CD4+ cytotoxic T cells and their use

Abstract

This specification provides CD4+ cytotoxic T cells, as well as methods for generating and using them.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 466,070 filed on 12 May 2023 and U.S. Provisional Application No. 63 / 637,675 filed on 23 April 2024, the respective disclosures of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Despite extensive research and development of cancer treatments, including immunotherapies such as PD-1 checkpoint blockade and CD19-directed chimeric antigen receptor (CAR)-T cell therapy, sustained remission remains difficult to achieve in many patients. Oncology immunology has traditionally focused on the role of cytotoxic CD8+ T cells in identifying and killing cancer cells. However, recent clinical studies have pointed to the existence and potential role of CD4+ cytotoxic T cells (CD4+ CTLs) in their direct cancer-killing ability. Nevertheless, methods for efficiently generating CD4+ CTLs, particularly those targeting multiple antigens and useful in adoptive cell therapy, are not yet available. Therefore, developing methods for generating CD4+ CTLs, and especially for their use in killing cancer cells and treating cancer, remains a pressing need. [Overview of the project]

[0003] This disclosure provides, in particular, CD4+ cytotoxic lymphocytes (CTLs) and related methods, uses, and techniques. In some embodiments, this disclosure provides a method for generating CD4+ CTLs using antigen-presenting cells (APCs), e.g., APCs derived from cancer cells, to activate CD4+ T cells. This method comprises contacting a population of CD4+ cells or CD4+ T cells with one or more target cells as described herein. In some embodiments, the method comprises contacting a population of CD4+ T cells or CD4+ T cells with one or more activators that promote the co-stimulation of antigen-presenting cells (e.g., cancer cell-derived APCs, e.g., major histocompatibility complex II (MHC II)-expressing cancer cells) and CD4+ T cells. In some embodiments, the co-stimulation is mediated by CD27-mediated signaling (e.g., CD70 / CD27 signaling). In some embodiments, the co-stimulation is mediated by OX40-mediated signaling (e.g., OX40L / OX40 signaling). In some embodiments, co-stimulation occurs via CD27-mediated signaling (e.g., CD70 / CD27 signaling) and OX40-mediated signaling (e.g., OX40L / OX40 signaling). Using such methods, CD4+ CTLs can be created that target cancer cells, including cancer cells that do not express CD70 and / or OX40L.

[0004] In some embodiments, a method for generating CD4+ CTLs is provided, which includes exposing CD4+ T cells to major histocompatibility complex II (MHC II) expressing cancer cells, and (i) one or more activators that promote CD27-mediated costimulation of CD4+ T cells and / or (ii) one or more activators that promote OX40-mediated costimulation of CD4+ T cells, for a period of time and under conditions suitable for the formation of at least one immune complex between CD4+ T cells and cancer cells and for the promotion of CD27-mediated costimulation and / or OX40-mediated costimulation of CD4+ T cells, thereby generating CD4+ CTLs.

[0005] Any or some of the embodiments described herein can be combined in any way.

[0006] For example, some embodiments provide a method that does not involve contacting CD4+ T cells with an OX40-mediated costimulator. Some embodiments provide a method that does not involve contacting CD4+ T cells with a CD27-mediated costimulator. Some embodiments provide a method that involves contacting CD4+ T cells with one or more factors that promote CD27-mediated costimulatory interaction of CD4+ T cells and one or more factors that promote OX40-mediated costimulatory interaction of CD4+ T cells.

[0007] In some embodiments, one or more activators that promote CD27-mediated costimulation include MHC II-expressing cancer cells, in which case the MHC II-expressing cancer cells are engineered or manipulated to express CD70 on their cell surface. In some embodiments, the provided method includes contacting CD4+ T cells or a population of CD4+ T cells with MHC II-expressing cancer cells that also express CD70. In some embodiments, the MHC II-expressing cancer cells are engineered to exogenously express CD70. In some embodiments, the MHC II-expressing cancer cells express both CD70 and OX40L.

[0008] In some embodiments, one or more activators that promote CD27-mediated co-stimulation include soluble CD70. In some embodiments, soluble CD70 includes a CD70 fusion protein (e.g., a CD70-Fc fusion protein).

[0009] In some embodiments, one or more activators that promote CD27-mediated co-stimulation include anti-CD27 agonist antibodies.

[0010] In some embodiments, one or more agents that promote OX40-mediated costimulation include MHC II-expressing cancer cells, where the MHC II-expressing cancer cells are engineered or have been engineered to express OX40 ligand on their cell surface. In some embodiments, the provided method includes contacting CD4+ T cells or a population of CD4+ T cells with MHC II-expressing cancer cells that also express OX40L. In some embodiments, the MHC II-expressing cancer cells are engineered to ectopically express OX40L. In some embodiments, the MHC II-expressing cancer cells express both CD70 and OX40L.

[0011] In some embodiments, one or more agents that promote OX40-mediated costimulation include soluble OX40 ligand. In some embodiments, the soluble OX40 ligand includes an OX-40L fusion protein (e.g., an OX40L-Fc fusion protein).

[0012] In some embodiments, one or more agents that promote OX40-mediated costimulation include an anti-OX40 agonistic antibody.

[0013] In some embodiments, one or more agents for costimulating CD4+ T cells include: a) MHC II-expressing cancer cells, where the MHC II-expressing cancer cells are engineered or have been engineered to express CD70 on their cell surface; b) soluble CD70, optionally, the soluble CD70 includes a CD70 fusion protein (e.g., a CD70-Fc fusion protein); c) an anti-CD27 agonistic antibody; d) MHC II-expressing cancer cells, where the MHC II-expressing cancer cells are engineered or have been engineered to express OX40 ligand (OX40L) on their cell surface; e) soluble OX40L, optionally, the soluble OX40L is an OX40L fusion protein (e.g., an OX40L-Fc fusion protein); and / or f) an anti-OX40 agonistic antibody.

[0014] In some embodiments, the provided method includes culturing CD4+ T cells with an anti-CD27 agonistic antibody. In some embodiments, the CD4+ cells are cultured with the anti-CD27 agonistic antibody for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, or longer. In some embodiments, the CD4+ T cells are cultured in the presence of the anti-CD27 agonistic antibody starting at the beginning of the culture (day 0). In some embodiments, the CD4+ T cells are cultured in the presence of the anti-CD27 agonistic antibody throughout the entire culture period. In some embodiments, the anti-CD27 agonistic antibody is present for a portion of the culture. In some embodiments, the anti-CD27 agonistic antibody is added at a point after the culture has started. In some embodiments, the CD4+ T cells are cultured in the presence of the anti-CD27 agonistic antibody starting on day 1, 2, 3, 4, 5, or 6 of the culture.

[0015] In some embodiments, the provided method includes culturing CD4+ T cells with an anti-OX40 agonistic antibody. In some embodiments, the CD4+ cells are cultured with the anti-OX40 agonistic antibody for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, or longer. In some embodiments, the CD4+ T cells are cultured in the presence of the anti-OX40 agonistic antibody starting at the beginning of the culture (day 0). In some embodiments, the CD4+ T cells are cultured in the presence of the anti-OX40 agonistic antibody throughout the entire culture period. In some embodiments, the anti-OX40 agonistic antibody is present for a portion of the culture. In some embodiments, the anti-OX40 agonistic antibody is added at a point after the culture has started. In some embodiments, the CD4+ T cells are cultured in the presence of the anti-OX40 agonistic antibody starting on day 1, 2, 3, 4, 5, or 6 of the culture.

[0016] In some embodiments, the CD4+ T cells for use in generating CD4+ CTLs by the method described herein are CD4+ T cells, naive CD4+ T cells, memory CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, Trp1-specific CD4+ T cells, tumor antigen-specific CD4+ T cells, neoantigen-specific CD4+ T cells, CD4+ T cells derived from peripheral blood mononuclear cells (PBMCs), CD4+ T cells derived from umbilical cord blood, CD4+ T cells derived from tumor-infiltrating lymphocytes, primary CD4+ T cells, CD4+ T cells derived from cell lines, in vitro amplified CD4+ cells, and / or in vitro activated CD4+ cells.

[0017] In some embodiments, CD4+ T cells are allogeneic, syngeneic, or autologous to APCs. In some embodiments, CD4+ T cells are allogeneic, syngeneic, or autologous to MHC II-expressing cancer cells.

[0018] In some embodiments, CD4+ T cells are selected from the group consisting of mammalian CD4+ T cells, rodent CD4+ T cells, mouse CD4+ T cells, primate CD4+ T cells, and human CD4+ T cells.

[0019] In some embodiments, the cancer cells used to generate CD4+ CTLs by the method described herein are allogeneic, syngeneic, or autologous relative to CD4+ T cells.

[0020] In some embodiments, APCs such as cancer cells are induced to express MHC II through contact with interferon-gamma (IFNγ). In some embodiments, cells are cultured in the presence of IFNγ at doses of 10 ng / ml to 400 ng / ml. In some embodiments, cells are cultured in the presence of IFNγ at doses of 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 150 ng / ml, 200 ng / ml, 250 ng / ml, 300 ng / ml, 350 ng / ml, or 400 ng / ml. In some embodiments, cells are cultured with IFNγ for 1, 2, 3, 4, 5 days, or longer. In some embodiments, IFNγ is human IFNγ. In some embodiments, cells are cultured in the presence of human IFNγ at a dose of 10 ng / ml to 200 ng / ml. In some embodiments, cells are cultured with human IFNγ for 2 to 4 days. In some embodiments, cells are cultured in the presence of human IFNγ at a dose of 20 ng / ml for 3 days. In some embodiments, IFNγ is mouse IFNγ. In some embodiments, cells are cultured in the presence of mouse IFNγ at a dose of 10 ng / ml to 400 ng / ml. In some embodiments, cells are cultured with mouse IFNγ for 2 to 5 days. In some embodiments, cells are cultured in the presence of mouse IFNγ at a dose of 100 ng / ml for 4 days.

[0021] In some embodiments, cancer cells are induced or have been induced to express MHC II by the expression of class II transactivators (CIITAs).

[0022] In some embodiments, cancer cells endogenously (e.g., spontaneously) express MHC-II. In some embodiments, cancer cells originate from cancer that endogenously (e.g., spontaneously) expresses MHC-II.

[0023] In some embodiments, the provided method involves contacting CD4+ cells with IL-2. While not bound by theory, it is thought that IL-2 treatment may be useful in certain embodiments (e.g., when certain T cells have relatively low affinity T cell antigen binding) to promote the survival and / or proliferation of CD4+ T cells so that differentiation into EOMES-mediated CD4+ cytotoxic T cells can be carried out.

[0024] In some embodiments, cancer cells do not endogenously (e.g., spontaneously) express CD70. In some embodiments, cancer cells originate from cancer that does not endogenously (e.g., spontaneously) express CD70.

[0025] In some embodiments, cancer cells do not endogenously (e.g., spontaneously) express OX40L. In some embodiments, cancer cells originate from cancer that does not endogenously (e.g., spontaneously) express OX40L.

[0026] In some embodiments, cancer cells endogenously (e.g., spontaneously) express CD70 and / or OX40L. In some embodiments, cancer cells originate from cancer that endogenously (e.g., spontaneously) expresses CD70 and / or OX40L.

[0027] In some embodiments, cancer cells originate from lymphoma and / or leukemia. In some embodiments, cancer cells originate from B-cell leukemia, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), classical Hodgkin lymphoma (cHL), and / or acute myeloid leukemia (AML). In some embodiments, cancer cells originate from solid tumors. In some embodiments, cancer cells originate from melanoma, bladder cancer, breast cancer, head and neck cancer, hepatocellular carcinoma (HCC), colon cancer, pancreatic cancer, kidney cancer, ovarian cancer, and / or brain cancer.

