***p<0.001. [Figure 5CF]This figure shows the evaluation of the therapeutic efficacy of allogeneic EBV-specific cytotoxic T cells against lymphoid malignancies in vivo. (A) A schematic diagram showing a 12-week human immune system rearrangement schedule in NRG mice using irradiated CD34+ cells, and mouse monitoring for graft-versus-host disease (GVHD). The schematic also shows that EBV virus (QIMR-WIL strain) was administered after rearrangement and monitored for 2 weeks for EBV incubation. HLA-matched T cells were administered on the indicated days after EBV infection (highlighted by red arrows), and mice were sacrificed 2 weeks after T cell therapy. (B) A statistical display highlighting the 12-week human immune system rearrangement, indicated by the presence of percentages of live (i) CD45+; (ii) CD45+ CD3+; and (iii) CD45+ CD19+ populations in each group. (C) Macroscopic morphology of the pancreas showing the size and presence of lymphoid malignancies in the pancreas of n=3 mice reconstituted with CB33A CD34+ umbilical cord blood cells between each treatment group. G1 represents administration of three consecutive doses of TI_005 (each at 96-hour intervals), while G2 represents switching to TI_002 for the third dose after two doses of TI_005. (D) Statistical display comparing pancreatic weight (gm) between each treatment group. (E) Macroscopic morphology of the pancreas showing the size and presence of lymphoid malignancies in the pancreas of n=3 mice reconstituted with CB03 CD34+ umbilical cord blood cells between each treatment group. According to the strategy shown in (C), G1 was treated with TI_002, while G2 was treated with TI_003. (F) Statistical display comparing pancreatic weight (gm) between each treatment group. The statistical significance of tumor weight data was analyzed by one-way ANOVA: **p<0.01, and ***p<0.001. [Figure 6A]This figure shows the effect of PD1 inhibition on the therapeutic efficacy of allogeneic EBV-specific cytotoxic T cells in vivo. (A) Heatmap representing the gene signatures of 326 genes observed in RNA isolated from tumor-infiltrating (TIL) CD8+ cells, performed using a NanoString Immune functional panel. TILs were isolated from SNU719-derived tumor xenografts from six independent mice (LT5-10) 5 days after a single dose of TI_001 treatment when the tumor size reached 40 mm2. Gene expression observed in TILs was compared to unstimulated (LT11) and EBV-pepmix stimulated (LT12). (B) Statistical representation of (i) the percentage of the CD3+CD8+ population; (ii) the percentage of the CD8+PD1+ population; (iii) the percentage of the CD8+LAG3+ population; and (iv) the percentage of the CD8+TIM3+ population of live TILs (as described in (A)), as well as compared with unstimulated T cells (T cell therapy). Error bars represent ±SEM from three independent experiments. P values ​​were calculated using one-way ANOVA. (C) Tumor growth; (D) Tumor weight (gm) at the ethical limits of tumor growth; (E) Statistical representation of SNU719-derived xenografts showing survival percentages after T cell therapy, anti-PD1 therapy, and combination therapy of T cell and anti-PD1 therapy observed in each treatment group compared with a mock (PBS) treatment control group. The statistical significance of tumor weight data was analyzed by one-way ANOVA. Mouse survival was monitored over the specified period, and the statistical significance of the data was analyzed by log-rank tests: ns not significant, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 6BE]This figure shows the effect of PD1 inhibition on the therapeutic efficacy of allogeneic EBV-specific cytotoxic T cells in vivo. (A) Heatmap representing the gene signatures of 326 genes observed in RNA isolated from tumor-infiltrating (TIL) CD8+ cells, performed using a NanoString Immune functional panel. TILs were isolated from SNU719-derived tumor xenografts from six independent mice (LT5-10) 5 days after a single dose of TI_001 treatment when the tumor size reached 40 mm2. Gene expression observed in TILs was compared to unstimulated (LT11) and EBV-pepmix stimulated (LT12). (B) Statistical representation of (i) the percentage of the CD3+CD8+ population; (ii) the percentage of the CD8+PD1+ population; (iii) the percentage of the CD8+LAG3+ population; and (iv) the percentage of the CD8+TIM3+ population of live TILs (as described in (A)), as well as compared with unstimulated T cells (T cell therapy). Error bars represent ±SEM from three independent experiments. P values ​​were calculated using one-way ANOVA. (C) Tumor growth; (D) Tumor weight (gm) at the ethical limits of tumor growth; (E) Statistical representation of SNU719-derived xenografts showing survival percentages after T cell therapy, anti-PD1 therapy, and combination therapy of T cell and anti-PD1 therapy observed in each treatment group compared with a mock (PBS) treatment control group. The statistical significance of tumor weight data was analyzed by one-way ANOVA. Mouse survival was monitored over the specified period, and the statistical significance of the data was analyzed by log-rank tests: ns not significant, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 7AB]This figure shows the combined use of MEK / 12 inhibitors and EBV-specific T cells. (A) Statistical representation of cell viability measured by the MTS assay, highlighting the effects of HLA-matched EBV-specific T cells and MEK1 / 2 inhibitors (AZD6244 and trametinib) individually and in combination on suppressing cell proliferation 48 hours after incubation with SNU719 cells. (B) Statistical representation of cell death measured by the Annexin V binding assay, observed in the presence of EBV-specific T cells and MEK1 / 2 inhibitors as individual and combination therapies, highlighting the level of cell death observed 48 hours after incubation with SNU719 cells. (C) The proliferation curves, using Xcellegence, highlight the rates of cell death observed individually and in combination with HLA-matched EBV-specific T cells and MEK1 / 2 inhibitors. P values ​​were calculated using one-way ANOVA: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 7C] This figure shows the combined use of MEK / 12 inhibitors and EBV-specific T cells. (A) Statistical representation of cell viability measured by the MTS assay, highlighting the effects of HLA-matched EBV-specific T cells and MEK1 / 2 inhibitors (AZD6244 and trametinib) individually and in combination on suppressing cell proliferation 48 hours after incubation with SNU719 cells. (B) Statistical representation of cell death measured by the Annexin V binding assay, observed in the presence of EBV-specific T cells and MEK1 / 2 inhibitors as individual and combination therapies, highlighting the level of cell death observed 48 hours after incubation with SNU719 cells. (C) The proliferation curves, using Xcellegence, highlight the rates of cell death observed individually and in combination with HLA-matched EBV-specific T cells and MEK1 / 2 inhibitors. P values ​​were calculated using one-way ANOVA: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 8AB]This figure shows the combined use of JQ1 and EBV-specific T cells. (A) Statistical representation of cell viability measured by the MTS assay, highlighting the effects of HLA-matched EBV-specific T cells and JQ1 in suppressing cell proliferation individually and in combination 48 hours after incubation with SNU719 cells. (B) Statistical representation of cell death measured by the annexin V binding assay, observed in the presence of EBV-specific T cells and JQ1 as individual and combination therapies, highlighting the level of cell death observed 48 hours after incubation with SNU719 cells. P values ​​were calculated using one-way ANOVA: ns- not significant, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 9] This figure shows the determination of IC50 values ​​for MEK1 / 2 inhibitors. Cell viability is displayed using the MTS assay after incubation of the shown cell lines with selumatinib (left panel) and trametinib (right panel) at 0.1 μM–5 μM for 48 hours. Data are expressed as mean ± SD from three independent experiments. [Figure 10] This figure shows the combined use of MEK1 / 2 inhibitors with HLA-matched allogeneic EBV-specific T cells. It highlights the effects of HLA-matched EBV-specific T cells at an effector-to-target ratio of 25:1, and the effects of the MEK1 / 2 inhibitors selmatinib (upper panel) and trametinib (lower panel) at a concentration of 1 μlM, on suppressing cell proliferation individually and in combination with (A)C17; (B)C666.1; (C)SNU719 and (D)YCCLE1 after 48 hours of incubation, as well as cell viability as measured by the MTS assay. Data are expressed as mean ± SD from three independent experiments. P-values ​​were calculated using one-way ANOVA: ns = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. [Figure 11AC]This figure shows the effects of dual combination therapy with MEK1 / 2 inhibitors and HLA-matched allogeneic EBV-specific T cells. (A) The cell proliferation curve highlights the rates of cell proliferation of HLA-matched EBV-specific T cells (effector-to-target ratio 25:1) individually and in combination, and of SNU719 (upper panel) and C666.1 (lower panel) observed in the presence of selmatinib (1 μM), as measured using Xcellegence. (B) Effects of individual and dual combination therapy on cell proliferation based on Ki67, and (C) effects on cell death based on activated caspase-3, of SNU719 expression (upper panel) and C666.1 (lower panel), as measured using flow cytometry. Data are expressed as mean ± SD from three independent experiments. P values ​​were calculated using one-way ANOVA: ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. [Figure 12AC] This figure shows the combined use of MEK1 / 2 inhibitors with HLA-incompatible allospecific T cells. It highlights the effects of HLA-incompatible T cells at an effector-to-target ratio of 25:1, and the effects of 1 μM MEK1 / 2 inhibitors (A) selmatinib and (B) trametinib, on suppressing cell proliferation individually and in combination 48 hours after incubation with SNU719 (upper panel) and C666.1 (lower panel), as well as cell viability measured by the MTS assay. (C) Cell viability measured by the MTS assay compares the effects of HLA-compatible and HLA-incompatible EBV-specific T cells (at an effector-to-target ratio of 25:1) alone and in combination with selmatinib and trametinib (1 μM) when incubated with SNU719 (upper panel) and C666.1 (lower panel). Data are presented as mean ± SD from three independent experiments. P-values ​​were calculated using one-way ANOVA: ns = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. [Figure 13AD]This figure shows the phenotypic determination of the effects of dual combination therapy with MEK1 / 2 inhibitors and HLA-matched allogeneic EBV-specific T cells on the intracellular pathway of cancer cells. Flow cytometry was used to display the cancer cell phenotypes 16 hours after individual and dual combination therapy with selmatinib (1 μM) and HLA-matched EBV-specific T cells (effector-to-target ratio 25:1) in SNU719 (upper panel) and C666.1 (lower panel) when (A) pERK1 / 2; (B) MHC class I; (C) pSTAT3; and (D) MYC expression. Data are expressed as mean ± SD from three independent experiments. P-values ​​were calculated using one-way ANOVA: ns = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. [Figure 14] This figure shows the determination of the IC50 values ​​of JQ1 inhibitors. Cell viability is displayed using the MTS assay after incubation of the shown cell lines with JQ1 0.5 μM–10 μM for 48 hours. Data are expressed as mean ± SD from three independent experiments. [Figure 15AD]This figure shows the effects of dual combination therapy with a JQ1 inhibitor and HLA-matched allogeneic EBV-specific T cells. The figures highlight the effects of (A) HLA-matched EBV-specific T cells and JQ1 (2.5 μM) at an effector-to-target ratio of 25:1, individually and in combination, on suppressing cell proliferation 48 hours after incubation with gastric cancer cells (SNU719, YCCLE1 (upper panel)) and nasopharyngeal cancer cells (C17, C666.1 (lower panel)), as measured by the MTS assay. The figures also show cell proliferation curves highlighting the rates of cell proliferation of SNU719 (upper panel) and C666.1 (lower panel) observed in the presence of (B) HLA-matched EBV-specific T cells (effector-to-target ratio 25:1) and JQ1 (2.5 μM), individually and in combination, as measured using Xcellegence. Effects of SNU719 expression (upper panel) and C666.1 (lower panel) individually and in combination on (C) Ki67-based cell proliferation and (D) active caspase-3-based cell death, as measured by flow cytometry. Data are expressed as mean ± SD from three independent experiments. P-values ​​were calculated using one-way ANOVA: ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. [Figure 16AB]This figure shows the combined use of a JQ1 inhibitor with HLA-incompatible allospecific T cells. (A) Cell viability, as measured by the MTS assay, highlighting the effects of HLA-incompatible T cells at an effector-to-target ratio of 25:1 and a JQ1 inhibitor at a concentration of 2.5 μM on suppressing cell proliferation individually and in combination after 48 hours of incubation with SNU719 (upper panel) and C666.1 (lower panel). (B) Cell viability, as measured by the MTS assay, comparing the effects of HLA-compatible and HLA-incompatible EBV-specific T cells (at an effector-to-target ratio of 25:1) alone and in combination with a JQ1 inhibitor (2.5 μM) when incubated with SNU719 (upper panel) and C666.1 (lower panel). Data are presented as mean ± SD from three independent experiments. P-values ​​were calculated using one-way ANOVA: ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. [Figure 17AD] This figure shows the phenotypic determination of the effects of dual combination therapy with JQ1 inhibitors and HLA-matched allogeneic EBV-specific T cells on the intracellular pathway of cancer cells. The figures display cancer cell phenotypes 16 hours after the use of individual and dual combination therapy with JQ1 (2.5 μM) and HLA-matched EBV-specific T cells (effector-to-target ratio 25:1) in SNU719 (upper panel) and C666.1 (lower panel) during the expression of (A) MYC; (B) pERK1 / 2; (C) pAKT; and (D) pSTAT3, performed using flow cytometry. Data are expressed as mean ± SD from three independent experiments. P-values ​​were calculated using one-way ANOVA: ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. [Figure 18AC]This figure shows the phenotypic determination of the effects of dual combination therapy with a JQ1 inhibitor and HLA-matched allogeneic EBV-specific T cells on immunomodulatory molecules. The figures display cancer cell phenotypes 16 hours after the use of individual and dual combination therapy with JQ1 (2.5 μM) and HLA-matched EBV-specific T cells (effector-to-target ratio 25:1) in SNU719 (upper panel) and C666.1 (lower panel) during the expression of (A) MHC class I; (B) PD-L1; and (C) CD47, performed using flow cytometry. Data are expressed as mean ± SD from three independent experiments. P-values ​​were calculated using one-way ANOVA: ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. [Figure 19AE]This figure shows the evaluation of the therapeutic efficacy of dual combination therapy with selmatinib and allogeneic EBV-specific cytotoxic T cells in vivo. (A) The effect of dual combination therapy with selmatinib and allogeneic T cells (TIG-001) on the growth of EBV-positive SNU719 xenografts in NRG mice was evaluated. Tumor-bearing mice were orally treated for 14 consecutive days with two doses of either HLA-matched T cells (2 × 10⁷ T cells per mouse, indicated by the black arrow) and selmatinib (12.5 mg / kg), either individually or in combination. Growth of each xenograft is expressed as mean tumor area ± SD from n=9 mice / group. (B) Macroscopic morphology of tumors isolated from mice in the indicated treatment group. (C) Representation of tumor weights from mice in the indicated treatment group as described in (B). Data are expressed as mean ± SD from n=3 mice per group. P-values ​​were calculated using one-way ANOVA. (D) Display of tumor infiltration survival percentage observed in individually treated allogeneic T cells (TIG-001) and allogeneic T cells (TIG-001) in combination with selmatinib, as determined by CD45, CD3, and CD8 expression using flow cytometry. P values ​​were calculated using Student's t-test. (E) Kaplan-Meier overall survival analysis of mice carrying the EBV-related tumors described in (A) after individual treatment and combination therapy with selmatinib and allogeneic T cells. Animal survival (n=6 mice / group) was monitored over the indicated period, and statistical significance was analyzed by log-rank test: ns=not significant, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Detailed description of the invention

