T-cell immunotherapy derived from autologous stem cell memory T cells

Abstract

This invention relates to a novel and specific T-cell therapy method based on the use of stem cell memory T cells (Tscm). This novel cellular immunotherapy may be applied not only to PML patients but also to other infections or cancers in which specific memory T-cell responses are functionally impaired.

Description

[Technical Field]

[0001] The present invention relates to the treatment of diseases in the field of medicine, particularly cancer or diseases caused by pathogens, preferably diseases caused by human polyomaviruses such as progressive multifocal leukoencephalopathy, using T-cell immunotherapy. [Background technology]

[0002] Progressive multifocal leukoencephalopathy (PML) is a demyelinating opportunistic disease with a poor prognosis associated with the replication of polyomavirus JC (JCV) in the central nervous system. PML is primarily observed during prolonged and severe cellular immunosuppression, mainly in patients with AIDS or malignant hematological disorders, or after immunosuppressive therapy, including novel and intensive immunosuppressive biotherapy. This serious disease is associated with a high mortality rate and significant neurological sequelae in survivors.

[0003] PML relates to impaired brain immune regulation of viral replication by cytotoxic memory CD8 T lymphocytes, which require functional memory CD4 T cells for optimal function. Impairment of the anti-JC virus CD8 T cell response involves several mechanisms, including anergy and functional exhaustion due to overexpression of inhibitory receptors such as PD1, LAG3, TIGIT, TIM3, CTLA4, and CD160.

[0004] There is no specific antiviral treatment for PML. The only treatment that shows some efficacy is, if possible, restoring the function of the anti-JCV T cell response, for example, by initiating effective antiretroviral therapy in HIV-infected patients or by discontinuing immunosuppressant therapy. However, such immune recovery may require a long period, during which time JCV continues to replicate, neurological lesions expand, and survival and neurological prognosis are impaired.

[0005] International Publication No. 2023 / 073062 describes a novel and specific T-cell therapy method based on the use of stem cell memory T cells (Tscm (memory stem T-cells)). This method is based on the important observation that in patients with severe and long-term immunosuppression, such as PML patients, this rare memory T cell subset maintains high functionality in terms of proliferation and differentiation, and can generate effective specific cytotoxic effectors against viruses or tumor antigens ex vivo, while more differentiated memory T cell subsets such as effector memory (Tem), central memory (Tcm), or effector (Teff) have poor functionality against viruses or tumor antigens. This novel cellular immunotherapy may be applicable not only to PML patients but also to other infections or cancers in which specific memory T-cell responses are functionally impaired.

[0006] The first step of this protocol is based on selecting a population of Tscm cells from a patient's cell sample that have cell surface phenotypes including (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and / or CD62L+, and (iv) CD95+. However, the feasibility of this cell therapy at the clinical stage is hindered by the use of at least five GMP clinical-grade antibodies for this selection. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2023 / 073062 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] Therefore, there is a strong need for alternative methods that efficiently generate an effective and sustained immune response to JCV in PML patients, and that simplify the process so that this treatment can be used on a large clinical scale. [Means for solving the problem]

[0009] In International Publication No. 2023 / 073062, the inventors described a novel and specific T-cell therapy method based on the use of stem cell memory T cells (Tscm). This method is based on the important observation that in patients with severe and long-term immunosuppression, such as PML patients, this rare memory T cell subset maintains high functionality in terms of proliferation and differentiation, and can generate effective specific cytotoxic effectors ex vivo against viral or tumor antigens, while more differentiated memory T cell subsets such as effector memory (Tem), central memory (Tcm), or effector (Teff) have poor functionality against viral or tumor antigens. This novel cellular immunotherapy can be applied not only to PML patients but also to other infections or cancers in which specific memory T cell responses are functionally impaired. Now, the inventors provide a simpler alternative to this method, improving its feasibility on a large clinical scale. This alternative is based on the use of a population containing Tscm and naive T cells instead of a population containing only Tscm, thereby simplifying the protocol for selecting the initial population of the aforementioned method.

[0010] Therefore, in the first aspect, the present invention is a) A step of selecting a population of T cells having a cell surface phenotype including CD4+ or CD8+, CD45RA+ and CCR7+ or CD62L+ from a cell sample of a subject suffering from cancer or a disease caused by a pathogen, particularly a cancer or disease caused by a pathogen in which a specific memory T cell response is functionally impaired. b) A step of culturing the T cell population in the presence of antigen-presenting cells carrying at least one target antigen or at least one peptide derived from at least one target antigen, and optionally in the presence of IL-7 and IL-15 or other stimulating cytokinins, and Optionally, c) A step of collecting the cells obtained in step b), particularly CD8+ and / or CD4+ cells, preferably CD8+ and CD4+ cells. This relates to an in vitro method for obtaining a population of cells, including antigen-specific T cells.

[0011] The T cell population selected in step a) includes both naive T cells (CD95-) and Tscm cells (CD95+). Preferably, the population contains at least 10%, preferably at least 30%, and more preferably at least 50% naive T cells relative to the total number of cells in the population.

[0012] The T cell population selected in step a) may further have a cell surface phenotype comprising PD1-, TIGIT-, LAG3-, TIM3-, CTLA4- and / or CD160-, preferably PD1-, TIGIT-, LAG3-, TIM3- and / or CTLA4-, more preferably PD1-, TIGIT-, LAG3- and / or TIM3-, and even more preferably PD1- and TIGIT-. In particular, the T cell population selected in step a) may further have a cell surface phenotype comprising PD1- and TIGIT-, and optionally LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-, more preferably LAG3- and TIM3-.