[0028] In some embodiments, cancer cells present at least one MHC II-antigen complex on their cell surface. In some embodiments, the antigen presented by the MHC II-antigen complex is a tumor-associated antigen and / or neoantigen.

[0029] In some embodiments, the CD4+ CTLs produced by the method provided herein express a T cell receptor (TCR) that specifically binds to the MHC II antigen complex expressed on the surface of cancer cells by cancer cells.

[0030] In some embodiments, the CD4+CTLs produced by the methods provided herein express granzymes, such as granzyme B. In some embodiments, the CD4+CTLs produced by the methods provided herein express perforin. In some embodiments, the CD4+CTLs produced by the methods provided herein express granzymes and perforin. In some embodiments, the CD4+CTLs produced by the methods provided herein express CD69. In some embodiments, the CD4+CTLs produced by the methods provided herein express granzymes (e.g., granzyme B), perforin, and CD69. In some embodiments, the CD4+CTLs produced by the methods provided herein are programmed by Eomes.

[0031] In some embodiments, the CD4+CTLs provided herein secrete cytokines. In some embodiments, the CD4+CTLs secrete IFNγ.

[0032] In some embodiments, the CD4+ CTLs provided herein induce antigen-specific T cell immune responses. In some embodiments, the CD4+ CTLs provided herein induce multi-antigen-specific T cell immune responses.

[0033] In some embodiments, the CD4+ CTLs provided herein can kill cancer cells.

[0034] In some embodiments, the CD4+ CTLs provided herein directly kill cancer cells that express an MHC II-antigen complex, which is specifically recognized by the CD4+ CTL, on their cell surface.

[0035] In some embodiments, the CD4+ CTLs provided herein are proliferative. In some embodiments, the CD4+ CTLs provided herein can be amplified in vitro and / or ex vivo.

[0036] In some embodiments, the CD4+CTLs provided herein persist in vivo. In some embodiments, the CD4+CTLs provided herein persist in vivo for at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 1.5 years, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years. In some embodiments, the CD4+CTLs provided herein persist in vivo for a period of any range including endpoints such as 6 months to 10 years, or 6 months to 12 months, 6 months to 2 years, or 6 months to 5 years.

[0037] In some embodiments, the CD4+ CTLs provided herein perform CD4+ helper functions. In some embodiments, the CD4+ helper function includes (i) licensing APCs to prime anti-cancer CD8+ CTLs, (ii) activating anti-cancer NK cells, (iii) activating anti-cancer macrophages, (iv) activating anti-cancer CD8+ T cells, and / or (v) activating anti-cancer immune cells. Such CD4+ CTL-mediated helper functions can thereby kill MHC-II-negative cancer cells.

[0038] In some embodiments, the method provided further includes culturing CD4+CTLs under conditions suitable for enabling the proliferation of CD4+CTLs. In some embodiments, CD4+CTLs are cultured for a period of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days. In some embodiments, CD4+CTLs are cultured for a period selected from the group consisting of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, and 14 days.

[0039] In some embodiments, culturing CD4+CTLs under conditions suitable for enabling their proliferation includes culturing them with IL-2. In some embodiments, IL-2 is added to the CD4+CTL culture after 36, 48, 60, 72, or 84 hours of incubation. In some embodiments, IL-2 is added to the CD4+CTL culture after approximately 2 to 4 days of incubation.

[0040] In some embodiments, the method provided further includes the step of treating the APC (e.g., cancer cells) to prevent further proliferation before contact with CD4+ T cells. In some embodiments, the method provided further includes irradiating the APC (e.g., cancer cells).

[0041] In some embodiments, the method provided further includes the step of isolating and / or purifying CD4+CTLs.

[0042] In some embodiments, the provided method is carried out ex vivo or in vivo. In some embodiments, CD4+ T cells are contacted ex vivo or in vivo.

[0043] In some embodiments, this disclosure provides compositions comprising CD4+CTL produced according to the method described herein.

[0044] In some embodiments, the provided composition contains CD4+ CTLs that are expressed by cancer cells and are specific to antigens presented by MHC II. In some embodiments, the provided composition contains CD4+ CTLs that are multispecific to antigens expressed by cancer cells and presented by MHC II.

[0045] In some embodiments, an immunogenic composition comprising CD4+CTL produced according to the method described herein is provided. In some embodiments, a vaccine comprising CD4+CTL produced according to the method described herein is provided. In some embodiments, the immunogenic composition and / or vaccine provided further comprises an adjuvant.

[0046] In some embodiments, an immunogenic composition or vaccine can induce an immune response against antigen-expressing cancer cells in a target. In some embodiments, the immune response is selected from the group consisting of i) a T cell response, a CD4+ helper T cell response, a CD4+ cytotoxic T cell response, and / or a CD8+ T cell response, and / or ii) T cell amplification, cytokine release, and / or cytotoxic killing.

[0047] In some embodiments, the Disclosure provides a method for killing cancer cells, the method comprising contacting the cancer cells with a composition comprising CD4+CTL as described herein. In some embodiments, the Disclosure provides a method for killing cancer cells, the method comprising contacting the cancer cells with an immunogenic composition or vaccine comprising CD4+CTL as described herein.

[0048] In some embodiments, the Disclosure provides a method for treating cancer, the method comprising administering to a subject a composition comprising CD4+CTL as described herein. In some embodiments, the Disclosure provides a method for treating cancer, the method comprising contacting cancer cells with an immunogenic composition or vaccine comprising CD4+CTL as described herein.

[0049] In some embodiments, the CD4+CTLs provided herein are suitable for transplantation into a subject. In some embodiments, the CD4+CTLs are allogeneic, syngeneic, or autologous to cancer cells. In some embodiments, the CD4+CTLs are allogeneic, syngeneic, or autologous to a subject.

[0050] In some embodiments, cancer cells originate from lymphoma and / or leukemia. In some embodiments, cancer cells originate from B-cell leukemia, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), classical Hodgkin lymphoma (cHL), and / or acute myeloid leukemia (AML). In some embodiments, cancer cells originate from solid tumors. In some embodiments, cancer cells originate from melanoma, bladder cancer, breast cancer, head and neck cancer, hepatocellular carcinoma (HCC), colon cancer, pancreatic cancer, kidney cancer, ovarian cancer, and / or brain cancer.

[0051] In some embodiments, a method is provided for treating cancer in a subject that has been or is scheduled to be treated with an immune checkpoint inhibitor and / or has been or is scheduled to be treated with regard to CD4+ regulatory T cell depletion. In some embodiments, the CD4+ CTLs provided herein come into contact with cancer cells in vivo, ex vivo, or in vitro. In some embodiments, the CD4+ CTLs are MHC matched to the subject having cancer.

[0052] In some embodiments, the provided method for treating cancer and / or killing cancer cells involves CD4+ CTLs coming into contact with cancer cells in vivo. In some embodiments, the CD4+ CTLs induce an antigen-specific T-cell immune response. In some embodiments, the CD4+ CTLs are administered in a therapeutically effective dose to treat the cancer.

[0053] In some embodiments, the subject is an animal model of cancer. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human, primate, rodent, or mouse.

[0054] In some embodiments, the provided method for treating cancer and / or killing cancer cells further comprises administering one or more adjuvants.

[0055] In some embodiments, the provided method for treating cancer and / or killing cancer cells comprises a further administration of one or more additional therapies, thereby the subject being treated with both CD4+ CTLs and one or more additional therapies.

[0056] In some embodiments, the provided method for treating cancer and / or killing cancer cells further comprises administering one or more immune checkpoint inhibitor therapies. In some embodiments, the immune checkpoint therapies control PD-1, PD-L1, PD-L2, LAG3, TIM3, CEACAM1, IDO, and / or CTLA4.

[0057] In some embodiments, the provided method for treating cancer and / or killing cancer cells includes a further administration of one or more immunocostimulatory therapies. In some embodiments, the immunocostimulatory therapy promotes costimulation of the CD27 and / or OX40 costimulatory pathways in CD4+ CTLs.

[0058] In some embodiments, one or more regulatory T cell (Treg) depletion or inhibitory therapies are further administered.

[0059] In some embodiments, one or more additional treatments are administered concurrently or sequentially with CD4+ CTLs.

[0060] The drawings included in this specification are for illustrative purposes only and are not limiting. [Brief explanation of the drawing]

[0061] [Figure 1]This diagram provides an exemplary schematic representation of tumor cell-mediated activation of CD4+ T cells to generate CD4+ CTLs. The left panel shows activation by B-cell malignancies expressing MHC-II and the co-stimulatory signal (CD70 and / or OX40L). The right panel shows activation by other cancer cells induced by IFN-γ to express MHC-II and the co-stimulatory signal (CD70 and / or OX40L). [Figure 2A] FACS analysis of MHC-II and CD70 in A20 cells with and without CD70 transfection is shown. [Figure 2B] This shows FACS analysis of Eomes, granzyme B, and IFN-γ in CD4+ T cells primed for 6 days with irradiated CD70-transfected A20 cells. [Figure 2C-1] This shows FACS analysis of Eomes, granzyme B, and IFN-γ in CD4+ T cells primed for 9 days with irradiated CD70-transfected A20 cells. [Figure 2C-2] This shows FACS analysis of Eomes, granzyme B, and IFN-γ in CD4+ T cells primed for 9 days with irradiated CD70-transfected A20 cells. [Figure 3] This study demonstrates the killing activity of CD4+ T cells against WT A20 and MHCII-null A20 target cells. [Figure 4] This diagram illustrates the exemplary activation of CD4+ T cells by tumor cells (e.g., melanoma) to produce CD4+ CTLs. The upper panel shows activation by melanoma with IFN-γ-induced expression of MHC-II and co-stimulatory signals (CD70 and / or OX40L). The lower panel shows activation by melanoma with treatment using anti-CD27 and / or anti-OX40 antibodies in relation to IFN-γ-induced expression of MHC-II and co-stimulatory signals. [Figure 5A] FACS analysis of MHC-II and CD70 in B16 cells with or without IFN-γ treatment or CD70 transfection is shown. [Figure 5B]This shows FACS analysis of the activation marker CD69 in Trp1-specific CD4+ T cells stimulated for 18 hours with a specified B16 variant. [Figure 5C] FACS analysis of Eomes expression in Trp1-specific CD4+ T cells primed with MHC-II+CD70+B16 cells for 6 days is shown, compared to naive CD4 cells derived from normal mice. [Figure 6] FACS analysis of naive polyclonal CD4 cells co-cultured with irradiated MHC-II+CD70+ or ​​MHC-II+OX40L+B16 cells is shown. (A) FACS analysis of Eomes expression in unprimed naive polyclonal CD4 cells (left), naive polyclonal CD4 cells primed with MHC-II+CD70+B16 cells (center), and naive polyclonal CD4 cells primed with MHC-II+OX40L+B16 cells (right). (B) FACS analysis of perforin expression is shown. (C) FACS analysis of granzyme B expression is shown. (D) FACS analysis of IFN-γ expression is shown. (E) FACS analysis of PD-1 expression is shown. Unprimed CD4 cells were used as a negative control. [Figure 7] FACS analysis of naive polyclonal CD4 cells co-cultured with MHC-II+B16 cells and αCD27 or αOX40 agonist antibodies is shown. (A) FACS analysis of Eomes expression in unprimed naive polyclonal CD4 cells (left), naive polyclonal CD4 cells primed with MHC-II+B16 cells and αCD27 agonist antibody (center), and naive polyclonal CD4 cells primed with MHC-II+B16 cells and αOX40 agonist antibody (right). (B) FACS analysis of perforin expression is shown. (C) FACS analysis of granzyme B expression is shown. Unprimed CD4 cells were used as a negative control. [Figure 8]The FACS analysis of naive polyclonal CD4 cells co-cultured with MHC-II+B16 cells and αCD27 or αOX40 agonist antibodies at different time points is shown. From left to right, the panels show CD4 cells co-cultured with MHC-II+B16 cells, with αCD27 agonist antibody from day 0 (leftmost panel), with αCD27 agonist antibody from day 3 (second from the left), with αOX40 agonist antibody from day 0 (second from the right), and with αOX40 agonist antibody from day 3 (rightmost panel). [Figure 9A] This diagram shows a schematic representation of the adoptive cell therapy (ACT) protocol used herein. B16 refers to B16 melanoma cells; TBI refers to whole-body irradiation. [Figure 9B] This shows the mean tumor volume in the specified mouse group (n=3 mice per group). Statistical analysis by one-way ANOVA, *, p<0.05. [Figure 9C] Figure 9A shows representative images of tumors treated with specified CD4 cells according to the ACT protocol at day 18. The left image shows a representative "no ACT" control mouse; the center image shows a representative mouse treated with naive CD4 cells; and the right image shows a representative mouse treated with CD4 CTLs. Tumor volume is circled in each image. [Figure 10A] This shows FACS analysis of HLA-II expression in patient-derived melanoma cells with and without IFN-γ treatment (hIFNg 20 ng / ml, 3 days). [Figure 10B] FACS analysis of patient-derived melanoma cells with and without CD70 transduction is shown.