[0029]

[0059] The present invention somewhat predicts the remarkable discovery that adoptive immunotherapy using "off-the-shelf" allogeneic EBV-specific T cells can treat or prevent a range of EBV-related or EBV-positive cancers. This therapeutic effect has been shown to be particularly effective when combinations of EBV-specific T cell populations specific to different EBV antigen epitopes are used. Furthermore, the inventors have shown that the combination of allogeneic EBV-specific T cells with immune checkpoint inhibitors, MEK1 / 2 inhibitors, and / or BET inhibitors can significantly improve the efficacy of such adoptive T cell therapy for EBV-related diseases, disorders, or conditions.

[0030]

[0060] Therefore, in a broader form, the present invention relates to a method for treating or preventing an EBV-related disease, disorder, or condition in a subject, comprising the step of administering to a population of allogeneic T cells that bind to or recognize an EBV antigen epitope to the subject, thereby treating or preventing an EBV-related disease, disorder, or condition in the subject.

[0031]

[0061] In one embodiment, the present invention relates to a method for treating or preventing EBV-related diseases, disorders, or conditions in a subject:

[0062] (a) the step of administering a therapeutically effective dose to a first population of allogeneic T cells that bind to or recognize the first epitope of the EBV antigen; and

[0063] (b) A step of administering a therapeutically effective dose to a second population of allogeneic T cells that bind to or recognize EBV antigen or a second epitope of further EBV antigen. Includes,

[0064] The present invention relates to methods for treating or preventing EBV-related diseases, disorders, or conditions in subjects.

[0032]

[0065] In related embodiments, the present invention relates to a pharmaceutical composition for treating or preventing EBV-related diseases, disorders, or conditions in a subject:

[0066] A first population of allogeneic T cells that bind to or recognize the first epitope of the EBV antigen;

[0067] A second population of allogeneic T cells that bind to or recognize a second epitope of EBV antigen or further EBV antigen;

[0068] Optionally, a pharmaceutically acceptable carrier, diluent and / or excipient. The present invention provides a composition containing [a certain substance].

[0033]

[0069] The following description applies equally to the two aforementioned aspects.

[0034]

[0070] Epstein-Barr virus, or EBV, is a common human pathogen that can cause or be associated with one or more diseases, disorders, or conditions in humans. Therefore, certain embodiments of the methods described above relate to the prevention and / or treatment of one or more diseases, disorders, or conditions caused by or associated with EBV infection in humans, such as EBV-associated cancer. EBV primarily infects human hosts through epithelial cells and B lymphocytes, and can subsequently establish long-term latent infection in the human host. Primary EBV infection accounts for over 90% of infectious mononucleosis (IM) cases worldwide, primarily infecting children and young adults through the spread of EBV-infected B cells. EBV is also associated with several cancers, including Burkitt lymphoma and Hodgkin lymphoma, gastric and nasopharyngeal cancer, lymphoma in HIV-infected individuals, and post-transplant lymphoproliferative disorder (PTLD). EBV has also been found to be involved in autoimmune diseases, particularly multiple sclerosis.

[0035]

[0071] In the context of this invention, “EBV-related disease, disorder, or condition” means any clinicopathology resulting from or associated with an infection caused by the Epstein-Barr virus. For this purpose, EBV-related disease, disorder, or condition may mean any disease directly or indirectly caused by EBV, as well as any condition that makes a patient susceptible to EBV-related infection. Examples of diseases in the former category include infectious mononucleosis, nasopharyngeal carcinoma, and Burkitt lymphoma. Diseases in the latter category (i.e., diseases that expose a patient to the risk of EBV infection) include acquired immunodeficiency syndrome and any condition that results in immunosuppression or impaired immune system function, such as in patients undergoing organ transplantation and certain cancer therapies. In one particular embodiment, EBV-related disease, disorder, or condition is appropriately multiple sclerosis or includes multiple sclerosis.

[0036]

[0072] The term "EBV-positive cells" refers to cells, including cancer cells, that express EBV or one or more EBV proteins in latent forms or other states.

[0037]

[0073] In preferred embodiments, an EBV-related disease, disorder, or condition is an EBV-related and / or positive cancer, or includes an EBV-related and / or positive cancer. As used herein, and unless otherwise specified, the terms “EBV-related cancer” or “EBV-positive cancer” refer to cancer associated with the Epstein-Barr virus (EBV). In certain embodiments, an EBV-positive cancer is a cancer in which more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, or more than 80% contains or expresses the EBV virus.

[0038]

[0074] As commonly used herein, the terms “cancer,” “tumor,” “malignant,” and “malignant tumor” refer to a disease or condition, or cells or tissues associated with a disease or condition, characterized by abnormal cell proliferation, differentiation, and / or migration, often accompanied by an abnormal molecular phenotype including carcinogenesis, expression of tumor markers, expression or loss of activity of tumor suppressor factors, and / or expression of abnormal (aberrant or abnormal) cell surface markers.

[0039]

[0075] Cancer may include, but is not limited to, any invasive or potentially invasive cancer, tumor, or other malignant tumor listed in the NCI Cancer Index at http: / / www.cancer.gov / cancertopics / alphalist, including all major cancer forms such as sarcomas, carcinomas, lymphomas, leukemias, and blastomas. These may include, but are not limited to, cancers of the reproductive system including breast cancer, lung cancer including lung adenocarcinoma, cancers of the ovarian system including ovarian cancer, cervical cancer, uterine cancer, and prostate cancer, cancers of the brain and nervous system, cancers of the head and neck, cancers of the colon, colorectal cancer, and stomach cancer, cancers of the liver, kidney cancer, skin cancers including melanoma and skin cancer, hematological cancers including lymphoid cancers and myelomonocytic cancers, cancers of the endocrine system including pancreatic cancer and pituitary cancer, and musculoskeletal cancers including bone cancers and soft tissue cancers. In certain embodiments, cancer may be a solid tumor or a leukemia or liquid tumor. Appropriately, cancer expresses, for example, overexpresses, one or more EBV antigens, such as those described earlier herein.

[0040]

[0076] In certain embodiments, EBV-associated cancer is selected from the group consisting of nasopharyngeal carcinoma, NKT cell lymphoma, Hodgkin lymphoma, post-transplant lymphoproliferative disorder, Burkitt lymphoma, diffuse large B-cell lymphoma, gastric cancer, parotid gland cancer, breast cancer, leiomyosarcoma, and any combination thereof. In certain embodiments, EBV-associated cancer is not a post-transplant lymphoproliferative disorder.

[0041]

[0077] As used herein, “isolated” means a substance that has been removed from its natural state or otherwise subjected to human manipulation. An isolated substance may substantially or essentially lack components normally associated with it in its natural state, or it may be manipulated to be in an artificial state together with components normally associated with it in its natural state. An isolated substance may be in recombinant, chemically synthesized, concentrated, purified, or partially purified form.

[0042]

[0078] As used herein, “treating,” “treat,” or “treatment” refers to a therapeutic intervention that, after the onset of symptoms or pathological signs of an EBV-related disease, disorder, or condition, improves, eliminates, or reduces them at least partially. The treatment does not necessarily have to be absolutely beneficial to the subject.

[0043]

[0079] As used herein, “preventing,” “prevent,” or “prevention” refers to a set of actions initiated before infection with EBV or its molecular components, or before exposure to EBV or its molecular components, and / or before the onset of symptoms or pathological signs of an EBV-related disease, disorder, or condition, in order to prevent and / or reduce symptoms or pathological signs of the disease or pathology. Such prevention should be understood not to be absolutely or completely beneficial to the subject.

[0044]

[0080] The term “therapeutic dose” refers to the amount of an specified agent, such as EBV-specific allogeneic T cells or a therapeutic agent, that is sufficient to achieve the desired effect in a subject being treated with the agent. For example, this may be the amount of a composition comprising a first population of allogeneic T cells, a second population of allogeneic T cells, and / or a therapeutic agent described herein, that is necessary to reduce, alleviate and / or prevent EBV-related diseases, disorders, or conditions, including EBV-related cancer, cancer metastasis, and recurrence. In some embodiments, the “therapeutic dose” is sufficient to reduce or eliminate the symptoms of an EBV-related disease, disorder, or condition. In other embodiments, the “therapeutic dose” is sufficient to achieve the desired biological effect, for example, an amount that is effective in reducing or preventing EBV-related cancer growth, recurrence, and / or metastasis.

[0045]

[0081] Ideally, the therapeutically effective dose of a drug is sufficient to induce the desired outcome without causing substantial cytotoxic effects on the target. The effective dose of a drug useful for reducing, alleviating, and / or preventing EBV-related diseases, disorders, or conditions will depend on the target being treated, the type and severity of any related disease, disorder, and / or condition (e.g., type of EBV-related disease, disorder, or condition), and the mode of administration of the therapeutic composition.

[0046]

[0082] It will be understood that the methods of this embodiment may include one or more further treatments, such as cancer treatment, in addition to those listed above. Such treatments may include, but are not limited to, drug therapy, chemotherapy, antibody, nucleic acid and other biomolecular therapies, radiotherapy, surgery, nutritional therapy, relaxation or meditation therapy, and other natural or holistic therapies. Generally, drugs, biomolecules (e.g., antibodies, inhibitory nucleic acids such as siRNA) or chemotherapeutic agents are referred to herein as “anti-cancer agents” or “anti-cancer agents.”

[0047]

[0083] "Administering" or "administration" means the introduction of allogeneic T cells and / or therapeutic agents or compositions disclosed herein into an animal subject via a specific selected route.

[0048]

[0084] Administration of allogeneic T cells and / or therapeutic agents, or compositions comprising allogeneic T cells and / or therapeutic agents, may be by any known parenteral, topical, or enteral route, including but not limited to intravenous, intramuscular, intraperitoneal, intracranial, transdermal, oral, intranasal, anal, and intraocular routes.

[0049]

[0085] Dosage forms include tablets, dispersants, suspensions, injections, solutions, syrups, lozenges, capsules, suppositories, aerosols, and transdermal patches. These dosage forms may also include injectable or implantable controlled-release devices specifically designed for this purpose, or other forms of implants modified to operate in this manner. Controlled release of therapeutic agents can be achieved by coating the therapeutic agent with hydrophobic polymers, including, for example, acrylic resins, waxes, higher aliphatic alcohols, polylactic acid and polyglycolic acid, and certain cellulose derivatives such as hydroxypropyl methylcellulose. Furthermore, controlled release can be achieved by using other polymer matrices, liposomes, and / or microspheres.

[0050]

[0086] The compositions of the present invention, suitable for oral or parenteral administration, may be presented as individual units such as capsules, pouches, or tablets, each containing a predetermined amount of one or more therapeutic agents of the present invention, as powders or granules, or as solutions or suspensions in aqueous or non-aqueous liquids, oil-in-water emulsions, or water-in-oil liquid emulsions. Such compositions can be prepared by any pharmaceutical method, all of which involve the step of associating one or more agents as described above with carriers constituting one or more required components. Generally, compositions are prepared by uniformly and tightly mixing the agents of the present invention with a liquid carrier or a fine powder solid carrier or both, and then, if necessary, forming the product into the desired form.

[0051]

[0087] The allogeneic T cells, therapeutic agents, and compositions described herein may be administered in a manner compatible with the administered formulation and in a pharmaceutically effective amount. The dose administered to the patient should, in the context of the present invention, be sufficient to produce a beneficial response in the patient over a suitable period of time. The amount of the drug(s) to be administered may depend on the subject being treated, including factors that will depend on the subject's age, sex, weight, and overall health, as well as the judgment of the practicing physician.