[0062] For any figure showing bar histograms, curves, or other data related to the legend, the bars, curves, or other data presented from left to right in each display directly correspond to the boxes in the legend from top to bottom or left to right, unless otherwise indicated.

[0063] A certain definition To facilitate understanding of the present invention, certain terms are first defined below. Additional definitions of the following terms and other terms are provided throughout this specification. Publications and other references referenced herein to provide background to the present invention and further details relating to its implementation are incorporated herein by reference.

[0064] In this application, unless otherwise evident from the context, (i) the terms “a” and “an” are used herein to refer to one or more (i.e., at least one) grammatical objects of an article; (ii) the term “or” may be understood to mean “and / or”; (iii) the terms “comprising” and “including” may be understood to encompass the itemized component or step, whether presented by itself or together with one or more additional components or steps; and (iv) where a scope is provided, endpoints are included.

[0065] The term “administering” means providing a pharmaceutical or composition to a subject, including, but not limited to, administration by a medical professional and by self-administration. This involves the physical introduction of a composition containing therapeutic factors to a subject, using any of the various methods and delivery systems known to those skilled in the art. In some embodiments, the routes of administration for CD4+CTL described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration, such as injection or infusion. As used herein, the term "parenteral administration" means, but is not limited to, methods of administration other than enteral and topical administration, usually by injection, including, but is not limited to, intravenous injection and infusion, intraperitoneal injection and infusion, intramuscular injection and infusion, intra-arterial injection and infusion, intrathecal injection and infusion, intralymphatic injection and infusion, intralesional injection and infusion, intracapsular injection and infusion, intraorbital injection and infusion, intracardiac injection and infusion, intradermal injection and infusion, transtracheal injection and infusion, subcutaneous injection and infusion, subcuticular injection and infusion, intra-articular injection and infusion, subcapsular injection and infusion, subarachnoid injection and infusion, intraspinal injection and infusion, epidural injection and infusion, intrasternal injection and infusion, and in vivo electroporation. Alternatively, the binding proteins described herein may be administered via non-parenteral routes such as topical, epidermal, or mucosal administration routes, for example, intranasal, oral, vaginal, rectal, sublingual, or topical. Furthermore, administration may be, for example, once, multiple times, and / or over a long period of time.

[0066] As used herein, the term “antibody” includes the whole antibody and any antigen-binding fragment (i.e., “antigen-binding moiety”) or one of its chains. In one embodiment, “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds, or its antigen-binding moiety. Each heavy chain has a heavy chain variable region (V as used herein). H The heavy chain constant region includes the heavy chain constant region (abbreviated as CH1, CH2, and CH3). In certain naturally occurring antibodies, each light chain has a light chain variable region (V as specified herein). L(Abbreviated as ) and includes the light chain constant region. The light chain constant region includes one domain, CL. V H and V L The region can be further subdivided into a highly variable region called the complementarity-determining region (CDR) and a more conserved region called the framework region (FR) that lies between them. H and V L It consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q).

[0067] As used herein, the term “antigen” is defined as a molecule that elicits an immune response. This immune response may involve either antibody production, activation of specific immune-qualified cells, or both.

[0068] As used herein, the term “autologous” is intended to refer to any material originating from the same subject that is to be later reintroduced. If cells originate from a subject that is to be later reintroduced, those cells are considered “autologous” to that subject.

[0069] As used herein, the term “allogeneic” refers to any material derived from genetically different subjects of the same species. A cell is considered “allogeneic” with respect to the subject if it originates from the same animal species as the subject but presents sequence variations at at least one locus of the subject compared to each of the subject's respective loci.

[0070] As used herein, the terms “cell,” “cell line,” and “cell culture” include offspring, which are all progeny. It is understood that due to intentional or accidental mutations, not all offspring may be identical.

[0071] Generally, the term “cytotoxic T cells (CTLs)” refers to T lymphocytes that can kill cells expressing MHC-presented antigens, such as virus-infected cells or transformed cancer cells. In this specification, cytotoxic T cells include the subtypes CD8+ T cells (CD8+ CTLs) and CD4+ T cells (CD4+ CTLs). CTLs are specific to peptide antigens presented in association with proteins encoded by MHC genes and expressed on the surface of cells. In some embodiments, CTLs lyse cancer cells.

[0072] As used herein with respect to nucleic acids and specific cells, the term “exogenous” refers to any nucleic acid found in nature that does not originate from that particular cell. Therefore, a nucleic acid that does not exist in nature is considered exogenous to a cell once it is introduced into that cell. A nucleic acid that exists in nature can also be exogenous to a particular cell. For example, a unique nucleic acid sequence isolated from a cell of subject X is exogenous to a cell of subject Y once that nucleic acid is introduced into the cell of subject Y.

[0073] The term "immune checkpoint" refers to a class of molecules on the surface of CD4+ and / or CD8+ T cells that fine-tune the immune response by downmodulating or inhibiting the antitumor immune response. Immune checkpoint proteins and their sequences are well known in the art and include, but are not limited to, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP, CD47, CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophyllin, IDO, CD39, CD73, and A2aR (see, for example, WO2012 / 177624). This term further encompasses biologically active protein fragments, as well as nucleic acids encoding full-length immune checkpoint proteins and their biologically active protein fragments. In some embodiments, this term further encompasses any fragments subject to the homology description provided herein. In one embodiment, the immune checkpoint protein is PD-1.

[0074] The term “immunotherapy” generally refers to any strategy for modulating the immune response in a beneficial manner, and includes treatment of subjects who are suffering from a disease, are at risk of suffering from a disease, or are at risk of recurrence of a disease, as well as any treatment that uses a specific part of the subject’s immune system to fight a disease such as cancer. The subject’s own immune system is stimulated (or suppressed), with or without the administration of one or more agents for that purpose. Immunotherapy designed to induce or amplify the immune response is called “activating immunotherapy.” Immunotherapy designed to reduce or suppress the immune response is called “suppressive immunotherapy.” In some embodiments, immunotherapy is specific to target cells, such as cancer cells. In some embodiments, immunotherapy may be “detargeted,” which refers to the administration of agents that do not selectively interact with immune system cells but modulate immune system function. Typical examples of detargeted therapies include, but are not limited to, chemotherapy, gene therapy, and radiotherapy.

[0075] The terms "MHC matching" or "HLA matching" refer to the matching of one or more MHC antigens (HLA antigens in humans) between donor cells and host recipients. As the number of MHC (HLA) matches in a donor-recipient pair increases (i.e., 1, 2, 3, 4, 5, and up to 6 allele matches), the likelihood of host recipient immune cells such as T cells and NK cells recognizing the donor cells as foreign decreases, thus reducing the likelihood of an immune response to the donor.

[0076] The terms “nucleic acid” or “polynucleotide” refer to deoxyribonucleic acid or ribonucleic acid in either single-stranded or double-stranded form and polymers thereof. Unless otherwise specified, the term encompasses nucleic acids containing known analogues of native nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly includes not only the explicitly indicated sequence but also its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences. Specifically, degenerate codon substitution can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al, Mol. Cell. Probes 8:91-98 (1994)).

[0077] The term “promoter” typically refers to a nucleic acid sequence located upstream (5') of the coding sequence, which directs the transcription of the nucleic acid sequence into mRNA. A promoter or promoter region typically provides recognition sites for RNA polymerase and other factors necessary for proper transcription initiation. As intended herein, a promoter or promoter region includes promoter mutations induced by means of insertion or deletion of a regulatory region, or by subjecting the promoter to random mutagenesis or site-directed mutagenesis. The activity or strength of a promoter can be measured by the amount of RNA it produces or the amount of protein accumulation in cells or tissues, compared to promoters whose transcriptional activity has been previously evaluated.

[0078] The term "subject" includes, but is not limited to, mammals, such as humans, and domesticated or livestock, such as cats, dogs, cattle, and horses. As used herein, the term "patient," unless otherwise specified, is intended to include any warm-blooded vertebrate domesticated animal (including, but not limited to, livestock, horses, cats, dogs, and other pets) and humans.

[0079] As used herein, the term “syngene” refers to any material derived from an object that is genetically similar or identical. Cells are considered “syngene” with respect to an object if they are sufficiently identical and immunologically compatible to be transplantable.

[0080] As used herein, the terms “transfection” or “transduction” refer to the process by which exogenous nucleic acids are transferred to or introduced into host cells. A transfected or transductioned cell is a cell that has been transfected or transductioned with exogenous nucleic acids. A “transfected or transductioned cell” includes primary host cells and their progeny.

[0081] A "vector" is a composition containing isolated nucleic acids that can be used to deliver those isolated nucleic acids into the interior of a cell. Many vectors are known in the art and include, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, circular RNA, plasmids, and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid compounds and non-viral compounds that facilitate the transport of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and lentiviral vectors.

[0082] Furthermore, this disclosure may employ conventional molecular biology, microbiology, and recombinant DNA techniques to the extent that they are within the skill of those skilled in the art. Such techniques are fully described in the literature. For example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein “Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (DNGlover ed.1985); Oligonucleotide Synthesis (MJ Gait ed.1984);Nucleic Acid Hybridization[BDHames&S.J.Higgins eds.(1985)];Transcription And Translation[BDHames&S.J.Higgins,eds.(1984)];Animal Cell Culture[RIFreshney,ed.(1986)];Immobilized Cells And Enzymes[IRL Press,(1986)];B.Perbal,A Practical Guide To Molecular Cloning(1984);FMAusubel et See al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994). [Modes for carrying out the invention]

[0083] CD4+ T cells are best known for their helper function, which allows them to recruit and / or enhance other immune effectors to fight cancer. (Toes et al., Semin.Immunol.10,443-448 (1998)). Specifically, CD4+ T cells can recognize tumor antigens presented by antigen-presenting cells (APCs) and assist in initiating an antitumor CD8+ T cell response through cross-priming, or activate tumor cell-killing macrophages and NK cells by producing IFN-γ. (See above). CD4+ T cells with cytotoxic function (CD4+ CTLs) are increasingly recognized to occur in both mice and humans in response to viral infections and in various cancers. Swain et al.,Nat.Rev.Immunol.12,136-148(2012);Cenerenti et al.,Front. Immunol.13,867189(2022);Oh et al.,Cell 181,1612-1625 e1613(2020);and Cachot et al.,Sci Adv.7(2021).

[0084] For example, the clinical response to PD-1 blockade in patients with classical Hodgkin lymphoma (cHL) and melanoma is associated with MHC-II expression on tumor cells, suggesting the therapeutic importance of CD4+ T cells. Roemer et al. J. Clin. Oncol. 36, 942-950 (2018); Rodig et al. Sci. Transl. Med. 10 (450) (2018); Johnson et al. Nat Commun 7: 10582 (2016).