[0052]

[0088] In certain embodiments, the methods for treating EBV-related diseases, disorders, or conditions described herein include administering a first and / or second population of allogeneic T cells and / or a therapeutic agent to a subject at least twice (e.g., two, three, four, five, six times, etc.). Such doses may be administered regularly, for example, daily, weekly, bi-weekly, monthly, etc., as needed.

[0053]

[0089] One particular broad application of the present invention is to provide a method for performing cellular immunotherapy or adoptive immunotherapy in subjects having EBV-related diseases, disorders, or conditions, such as those described herein, comprising the step of administering a therapeutically effective amount of allogeneic T cells and optionally pharmaceutically acceptable carriers, diluents, or excipients to the subject.

[0054]

[0090] The terms "cellular immunotherapy" or "adoptive immunotherapy" refer to the introduction of immune cells, such as T cells, for the treatment of cancer or infectious diseases (see, for example, June, CH., ed., 2001, In: Cancer Chemotherapy and Biotherapy: Principles and Practice; Lippincott Williams & Wilkins, Baltimore; Vonderheide et al., 2003, Immun. Research 27: pp. 1-15). For this purpose, adoptive immunotherapy will be understood as a strategy typically aimed at replacing, repairing, or enhancing the biological function of a tissue or system, such as the immune system, using autologous or allogeneic cells, such as T cells.

[0055]

[0091] As used herein, the term “allogeneic” refers to cells or tissues, such as T cells, that originate from individuals of the same species but are genetically different and therefore generally immunologically incompatible. Thus, the term “allogeneic cells” refers to cell types that are antigenically different but still belong to the same species. Typically, the term “allogeneic” is used to define cells, such as T cells, that are transplanted from a donor to a recipient of the same species.

[0056]

[0092] As used herein, the term “T cell” (i.e., T lymphocyte) is intended to include all cells within the T cell lineage, including mammalian (e.g., human) thymocytes, immature T cells, mature T cells, etc. Various T cell populations, such as helper T cells, regulatory T cells, cytotoxic T cells, natural killer T cells, and memory T cells, can be defined based on their cytokine profiles and their functions. Preferably, T cells are mature T cells expressing either CD4 or CD8, but not both, and a T cell receptor. The T cell receptor (TCR) will be understood to be a molecule found on the surface of a T cell that is involved in recognizing antigenic peptides bound to MHC or HLA molecules. Appropriately, allogeneic T cells include CD4+ helper T cells and / or CD8+ cytotoxic T cells. In this regard, allogeneic T cells as described herein may be in a mixed population of CD4+ helper T cells / CD8+ cytotoxic T cells.

[0057]

[0093] According to the present invention, a population of allogeneic T cells, including EBV-specific T cells, for example, a first and / or second population of allogeneic T cells, is administered to a human patient. The population of allogeneic T cells administered to the human patient is appropriately constrained by an HLA allele shared with EBV-positive cells of an EBV-related disease, disorder, or condition. In one particular embodiment, both the first population of allogeneic T cells and the cells of an EBV-related disease, disorder, or condition share a first human leukocyte antigen (HLA) allele encoding a first MHC protein, or are constrained by said HLA allele. In another embodiment, both the second population of allogeneic T cells and the cells of an EBV-related disease, disorder, or condition contain a second HLA allele encoding a second MHC protein, or are constrained by said HLA allele. In some embodiments, this HLA allele constraint is ensured by confirming the HLA assignment of cells of EBV-related disease, disorder, or condition, such as cancer cells, and selecting a population of allogeneic T cells that includes EBV-specific T cells (or T cell lines from which a population of allogeneic T cells is derived) that are constrained by the HLA alleles of such cells. The HLA assignment (i.e., HLA locus type) can be confirmed (i.e., typed) by any method known in the art. Non-limiting exemplary methods for confirming HLA assignment can be found in the ASHI Laboratory Manual, Edition 4.2 (2003), which is incorporated herein by reference.

[0058]

[0094] In certain embodiments, the first and / or second populations of allogeneic T cells share one or more HLA alleles (e.g., one, two, three, four, five, six, seven, or eight HLA alleles) with EBV-positive cells of EBV-related disease, disorder, or condition. In this regard, it is assumed that the first and second populations of allogeneic T cells may share one or more of the same HLA alleles with cells of EBV-related disease, disorder, or condition. In fact, in certain embodiments, the first population of allogeneic T cells shares one or more HLA alleles (e.g., one, two, three, four, five, six, seven, or eight HLA alleles) with the second population of allogeneic T cells. Ideally, the first population of allogeneic T cells appropriately includes one or more HLA alleles, e.g., the first HLA allele, that are shared with cells of EBV-related disease, disorder, or condition, and are not shared (i.e., different) with the HLA alleles of the second population of allogeneic T cells, e.g., the second HLA allele. Similarly, the second population of allogeneic T cells appropriately includes one or more HLA alleles, e.g., the second HLA allele, that are shared with cells of EBV-related disease, disorder, or condition, and are not shared (i.e., different) with the HLA alleles included by the first population of allogeneic T cells, e.g., the first HLA allele. For this purpose, the first and second populations of allogeneic T cells preferably do not have or include the same or identical complements of HLA alleles. Furthermore, the first population of allogeneic T cells does not appropriately recognize or bind to the second epitope, and / or the second population of allogeneic T cells does not appropriately recognize or bind to the first epitope.

[0059]

[0095] As used herein, the term “Major Histocompatibility Complex” (MHC) refers to an antigen-presenting molecule, protein, or polypeptide that functions as part of the immune system to bind antigens and other peptide fragments and present them on the cell surface for recognition by antigen-recognition molecules such as TCRs. When used in reference to human MHC, MHC may be used interchangeably with the term “Human Leukocyte Antigen” (HLA); therefore, MHC refers to all HLA subtypes, including the classical MHC alleles or the genes disclosed herein, in addition to any variants, isoforms, isotypes, and other bioequivalents thereof: HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-DM, HLA-DO, HLA-DP, HLA-DQ, and HLA-DR. MHC class I (MHC-I) and MHC class II (MHC-II) molecules utilize different antigen processing pathways. Generally, peptides derived from intracellular antigens are presented to CD8+ T cells by MHC class I molecules, which are expressed in virtually all cells, while peptides derived from extracellular antigens are presented to CD4+ T cells by MHC-II molecules. However, several exceptions to this general principle have been observed.

[0060]

[0096] In certain embodiments disclosed herein, specific EBV-specific antigens, peptides, and / or epitopes are identified and presented in antigen-MHC complexes in the context of MHC class I or II proteins appropriate for cells with EBV-related disease, disorder, or condition. For example, a first MHC protein may appropriately present a first epitope of the EBV antigen to cells with EBV-related disease, disorder, or condition for recognition by a first population of allogeneic T cells, while a second MHC protein may present a second epitope of the EBV antigen or further EBV antigen to cells with EBV-related disease, disorder, or condition for recognition by a second population of allogeneic T cells. From this perspective, the genetic structures of allogeneic T cells described herein may be evaluated to determine which HLA / MHC alleles are appropriate for a particular set of EBV antigens and / or EBV-related disease, disorder, or condition.

[0061]

[0097] Appropriately, the EBV antigen and / or further EBV antigens may be any known in the Art. Exemplary EBV antigens include the proteins EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, and LMP2. In certain embodiments, the EBV antigen and / or further EBV antigens are EBNA1, LMP1, and / or LMP2, or comprise EBNA1, LMP1, and / or LMP2.

[0062]

[0098] The allogeneic T cells described herein appropriately possess antigen specificity to EBV antigens and / or further EBV antigens. When used herein, the terms “antigen specificity” and “induces antigen-specific response” mean that the allogeneic T cells specifically bind to an antigen and are immunologically recognizable such that the binding of the allogeneic T cells to the antigen induces an immune response. By inducing an antigen-specific response to EBV-positive cells of EBV-related disease, disorder, or condition, without being bound by any particular theory or mechanism, the EBV-specific allogeneic T cells described herein are thought to provide one or more of the following: targeting and destruction of EBV-positive cells, e.g., EBV-positive cancer cells; reduction or elimination of cancer cells; promotion of immune cell infiltration into tumor sites; and enhancement / expansion of anti-cancer responses.

[0063]

[0099] As commonly used herein, “epitope” is an antigen protein fragment comprising a continuous or discontinuous sequence of amino acids in a protein, and an epitope can be recognized or bound by an element of the immune system, such as an antibody or other antigen receptor, such as an MHC protein. It will be understood by those skilled in the art that most EBV antigens may have multiple epitopes or antigenic determinants.

[0064]

[0100] From the above perspective, the first epitope may be an antigenic protein fragment of the EBV protein, but the second epitope is appropriately an antigenic protein fragment different from the same EBV protein from which the first epitope originates, or a further EBV protein.

[0101] As used herein, “protein” refers to an amino acid polymer, and amino acids may include D-amino acids, L-amino acids, and natural and / or unnatural amino acids. As typically used herein, “peptide” refers to a protein containing 60 or fewer consecutive amino acids. As typically used herein, “polypeptide” refers to a protein containing more than 60 consecutive amino acids. The term “protein” should also be understood, though not limited to, protein-containing molecules such as glycoproteins and lipoproteins.

[0065]

[0102] In some embodiments, the allogeneic T cells and / or therapeutic agents described herein (including combinations thereof) may be administered to a subject in the form of a composition comprising a pharmaceutically acceptable carrier, diluent, or excipient.

[0066]

[0103] It will be understood that pharmaceutically acceptable carriers, diluents and / or excipients may include any solid, semi-solid, gel or liquid fillers, diluents or encapsulating materials that can be safely used in systemic administration. Depending on the specific route of administration, carriers, diluents and / or excipients may be selected from, but are not limited to, sugars, starches, cellulose and their derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, arginine, isotonic salines, pyrogen-free water, wetting or emulsifying agents, bulk agents, flow enhancers, coating agents (e.g., enteric coating agents), emollients, binders, fillers, disintegrants, lubricants, pH buffers (e.g., phosphate buffers) and / or flavoring agents. The composition may be administered to humans in any one or more dosage forms, including tablets, dispersants, suspensions, injectable solutions, syrups, lozenges, capsules, suppositories, aerosols, transdermal patches, etc.

[0067]

[0104] A useful reference for describing pharmaceutically acceptable carriers, diluents, and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co., NJUSA, 1991), which is incorporated herein by reference.

[0068]

[0105] In certain embodiments, a second population of allogeneic T cells is administered (i) before; (ii) after; or (iii) concurrently with the administration of a first population of allogeneic T cells. In one embodiment, the administration of a first population of allogeneic T cells and the administration of a second population of allogeneic T cells (either sequentially or concurrently) results in the treatment or prevention of EBV-related disease, disorder, or condition that surpasses such treatment or prevention from the administration of either the first or second population of allogeneic T cells in the absence of the other.

[0069]

[0106] In certain embodiments, the method described above further includes the first step of generating a first and / or second population of allogeneic T cells in vitro. The first and second populations of allogeneic T cells, including EBV-specific T cells, to be administered to a human patient may be generated by methods known in the Art, or selected from an existing bank (collection) of cryopreserved T cell lines (each T cell line containing EBV-specific T cells) generated by methods known in the Art, and may be thawed and preferably expanded before administration.

[0070]

[0107] In certain embodiments, the step of generating a population of allogeneic T cells in vitro includes sensitizing (i.e., stimulating) allogeneic T cells to produce EBV-specific T cells with one or more EBV antigens. The allogeneic T cells used to generate a population of allogeneic T cells in vitro can be isolated from an allogeneic T cell donor by any method known in the art. In certain embodiments, allogeneic T cells are enriched from peripheral blood lymphocytes isolated from PBMCs of an allogeneic T cell donor.

[0071]

[0108] In certain embodiments, the step of sensitizing allogeneic T cells includes loading or transforming antigen-presenting cells, such as dendritic cells, cytokine-activated monocytes, or peripheral blood mononuclear cells, with at least one immunogenic peptide derived from one or more EBV antigens. For this purpose, antigen-presenting cells may be loaded or transformed with, for example, a pool of polytopes containing overlapping peptides derived from one or more EBV antigens. In one particular embodiment, the step of generating a population of allogeneic T cells in vitro includes sensitizing allogeneic T cells using peripheral blood mononuclear cells.

[0072]

[0109] Appropriately, the method described above includes a further step of administering the therapeutic agent to the subject. Similarly, the composition described above may further contain a therapeutic agent. As used herein, the term “therapeutic agent” means a compound or molecule used to image, affect, treat, address, prevent or improve an undesirable condition or disease in a subject, such as an EBV-related disease, disorder or condition.

[0073]

[0110] The therapeutic agent may be any known in the art. In some embodiments, the therapeutic agent is an anti-cancer treatment or anti-cancer agent, or comprises an anti-cancer treatment or anti-cancer agent. Generally, drugs, biomolecules (e.g., antibodies, inhibitory nucleic acids, e.g., siRNA) or chemotherapeutic agents are referred to herein as “anti-cancer therapeutic agents.” These are just a few examples, but include: chemotherapeutic agents, e.g., paclitaxel, doxorubicin, methotrexate, irinotecan, dacarbazine, temozolomide, and cisplatin; biotherapeutic agents or immunotherapeutic agents, e.g., anti-PD-1 antibodies (e.g., nivolumab) and anti-CTLA4 antibodies (e.g., ipilimumab); and / or molecularly targeted agents, e.g., MAPK pathway (i.e., Ras-Raf-MEK-ERK signaling) inhibitors and BET inhibitors.