[0085] Studies of CD19 CAR-T cell therapy in long-term surviving patients revealed two distinct phases of the antitumor response: CD8+ CAR-T cells were dominant in the initial phase, followed by the dominance (almost 100%) of cytotoxic CD4+ CAR-T cells over several years, suggesting that CD4+ T cells may provide sustained and long-lasting immunity in vivo. Melenhorst et al. Nature 602, 503-509 (2022). In PTLD trials, clinical response was found to be significantly correlated with the frequency of CD4+ T cells in the injected T cells; CD4+ CTLs demonstrated superior therapeutic efficacy compared to CD8+ CTLs in a mouse B-cell lymphoma model. Haque et al. Blood (2007); Choi et al. PNAS (2018). CD4+ CTLs can attack other cancers that upregulate MHC-II upon exposure to IFN-γ, including melanoma, bladder cancer, breast cancer, head and neck cancer, and hepatocellular carcinoma, as demonstrated in mouse models and patients. (Quezada et al. J.Exp.Med. 207, 637-650 (2010); Xie et al. J.Exp.Med. 207, 651-667 (2010); Kitano et al. Cancer Immunol. Res. 1, 235-244 (2013); Oh et al., Cell 181, 1612-1625 e1613 (2020); and Cachot et al., Sci Adv. 7 (2021).) In summary, these results suggest that CD4+ CTLs may be a useful cell-based therapy for treating cancer.

[0086] In recent years, methods for inducing CD4+ CTL responses using B cells expressing the signaling protein LMP1, encoded by the Epstein-Barr virus (EBV) with various effects, have been described (Choi et al. PNAS 115, E686-E695 (2018) and Choi et al. Nature 590, 157-162 (2021)). While this method is highly effective in cases where LMP1 is expressed, not all cancer cells express LMP1. Therefore, the identification of other CD4+ CTL generation methods, such as the signal 2 pathway that leads to CD4+ CTL generation, has been desired.

[0087] Furthermore, the development of CD4+CTLs effective against solid tumors remains an ongoing challenge.

[0088] This disclosure provides, in particular, CD4+ cytotoxic T lymphocytes (CTLs) and related methods, uses, and techniques. This disclosure demonstrates that the provided CD4+ CTLs are effective across tumor types, including solid tumors.

[0089] A method for generating CD4+ cytotoxic lymphocytes from CD4+ T cells. In some embodiments, a method for generating CD4+ CTLs is provided, which includes exposing CD4+ T cells to major histocompatibility complex II (MHC II) expressing cancer cells, and one or more activators that promote (i) CD27-mediated costimulation and / or (ii) OX40-mediated costimulation of CD4+ T cells, for a period of time and under conditions suitable for the formation of at least one immune complex between CD4+ T cells and cancer cells and for the promotion of CD27-mediated costimulation and / or OX40-mediated costimulation of CD4+ T cells, thereby generating CD4+ CTLs.

[0090] Some embodiments provide a method that does not involve contacting CD4+ T cells with an OX40-mediated costimulator. Some embodiments provide a method that does not involve contacting CD4+ T cells with a CD27-mediated costimulator. Some embodiments provide a method that involves contacting CD4+ T cells with one or more factors that promote CD27-mediated costimulatory interaction and one or more factors that promote OX40-mediated costimulatory interaction. Some embodiments provide a method that further involves contacting CD4+ T cells with IL-2.

[0091] For example, a method for generating CD4+ CTLs is provided, which comprises contacting CD4+ T cells with major histocompatibility complex II (MHC II) expressing cancer cells and (i) one or more activators that promote CD27-mediated costimulation of CD4+ T cells, under conditions and for a period of time suitable for the formation of at least one immune complex between CD4+ T cells and cancer cells and the promotion of CD27-mediated costimulation of CD4+ T cells, thereby generating CD4+ CTLs. In some embodiments, the method provided further comprises contacting with IL-2.

[0092] In some embodiments, one or more activators that promote CD27-mediated costimulation include MHC II-expressing cancer cells, in which case the MHC II-expressing cancer cells are engineered or manipulated to express CD70 on their cell surface. In some embodiments, the provided method includes contacting CD4+ T cells or a population of CD4+ T cells with MHC II-expressing cancer cells that also express CD70. In some embodiments, MHC II-expressing cancer APCs, such as cancer cells, are engineered or manipulated to exogenously express CD70. In some embodiments, the MHC II-expressing cancer cells express both CD70 and OX40L.

[0093] In some embodiments, a method is provided for generating CD4+ CTLs, which comprises contacting CD4+ T cells with one or more populations of cancer cells that present antigens by class II MHC molecules and express CD70 and / or OX40L, under conditions and for a period of time suitable for the formation of at least one immune complex between the CD4+ T cells and the cancer cells and for promoting the co-stimulation of the CD4+ T cells, thereby generating CD4+ CTLs. In some embodiments, the provided method further comprises contacting with IL-2.

[0094] In some embodiments, a method is provided for generating CD4+ CTLs, the method comprising contacting CD4+ T cells with one or more populations of cancer cells presenting antigens by class II MHC molecules, and one or more soluble activators that promote cellular co-stimulation mediated by (i) CD27 / CD70 signaling and / or (ii) OX40L / OX40 signaling, under conditions and for a period of time suitable for the formation of at least one immune complex between CD4+ T cells and cancer cells and for the promotion of CD4+ T cell co-stimulation, thereby generating CD4+ CTLs. In some embodiments, the one or more soluble activators include agonistic antibodies. In some embodiments, the one or more soluble activators include fusion proteins (e.g., Fc fusion proteins). In some embodiments, the method provided further comprises contact with IL-2.

[0095] In some embodiments, the CD4 T cells for use in generating CD4+ CTLs by the method described herein are naive CD4+ T cells, memory CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, Trp1-specific CD4+ T cells, tumor antigen-specific CD4+ T cells, neoantigen-specific CD4+ T cells, CD4+ T cells derived from peripheral blood mononuclear cells (PBMCs), CD4+ T cells derived from umbilical cord blood, CD4+ T cells derived from tumor-infiltrating lymphocytes, primary CD4+ T cells, CD4+ T cells derived from cell lines, in vitro amplified CD4+ cells, and / or in vitro activated CD4+ cells.

[0096] In some embodiments, CD4+ T cells are allogeneic, syngeneic, or autologous to APCs. In some embodiments, CD4+ T cells are allogeneic, syngeneic, or autologous to MHC II-expressing cancer cells.

[0097] In some embodiments, CD4+ T cells are selected from the group consisting of mammalian CD4+ T cells, rodent CD4+ T cells, mouse CD4+ T cells, primate CD4+ T cells, and human CD4+ T cells.

[0098] CD4+CTL In some embodiments, this disclosure provides CD4+CTL produced by the methods described herein.

[0099] In some embodiments, the CD4+CTLs provided herein express a granzyme, such as granzyme B. In some embodiments, the CD4+CTLs produced by the method provided herein express perforin. In some embodiments, the CD4+CTLs produced by the method provided herein express both a granzyme and perforin. In some embodiments, the CD4+CTLs provided herein express CD69. In some embodiments, the CD4+CTLs provided herein express a granzyme (e.g., granzyme B), perforin, and CD69. In some embodiments, the CD4+CTLs provided herein are programmed by Eomes.

[0100] In some embodiments, the CD4+CTLs provided herein secrete cytokines. In some embodiments, the CD4+CTLs secrete IFNγ.

[0101] In some embodiments, the CD4+ CTLs provided herein induce antigen-specific T cell immune responses. In some embodiments, the CD4+ CTLs may target multiple tumor antigens. In some embodiments, the CD4+ CTLs provided herein induce multi-antigen-specific T cell immune responses.

[0102] In some embodiments, the CD4+ CTLs provided herein are effectively used as monotherapy for treating cancer.

[0103] In some embodiments, the CD4+ CTLs provided herein can kill cancer cells. In some embodiments, the CD4+ CTLs can directly kill MHC-II+ cancer cells. In some embodiments, the CD4+ CTLs provided herein directly kill cancer cells that express an MHC-II antigen complex specifically recognized by the CD4+ CTL on their cell surface.

[0104] In some embodiments, the CD4+ CTLs provided herein are proliferative. In some embodiments, the CD4+ CTLs provided herein can be amplified in vitro and / or ex vivo.

[0105] In some embodiments, CD4+ CTLs provide long-term tumor immunity. In some embodiments, the CD4+ CTLs provided herein persist in vivo. In some embodiments, the CD4+ CTLs provided herein persist in vivo for at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 1.5 years, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years. In some embodiments, the CD4+ CTLs provided herein persist in vivo for a period of any range including endpoints such as 6 months to 10 years, or 6 months to 12 months, 6 months to 2 years, or 6 months to 5 years.

[0106] In some embodiments, the CD4+ CTLs provided herein perform CD4+ helper functions. In some embodiments, the CD4+ helper functions include (i) licensing APCs to prime anti-cancer CD8+ CTLs (e.g., by secreting cytokines, activating APCs, and / or mobilizing APCs), (ii) activating anti-cancer NK cells, (iii) activating anti-cancer macrophages, and / or (iv) activating anti-cancer immune cells.

[0107] CD4+ CTLs produced by the methods described herein may be functionally characterized using methodologies for assaying T cell activity, including determining T cell binding, activation, or induction, and also determining antigen-specific T cell responses. Examples include T cell proliferation, T cell cytokine release, antigen-specific T cell stimulation, MHC-restricted T cell stimulation, and CTL activity (e.g., from pre-loaded target cells). 51 This includes detecting Cr release or detecting active caspase-3 in target cells, changes in T cell phenotypic marker expression, and determination of other measures of T cell function.

[0108] Procedures for performing these and similar assays can be found, for example, in Lefkovits (Immunology Methods Manual: His Comprehensive Sourcebook of Techniques, 1998), as well as in Current Protocols in Immunology, Weir, (1986) Handbook of Experimental Immunology, Blackwell Scientific, Boston, MA; Mishell and Shigii (eds.) (1979) Selected Methods in Cellular Immunology, Freeman Publishing, San Francisco, CA; Green and Reed (1998) Science 281:1309, and the references cited therein.

[0109] Cytotoxic assays to determine CTL activity may be performed using any one of several techniques and methods routinely used in the art (e.g., Henkart el al., “Cytotoxic T-Lymphocytes” in Fundamental Immunology, Paul (ed.) (2003 Lippincott Williams & Wilkins, Philadelphia, PA), pages 1127-50, and the references cited therein).

[0110] In some embodiments, the CD4+CTLs provided herein have MHC-II-restricted perforin / granzyme-mediated cytotoxicity. In some embodiments, the CD4+CTLs provided herein are characterized by the expression of certain markers, including, for example, CD69, perforin, and / or granzymes such as granzyme B. In some embodiments, the generation of CD4+CTLs is dependent on (i.e., programmed by) the transcription factor eomesodermine (Eomes).

[0111] In some embodiments, CD4+ CTLs produced by the method of this disclosure can kill unmodified cells (e.g., unmodified tumor cells) that express antigens recognized by TCRs expressed by CD4+ CTLs when presented in relation to MHC II molecules. In some embodiments, CD4+ CTLs produced by the method of this disclosure can be used in adoptive cell therapy.

[0112] costimulatory factor Productive activation of CD4+ T cells (e.g., naive CD4+ T cells) requires two signals: signal 1, through TCR recognition of antigens presented on MHC complexes, and signal 2, via a co-stimulatory pathway. This disclosure provides insight that tumor antigen-specific CD4+ CTLs can be induced using tumor B cells (which naturally present endogenous antigens on MHC-II and enable signal 1), provided that co-stimuli are also available that can drive CD4+ CTL differentiation (signal 2).

[0113] The term “immunostimulatory molecule” or “costimulatory factor” refers to a molecule that provides non-antigen-specific signals for T cell proliferation and functional differentiation. Representative immunocostimulatory molecules include, but are not limited to, CD80 / B7-1, CD86 / B7-2, CD70, CD27, OX40 ligand, OX40, 4-1BB ligand, 4-1BB, and GITR. The methods provided encompass the insight that costimulation via CD27-mediated signaling (e.g., CD70 / CD27 signaling) and / or OX40-mediated signaling (e.g., OX40L / OX40 signaling) can promote the activation of CD4+ T cells and their differentiation into CD4+ CTLs. In some embodiments, costimulation is mediated via CD27-mediated signaling (e.g., CD70 / CD27 signaling). In some embodiments, costimulation is mediated via OX40-mediated signaling (e.g., OX40L / OX40 signaling). In some embodiments, co-stimulation is mediated via CD27-mediated signaling (e.g., CD70 / CD27 signaling) and OX40-mediated signaling (e.g., OX40L / OX40 signaling).

[0114] This disclosure provides additional insight that CD27-mediated co-stimulation (e.g., co-stimulation via CD70 / CD27 signaling) is particularly useful in driving CD4+ CTL differentiation. In some embodiments, co-stimulation for activating CD4+ CTL differentiation is via both CD27-mediated co-stimulation (e.g., co-stimulation via CD70 / CD27 signaling) and OX40-mediated co-stimulation (e.g., co-stimulation via OX40L / OX40 signaling).

[0115] Table 1 provides representative and non-limiting examples of nucleic acid and amino acid sequences of exemplary co-stimulatory molecules.

[0116] In some embodiments, the co-stimulatory factor is encoded by a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleic acid sequences described herein (e.g., listed in Table 1).

[0117] In some embodiments, the co-stimulatory factor includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequences described herein (e.g., listed in Table 1). In some embodiments, the co-stimulatory factor includes an amino acid sequence described herein (e.g., listed in Table 1).

[0118] In some embodiments, the costimulatory molecule is or includes CD70 (also known as CD27L; LPFS3; CD27-L; CD27LG; ​​TNFSF7; TNLG8A). CD70 is a ligand belonging to the tumor necrosis factor (TNF) family and functions to enhance the T cell-regulated immune response by activating the costimulatory receptor CD27.

[0119] In some embodiments, the CD27-activating costimulator includes a nucleic acid sequence encoding CD70. In some embodiments, the CD27-activating costimulator includes a CD70 amino acid sequence.

[0120] In some embodiments, the CD70 sequence is of human origin. Exemplary human CD70 nucleic acid and amino acid sequences are available in the GenBank database, for example, NM_001252.5, NP_001243.1, and NM_001330332.2 and NP_001317261.1. In some embodiments, the CD70 sequence is of mouse origin. Exemplary mouse CD70 nucleic acid and amino acid sequences are available in the GenBank database, for example, NM_011617.2 and NP_035747.1. Nucleic acid and polypeptide sequences of CD70 orthologs in other organisms are publicly known in the art and are all included within the scope of this disclosure.

[0121] In some embodiments, the nucleic acid sequence encoding CD70 has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of sequence numbers 2, 4, and 6. In some embodiments, the nucleic acid sequence encoding CD70 includes the sequence described in any one of sequence numbers 2, 4, and 6.

[0122] In some embodiments, the CD70 amino acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 1, 3, and 5. In some embodiments, the CD70 amino acid sequence includes the sequence described in any one of SEQ ID NOs: 1, 3, and 5.

[0123] In some embodiments, the co-stimulatory molecule is or includes CD27 (also known as S152, S152.LPFS2, T14, TNFRSF7, and Tp55). CD27 is a 55 kDa type I transmembrane protein belonging to the tumor necrosis factor receptor (TNFR) family that co-stimulates T cell activation after binding to its ligand, CD70. In humans, CD27 is constitutively expressed by IT cells and also expressed in activated T cells (e.g., transiently upregulated).

[0124] In some embodiments, the CD27-activating costimulator includes a nucleic acid sequence encoding CD27. In some embodiments, the CD27-activating costimulator includes a CD27 amino acid sequence. In some embodiments, CD27 is of human origin. Exemplary human CD27 nucleic acid and amino acid sequences are available in the GenBank database as NM_001242.5, NP_001233.2, NM_001413263.1, NP_001400192.1, NM_001413264.1, NP_001400193.1, NM_001413265.1, and NP_001400194.1. In some embodiments, the CD27 sequence is of mouse origin. Exemplary mouse CD27 nucleic acid and amino acid sequences are publicly available in the GenBank database, for example, NM_001033126.2, NP_001028298.1, NM_001042564.1, NP_001036029.1, NM_001286753.1, and NP_001273682.1. In addition, nucleic acid and polypeptide sequences of CD27 orthologs in other organisms are publicly known in the art.

[0125] In some embodiments, the nucleic acid sequence encoding CD27 has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 8. In some embodiments, the nucleic acid sequence encoding CD27 includes the sequence described in SEQ ID NO: 8.

[0126] In some embodiments, the CD27 amino acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 7. In some embodiments, the CD27 amino acid sequence includes the sequence described in SEQ ID NO: 7.

[0127] In some embodiments, the co-stimulatory molecule is or comprises OX40L (also known as TNFSF4, CD134L, CD252, GP34, OX-40L, OX4OL, TNLG2B, and TXGP1). In some embodiments, the OX40-activating co-stimulatory factor comprises a nucleic acid sequence encoding OX40L. In some embodiments, the OX40-activating co-stimulatory factor comprises the OX40L amino acid sequence.

[0128] OX40L is predominantly expressed in antigen-presenting cells. Exemplary OX40L nucleic acid and amino acid sequences are available in the GenBank database. In some embodiments, OX40L is of human origin. Exemplary human OX40L nucleic acid and amino acid sequences are available in the GenBank database, for example, NM_001297562.2, NP_001284491.1, NM_003326.5, and NP_003317.1.

[0129] In some embodiments, the OX40L sequence is mouse-derived. Exemplary mouse OX40L nucleic acid and amino acid sequences are publicly available in the GenBank database, for example, NM_009452.2 and NP_033478.1. In addition, nucleic acid and polypeptide sequences of OX40L orthologs in other organisms are publicly known in the art and are all included within the scope of this disclosure.

[0130] In some embodiments, the nucleic acid sequence encoding OX40L has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 10. In some embodiments, the OX40L nucleic acid sequence includes the sequence described in SEQ ID NO: 10.

[0131] In some embodiments, the OX40L amino acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 9. In some embodiments, the OX40L amino acid sequence includes the sequence described in SEQ ID NO: 9.

[0132] In some embodiments, the co-stimulatory molecule is or includes OX40 (also known as TNFRSF4, ACT35; CD134; IMD16; TXGP1L). OX40 is not constitutively expressed in untreated T cells but is induced after T cell receptor (TCR) involvement. OX40 expression is induced in activated CD4+ T cells but is generally not expressed in resting naive T cells or most resting memory T cells.

[0133] In some embodiments, the OX40 activating costimulator includes a nucleic acid sequence encoding OX40. In some embodiments, the OX40 activating costimulator includes an OX40 amino acid sequence.

[0134] Exemplary OX40 nucleic acid and amino acid sequences are publicly available in the GenBank database. In some embodiments, OX40 is of human origin. Exemplary human OX40L nucleic acid and amino acid sequences are publicly available in the GenBank database, for example, NM_001410709.1, NP_001397638.1, NM_003327.4, and NP_003318.1. In some embodiments, the OX40L sequence is of mouse origin. Exemplary mouse OX40 nucleic acid and amino acid sequences are publicly available in the GenBank database, for example, NM_011659.2 and NP_035789.1. In addition, nucleic acid and polypeptide sequences of OX40 orthologs in other organisms are publicly known in the art and are all included within the scope of this disclosure.

[0135] In some embodiments, the nucleic acid sequence encoding OX40 has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 12. In some embodiments, the OX40 nucleic acid sequence includes the sequence described in SEQ ID NO: 12.

[0136] In some embodiments, the OX40 amino acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 11. In some embodiments, the OX40 amino acid sequence includes the sequence described in SEQ ID NO: 11.

[0137] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

[0138] Co-stimulation can be mediated by any method known in the art. In some embodiments, co-stimulation is mediated by an agonist antibody that specifically binds to an immunocostimulatory molecule. In some embodiments, co-stimulation is at least partially mediated by an anti-CD27 agonist antibody and / or an anti-OX40 agonist antibody.

[0139] In some embodiments, one or more co-stimulatory factors include anti-CD27 agonist antibodies. Exemplary anti-CD27 agonist antibodies are known in the art. For example, WO2012 / 004367 describes an anti-human agonist antibody called hCD27.15, and WO2011 / 130434 describes an anti-human CD27 antibody called 1F5. In some embodiments, the anti-mouse CD27 antibody is BE0348, clone RM27-3E5 (BioXCell).

[0140] In some embodiments, one or more co-stimulatory factors include anti-OX40 agonist antibodies. Exemplary anti-OX40 agonist antibodies are known in the art, for example, INCAGN01949, 9B12, MEDI0562, and BMS-986178. In some embodiments, the anti-mouse OX40 (CD134) antibody is BE0031, clone OX-86 (BioXCell).

[0141] In some embodiments, co-stimulation is mediated by soluble fusion proteins of immunoco-stimulatory molecules. In some embodiments, co-stimulation is mediated by CD70 fusion proteins (e.g., CD70-Fc fusion proteins) and / or OX40L fusion proteins (e.g., OX40L-Fc fusion proteins).

[0142] In some embodiments, co-stimulation is mediated by a CD70 fusion protein (e.g., CD70-Fc fusion protein). In some embodiments, the CD70 fusion protein binds to its co-stimulatory surface receptor CD27, which is expressed on T lymphocytes. Exemplary CD70-Fc fusion proteins are commercially available: Ag28070 (Proteintech®) and CD70-Fc (SKU number: FCL2530, G&P Biosciences®).

[0143] In some embodiments, antigen-presenting cells (e.g., antigen-presenting cancer cells) are supplemented with CD27 activators (e.g., CD70, e.g., ectopically expressed CD70) and / or OX40 activators (e.g., OX40L, e.g., ectopically expressed OX40L) to activate CD4+ T cells and form CD4+ CTLs.

[0144] antigen presenting cells The term “antigen-presenting cell” refers to any of the various cells capable of displaying, acquiring, and / or presenting at least one antigen or antigenic fragment on its cell surface. Generally, antigen-presenting cells (APCs) can be any cell that induces and / or enhances an immune response to an antigen or antigenic composition. Full activation of T cells can be achieved by the antigen displayed by the APC in the form of an MHC-bound peptide that gives specificity to the response, and by a co-stimulatory signal that facilitates the development of an effective immune response of antigen-nonspecific and adaptive immunity. T cell co-stimulation increases T cell proliferation, differentiation, and survival. T cell activation without co-stimulation can lead to the development of T cell anergy, T cell deletion, or immune tolerance. Additional molecules expressed by APCs or other immune cells that can assist or enhance the immune response include secretory molecules, such as cytokines and cytotoxic molecules. Some cells, such as dendritic cells, are professional APCs. In some embodiments, APCs are derived from cancer cells. In some embodiments, APCs are manipulated (for example, cancer cells manipulated to express MHC II, as well as CD70 and / or OX40L).