[0074]

[0111] In certain embodiments, the therapeutic agent is selected from the group consisting of immunotherapeutic agents, mitogen-activated protein kinase (MAPK) pathway inhibitors, BET inhibitors, and any combination thereof.

[0075]

[0112] As used herein, the term “immunotherapy agent” refers to any agent capable of inducing, enhancing, or suppressing an immune response in a subject. In certain embodiments, the immunotherapy agent may be an immune checkpoint modulator. As used herein, the term “immune checkpoint modulator” refers to a molecule capable of completely or partially reducing, inhibiting, interfering with, or modulating one or more immune checkpoint proteins that control T cell activation or function. In certain embodiments, the immune checkpoint modulator is an immune checkpoint inhibitor.

[0076]

[0113] Non-exclusive examples of immune checkpoint proteins include cytotoxic T lymphocyte-associated antigens (CTLAs; e.g., CTLA4) and their ligands CD80 and CD86; programmed cell death proteins (PDs; e.g., PD-1) and their ligands and PDL2; indoleamine-pyrrole 2,3-dioxygenase-1 (ID01); T cell membrane proteins (TIMs; e.g., TIM3); adenosine A2a receptors (A2aR); lymphocyte activating genes (LAGs; e.g., LAG3); killer immunoglobulin receptors (KIRs); and CD96. These proteins are typically understood to be involved in co-stimulatory or inhibitory T cell responses. Immune checkpoint proteins can broadly regulate and maintain self-tolerance and the duration and magnitude of physiological immune responses.

[0077]

[0114] In certain embodiments, the immune checkpoint modulator (e.g., an immune checkpoint inhibitor) may be a small molecule, antibody, recombinant binding protein, or peptide that binds to or inhibits the biological activity of an immune checkpoint protein.

[0078]

[0115] Non-limiting examples of immune checkpoint modulators (e.g., immune checkpoint inhibitors) include CTLA4 inhibitors (e.g., ipilimumab), PD1 inhibitors (e.g., nivolumab), PDL1 inhibitors (e.g., atezolizumab, avelumab, durvalumab), LAG3 inhibitors, KIR inhibitors, B7-H3 ligands, B7-H4 ligands, CD96 inhibitors, and TIM3 inhibitors. In certain embodiments, the immune checkpoint inhibitor is selected from the group consisting of anti-PD1 antibodies, anti-PDL1 antibodies, anti-CTLA4 antibodies, anti-LAG3 antibodies, anti-TIM3 antibodies, anti-CD96 antibodies, and any combination thereof.

[0079]

[0116] In one particular embodiment, the immune checkpoint inhibitor is a PD1 inhibitor or comprises a PD1 inhibitor, more specifically an anti-PD1 antibody or comprises an anti-PD1 antibody. Exemplary PD1 inhibitors and anti-PD1 antibodies include pembrolizumab, nivolumab, semiprimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, AMP-224, and AMP-514.

[0080]

[0117] As used herein, “antibody” is either an immunoglobulin protein (including its fragments) or comprises an immunoglobulin protein (including its fragments). The term “immunoglobulin” includes any antigen-binding protein product of the mammalian immunoglobulin gene complex, including immunoglobulin isotypes IgA, IgD, IgM, IgG, and IgE and their antigen-binding fragments. The term “immunoglobulin” includes recombinant, chimeric, or humanized immunoglobulins, whether naturally occurring or produced by human intervention (e.g., recombinant DNA technology), or immunoglobulins comprising modified or variant amino acid residues, sequences, and / or glycosylation.

[0081]

[0118] The present invention also includes antibody fragments, such as Fc, Fab, or F(ab)2 fragments, or single-chain Fv antibodies (scFv). The present invention is also intended to include polyvalent recombinant antibody fragments containing multiple scFv, so-called diabodies, triabodies, and / or tetrabodies, as well as dimerized activated demibodies (e.g., International Publication No. 2007 / 062466). For example, such antibodies can be prepared according to the methods described by Holliger et al., 1993 Proc Natl Acad Sci USA 90:6444-6448; or Kipriyanov, 2009 Methods Mol Biol 562:177-93, which are incorporated herein by reference in their entirety.

[0082]

[0119] Generally, antibodies and antibody fragments may be polyclonal or monoclonal. It is also understood that antibodies can be produced as recombinant synthetic antibodies or antibody fragments by expressing nucleic acids encoding the antibody or antibody fragment in a suitable host cell. Non-limiting examples of recombinant antibody expression and selection methods, including phage display methods, are provided in Chapter 17 of *Current Protocols in Immunology* by Coligan et al., and in *Protein Engineering*, *Design & Selection*, 22, p. 169, 2009.

[0083]

[0120] As used herein, the term “MAPK inhibitor” refers to any compound or chemical substance that, when administered to a subject, results in the inhibition of the MAPK pathway in one or more cells of that subject, such as cancer cells. MAPK inhibitors include, but are not limited to, low molecular weight inhibitors, antibodies or antibody fragments, antisense constructs, small inhibitory RNAs (i.e., RNA interference by dsRNA; RNAi), and ribozymes. In some embodiments, the MAPK inhibitor is a small organic molecule. Examples of MAPK inhibitors include RAS inhibitors, RAF inhibitors, MEK inhibitors, ERK inhibitors, JNK inhibitors, and / or p38 inhibitors.

[0084]

[0121] MAPK pathway inhibitors may be any known in the art, including specific inhibitors of Ras (i.e., HRas, KRas, and / or NRas), Raf (i.e., A-Raf, B-Raf, and / or C-Raf), mitogen-activated protein kinase kinases (i.e., MEK1 / 2), and / or extracellular signal-regulated kinases (i.e., ERK1 / 2) (including their variant variants) function and / or signal transduction. For example, such MAPK pathway inhibitors may be selected from the following:

[0122] i) MEK inhibitors: AZD6244, R04987655, R05126766, TAK-733, MSC1936369B (AS703026), GSK1 120212, BAY86-9766, GDC-0973, GDC-0623, PD325901, ARRY-438162, CM 040, E6201, ARRY300;

[0123] ii) Raf and / or BRaf selective inhibitors: PLX4032, GSK21 18436, sorafenib (BAY-43-9006), BMS-908662 (XL-281), RAF265, RG-7256 (RO5212054, PLX3603), R05126766, ARQ-736, E-3810, DCC-2036;

[0124] iii) ERK inhibitors: Urixertinib (BVD-523), SCH772984, DEL-22379, MK-8353 (SCH900353), AZD0364, VX-11e, CC-90003;

[0125] iv) Ras inhibitors: MCI-062, saliracib, BAY 293, ARS-1620.

[0085]

[0126] It is assumed that the BET inhibitor may be any known in the art. As used herein, the term “BET inhibitor” refers to a compound that binds to BET and inhibits and / or reduces the biological activity of BET. In some embodiments, the BET inhibitor substantially or completely inhibits the biological activity of BET. In some embodiments, the biological activity is the binding of BET to chromatin (e.g., DNA-related histones) and / or another acetylated protein. Suitablely, the BET inhibitor inhibits one or more of BRD2, BRD3, BRD4, and BRDT. Examples of BET inhibitors include modulators of bromodomain-containing proteins, e.g., benzimidazole derivatives disclosed in U.S. Publication No. 2014 / 0336190. Examples of BET inhibitors include I-BET 151 (GSK1210151A), I-BET 762 (GSK525762), OTX-015, TEN-010, CPI-203, CPI-0610, olinone, RVX-208, LY294002, AZD5153, MT-1, and MS645.

[0086]

[0127] In another aspect, the present invention relates to a method for treating or preventing an EBV-related disease, disorder, or condition in a subject:

[0128] (a) A step of administering to a population of allogeneic T cells that bind to or recognize the EBV antigen epitope; and

[0129] (b) A step in which the therapeutic agent is administered to a target, selected from the group consisting of immunotherapeutic agents, MAPK pathway inhibitors, BET inhibitors, and any combination thereof. Includes,

[0130] The present invention relates to methods for treating or preventing EBV-related diseases, disorders, or conditions in subjects.

[0087]

[0131] In related embodiments, the present invention relates to a pharmaceutical composition for treating or preventing EBV-related diseases, disorders, or conditions in a subject:

[0132] A population of allogeneic T cells that bind to or recognize the EBV antigen epitope;

[0133] A therapeutic agent selected from the group consisting of immunotherapeutic agents, MAPK pathway inhibitors, BET inhibitors, and any combination thereof;

[0134] Optionally, a pharmaceutically acceptable carrier, diluent and / or excipient. The composition contains [this].

[0088]

[0135] "Pharmacologically acceptable carrier" means a pharmaceutical vehicle consisting of a substance that is not biologically or otherwise undesirable. That is, the substance can be administered to a subject together with a selected activator without causing any or substantial adverse reactions. Carriers may include excipients and other additives, such as diluents, surfactants, colorants, wetting agents or emulsifiers, pH buffers, preservatives, and transfection agents.

[0089]

[0136] Similarly, the “pharmaceutically acceptable” salts, esters, amides, prodrugs, or derivatives of the compounds provided herein are salts, esters, amides, prodrugs, or derivatives that are not biologically or otherwise undesirable.

[0090]

[0137] The following description applies equally to the two aforementioned aspects.

[0091]

[0138] Appropriately, a population of allogeneic T cells and cells of EBV-related disease, disorder, or condition share human leukocyte antigen (HLA) alleles encoding MHC proteins. In certain embodiments, the MHC proteins present epitopes of the EBV antigen to cells of EBV-related disease, disorder, or condition.

[0092]

[0139] The immunotherapy agent, MAPK pathway inhibitor, and / or BET inhibitor may be any known in the art, such as those described above. In certain embodiments, the MAPK pathway inhibitor is or comprises a MEK1 / 2 inhibitor. In other embodiments, the immunotherapy agent is or comprises an immune checkpoint inhibitor, such as an anti-PD1 antibody.

[0093]

[0140] Ideally, the population of allogeneic T cells is administered before, simultaneously with, and / or after the administration of the therapeutic agent. Therefore, in certain embodiments, the subject is administered the therapeutic agent, followed by the administration of allogeneic T cells. In alternative embodiments, the individual is administered allogeneic T cells, followed by the administration of the therapeutic agent. In further alternative embodiments, the allogeneic T cells are administered simultaneously with the therapeutic agent.

[0094]

[0141] In one embodiment, the method of this embodiment further includes a first step of generating a population of allogeneic T cells in vitro, for example, by the methods described above.

[0095]

[0142] Appropriately, the EBV antigen and / or additional EBV antigens are selected from the group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2 and any combination thereof. More specifically, the EBV antigen and / or additional EBV antigens are appropriately EBNA1, LMP1 and / or LMP2, or include EBNA1, LMP1 and / or LMP2.

[0096]

[0143] Appropriately, EBV-related disease, disorder, or condition is or includes EBV-related cancers such as those described above. In one embodiment, EBV-related cancer is selected from the group consisting of nasopharyngeal carcinoma, NKT cell lymphoma, Hodgkin lymphoma, post-transplant lymphoproliferative disorder, Burkitt lymphoma, diffuse large B-cell lymphoma, gastric cancer, and any combination thereof.

[0097]

[0144] In yet another aspect, the present invention relates to the use of a first population of allogeneic T cells that bind to or recognize a first epitope of EBV antigen, such as those described herein, in the manufacture of a pharmaceutical product for the treatment or prevention of EBV-related disease, disorder or condition in a subject; the first population of allogeneic T cells is: (a) a second population of allogeneic T cells that bind to or recognize a second epitope of EBV antigen or further EBV antigen, such as those described herein; and / or (b) a therapeutic agent administered in combination with a therapeutic agent selected from the group consisting of immunotherapeutic agents, MAPK pathway inhibitors, BET inhibitors and any combination thereof, such as those described herein.

[0098]

[0145] In the final aspect, the present invention provides a first population of allogeneic T cells that bind to or recognize a first epitope of the EBV antigen, such as those described herein, for use in the treatment or prevention of EBV-related diseases, disorders or conditions in a subject; the first population of allogeneic T cells is: (a) a second population of allogeneic T cells that bind to or recognize a second epitope of the EBV antigen or a further EBV antigen, such as those described herein; and / or (b) a therapeutic agent administered in combination with a therapeutic agent selected from the group consisting of immunotherapeutic agents, MAPK pathway inhibitors, BET inhibitors and any combination thereof, such as those described herein.

[0099]

[0146] In the embodiments described above, the term "subject" includes, but is not limited to, mammals, including humans, performance animals (such as horses, camels, and greyhounds), livestock (such as cattle, sheep, and horses), and companion animals (such as cattle and dogs). In one particular embodiment, the subject is human.

[0100]

[0147] See the following non-limiting examples for details on preferred embodiments that can be practically implemented. [Examples]

[0101] Example 1

[0148] Allogeneic "off-the-shelf" T-cell therapy has emerged as a powerful tool for treating infectious complications in transplant recipients. These allogeneic antigen-specific T cells can be expanded from peripheral blood lymphocytes collected from a large panel of healthy donors offering a diverse HLA applicability, cryopreserved, and administered to HLA-matched transplant patients in need. In this embodiment, we provide a preclinical evaluation of allogeneic EBV-specific T cells as a therapeutic tool for the treatment of multiple cancers. Furthermore, we have shown that the combination of allogeneic antigen-specific T cells and antibodies blocking the PD1 / PD-L1 axis significantly improves the efficacy of adoptive T-cell therapy for EBV cancer.