[0145] The term "MHC" refers to "major histocompatibility complex." In humans, MHC genes are known as HLA ("human leukocyte antigen") genes. While there is no consistent convention, some literature uses HLA to refer to the HLA protein molecule and MHC to refer to the gene encoding the HLA protein. Therefore, the terms "MHC" and "HLA" are used interchangeably herein. The human HLA lineage has an equivalent in mice, namely the H2 lineage. The most studied HLA genes are the nine so-called classical MHC genes: HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1. In humans, MHC contains at least three regions: classes I, II, and III. The A, B, and C genes belong to MHC class I, and the six D genes belong to class II. MHC class I molecules consist of a single polymorphic chain containing three domains (alpha-1, 2, and 3), which associates with beta-2 microglobulin on the cell surface. Class II molecules consist of two polymorphic chains, each containing two domains (alpha-1 and 2, and beta-1 and 2). Class I MHC molecules are expressed on virtually all nucleated cells. Peptide fragments presented in association with class I MHC molecules are recognized by CD8+ T lymphocytes (traditionally called cytotoxic T lymphocytes or CTLs). CD8+ T lymphocytes frequently mature into cytotoxic effectors capable of lysing cells with stimulating antigens. Class II MHC molecules are primarily expressed on activated lymphocytes and professional APCs. CD4+ T lymphocytes (traditionally called helper T lymphocytes or HTLs) are activated by recognition of specific peptide fragments presented by class II MHC molecules, which are typically found on APCs such as macrophages, dendritic cells, or B cells. CD4+ T lymphocytes proliferate and secrete cytokines that either support antibody-mediated responses through the production of IL-4, support cell-mediated responses through the production of IL-2 and IFN-gamma, or acquire direct killing activity (cytotoxicity).

[0146] In some embodiments, APCs (e.g., antigen-presenting cancer cells) are induced or have been induced to express MHC II through contact with interferon-gamma (IFNγ). Many cancers, including melanoma, bladder cancer, breast cancer, head and neck cancer, and hepatocellular carcinoma (HCC), are known to induce MHC II expression via IFNγ exposure. In some embodiments, APCs (e.g., antigen-presenting cancer cells) are induced or have been induced to express MHC II by the expression of class II transactivator (CIITA).

[0147] In some embodiments, APCs (e.g., antigen-presenting cancer cells) endogenously (e.g., spontaneously) express MHC-II. In some embodiments, APCs (e.g., antigen-presenting cancer cells) are derived from cancer that endogenously (e.g., spontaneously) expresses MHC-II.

[0148] Cells may be transfected or transductioned using any suitable method, for example, APCs (e.g., cancer cells) are included by the methods described herein. Methods for delivering polynucleotides to host cells include, for example, the use of cationic polymers, lipid-like molecules, and certain commercial products, such as in vivo-jetPEI®. Other methods include ex vivo transduction, injection, electroporation, DEAE-dextran, sonication loading, liposome-mediated transfection, receptor-mediated transduction, microparticle guns, and transposon-mediated transfer. Further methods for transfecting or transductioning host cells employing vectors are described in more detail herein.

[0149] In some embodiments, APCs (e.g., antigen-presenting cancer cells) are brought into contact with IL-2.

[0150] cancer The terms "cancer," "tumor," or "hyperproliferative" refer to the presence of cells that have characteristics typical of cancerous cells, such as uncontrolled growth, immortality, potential for metastasis, rapid growth and proliferation rates, and certain characteristic morphological features.

[0151] In some embodiments, the cancer is B-cell carcinoma. B-cell malignancies include numerous genetically, phenotypically, and clinically distinct diseases, including Hodgkin lymphoma (HL), most non-Hodgkin lymphomas (NHL), chronic lymphocytic leukemia (CLL), and B-cell acute lymphoblastic leukemia (B-ALL). NHL can be further classified into many subtypes, including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, and others.

[0152] In some embodiments, CD4+ CTLs produced by the method of the present disclosure are suitable for killing and / or treating CD70-deficient cancers. In some embodiments, CD4+ CTLs produced by the method of the present disclosure are suitable for killing and / or treating CD70-deficient B-cell tumors.

[0153] In some embodiments, CD4+ CTLs produced by the methods of this disclosure are suitable for killing and / or treating CD70-deficient solid tumors.

[0154] In some embodiments, CD4+CTLs produced by the methods disclosed herein are suitable for killing and / or treating cancers having intact CD70.

[0155] In some embodiments, the method can generate CD4+ CTLs against a wide range of endogenous tumor antigens without the need to identify them in each patient.

[0156] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is melanoma, bladder cancer, breast cancer, head and neck cancer, and / or hepatocellular carcinoma (HCC), colon cancer, pancreatic cancer, kidney cancer, ovarian cancer, or brain cancer.

[0157] In some embodiments, cancer presents at least one MHC II-antigen complex on its cell surface. In some embodiments, the antigen presented by the MHC II-antigen complex is a tumor-associated antigen and / or neoantigen.

[0158] In some embodiments, cancer cells endogenously (e.g., spontaneously) express CD70. In some embodiments, cancer cells do not endogenously (e.g., spontaneously) express CD70. For example, some B-cell carcinomas are known to initially express CD70 and then lose CD70 expression. In some embodiments, cancer-derived cells for use in the provided method are engineered to exogenously express CD70.

[0159] In some embodiments, cancer cells endogenously (e.g., naturally) express OX40L. In some embodiments, cancer cells do not endogenously (e.g., naturally) express OX40L. In some embodiments, cancer-derived cells for use in the provided method are engineered to exogenously express OX40L.

[0160] Nucleic acids and vectors Provided herein are nucleic acid molecules useful in the methods provided herein. For example, in some embodiments, nucleic acid molecules encoding co-stimulatory factors (e.g., factors that promote CD27-mediated and / or OX40-mediated co-stimulation) are provided. In some embodiments, nucleic acid molecules encoding factors that promote CD27-mediated co-stimulation are provided, for example, in this case the factor is a CD70 polypeptide (e.g., a fusion polypeptide, e.g., CD70-Fc) or an anti-CD27 agonist antibody. In some embodiments, nucleic acid molecules encoding factors that promote OX40-mediated co-stimulation are provided, for example, in this case the factor is an OX40L polypeptide (e.g., a fusion polypeptide, e.g., OX40L-Fc) or an anti-OX40 agonist antibody. In some embodiments, nucleic acid molecules encoding factors that promote OX40-mediated co-stimulation are provided, for example, in this case the factor is an OX40L polypeptide or an agonist OX40 antibody. Methods for gene delivery to cells are well known in the art and are further described in the APC section above, including, for example, mRNA transfection, which has been found to be particularly effective in the examples for the expression of exogenous nucleic acids in human cancer cells.

[0161] In some embodiments, the nucleic acids provided are recombinant. As used herein, the term “recombinant” means (i) a molecule constructed outside of a living cell by conjugating a native or synthetic nucleic acid segment to a nucleic acid molecule that can replicate in a living cell, or (ii) a molecule resulting from replication of one of those described in (i) above. For the purposes of this specification, replication may be in vitro / ex vivo replication or in vivo replication.

[0162] In some embodiments, the nucleic acid comprises a codon-optimized nucleotide sequence. Without being bound to a particular theory or mechanism, codon optimization of the nucleotide sequence is thought to improve the translation efficiency of the mRNA transcript. Codon optimization of the nucleotide sequence may involve replacing natural codons with other codons that encode the same amino acid but can be translated by tRNAs that are more readily available intracellularly, thus improving translation efficiency. Optimization of the nucleotide sequence may also reduce secondary mRNA structures that interfere with translation, thus improving translation efficiency.

[0163] Compositions and Specifications Compositions comprising CD4+ CTLs generated by the methods of the present disclosure, and their use are provided herein.

[0164] In some embodiments, the compositions comprising CD4+ CTLs disclosed herein further comprise a suitable injection vehicle. The suitable injection vehicle may be any isotonic vehicle formulation, typically normal saline, Normosol®-R (Abbott) or Plasma-Lyte® A (Baxter), 5% aqueous dextrose, lactated Ringer's solution may be utilized. The injection vehicle may be supplemented with human serum albumin or other human serum components. Unit doses or compositions comprising an effective amount of CD4+ CTLs are also contemplated.

[0165] The amount of CD4+ CTLs in the composition or unit dose is at least 1 cell, or more typically 10 2 or more cells, for example, up to 10 6 , up to 10 7 , up to 10 8 , up to 10 9 , or 10 10 or more cells. In some embodiments, the CD4+ CTLs are in the range of about 10 6 ~ about 10 10 cells / m 2 , for example, about 10 5 ~ about 10 9 cells / m 2They are administered within a range of . The number of CD4+CTLs will vary depending on the final use for which the composition or unit dose is intended, as well as the type of cells contained therein. For example, in some embodiments, CD4+CTLs expressing a TCR specific to a particular antigen will constitute a cell population containing at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of such CD4+CTLs. For the uses provided herein, the CD4+CTLs are generally in volumes of 1 liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. In embodiments, the desired density of CD4+CTLs is typically 10 4 Higher than cells / ml, generally 10 7 Higher than cells / ml, generally 10 8 The number of cells / ml or higher. CD4+ CTLs may be administered as a single infusion or in multiple infusions over a period of time. A clinically relevant number of immune cells is 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 It can be distributed across multiple infusions that are cumulatively equal to or exceed the number of CD4+ CTLs.

[0166] The effective dose of a pharmaceutical composition refers to a sufficient amount in the required dosage and duration to achieve the desired clinical outcome or beneficial treatment as described herein. The effective dose may be delivered in one or more doses.

[0167] This specification provides methods for preventing and / or treating non-malignant disorders, hyperproliferative disorders, or recurrences of hyperproliferative disorders, and / or for inducing an immune response against cells of interest, such as hyperproliferative cells, the methods comprising contacting undesirable cells expressing an antigen(s) with CD4+CTLs described herein that target such antigen(s) (e.g., adoptive cell therapy). In some embodiments, the methods comprise administering to a subject a therapeutically effective amount of a composition containing CD4+CTLs, for example, through autologous, allogeneic, or syngeneic transplantation. In some embodiments, the CD4+CTLs described herein may be used to determine the responsiveness to CD4+CTL therapy for such disorders.

[0168] Methods for preparing cells for adoptive cell therapy (e.g., isolation, purification, irradiation, etc.) and administering them are publicly known and may be used in connection with the provided methods and compositions (e.g., U.S. Patent Publication No. 2003 / 0170238, U.S. Patent No. 4,690,915, Rosenberg (2011) Nat. Rev. Clin. Oncol. 8:577-585, Themeli et al. (2013) Nat. Biotechnol. 31:928-933, Tsukahara et al. (2013) Biochem. Biophys. Res. Commun. 438:84-89, and Davila et al. (2013) PloS ONE 8:e61338).

[0169] In addition, the CD4+CTL compositions described herein may also be administered in combination with one or more additional activators or therapies to further modulate the desired activity. Additional activators and therapies include, but are not limited to, Treg modulation and / or depletion therapy, cancer immunotherapy, immune checkpoint inhibitor (e.g., blockade) therapy, chemotherapeutic agents, hormones, anti-angiogenic agents, radiolabeled compounds, surgery, cryotherapy, and / or radiotherapy. The preceding treatment method may be administered in combination with other forms of conventional treatment (e.g., standard treatments for cancer well known to those skilled in the art). The CD4+CTL compositions described herein may be administered before, after, or consecutively with additional activators and / or therapies. For example, the CD4+CTL composition may be administered with a therapeutically effective dose of a chemotherapeutic agent. In another embodiment, the CD4+CTL composition is administered in combination with an additional therapy, such as immune checkpoint inhibitor therapy, to enhance the activity and efficacy of the additional therapy. Physicians' Desk Refiner (PDR) discloses dosages of additional activators or therapies used in the treatment of various cancers. The medication regimen and dosage for treatment intervention may be determined by the physician, depending on the specific disorder being treated, the degree of the disorder, and other factors well known to a physician skilled in the art.