[0102] Materials and methods cell culture

[0149] The EBV-associated cell lines used in this study were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, USA), cultured, and maintained according to ATCC recommendations. Table 1 lists each EBV-associated cell line used in the study and their respective HLAs. Cultures of these cell lines were incubated and maintained at 37°C, 20% oxygen, and 5% CO2. All tissue culture plasticware was purchased from Corning® Stone, Staffordshire, UK (flasks and plates) and Costar® Washington, DC, USA (plastic pipettes). All cell lines were regularly tested for mycoplasma infection and validated using short tandem repeat (STR) profiling by the scientific team at QIMR Berghofer Medical Research Institute.

[0103] RNA extraction and quantitative real-time PCR

[0150] RNA was extracted from each cell line using the QIAgen RNeasy® kit (Valencia, CA, USA) according to the manufacturing instructions. 1 × 10 6Each cell line was plated, recovered using trypsin-EDTA (Sigma Aldrich®), washed (PBS, twice), and then an appropriate volume of RLT buffer, 4°C (supplied in the kit) was added, followed by the subsequent steps as indicated by the manufacturer. After RNA extraction, the DNAse digestion step was performed using the DNAse enzyme provided in the iScript® cDNA kit (Bio-Rad Laboratories Inc.). RNA quality and quantity were obtained using an ND-1000 spectrophotometer (Thermo-Scientific). Reverse transcription was performed using iScript® Reverse Transcriptase (Bio-Rad Laboratories Inc.) according to the manufacturer's instructions. The cycle conditions used were: priming at 25°C for 5 minutes, reverse transcription at 46°C for 20 minutes, and reverse transcriptase activation at 95°C for 1 minute. qRT-PCR was performed in a 384-well plate using a Biorad CFX384 Touch® real-time PCR detection system. The primers consisted of EBV-related genes LMP1, LMP2, and EBNA1 obtained from their respective publications. The master mix composition in a total volume of 10 μL was: 5 μL of Sybr green, 1 mM of each primer, 1 μL of diluted cDNA, and 3 μL of H2O, for three double-acted biological replicas. The cycle conditions used were: 95°C for 5 minutes, followed by 40 cycles of: 95°C for 10 seconds, 60°C for 10 seconds, and 72°C for 5 seconds, and a final extension step of 72°C for 5 minutes. t The values ​​were calculated using the attached Biorad CFX384 software, version 1.5.0.39, and then normalized to 18sRNA and HPRT to obtain ΔΔC. tThe calculations were performed using the specified method. For each biological cDNA sample analysis, a negative control containing a cDNA solution not treated with reverse transcriptase was used to ensure no genomic DNA contamination. In addition, a standard negative control containing only H2O was included in each primer set. Primers are LMP1:FP-5'-CAGTCAGGCAAGCCTATGA3', RP-5'CTGGTTCCGGTGGAGATGA3';LMP2:5'-AGCTGTAACTGTGGTTTCCATGAC-3 ', RP-5'-GCCCCCTGGCGAAGAG-3';EBNA1:FP-5'-TACAGGACCTGGAAATGGCC-3', RP-5'-TCTTTGAGGTCCACTGCCG-3';HPRT1:FP- 5'-CCTGGCGTCGTGATTAGTGAT-3', RP-5'-AGACGTTCAGTCCTGTCCATAA-3';18sRNA:5'-CGAAAGCATTTACCAAGGAC-3', RP-5'-TTATTGTGTCTGGACCTGG-3'.

[0104] T cell generation

[0151] To generate an LMP / EBNA1-specific "ready-made" T cell bank, peripheral blood mononuclear cells (PBMCs) were collected from 100–300 mL of venous blood from seropositive donors to cover a broad HLA spectrum. Thirty percent of the PBMCs were then infected with the AdE1-LMPpoly vector [11, 12], consisting of 16 HLA-restricted LMP1 and 2-epitope polyepitopes fused to a truncated gly / ala-deleted EBNA1 gene (MOI of 10:1). These transfected PBMCs were then irradiated and co-cultured with the remaining PBMCs for two weeks. The cultures were supplemented with fresh growth medium and 120 IU / mL recombinant IL-2 every 3–4 days (Komtur Pharmaceuticals). Expanded T cells were tested for antigen specificity and microbial contamination before release for infusion. Each T cell used to target HLA-matched EBV-associated cancer cell lines is listed in Table 1.

[0105] Intracellular cytokine assay

[0152] To analyze the frequency of LMP1 and 2- and EBNA1 specificity in AdE1-LMPpoly vector-transfected T cell products, the T cell products were stimulated for 4 hours in the presence of GolgiPlug (BD Biosciences) with a pool of distinct epitopes derived from LMP1 and 2- or EBNA1, or with a duplicate set of peptides encompassing the entire EBNA1 protein (all mimotope-derived, GenScript, or JPT Technologies). For multiparametric analysis, cells were stimulated for 4 hours in the presence of GolgiPlug and GolgiStop (BD Biosciences) with the peptides listed above and anti-CD107a-FITC (BD Biosciences). The cells were then washed, stained with anti-CD8-PerCPCy5.5 (eBioscience) and anti-CD4-PECy7 (BD Biosciences), fixed, permeabilized with Cytofix / Cytoperm (BD Biosciences), washed again, and stained with anti-IFN-γ-AF700 (all from BD Biosciences)

[11] . After further washing, the cells were resuspended in PBS and acquired using a BD LSR Fortessa (BD Biosciences) with FACSDiva software. Post-acquisition and Boolean analysis were performed using FlowJo software (TreeStar).

[0106] Cell viability assay

[0153] Cell viability assays were performed triplicately on three biological replicas for each EBV-associated cancer cell line using CellTiter 96® AQueous one-cell viability assay reagent (Promega, WI, USA). Briefly, cancer cells (target cells) were plated in 96-well tissue culture plates (BD Falcon®) with a total medium volume of 200 μL at a density of 5000 cells per well. Effector AdE1-LMPpoly-transfected T cells were freshly thawed at 37°C and 50% CO2 in RPMI-1640 containing 10% FCS and recombinant IL-2 120 IU / mL. After 24 hours of plating and incubation at 37°C and 50% CO2, effector T cells were mixed with target cells at effector-to-target (E:T) gradient ratios ranging from 5:1 to 100:1. T ) T cells used for each (T o The exact number of PBS-treated target cells (E s ) and a single medium (M0) were used independently as controls. After 24 hours of incubation at 37°C and 50% CO2, MTS was added to each well (diluted 1:100 in the medium), incubated for 1 hour, and then the plates were centrifuged at 1,200 × g at room temperature for 5 minutes. The absorbance of the mixture at an optical density of 490 nm was measured using a microplate reader. Relative cell viability was calculated using the following formula.

[0107]

number

[0108] Cytotoxic assay

[0154] The cytotoxicity assay was performed in triplicate for three biological replicates for each EBV-related cancer cell line using the CytoTox 96® Nonradioactive Cytotoxic Assay Kit (Promega, WI, USA)

[14] . Briefly, cancer cells (target cells) were plated at a density of 5000 cells per well in a 96-well tissue culture plate with a total medium volume of 200 μL, and conditions similar to those for the cell viability assay were maintained as previously described. Along with that, the inventors also seeded the exact number of target cells (E M ). After mixing the effector and target cells at 37 °C for 24 hours, 10× lysis buffer was added to the E M wells and incubated at 37 °C and 50% CO2 for 45 minutes. After complete lysis of the target cells, the plate was centrifuged at 1,200 × g for 5 minutes at room temperature, and 50 μl of the supernatant from each well was transferred to another plate. The assay buffer was mixed with the substrate mix and aliquoted into each well. After stopping with the stop solution, the absorbance of the mixture at an optical density of 490 nm was measured using a microplate reader. The relative lysis in the experimental and control wells was calculated as follows.

[0109]

Number

[0110] Multicolor profiling of cancer cell phenotypes

[0155] Each EBV-related cancer cell was seeded at 1 × 10 5Cells were plated at cell density and, after 24 hours, mixed with T cells in a 50:1 effector-versus-target (E:T) ratio and incubated at 37°C and 50% CO2 for 24 hours. To evaluate the effect of T cells on cancer cells, cells were then incubated at 4°C with the following antibodies: human anti-CD45-V500, anti-CD3-AF700, anti-Ki67-BV421, anti-BCL2-FITC, and anti-active caspase 3-BV605. Cells were acquired using a BD LSR Fortessa (BD Biosciences) with FACSDiva software, and post-acquisition analysis was performed using FlowJo software (TreeStar).

[0111] Multicolor profiling of AdE1-LMPpoly-transfected T cell phenotypes

[0156] Freshly thawed effector AdE1-LMPpoly-transfected T cells were used in a 50:1 effector-versus-target (E:T) ratio to target cells (1 × 10⁶). 5The cells were mixed with ) and incubated at 37°C and 50% CO2 for 24 hours. For evaluation of surface phenotype, the cells were then incubated at 4°C with the following antibody panel: (i) human anti-CD45-V500, anti-CD3-AF700, anti-CD4-PECy7 and anti-CD8-PerCPCy5.5; (ii) human anti-CD45-V450, anti-CD4-AF700, anti-CD8-PerCPCy5.5, anti-CD14-eFluor450, anti-CD19-eFluor450; anti-PD-1-BV786; (iii) MHC-class I antibody (clone W6 / 32) (homemade, produced in mice), LIVE / DEAD Fixable Near-IR Dead Cell Stain (Thermo Fisher Scientific, MA). For intracellular analysis, cells were treated with TF fixation / permeabilization buffer (BD Biosciences) and then stained in the presence of Perm / Wash containing the following antibodies: (i) anti-perforin-BV421, anti-granzyme B-AF700, and anti-granzyme K-FITC. Cells were acquired using a BD LSR Fortessa (BD Biosciences) with FACSDiva software, and post-acquisition analysis was performed using FlowJo software (TreeStar).

[0112] Animal breeding facilities

[0157] All animal experiments were approved by the QIMR Berghofer Medical Research Institute, Animal Ethics Committee (No. A0707-606M), and were conducted in strict accordance with the Australian Code of Animal Care and Use for Scientific Purposes. All experimental animals were maintained with a mixed strain (129SV / EX C57BL / 6) and housed in OptiMICE® casings (Centennial, Colorado, USA) at the Queensland Institute of Medical Research Animal Facility at 25°C with a 12-hour light-dark cycle. Dry granular diets were sterilized by irradiation. Mice were given free access to food and sterile water.

[0113] In vivo evaluation of the therapeutic efficacy of allogeneic EBV-specific T cells.

[0158] This study used a total of 12-24 female NOD / SCID mice (depending on the experiment) aged 7-8 weeks. The mice were irradiated with 0.8 Gy cobalt-60, and after 4 hours, 5 × 10⁻¹⁴ ray growths were observed. 6 Each EBV-associated cancer cell was subcutaneously injected using a 29-gauge needle. Mice were monitored three times a week for tumor growth, body weight, and physical score. Once the tumors became palpable, mice were randomized to one group and given either PBS or 20 × 10⁶ doses. 6 The mice were treated with respective doses of tumor HLA-matched allogeneic EBV-specific T cells. The tumor size of these mice was measured three times weekly using Vernier calipers. To calculate the tumor area, the following formula was used based on two-dimensional caliper measurements, as previously described

[13] : Tumor area = B × S (B = maximum tumor measurement, and S = minimum major measurement).

[0114] In vivo evaluation of the therapeutic efficacy of allogeneic EBV-specific T cells in a humanized mouse model.

[0159] Fresh human CD34+ umbilical cord blood cells were obtained from healthy full-term newborns after obtaining written parental consent and concentrated using immunomagnetic beads according to the manufacturer's instructions (CD34+ Selection Kit, Miltenyi Biotec, Bergisch-Glatbach, Germany). Female NRG mice aged 7-8 weeks were irradiated with 275 cGy twice, 3-4 hours apart, and then 5 × 10⁶ cells per mouse were administered. 4CD34+ cells (HLA matching AdE1-LMPpoly-transfected T cells used for treatment) were intravenously injected using a 29-gauge needle. Mice were monitored twice weekly for body weight, physical score, and adverse reactions, including graft-versus-host disease (GVHD). In addition, tail vein blood collection was performed at weeks 4, 8, 10, and 12, during which 100-200 μL of blood was collected from each mouse in EDTA tubes while monitoring the rearrangement of the human immune system. Surface phenotyping was performed using human anti-CD45-V500, mouse anti-CD45-V450, anti-CD3-APC, anti-CD4-AF700, anti-CD8-PerCPCy5.5, anti-CD8-PerCPCy5.5, anti-CD14-FITC, anti-CD19-PeCy5, anti-CD23-BV786, and anti-CD56-BV650 to evaluate rearrangement from human CD34+ umbilical cord blood cells. In the 12th week of reconstitution, humanized NRG mice were given EBV B95-8 in 100 μL of PBS. 6 EBV particles were administered intravenously using a 29-gauge needle under non-anesthetic conditions. On day 13 after EBV infection, mice were conditioned in PBS or 20 × 10⁶ 6 Mice were treated with either tumor-HLA-matched T cells or switched AdE1-LMPpoly-transfected T cells, each at their respective doses. The HLA of each umbilical cord blood cell and the corresponding T cells used to treat lymphoid malignancies are listed in Table 1. Mice were monitored for 14 days after T cell therapy, then sacrificed, and their pancreases were analyzed for tumor volume.

[0115] Immunohistochemical examination

[0160] For histological examination, tissue was collected, washed (three times in PBS), fixed with 4% formaldehyde in PBS, and stored in 70% ethanol before processing. The tissue was then embedded in paraffin blocks, and 5 μm thick sections were prepared for staining. The tissue was embedded in paraffin, and 4 μm sections were mounted on Superfrost Plus slides using Sakura Tissue-Tek® (Sakura Finetek, Tokyo, Japan). Immunohistochemical analysis was performed with the help of facilities at the QIMR Berghofer Medical Research Institute. Antigen was recovered using 2.94 g trisodium citrate in 1 L MQ (pH 6.0) buffer and microwaved. Tissue sections were permeabilized with 0.2% Triton X-100 / PBS for 5 minutes, followed by 0.05% Triton X-100 / PBS for 10 minutes. Tissue sections were treated with 3% (vol / vol) H2O2, then immunostained with 2% BSA anti-CD3 (1:40 Dako M7254) antibody, followed by secondary antibody (VEMP7402) Dako EnVision® (Agilent, system, Wokshaw, WI, USA), and counterstained with hematoxylin. Slides were scanned with Aperio® Scanscope® XT (Aperio®, Vista, USA) using a 20X or 40X objective.

[0116] Genetic signature profiling using NanoString and invasive profiling

[0161] Six 8-week-old female NOD / SCID mice were irradiated with 0.8 Gy Cobalt-60, and after 4 hours, 5 × 10⁻⁶ mice were exposed to the light. 6 SNU719 cells were subcutaneously injected using a 29-gauge needle. Tumor size: 40 mm 2 Once it reaches 20x10, move the mouse to 20x10 6The tumors were treated with TI_001 T cells. After 5 days, the tumors were harvested and FACS was used to sort the T cells for the viable CD8+ population using human anti-CD45-V500, mouse anti-CD45-V450, anti-CD3-APC, anti-CD4-PE, and anti-CD8-PerCPCy5.5. The sorted viable CD8 cells from individual mice were sorted using the Qiagen RNAeasy kit as described above. + RNA was isolated from the population. Gene expression analysis was performed using a customized 326-NanoString Immune gene expression panel. 50 ng of total RNA per mouse sample was used in a final volume of 5 μl and mixed with a 3' biotinylated capture probe along with a 5' reporter probe tagged with a fluorescent barcode from a custom gene expression code set. The probes and target transcripts were hybridized at 65°C for 12–16 hours. The hybridized samples were run on a NanoString nCounter preparation station using the recommended manufacturer protocol to remove excess capture and reporter probes, and the transcript-specific triple complexes were immobilized on streptavidin-coated cartridges. Samples were scanned at maximum scan resolution using an nCounter Digital Analyzer. Data were processed using nSolver Analysis Software and the nCounter Advanced Analysis module. Gene expression analysis data were normalized using the geometric mean of housekeeping genes selected by the GeNorm algorithm.

[0117] statistical analysis

[0162] Using GRAPHPAD PRISM v6.0 (GraphPAd Software, La Jolla, CA, USA), one-way or two-way ANOVA (as indicated in the figure caption) was performed using Student's t-test, Bonferroni post-hoc test, or Mann-Whitney U test, and p-values ​​were calculated as shown in the figure caption. Asterisks indicate statistical significance. * p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001), ns=not significant.

[0118] result In vitro recognition of multiple cancers by homogeneous "off-the-shelf" EBV-specific T cells.

[0163] To evaluate the expression of EBV coding genes (LMP1, LMP2, and EBNA1) in multiple EBV-related malignancies, including NPC, gastric cancer, NKT lymphoma, and BLCL, the inventors used qRT-PCR to analyze the transcription levels of each of these genes. The inventors observed that in all EBV-related malignancies, LMP2 and EBNA1 were consistently expressed at higher levels compared to LMP1 (Figure 1A). The inventors evaluated the sensitivity of these EBV-positive cancer cells to allogeneic "off-the-shelf" EBV-specific T cells specific to LMP1, LMP2, and / or EBNA1 (Figure 1B; Table 2). The data shown in Figures 1C-D demonstrate that gastric cancer (SNU719), NPC (C17 and C661), and NKT lymphoma (SNKT16) cells were efficiently recognized by allogeneic HLA-matched EBV-specific T cells at various effector-to-target ratios. Furthermore, the annexin V binding assay also showed increased annexin V binding ability, which induced targeted cell death, compared to mock-treated controls (Figure 1D-E).

[0119]

[0164] In the next set of experiments, the inventors analyzed the effect of allogeneic EBV-specific T cells on phenotypic changes in EBV-associated cancer cells. The inventors observed a significant decrease (p<0.001) in the proliferation rates of both gastric cancer, SNU719 cells, and NPC, C17 cells 24 hours after exposure to HLA-matched allogeneic EBV-specific T cells, as indicated by Ki67 staining (Figure 2A). Furthermore, in addition to a significant decrease (p<0.01) in BCL2 staining, the inventors also observed a significant increase (p<0.001) in active caspase 3 staining. This suggests an increase in cell death induced by EBV-specific T cells (Figure 2A). The inventors also investigated the effect of EBV-associated cancer cells on allogeneic EBV-specific T cells. The inventors observed a significant increase in the CD8+ T cell population in the presence of EBV-associated cancer cells (p<0.01) (Figure 2B). In line with this data, the inventors have found that when exposed to EBV-positive cancer cells, CD8 + A significant increase in Ki67 expression was observed in T cells (Figure 2B). Furthermore, these T cells also exhibited high levels of effector function, indicated by significantly increased expression of GzmB, GzmK, and Perf staining (Figure 2B). Overall, these data demonstrate that allogeneic EBV-specific T cells can efficiently recognize multiple types of HLA-matched EBV-associated cancer cells in vitro.

[0120] Evaluation of the in vivo therapeutic efficacy of allogeneic EBV-specific cytotoxic T cells.

[0165] Having established in vitro efficient recognition of multiple EBV-related cancers by allogeneic EBV-specific T cells, the inventors then evaluated the therapeutic efficacy of these effector cells in vivo. In the first set of experiments, immunodeficient NOD-SCID mice were inoculated (subcutaneously) with EBV-related NPC tumors, C17, and C666.1 after irradiation. The tumors in each animal were 25 mm. 2 When these animals reached a certain stage, they were treated with HLA-matched allogeneic EBV-specific T cells. These animals received single or double infusions of allogeneic HLA-matched EBV-specific T cells (2 × 10⁶ infusions per animal). 7The patients were treated with either allogeneic HLA A2 / B40-restricted or HLA A11 / B58-restricted LMP2-specific T cells (2 × 10⁶). The data shown in Figure 3 represent allogeneic HLA A2 / B40-restricted or HLA A11 / B58-restricted LMP2-specific T cells (2 × 10⁶). 7 A single infusion of T cells was sufficient to significantly reduce tumor growth and also improved the overall survival of tumor-bearing mice (Figures 3A and B). When these animals were treated with two infusions of allogeneic EBV-specific T cells, further improvements in tumor burden and overall survival were observed (Figures 3C and D).

[0121]

[0166] In the next set of experiments, the inventors expanded the therapeutic efficacy analysis to gastric cancer. In these experiments, immunodeficient NOD-SCID mice were inoculated with EBV-associated gastric cancer, SNU719, and each animal's tumor was 25 mm. 2When this stage was reached, the animals were treated with HLA A24-restricted allogeneic LMP2-specific T cells. Consistent with data obtained from NPC tumor models, treatment of these animals with HLA-matched allogeneic LMP2-specific T cells resulted in a significant reduction in tumor volume and improved overall survival (Figure 4A). Interestingly, the inventors noticed that tumor growth was significantly reduced after the first T cell infusion, but this therapeutic benefit was less pronounced after the second T cell infusion. Possible explanations for this phenomenon include the presence of an immunosuppressive tumor microenvironment that negatively impacts clinical outcomes [16, 17] or HLA loss that leads to immune evasion

[18] . Therefore, the inventors hypothesized that EBV-associated tumors neutralize the therapeutic effects of allogeneic EBV-specific T cells by altering HLA expression or regulating EBV gene expression, which can prevent the presentation of EBV epitopes to CD8+ T cells. To investigate this hypothesis, the inventors established two mouse groups carrying gastric cancer (SNU719) and treated one mouse group (G1) with three consecutive doses of the same HLA-matched allogeneic EBV-specific T cells (HLA A24-restricted LMP2-specific T cells). Simultaneously, the inventors also treated the other group (G2) with two consecutive doses of the same HLA-A24-restricted allogeneic EBV-specific T cells, but switching the third dose to a different T cell line (HLA B7-restricted, EBNA1-specific) (Figure 4B). The inventors observed that after the third dose of T cells, the G2 group showed a significant reduction in tumor growth and improved overall survival compared to the G1 group (Figure 4B). Interestingly, compared to the animals in the G2 group, the G1 group showed the least impact on tumor growth after the third dose and a much higher tumor burden (Figure 4B). Overall, these data clearly demonstrate that allogeneic HLA-matched T cells can efficiently block tumor growth and improve overall survival in mice carrying EBV-associated epithelial tumors.

[0122] Allogeneic EBV-specific T-cell adoptive immunotherapy effectively inhibits the growth of EBV-associated lymphoid malignancies.

[0167] As previously mentioned, EBV is pathogenically involved in several diseases, including lymphoproliferative disorders (LPDs), AID-associated lymphomas, and other malignant lymphomas, namely Hodgkin lymphoma and Burkitt lymphoma, in immunocompromised patients such as PTLD [19-22]. To further demonstrate the potential therapeutic efficacy of allogeneic EBV-specific T cells, we utilized a humanized mouse model with a functionally reconstituted human immune system. The reconstitution of the human immune system was achieved using NOD-Rag1 null IL2rg null The treatment was established using intravenous administration of umbilical cord blood-derived CD34+ stem cells in mice (referred to as NRG), and these animals were regularly monitored as outlined in Figures 5A and B. After 12 weeks, these mice were infected with EBV (QIMR-WIL strain) and, after the onset of EBV-LPD, were adoptively treated with HLA-matched allogeneic EBV-specific T cells. Two independent sets of experiments were performed. In the first set, EBV-LPD-carrying NRG mice were divided into three groups (6 mice in each group) and either received mock treatment or were infused with T cell therapy (referred to as G1 and G2). Animals in group G1 were given HLA A2 and A24-restricted allogeneic LMP1 and LMP2-specific T cells (2 × 10⁶). 7 The animals in group G2 were treated with three doses of T cells (2 × 10⁶). 7 Two administrations of T cells ( / dose), and HLA A2, B40, and Cw3-restricted LMP1, LMP2, and EBNA1-specific T cells (2 × 10⁶). 7 A single dose of T cells was administered. The data shown in Figures 5C-D show that the animals in group G1 showed a significant reduction in tumor burden compared to treated mice. However, the animals in group G2, treated with a combination of two different allogeneic EBV-specific T cells, also showed a significant reduction in tumor burden compared to mock-treated animals and G1 animals. These observations were further confirmed in a second set of independent experiments, where animals treated with allogeneic EBV-specific T cells also showed significantly lower tumor burden (Figures 5E-F). These data further demonstrate that allogeneic HLA-matched EBV-specific T cells can efficiently block tumor growth and improve overall survival in mice carrying mouse EBV-associated lymphoid malignancies.

[0123] Blocking the PD1 / PD-L1 axis increases the therapeutic efficacy of allogeneic EBV-specific T cells.

[0168] To further illustrate the in vivo interaction between adoptive allogeneic EBV-specific T cells and tumor cells, we isolated invasive T cells from SNU719 tumors and analyzed their transcriptional signatures using NanoString technology. Furthermore, we confirmed the expression of several checkpoint molecules using specific antibodies. The data shown in Figure 6A illustrate heatmaps of gene expression in T cell therapy products and purified tumor-infiltrating human T cells. This analysis showed that several cellular genes involved in effector cell function and transcription factors associated with effector function were downregulated in tumor-infiltrating human lymphocytes. In contrast, the expression of several checkpoint molecules was upregulated in these T cells. To confirm the expression of these checkpoint molecules, we stained tumor-infiltrating human lymphocytes with specific antibodies and compared their expression to that of T cell therapy administered to tumor-bearing mice. This analysis confirmed NanoString expression and showed that tumor-infiltrating human lymphocytes expressed high levels of PD-1, LAG3, and TIM3 (Figure 6B).

[0124]

[0169] Based on these observations, the inventors hypothesized that a combination of checkpoint inhibitors and T-cell therapy could provide a better therapeutic benefit for EBV-associated tumors. To test the inventors' hypothesis that an upregulated PD1 / PD-L1 axis influences cancer cells in acquiring adaptive immune resistance, the inventors blocked PD1 in vivo using an anti-PD1 antibody (nivolumab) in combination with allogeneic EBV-specific T cells. Specifically, nivolumab was administered 24 hours after T-cell infusion, and the inventors observed that the combination group showed a significant reduction in SNU719 gastric cancer growth compared to monotherapy and mock therapy groups (p<0.0001) (Figure 6C). Furthermore, the inventors observed a significant reduction in tumor size compared to the monotherapy group, indicated by a reduction in tumor weight observed in the combination group (Figure 6D). Importantly, the inventors observed that the combination therapy group showed significantly improved post-therapy survival compared to monotherapy or mock therapy groups (p<0.0001) (Figure 6E). In particular, the combination therapy group showed approximately twice the survival rate (p<0.01) compared to the T cell-only therapy group (Figure 6E). Overall, these data highlight that inhibition of the PD1 / PD-L1 signaling pathway enables the increase of allogeneic EBV-specific CD8+ T cells in vivo, and therefore this pathway negatively determines the fate of allogeneic CD8+ T cells in eliminating cancer cells.