[0170] For prophylactic use, the dose should be sufficient to prevent, delay the onset of, or reduce the severity of a disease or disorder associated with it. The prophylactic benefit of immunogenic compositions administered according to the methods described herein can be determined by conducting preclinical (including in vitro, ex vivo, and in vivo animal studies) and clinical studies and analyzing the data obtained therefrom using appropriate statistical, biological, and clinical methods and techniques, all of which can be readily carried out by those skilled in the art.

[0171] In some embodiments, the subjects receiving a unit dose of CD4+ CTLs are those who have undergone or have previously undergone a transplant, such as a hematopoietic stem cell transplant (HCT; including myeloablative and non-myeloablative HCT). In any of the embodiments described above, the hematopoietic cells used in the HCT may be “universal donor” cells modified (e.g., by chromosomal gene knockout according to the method described herein) to reduce or eliminate the expression of one or more endogenous genes encoding polypeptide products selected from MHC, antigens, and binding proteins.

[0172] Techniques and regimens for performing cell transplantation are known in the art and may include the transplantation of any suitable donor cells, such as cells derived from umbilical cord blood, bone marrow, or peripheral blood, hematopoietic stem cells, recruited stem cells, or cells derived from amniotic fluid. Therefore, in some embodiments, CD4+ CTLs may be administered together with or immediately after cell transplantation therapy.

[0173] In addition, kits and devices comprising the CD4+CTL described herein are further provided. For example, a kit or device may contain the CD4+CTL composition alone or together with additional reagents, such as adjuvants, detection reagents, and combinations thereof, packaged in a suitable container, and may further include instructions for using such reagents. A kit or device may also include other components, such as an administration tool packaged in a separate container. A kit or device may be advertised, distributed, or sold as a unit for performing the methods described herein. [Examples]

[0174] Example 1: CD70-based method for generating CD4+ CTLs using B lymphoma cells This embodiment describes an exemplary CD70-based method for generating CD4+ CTLs using exemplary mouse B lymphoma cells (A20 cells). Specifically, it was found that A20 cells ectopically expressing CD70 (amino acid sequence NP_035747.1 available from GenBank) were sufficient to generate CD4+ CTLs when co-cultured in vitro with naive CD4 T cells.

[0175] We used the mouse B-cell lymphoma model A20 (BALB / c background) because these cells express MHC-II and do not have basal expression of CD70 or OX40L. To manipulate A20 cells to ectopically express CD70, we performed transduction with VSVG pseudotyped retrovirus carrying GFP or CD70-GFP, and GFP was expressed 48 hours after viral transduction. + Cells were sorted by FACS. Figure 2A shows the FACS analysis of MHC-II and CD70 in A20 cells carrying GFP or CD70-GFP.

[0176] Naive mouse spleen CD4+ T cells were purified using a naive CD4 T cell isolation kit with magnetically activated cell sorting (MACS; Miltenyi Biotec) according to the manufacturer's protocol. The mouse naive CD4+ T cells were co-cultured with CD70-GFP-expressing A20 cells or GFP-expressing A20 cells in a 2:1 ratio for 6 or 9 days to generate CD4+ cytotoxic T cells (CD4 CTLs).

[0177] This disclosure includes the recognition that CD4+ CTLs exert MHC-II-restricted perforin / granzyme B-mediated cytotoxicity; and that their functional differentiation (including the expression of perforin and granzyme B) is dependent on the transcription factor Eomes. Representative exemplary CD4+ CTLs were generated by co-culturing naive CD4+ T cells with CD70-GFP-expressing A20 cells. Briefly, T cells were stained for CD4, the transcription factor Eomes, cytotoxic molecules (granzyme B and perforin), and effector cytokines (IFNγ and TNFα). The results of FACS analysis of Eomes, granzyme B, and IFN-γ in CD4 cells primed with irradiated CD70-transfected A20 cells are shown in Figures 2B and 2C, respectively, after 6 and 9 days of culture. These results demonstrate that ectopic expression of CD70 in A20 cells is sufficient to induce CD4+ CTL formation in vitro, as indicated by the expression of Eomes, granzyme B (and IFN-γ) in a significant proportion of the generated CD4 cells.

[0178] CD4+ CTL generation was further evaluated using a killing assay. Target cells were labeled with CellTrace® Violet (Invitrogen) before use. CD4+ T cells were divided into 2 × 10⁶ cells. 3 Target cells were co-cultured in 96-well round-bottom plates at various effector / target ratios. The plates were then spun down at 8g for 2 minutes, followed by incubation at 37°C for 4–6 hours. The cultures were stained for active caspase-3 (BD ​​Biosciences). + CellTrace(TM) + The cells were defined as apoptotic target cells. The results are shown in Figure 3. CD4+ T cells primed with A20 cells ectopically expressing CD70 were able to induce specific killing, unlike A20 cells lacking CD70.

[0179] These results demonstrate that exemplary B lymphoma cells ectopically expressing CD70 are sufficient to enable the differentiation of naive CD4+ T cells into CD4+ CTLs.

[0180] Example 2: CD70 and MHC-II methods for generating CD4+ CTLs using melanoma cells In this example, as shown in Figure 4, we describe an exemplary CD70-based method for generating CD4+ CTLs using exemplary melanoma cells induced to express MHC-II. Specifically, CD70 was ectopically expressed in interferon-gamma (IFNγ) treated mouse melanoma cells (B16-F10 cells). B16 cells do not express CD70 in nature. Exemplary CD70-expressing MHC-II+ melanoma cells were found to be sufficient to induce CD4+ CTLs in vitro.

[0181] CD70 cDNA (encoding the amino acid sequence of NP_035747.1, available from GenBank) was cloned into an MSCV-GFP retroviral vector to create MSCV-CD70-GFP. The MSCV-CD70-GFP or MSCV-GFP retroviral vector was co-transfected into HEK293T cells with VSVG packaging plasmids (pCMV-VSVG and pKat). After 48 hours, the viral supernatant was collected, filtered through a 0.45 μm filter, and then used immediately to infect cells or stored at -80°C for later use. For transduction of B16-F10 cells, cells were infused with VSVG pseudotyped retrovirus carrying GFP or CD70-GFP (10 μg ml). -1 Cells were infected for 48 hours in the presence of polyblen (Sigma), and GFP+ cells were sorted by FACS.

[0182] It is well known that B16 cells upregulate MHC-II when exposed to IFN-γ. In this example, 3-day treatment with IFN-γ in cell culture induced MHC-II expression in all B16 cells. Mouse IFN-γ (BioLegend, catalog number 575306). Figure 5A shows FACS analysis of MHCII and CD70 expression in GFP or CD70-GFP transduction B16 cells treated with or without IFN-γ (100 ng / mL) for 3 days.

[0183] We evaluated T cell stimulation of exemplary CD4+ T cells expressing a transgenic TCR specific to a class II (I-Ab) restricted epitope, derived from tyrosinase-related protein 1 (Trp1), a TAA expressed in B16 cells (MHC-II+, CD70+B16-F10 cells).

[0184] Trp1 mouse CD4+ T cells were enriched from splenocytes using MACS (StemCell Technologies) with a negative selection method employing an antibody cocktail (anti-CD11b, anti-CD19, anti-B220, anti-GR-1, and anti-TER-119), and then FACS sorting was performed for CD4+CD25-CD69-. The purified Trp1 CD4 cells were then primed in vitro as follows. For the CD69 activation assay, naive Trp1 CD4 cells were cultured for 18 hours in 48-well flat-bottom plates with different B16-stimulated cells. The results are shown in Figure 5B. CD69 expression was then analyzed by FACS. For CD4 CTL generation, naive Trp1 CD4 cells were cultured for 6 days in 24-well flat-bottom plates with irradiated (30 Gy) IFN-γ-pre-treated CD70-transfected B16-F10-stimulated cells. Next, Eomes expression was analyzed by FACS; naive CD4 T cells from wild-type mice were used as a control. The results are shown in Figure 5C.

[0185] These results indicate that naive Trp1-CD4 cells recognize B16 cells in an MHC-II-dependent manner, and (IFN-γ treated "MHC-II enriched") MHC-II +We show that ectopic expression of CD70 in B16 cells leads to more potent activation of naive Trp1-CD4 cells (Figure 5B), and that priming of these cells induces the expression of Eomes, a master regulator of CD4 CTL differentiation (Figure 5C).

[0186] These results indicate that MHC-II expressing CD70 + This supports the idea that activation of CD4+ T cells using B16 cells can lead to efficient generation of CD4+ CTLs.

[0187] Example 3: Creation of multispecific CD4+ CTLs through co-stimulation with ectopically expressed ligands This embodiment confirms that CD4+ CTLs can be generated using co-stimulation through ectopic expression of CD70 or OX40L ligand, as shown in Figure 4. CD70 or OX40L was overexpressed in exemplary melanoma cells (interferon-gamma (IFNγ) treated B16 cells) that were also induced to express MHC-II. Specifically, naive polyclonal CD4 cells were overexpressed in irradiated MHC-II + CD70 + or MHC-II + OX40L + B16 cells were co-cultured with these cells for 10 days, and mouse IL-2 (final concentration 20 ng / ml) was added after 72 hours (to promote T cell amplification), followed by analysis for specified molecules (unprimed CD4+ cells were used as negative controls). As shown in Figures 6A–6E, both exemplary OX40L-expressing MHC-II+ melanoma cells and exemplary CD70-expressing MHC-II+ melanoma cells were found to be sufficient to induce CD4+ CTLs in vitro.

[0188] These results indicate that MHC-II expressing either OX40L or CD70 + This supports the idea that activation of CD4+ T cells using B16 cells can lead to efficient generation of CD4+ CTLs.

[0189] Example 4: Creation of multispecific CD4+ CTLs through co-stimulation with agonistic antibodies This example confirms that CD4+ CTLs can be generated using co-stimulation via agonistic antibodies against CD27 or OX40, as shown in Figure 4. Exemplary melanoma cells (interferon-gamma (IFNγ) treated B16 cells) induced to express MHC-II were supplemented with agonistic antibodies against CD27 or OX40. The anti-mouse CD27 and anti-mouse OX40 (CD134) in this example were obtained from BioXCell, catalog number BE0348, clone RM27-3E5 and catalog number BE0031, clone OX-86, respectively. Specifically, naive polyclonal CD4 cells were induced to express MHC-II + B16 cells were co-cultured for 10 days with exemplary αCD27 or αOX40 agonist antibodies (both at a final concentration of 10 μg / ml), and mouse IL-2 (final concentration of 20 ng / ml) was added after 72 hours, followed by analysis for specified molecules. Unprimed CD4 cells were used as negative controls. The results are shown in Figure 7. Exemplary MHC-II+ melanoma cells supplemented with anti-CD27 or anti-OX40 agonist antibodies were both found to be sufficient to induce CD4+ CTLs in vitro.

[0190] This disclosure includes the recognition that the advantage of using agonistic antibodies for co-stimulation is the ability to adjust the timing of stimulation. The effects of altering the timing of stimulation using anti-CD27 or anti-OX40 agonistic antibodies were also evaluated.

[0191] Specifically, naive polyclonal CD4 cells are MHC-II +B16 cells were co-cultured, and the above-mentioned αCD27 or αOX40 agonist antibody was added from day 0 or day 3, mouse IL-2 was added after day 3, and the specified molecule was analyzed on day 10. MHCII+ B16 cells were B16-F10 (from ATCC) treated with mouse IFNg (100 ng / ml, 4 days). Mouse IL-2 (final concentration 20 ng / ml) was added 72 hours after co-culture; αCD27 or αOX40 agonist antibody was added at the specified time (final concentration 10 ug / ml). Surprisingly, as shown in Figure 8, MHC-II + In co-culture with B16 cells, increased activation was observed when exposure to αCD27 or αOX40 agonistic antibodies was started on day 3, compared to when exposure started on day 0.

[0192] These results indicate that MHC-II + This supports the idea that activation of CD4+ T cells using B16 cells and agonistic αCD27 or αOX40 antibodies can lead to efficient generation of CD4+ CTLs.

[0193] Example 5: Adoptive cell therapy (ACT) using multispecific CD4 CTLs generated by the provided method. This embodiment describes an exemplary use of CD4+ CTLs generated using the method described herein in adoptive cell therapy. Specifically, exemplary multispecific CD4 CTLs primed with MHC-II+CD70+B16 cells were used in adoptive cell therapy in an in vivo mouse model. Mice were treated with the ACT protocol provided in Figure 9A. Mice were subcutaneously administered B16 cells (2 × 10^5 cells) on day 0. On day 8, mice were irradiated (5 Gy total body irradiation (TBI)) and intravenously administered CD4 T cells (4 × 10^5 cells). Figure 9B shows the mean tumor volume over time in a specified group of mice (n=3 mice per group). Compared to mice without ACT or naive CD4 treatment, tumor volume was significantly reduced over time. Statistical analysis by one-way ANOVA. *, p<0.05. This effect is also shown in Figure 9C, which provides a representative image of tumors treated with specified CD4 cells at day 18.

[0194] These results demonstrate that adoptive cell therapy (ACT) using multispecific CD4 CTLs primed with MHC-II+CD70+B16 cells clearly restricts the growth of pre-established B16 tumors.

[0195] Example 6: Upregulation of HLA-II and ectopic expression of CD70 in patient-derived melanoma cells This example confirms that exemplary patient-derived cancer cells (melanoma cells) can be induced to upregulate HLA-II and ectopically express CD70. As shown by the FACS analysis of HLA-II expression in Figure 10A, 3-day IFN-γ treatment with 20 ng / ml hIFN-γ upregulates HLA-II expression in five different patient-derived melanoma cells. Human IFN-γ (MedChemExpress, catalog no. HY-P7025) patient-derived melanoma cells were further transductioned with retroviral vectors. CD70-encoding cDNA was cloned into the MSCV-IRES-GFP retroviral vector. Each retroviral vector was co-transfected with a VSVG packaging plasmid and HEK293T cells. After 48 hours, the viral supernatant was collected, filtered through a 0.45 μm filter, and then used immediately to infect cells or stored at -80°C for later use. FACS analysis of patient-derived melanoma cells confirmed CD70 expression in CD70 transduction cells. Figure 10B. These results demonstrate that patient-derived melanoma cells can upregulate HLA-II upon IFN-γ exposure and be induced to express CD70 by gene transduction. These results support the use of primary cancer cells for use in the method for generating CD4+ CTLs described herein.

[0196] Built-in by reference All publications, patents, and patent applications referenced herein are incorporated herein by reference in whole, as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. In the event of any conflict, this application, including any definitions herein, shall prevail.

[0197] Any polynucleotide and polypeptide sequences that reference accession numbers corresponding to entries in public databases, such as those maintained by The Institute for Genomic Research (TIGR) at tigr.org on the World Wide Web and / or the National Center for Biotechnology Information (NCBI) at ncbi.nlm.nih.gov on the World Wide Web, are also incorporated in their entirety by reference.

[0198] Equivalents Those skilled in the art will be able to recognize or confirm many equivalents to the specific embodiments of the invention described herein simply by using routine experiments. Such equivalents are intended to be covered by the following claims.

Claims

1. A method for generating cytotoxic lymphocytes (CD4+ CTLs), comprising contacting CD4+ T cells with major histocompatibility complex II (MHC II) expressing cancer cells, and (i) one or more activators that promote CD27-mediated costimulation of the CD4+ T cells and / or (ii) one or more activators that promote OX40-mediated costimulation of the CD4+ T cells, under conditions and for a period of time suitable for the formation of at least one immune complex between the CD4+ T cells and the cancer cells and for the promotion of CD27-mediated costimulation of the CD4+ T cells and / or OX40-mediated costimulation of the CD4+ T cells, thereby generating the CD4+ CTLs.

2. The method according to claim 1, wherein the method does not involve contacting CD4+ T cells with an OX40-mediated costimulator.

3. The method according to claim 1, wherein the method does not involve contacting CD4+ T cells with a CD27-mediated costimulator.

4. The method according to claim 1, wherein the method comprises contacting CD4+ T cells with one or more activators that promote CD27-mediated costimulation of the CD4+ T cells and one or more activators that promote OX40-mediated costimulation of the CD4+ T cells.

5. The method according to any one of claims 1 to 4, wherein the CD4+ T cells are naive CD4+ T cells, memory CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, Trp1-specific CD4+ T cells, tumor antigen-specific CD4+ T cells, neoantigen-specific CD4+ T cells, CD4+ T cells derived from peripheral blood mononuclear cells (PBMCs), CD4+ T cells derived from umbilical cord blood, CD4+ T cells derived from tumor-infiltrating lymphocytes, primary CD4+ T cells, CD4+ T cells derived from cell lines, CD4+ cells amplified in vitro, and / or CD4+ cells activated in vitro.

6. The method according to any one of claims 1 to 5, wherein the CD4+ T cells are allogeneic, syngeneic, or autologous to the cancer cells.

7. The method according to any one of claims 1 to 6, wherein the CD4+ T cells are selected from the group consisting of mammalian CD4+ T cells, rodent CD4+ T cells, mouse CD4+ T cells, primate CD4+ T cells, and human CD4+ T cells.

8. The method according to any one of claims 1 to 7, wherein the cancer cells are induced to express MHC II through contact with interferon-gamma (IFNγ).

9. The method according to any one of claims 1 to 8, wherein the cancer cells endogenously express MHCII.

10. The method according to any one of claims 1 to 9, wherein the cancer cells do not express CD70, or in some cases, the cancer cells do not naturally express CD70.

11. The method according to any one of claims 1 to 10, wherein the cancer cells are derived from a cancer selected from the group consisting of B-cell leukemia, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), classical Hodgkin lymphoma (cHL), acute myeloid leukemia (AML), solid tumors, melanoma, bladder cancer, breast cancer, head and neck cancer, hepatocellular carcinoma (HCC), colon cancer, pancreatic cancer, kidney cancer, ovarian cancer, and brain cancer.

12. The method according to any one of claims 1 to 11, wherein the cancer cells present at least one MHC II antigen complex on their cell surface, and optionally the antigen is a tumor-associated antigen and / or neoantigen.

13. One or more of the factors in i) and one or more of the factors in ii) are a) MHC II-expressing cancer cells which are manipulated or have been manipulated to express CD70 on their cell surface; b) Soluble CD70, which optionally comprises a CD70 fusion protein (e.g., a CD70-Fc fusion protein); c) Anti-CD27 agonist antibody; d) MHC II-expressing cancer cells which are manipulated or have been manipulated to express OX40 ligand (OX40L) on their cell surface; e) Soluble OX40L, which optionally comprises an OX40L fusion protein (e.g., an OX40L-Fc fusion protein); and / or f) Anti-OX40 agonist antibody The method according to any one of claims 1 to 12, including the method described in any one of claims 1 to 12.

14. The aforementioned CD4+CTL is a) Expressing a T cell receptor (TCR) that specifically binds to the MHC II antigen complex expressed on the surface of cancer cells by the cancer cells; b) Expressing a granzyme, which in some cases is granzyme B; c) Expresses CD69; d) Express perforin; e) Programmed by Eomes; f) Secretes cytokines, in some cases the cytokine is IFNγ; g) Induce an antigen-specific T cell immune response; h) It can kill cancer cells; i) Directly kill cancer cells that express the MHC II-antigen complex, which is specifically recognized by the CD4+ CTL, on their cell surface; j) To multiply; k) to persist in vivo; and / or l) Execute CD4+ helper function. The method according to any one of claims 1 to 13.

15. The method according to any one of claims 1 to 14, wherein the duration is at least 6 months to 10 years.

16. The method according to any one of claims 1 to 15, wherein the CD4+ helper function comprises (i) licensing antigen-presenting cells (APCs) to prime anti-cancer CD8+ CTLs, (ii) activating anti-cancer NK cells, (iii) activating anti-cancer macrophages, and / or (iv) activating anti-cancer immune cells.

17. The method according to any one of claims 1 to 16, further comprising culturing the CD4+CTL under conditions suitable for enabling the proliferation of the CD4+CTL, wherein the culturing period is optionally selected from the group consisting of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, and 14 days.

18. The method according to any one of claims 1 to 17, further comprising the step of treating the APC (e.g., cancer cells) to prevent further proliferation before contact with the CD4+ T cells, wherein the treatment is optionally irradiation.

19. The method according to any one of claims 1 to 18, further comprising the step of purifying the CD4+CTL.

20. The method according to any one of claims 1 to 19, wherein the CD4+ T cells are brought into contact ex vivo or in vitro.

21. A composition comprising CD4+ CTLs produced according to the method of any one of claims 1 to 20, wherein the CD4+ CTLs are optionally multispecific to an antigen expressed by the cancer cells and presented by MHC II.

22. An immunogenic composition or vaccine comprising CD4+ CTL produced according to the method of any one of claims 1 to 20, wherein the immunogenic composition or vaccine optionally further comprises an adjuvant.

23. The immunogenic composition or vaccine according to claim 22, wherein the immunogenic composition or vaccine can induce an immune response against antigen-expressing cancer cells in a target, and the immune response may be selected from the group consisting of i) a T cell response, a CD4+ helper T cell response, a CD4+ cytotoxic T cell response, and / or a CD8+ T cell response, and / or ii) T cell amplification, cytokine release, and / or cytotoxic killing.

24. A method for killing cancer cells, comprising contacting the cancer cells with the composition according to claim 21, or the immunogenic composition or vaccine according to claim 22 or 23.

25. A method for treating cancer, comprising administering to a subject the composition described in claim 21, or the immunogenic composition or vaccine described in claim 22 or 23.

26. The method according to claim 24 or 25, wherein the CD4+ T cells are allogeneic, syngeneic, or autologous to the cancer cells.

27. The method according to any one of claims 24 to 26, wherein the cancer cells are derived from a cancer selected from the group consisting of B-cell leukemia, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), classical Hodgkin lymphoma (cHL), acute myeloid leukemia (AML), solid tumors, melanoma, bladder cancer, breast cancer, head and neck cancer, hepatocellular carcinoma (HCC), colon cancer, pancreatic cancer, kidney cancer, ovarian cancer, and brain cancer.

28. The method according to any one of claims 24 to 27, wherein the subject is treated or scheduled to be treated with an immune checkpoint inhibitor and / or is treated or scheduled to be treated with regard to CD4+ regulatory T cell depletion.

29. The method according to any one of claims 1 to 28, wherein the CD4+ CTLs come into contact with the cancer cells in vivo, ex vivo, or in vitro.

30. The method according to claim 29, wherein the CD4+CTL is MHC-matched to the subject having the cancer.

31. The CD4+ CTLs come into contact with the cancer cells in vivo. a) The CD4+ CTLs induce an antigen-specific T cell immune response; b) The CTL is administered in a therapeutically effective dose to treat the cancer; c) The subject is an animal model of cancer and / or a mammal, and the mammal may be a human, primate, rodent, or mouse; d) One or more adjuvants are administered; e) One or more additional anticancer therapies are administered; f) One or more immune checkpoint inhibitor therapies are administered, and if applicable, the immune checkpoint therapy controls PD-1, PD-L1, PD-L2, LAG3, TIM3, CEACAM1, IDO, and / or CTLA4; g) One or more immunocostimulatory therapies are administered, and if applicable, the immunocostimulatory therapies promote the costimulation of the CD27 and / or OX40 costimulatory pathways in the CD4+ CTL; h) One or more regulatory T cell (Treg) depletion or inhibitory therapies are administered further. The method according to any one of claims 1 to 30, wherein, depending on the circumstances, one or more of the treatments are administered simultaneously with or sequentially with the CD4+CTLs.

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