[0125] [Table 1]

[0126] [Table 2]

[0127] Example 2 Efficacy of dual combination therapy with MEK1 / 2 and BET inhibitors and EBV-specific T cells

[0170] Targeted therapies that inhibit molecular or biochemical pathways essential for tumor growth and maintenance have demonstrated their critical role in influencing the immune contexture of tumors. Recent studies have shown that targeted therapies affect specific immune cell populations, namely cytotoxic T lymphocytes and T reg It has been shown that immune responses can also be modulated, including attenuation of function

[23] . Targeted therapies affect T cell priming and direct T cell differentiation into memory and effector phenotypes, in addition to increasing antigen tumor presentation by dendritic cells and enabling better sensitization of tumor cells to immune-mediated destruction

[23] . Moreover, the potential interactions between immunotherapy and targeted therapy are not fully understood, and the synergistic and damaging profiles of combination approaches will likely be highly dependent on timing, order, and dosage

[23] .

[0128]

[0171] Of particular interest is the mitogen-activated protein kinase (MAPK) pathway, which is known to upregulate the production of IL-8 and VEGF, and in turn induce an inhibitory effect on T cell function and recruitment

[24] . Recent studies have shown that MEK1 / 2 inhibition selectively blocks naive (but antigen-unexperienced) effector T cell activation

[25] . Furthermore, independent studies have shown that BRAF inhibitors have immunosensitizing capabilities through upregulation of tumor antigen expression and presentation; an example was given in the case of melanoma where MAPK upregulation results in upregulation of melanocyte differentiation antigen (MAD) [26, 27]. Recent studies based on mouse models have shown improved efficacy of pmel-1 ACT and BRAF inhibitor dabrafenib in combination with trametinib (a MEK inhibitor), as the triple combination increased T cell infiltration into tumors, improved in vivo cytotoxicity, and resulted in complete tumor regression in a syngeneic BRAFV600E-driven melanoma mouse model

[28] . Furthermore, Kang et al. showed that trametinib enhances MHC class I expression in a STT3-dependent manner in human HNSCC cell lines, enabling better CD8+ T cell infiltration.

[29]

[0129]

[0172] Independently, epigenetic modifications of DNA using small molecule epigenetic modifiers can also alter immunogene signatures and affect antigen processing, presentation, and immune evasion [30, 31]. Recently, Kagoya et al. showed that the bromodomain and extra-terminal (BET) motif protein inhibitor, JQ1, maintains the functional properties of stem cell-like memory and central memory in CD8+ T cells

[32] . Since BATF directs the T cell differentiation process to the effector memory phenotype, BRD4 (BE protein) mechanistically directly regulates BATF (transcription factor) expression in CD8+ T cells. Therefore, JQ1-treated CD8+ T cells showed enhanced antitumor effects in a mouse adaptive T cell model for melanoma

[32] . Furthermore, JQ1 has been associated with downregulation of MYC in several cancers

[33] . MYCs have been shown to strongly regulate the tumor microenvironment by transcriptionally regulating immune modulators such as PD-L1 and CD47

[34] . Therefore, MYC inhibition may strongly promote immune-mediated tumor elimination by downregulating the adversarial tumor microenvironment.

[0130] result Combination therapy with MEK1 / 2 inhibitors and EBV-specific T cells

[0173] The inventors treated SNU719 cells (gastric cancer cells) with MEK1 / 2 inhibitors (AZD6244 (or selumetinib) and trametinib) at concentrations of 0.5 μM and 1.0 μM, respectively, either as standalone therapy or in combination with EBV-specific T cells in an effector-to-target ratio (25:1). The inventors observed that the cell viability of SNU719 cells was significantly reduced in the presence of the MEK1 / 2 inhibitor and EBV-specific T cells compared to standalone therapy (Figure 7A). Similarly, the inventors observed that the dual combination group resulted in significantly higher binding of annexin V among SNU719 cells compared to the standalone therapy group (Figure 7B). Furthermore, using Xcellegence, the inventors observed that the dual combination resulted in faster cell death among SNU719 cells compared to standalone therapy with either the MEK1 / 2 inhibitor or EBV-specific T cells (Figure 7C). Overall, these results highlight the efficacy of the dual combination of MEK1 / 2 inhibition and EBV-specific T cells in vitro.

[0131] Combination therapy with JQ1 inhibitors and EBV-specific T cells

[0174] The inventors treated SNU719 cells with a 5.0 μM JQ1 inhibitor either as a standalone treatment or in combination with EBV-specific T cells in an effector-to-target ratio (25:1). The inventors observed that the cell viability of SNU719 cells was significantly reduced in the presence of the JQ1 inhibitor and EBV-specific T cells compared to standalone treatment (Figure 8A). Similarly, the inventors observed that the dual combination group resulted in significantly higher binding of annexin V among SNU719 cells compared to the standalone treatment group (Figure 8B). Overall, these results highlight the efficacy of dual combination therapy with BET inhibition and EBV-specific T cells in vitro.

[0132] Example 3

[0175] To determine the upregulation of the MAPK pathway in EBV-associated solid tumors, we first investigated the IC of MEK1 / 2 inhibitors (AZD6244 or celumetanib and trematinib) in nasopharyngeal and gastric cancer cell lines. 50Values ​​were identified (at drug concentrations ranging from 0.1 μM to 5 μM). Both inhibitors reduced cell viability by approximately 50% using the MTS assay (IC). 50 Since this was achieved at a concentration of approximately 2.5 μM, the inventors of the present invention have found that NPC43( EBV- Compared to other cell lines, EBV-associated cell lines were observed to uniformly exhibit a high dependence on the MAPK pathway (Figure 9). In the following experimental set, the inventors sought to determine the effect of MEK1 / 2 inhibition in combination with allogeneic EBV-specific T cells on EBV-associated solid tumors. To this end, the inventors treated each EBV-associated cell line (both gastric and nasopharyngeal cancer cell lines) individually or in combination with MEK1 / 2 inhibitors (both selmetanib and trematinib) and EBV-specific T cells. For in vitro treatment, the inventors observed the sub-IC of each MEK1 / 2 inhibitor (1 μM) along with a 25:1 effector-to-target ratio of each HLA-matched allogeneic EBV-specific T cell. 50 Value (or IC) 25 The inventors selected a value. Compared to individual treatment, the inventors observed that the cell viability of each nasopharyngeal (C17 and C666.1) and gastric cancer cells (SNU719 and YCCLE1) was significantly reduced in the presence of a combination of MEK1 / 2 inhibitors and EBV-specific T cells (Figure 10).

[0133]

[0176] Furthermore, to confirm the combined effect on the proliferation of EBV-associated cancer cells, the inventors challenged SNU719 and C666.1 cells individually and in dual combination using Xcellegence. The inventors observed that dual combination resulted in faster cell death of SNU719 and C666.1 cells compared to individual treatment with either a MEK1 / 2 inhibitor (selmatinib) or HLA-matched EBV-specific T cells (Figure 11A). The inventors also phenotypic characterized SNU719 and C666.1 cells using flow cytometry 16 hours after individual and dual treatment in vitro. The inventors found that dual treatment resulted in a significant loss of cell proliferation compared to individual treatment (Ki67). + We observed that it caused significant cell death, as indicated by staining and by active caspase-3 staining (Figure 11B-C).

[0134]

[0177] Next, the inventors sought to determine the specificity of the combination therapy, and to that end, they challenged SNU719 and C666.1 cells with HLA-mismatched allogeneic T cells together with MEK1 / 2 inhibitors (both selmetanib and trematinib) individually and in combination. The inventors observed that HLA-mismatched allogeneic T cells did not have a significant effect on the cell viability of EBV-related cancer cells individually (Figure 12A-B). Interestingly, the inventors observed a significant decrease in the cell viability of cancer cells in the presence of the dual combination of MEK1 / 2 inhibitors and HLA-mismatched allogeneic T cells compared to DMSO-treated (control) cancer cells (Figure 12A-B). However, the decrease in cell viability observed in the presence of the dual combination did not show a significant decrease in cell viability compared to treatment with MEK1 / 2 inhibitors alone (Figure 12A-B). This data highlights that the decrease in cell viability observed in the presence of the dual combination was induced solely by inhibition of the MAPK pathway, and that the addition of HLA-incompatible T cells did not enhance the efficacy of the combination. To further confirm this concept, we compared the decrease in cell viability of EBV-associated cancer cells (SNU719 and C666.1) induced by each HLA-compatible and HLA-incompatible T cell alone and in combination with MEK1 / 2 inhibitors (both celumetanib and trematinib). We observed that HLA-compatible T cells alone induced significant cell death compared to HLA-incompatible T cells, and this effect was significantly amplified in the presence of either celumetanib or trematinib (Figure 12C).

[0135]

[0178] In the next set of experiments, the inventors investigated the effects of dual combination therapy on the intracellular pathways in cancer cells and sought to explain the rationale for the effective combination of MEK1 / 2 inhibition and T-cell therapy. The inventors observed that the presence of a MEK1 / 2 inhibitor (selmatinib) resulted in a significant decrease in pERK1 / 2 in cancer cells 16 hours after treatment (Figure 13A). As a result, the inventors observed upregulation of MHC class I expression in the presence of selmatinib alone and in combination with each HLA-matched T cell (Figure 13B). To confirm the mechanism by which MHC class I expression is upregulated in these cancer cells, the inventors investigated the effects of MEK1 / 2 inhibition on pSTAT3 and MYC expression. The inventors observed that the presence of salumatinib, which has been shown to be associated with driving MHC class I expression [29, 35, 36], resulted in upregulation of pSTAT3 expression (Figure 13C). Furthermore, the inventors observed that MEK1 / 2 inhibition also leads to downregulation of MYC expression (Figure 13D). The presence of MYC expression has been shown to downregulate MHC class I expression in EBV-related cancers, and therefore indirect inhibition of MYC expression may help MHC class I in these cancers

[37] . Thus, inhibition of the MEK1 / 2 pathway leads to upregulation of MHC class I expression via upregulation of the STAT3 pathway and downregulation of MYC, enabling better therapeutic suitability in the presence of HLA-matched allogeneic T cells. Overall, these results highlight the efficacy of the dual combination of MEK1 / 2 inhibition and EBV-specific T cells in vitro.

[0136]

[0179] Similarly, the inventors have demonstrated the IC of JQ1 inhibitors in nasopharyngeal and gastric cancer cell lines. 50 The values ​​were first determined (at drug concentrations ranging from 0.5 μM to 10 μM), and the inhibitor was used to target the MYC-dependent endogenous cancer pathway. The inventors found that among EBV-associated cell lines, NPC43 ( EBV- Compared to other cell lines, the JQ1 inhibitor reduced cell viability by approximately 50% (IC). 50We observed that it showed a concentration of approximately 2.5 μM (Figure 14). To characterize the effect of JQ1 inhibition in combination with allogeneic EBV-specific T cells on EBV-associated cancer cells, we used IC along with a 25:1 effector-to-target ratio for each HLA-matched allogeneic EBV-specific T cell. 25 The value JQ1 (2.5 μM) was used in vitro. The inventors observed that the cell viability of each gastric cancer cell (SNU719 and YCCLE1) and nasopharyngeal cells (C17 and C666.1) was significantly reduced in the presence of a combination of a JQ1 inhibitor and EBV-specific T cells compared to individual treatment (Figure 15A). Furthermore, using Xcellegence, the inventors observed that the dual combination resulted in faster cell death of SNU719 and C666.1 cells compared to individual treatment (Figure 15B). The inventors also phenotypic characterized SNU719 and C666.1 cells using flow cytometry 16 hours after individual and dual treatment in vitro. The inventors found that dual treatment resulted in a significant loss of cell proliferation compared to individual treatment (Ki67). + We observed that it caused significant cell death, as indicated by staining and by active caspase-3 staining (Figure 15B-C).

[0137]

[0180] Next, the inventors sought to determine the specificity of the combination, and for this purpose, challenged SNU719 and C666.1 cells with HLA-mismatched allogeneic T cells together with JQ1 inhibitors individually and in combination. Consistent with the inventors' previous data using MEK1 / 2 inhibitors, the inventors observed a significant decrease in the cell viability of cancer cells in the presence of the combination of JQ1 inhibitors and HLA-mismatched allogeneic T cells compared to DMSO-treated (control) cancer cells (Figure 16A). However, the decrease in cell viability observed in the presence of the double combination did not show a significant decrease in cell viability compared to treatment with JQ1 inhibitor alone (Figure 16A). The inventors observed that HLA-matched T cells alone induced significant cell death compared to HLA-mismatched T cells, and that this effect was significantly amplified in the presence of JQ1 (Figure 16B). In the next set of experiments, the inventors sought to explain the rationale for the effective combination of JQ1 inhibition and T cell therapy. The inventors observed that the presence of a JQ1 inhibitor resulted in a significant decrease in MYC expression in cancer cells 16 hours after treatment (Figure 17A). This resulted in downregulation of pERK1 / 2, pAKT, and pSTAT3 expression (Figures 17B-C). Furthermore, the inventors observed that the downregulation of MYC also led to an enhancement of MHC class I in these cancers, as discussed above

[37] (Figure 18A). In particular, the inventors also observed downregulation of immunomodulatory molecules such as PD-L1 and CD47 expression (Figures 18B-C). Therefore, the loss of MYC expression enhances the efficacy of cytotoxic T cells by enhancing MHC class I expression, which downregulates immune evasion molecules, making the combination highly effective. Overall, these results highlight the efficacy of the dual combination of JQ1 inhibition and EBV-specific T cells in vitro.

[0138]

[0181] In the next set of experiments, the inventors sought to investigate the effects of the above small molecule in combination with HLA-matched allogeneic EBV-specific T cells in vivo. The inventors treated a SNU719-derived xenograft tumor model in female NRG mice using a combination of selmatinib and an HLA-matched T cell product (TIG-001). The xenograft was approximately 25 mm. 2Once the size is reached, the inventors administer two injections of TIG-001 in combination with selmatinib (12.5 mg / kg) administered orally for 14 days (2 × 10 per injection). 7 Mice were treated with (cells). The inventors observed that the dual combination resulted in a significant reduction in tumor progression compared to individual treatments (Figure 19A). The inventors compared a control group of 150 mm 2 When the ethical size limit was reached, three mice were sacrificed from each group, and comparatively, the inventors observed that the tumor size in the dual combination group was significantly smaller than that of the individual treatment group (Figure 19B-C). Next, the inventors used flow cytometry to determine the phenotype of human-derived lymphocyte infiltration in these tumors. The inventors observed that the combination group showed a significantly higher number of viable CD45+CD3+CD8+ cells compared to the individual treatment T cell group (Figure 19D). The inventors also performed long-term monitoring of these mice after therapy (mice from each group were 150 mm 2 (Tumor progression was monitored up to the ethical size limit), and we observed that the dual combination group showed better survival rates compared to the individual treatment group (Figure 19E).

[0139]

[0182] Any reference cited herein should not be construed as an acknowledgment that such reference is available as “prior art” of this application.

[0140]

[0183] Throughout this specification, the objective has been to describe preferred embodiments of the invention without limiting it to any one embodiment or particular collection of features. Those skilled in the art will therefore understand that various modifications and changes can be made in particular embodiments in light of the exemplary disclosure provided without departing from the scope of the invention. All such modifications and changes are intended to be included within the scope of the appended claims.

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Claims

1. A method for treating or preventing EBV-related diseases, disorders, or conditions in a subject, (a) administering to the subject a first population of allogeneic T cells that bind to or recognize a first epitope of the EBV antigen; and (b) The step of administering to the subject a second population of allogeneic T cells that bind to or recognize a second epitope of the EBV antigen or a further EBV antigen. Includes, A method for treating or preventing the EBV-related disease, disorder, or condition in the subject.

2. A pharmaceutical composition for treating or preventing EBV-related diseases, disorders, or conditions in a subject, A first population of allogeneic T cells that bind to or recognize the first epitope of the EBV antigen; A second population of allogeneic T cells that bind to or recognize a second epitope of EBV antigen or further EBV antigen; Optionally, a pharmaceutically acceptable carrier, diluent and / or excipient. A composition containing the following:

3. The method according to claim 1 or the composition according to claim 2, wherein both the first population of allogeneic T cells and the cells of EBV-related disease, disorder, or condition contain a first human leukocyte antigen (HLA) allele encoding a first MHC protein, or are constrained by a first human leukocyte antigen (HLA) allele encoding a first MHC protein.

4. The method or composition according to claim 3, wherein the first MHC protein presents the first epitope of the EBV antigen to cells of the EBV-related disease, disorder, or condition.

5. The method or composition according to any one of claims 1 to 4, wherein both the second population of allogeneic T cells and the cells of the EBV-related disease, disorder, or condition contain a second HLA allele encoding a second MHC protein, or are constrained by a second HLA allele encoding a second MHC protein.

6. The method or composition according to claim 5, wherein the second MHC protein presents the second epitope of the EBV antigen or the further EBV antigen to cells of the EBV-related disease, disorder, or condition.

7. The method according to any one of claims 1 and 3 to 6, wherein the second population of allogeneic T cells is administered before, simultaneously with, and / or after the administration of the first population of allogeneic T cells.

8. The method according to any one of claims 1 and 3 to 7, further comprising the first step of generating the first and / or second population of allogeneic T cells in vitro.

9. The method according to any one of claims 1 and 3 to 8, further comprising the step of administering a therapeutic agent to the subject.

10. The method according to claim 9, wherein the therapeutic agent is selected from the group consisting of immunotherapeutic agents, MAPK pathway inhibitors, BET inhibitors, and any combination thereof.

11. A pharmaceutical combination comprising the pharmaceutical composition described in claim 2 together with an immunotherapy agent, a MAPK pathway inhibitor, a BET inhibitor, or a combination thereof.

12. A method for treating or preventing EBV-related diseases, disorders, or conditions in a subject, (a) the step of administering to the subject a population of allogeneic T cells that bind to or recognize the epitope of the EBV antigen; and (b) A step of administering to the subject a therapeutic agent selected from the group consisting of immunotherapeutic agents, MAPK pathway inhibitors, BET inhibitors, and any combination thereof. Includes, A method for treating or preventing the EBV-related disease, disorder, or condition in the subject.

13. A pharmaceutical composition for treating or preventing EBV-related diseases, disorders, or conditions in a subject, A population of allogeneic T cells that bind to or recognize the EBV antigen epitope; A therapeutic agent selected from the group consisting of immunotherapeutic agents, MAPK pathway inhibitors, BET inhibitors, and any combination thereof; Optionally, a pharmaceutically acceptable carrier, diluent and / or excipient. A composition containing the following:

14. The method according to claim 12 or the composition according to claim 13, wherein both the population of allogeneic T cells and the cells of the EBV-related disease, disorder, or condition contain or are constrained by a human leukocyte antigen (HLA) allele encoding an MHC protein.

14. The method or composition according to claim 14, wherein the MHC protein presents the epitope of the EBV antigen to cells of the EBV-related disease, disorder, or condition.

15. The method, combination, or composition according to any one of claims 10 to 15, wherein the immunotherapy agent is an immune checkpoint inhibitor such as a PD1 inhibitor (e.g., anti-PD1 antibody), a PD-L1 inhibitor (e.g., anti-PD-L1 antibody), a CTLA4 inhibitor (e.g., anti-CTLA4 antibody), a LAG3 inhibitor (e.g., anti-LAG3 antibody), a TIM3 inhibitor (e.g., anti-TIM3 antibody), or a CD96 inhibitor (e.g., anti-CD96 antibody), or includes the same.

16. The method or composition according to claim 15, wherein the immune checkpoint inhibitor is an anti-PD1 antibody, or comprises an anti-PD1 antibody.

17. The method or composition according to claim 16, wherein the anti-PD1 antibody is selected from pembrolizumab, nivolumab, or semiprimab.

19. The method or composition according to claim 15, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody, or comprises an anti-PD-L1 antibody.

20. The method or composition according to claim 19, wherein the anti-PD1 antibody is selected from atezolizumab, avelumab, or durvalumab.

21. The method, combination, or composition according to any one of claims 10 to 16, wherein the MAPK pathway inhibitor is a MEK1 / 2 inhibitor, or comprises a MEK1 / 2 inhibitor.

22. The method or composition according to claim 21, wherein the MEK1 / 2 inhibitor is selected from celmetanib and / or trematinib.

23. The method, combination, or composition according to any one of claims 10 to 16, wherein the BET inhibitor is JQ1.

24. The method according to any one of claims 12 and 14 to 23, wherein the population of allogeneic T cells is administered before, simultaneously with, and / or after the administration of the therapeutic agent.

25. The method according to any one of claims 12 and 14-24, further comprising the first step of generating the population of allogeneic T cells in vitro.

26. The method or composition according to any one of the claims, wherein the EBV antigen and / or the further EBV antigen is selected from the group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2 and any combination thereof.

27. The method or composition according to claim 26, wherein the EBV antigen and / or the further EBV antigen is EBNA1, LMP1 and / or LMP2, or comprises EBNA1, LMP1 and / or LMP2.

28. The method or composition according to any one of the claims, wherein the EBV-related disease, disorder or condition is EBV-related cancer, or includes EBV-related cancer.

23. The method or composition according to claim 22, wherein the EBV-related cancer is selected from the group consisting of nasopharyngeal carcinoma, NK T-cell lymphoma, Hodgkin lymphoma, post-transplant lymphoproliferative disorder, Burkitt lymphoma, diffuse large B-cell lymphoma, gastric cancer, and any combination thereof.

24. The use of a first population of allogeneic T cells that bind to or recognize a first epitope of the EBV antigen in the manufacture of a pharmaceutical product for the treatment or prevention of EBV-related disease, disorder, or condition in a subject, Use wherein the first population of allogeneic T cells is administered in combination with (a) a second population of allogeneic T cells that bind to or recognize a second epitope of the EBV antigen or further EBV antigen; and / or (b) a therapeutic agent selected from the group consisting of immunotherapeutic agents, MAPK pathway inhibitors, BET inhibitors and any combination thereof.

25. A first population of allogeneic T cells that bind to or recognize the first epitope of the EBV antigen, for use in the treatment or prevention of EBV-related diseases, disorders, or conditions in the subject, (a) a second population of allogeneic T cells that bind to or recognize the EBV antigen or a second epitope of further EBV antigens; and / or (b) a first population of allogeneic T cells administered in combination with a therapeutic agent selected from the group consisting of immunotherapeutic agents, MAPK pathway inhibitors, BET inhibitors and any combination thereof.

26. A method for treating or preventing EBV-related cancer or tumors in a subject, (a) the step of administering to the subject a population of allogeneic T cells that bind to or recognize the epitope of the EBV antigen; and (b) A step of administering to the subject a therapeutic agent selected from the group consisting of immunotherapeutic agents, MAPK pathway inhibitors, BET inhibitors, and any combination thereof. Includes, A method for treating or preventing the EBV-related disease, disorder, or condition in the subject.

27. A pharmaceutical composition for treating or preventing EBV-related cancer or tumors in a subject, A population of allogeneic T cells that bind to or recognize the EBV antigen epitope; A therapeutic agent selected from the group consisting of immunotherapeutic agents, MAPK pathway inhibitors, BET inhibitors, and any combination thereof; Optionally, a pharmaceutically acceptable carrier, diluent and / or excipient. A composition containing the following:

28. The method according to claim 26 or the composition according to claim 27, wherein both the population of allogeneic T cells and the cells of the EBV-related disease, disorder, or condition contain or are constrained by a human leukocyte antigen (HLA) allele encoding an MHC protein.

29. The method or composition according to claim 28, wherein the MHC protein presents the epitope of the EBV antigen to cancer cells or tumor cells.

30. The method, combination, or composition according to any one of claims 26 to 29, wherein the immunotherapy agent is an immune checkpoint inhibitor such as a PD1 inhibitor (e.g., anti-PD1 antibody), a PD-L1 inhibitor (e.g., anti-PD-L1 antibody), a CTLA4 inhibitor (e.g., anti-CTLA4 antibody), a LAG3 inhibitor (e.g., anti-LAG3 antibody), a TIM3 inhibitor (e.g., anti-TIM3 antibody), or a CD96 inhibitor (e.g., anti-CD96 antibody), or includes the same.

31. The method or composition according to claim 30, wherein the immune checkpoint inhibitor is an anti-PD1 antibody, or comprises an anti-PD1 antibody.

32. The method or composition according to claim 31, wherein the anti-PD1 antibody is selected from pembrolizumab, nivolumab, or semiprimab.

33. The method or composition according to claim 30, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody, or comprises an anti-PD-L1 antibody.

34. The method or composition according to claim 33, wherein the anti-PD1 antibody is selected from atezolizumab, avelumab, or durvalumab.

35. The method, combination, or composition according to any one of claims 26 to 29, wherein the MAPK pathway inhibitor is a MEK1 / 2 inhibitor, or comprises a MEK1 / 2 inhibitor.

36. The method or composition according to claim 35, wherein the MEK1 / 2 inhibitor is selected from celmetanib and / or trematinib.

37. The method or composition according to any one of claims 26 to 29, wherein the BET inhibitor is JQ1.

38. The method according to any one of claims 26 and 28-37, wherein the population of allogeneic T cells is administered before, simultaneously with, and / or after the administration of the therapeutic agent.

39. The method according to any one of claims 26 and 28-38, further comprising the first step of generating the population of allogeneic T cells in vitro.

40. The method or composition according to any one of claims 26 to 39, wherein the EBV antigen and / or the further EBV antigen is selected from the group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2 and any combination thereof.

41. The method or composition according to claim 40, wherein the EBV antigen and / or the further EBV antigen is EBNA1, LMP1 and / or LMP2, or comprises EBNA1, LMP1 and / or LMP2.

42. The method or composition according to any one of claims 26 to 41, wherein the cancer or tumor is selected from the group consisting of nasopharyngeal carcinoma, NK T-cell lymphoma, Hodgkin lymphoma, post-transplant lymphoproliferative disorder, Burkitt lymphoma, diffuse large B-cell lymphoma, gastric cancer, and any combination thereof.

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