[0013] Alternatively, the above method a) A step of selecting a population of Tscm cells from the target cell sample that have a cell surface phenotype including CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, PD1-, and TIGIT-, b) A step of culturing the T cell population in the presence of antigen-presenting cells carrying at least one immunogenic peptide derived from at least one target antigen, and optionally in the presence of IL-7 and IL-15 or other stimulating cytokinins, and c) A step of selecting CD8+ cells and optionally CD4+ cells from the cell population obtained in step b), preferably CD8+ cells and CD4+ cells from the cell population obtained in step b), It may include.

[0014] The T cell population selected in step a) includes both naive T cells (CD95-) and Tscm cells (CD95+). Preferably, the population includes at least 10%, preferably at least 30%, more preferably at least 50% naive T cells based on the total cell number of the population.

[0015] Optionally, the T cell population selected in step a) has a cell surface phenotype that further includes LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-.

[0016] The T cell population selected in step a) may have a cell surface phenotype that further includes CD3+ and / or CD45RO-, preferably CD3+ and CD45RO-.

[0017] In particular, the T cell population selected in step a) may include cells having a cell surface phenotype including CD4+, CD8-, CD45RA+, CCR7+, PD1- and TIGIT- and cells having a cell surface phenotype including CD4-, CD8+, CD45RA+, CCR7+, PD1- and TIGIT-.

[0018] More specifically, the T cell population selected in step a) may include cells having a cell surface phenotype including CD4+, CD8-, CD45RA+, CCR7+, PD1-, TIGIT-, LAG3- and TIM3-, preferably CD3+, CD45RO-, CD4+, CD8-, CD45RA+, CCR7+, PD1-, TIGIT-, LAG3- and TIM3- and cells having a cell surface phenotype including CD4-, CD8+, CD45RA+, CCR7+, PD1-, TIGIT-, LAG3- and TIM3-, preferably CD3+, CD45RO-, CD4-, CD8+, CD45RA+, CCR7+, PD1-, TIGIT-, LAG3- and TIM3-.

[0019] The antigen-presenting cell can be a dendritic cell, a monocyte, a monocyte-derived dendritic cell, peripheral blood mononuclear cells (PBMCs), a B-lymphoblastoid cell line cell transformed by Epstein-Barr virus (EBV-BLCL cell), or an artificial antigen-presenting cell (AAPC). Preferably, the antigen-presenting cell can be a dendritic cell, a monocyte, peripheral blood mononuclear cells (PBMCs), a B-lymphoblastoid cell line cell transformed by Epstein-Barr virus (EBV-BLCL cell), or an artificial antigen-presenting cell (AAPC).

[0020] Preferably, the antigen-presenting cell is autologous to the subject.

[0021] Preferably, the antigen-presenting cell is a monocyte or a dendritic cell, more preferably, an autologous monocyte or dendritic cell to the subject.

[0022] The at least one target antigen can be a pathogen antigen, preferably an antigen of a virus, a bacterium, or a fungus, or an antigen expressed by tumor cells such as a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA).

[0023] The subject can have cancer.

[0024] The at least one target antigen can be an antigen expressed by tumor cells such as a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA).

[0025] The subject can have a disease caused by human polyomavirus, preferably progressive multifocal leukoencephalopathy, Merkel cell carcinoma, or BK virus-associated nephropathy.

[0026] Preferably, the at least one target antigen is an antigen of a human polyomavirus, selected from the group consisting of polyomavirus JC, polyomavirus MPCyV, or polyomavirus BK. More preferably, the at least one target antigen is an antigen of polyomavirus JC or MPCyV, particularly an antigen of polyomavirus JC.

[0027] In step b), the T cell population may be cultured in the presence of IL-7 and IL-15, and / or cultured for 8 to 20 days, preferably 10 to 18 days, more preferably 12 to 16 days.

[0028] The cell sample may be a bone marrow cell sample, a blood cell sample, a fractionated or unfractionated whole blood sample, apheresis recovery of a fractionated or unfractionated cells, tumor-infiltrating lymphocytes, PBMCs, or a population rich in T cells from a blood sample or PBMCs. Preferably, the cell sample may be a population rich in PBMCs, or T cells from a blood sample or PBMCs.

[0029] The present invention also relates to an isolated cell population containing antigen-specific CD8+ T cells and optionally antigen-specific CD4+ T cells, which can be obtained by the method of the present invention for obtaining a population of cells containing antigen-specific T cells. The isolated population may include, or may consist of, Tscm cells, T effector (Teff) cells, T central memory (Tcm) cells, and T effector memory (Tem) cells.

[0030] The isolated cell populations of the present invention, Tscm, Tcm, and Tem cells, may represent up to 90% of the total cells, preferably 50% to 90%, and Teff cells may represent 10% to 50% of the total cells, preferably 10% to 20%.

[0031] The present invention also relates to an in vitro method for obtaining memory stem cell T cells (Tscm cells) and naive T cell populations, comprising selecting a T cell population having a cell surface phenotype including CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, PD1- and TIGIT- and optionally LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-, from a cell sample derived from a subject, wherein the population includes both CD95- and CD95+ cells. Preferably, the population contains at least 10%, preferably at least 30%, and more preferably at least 50% naive T cells relative to the total number of cells in the population.

[0032] The subjects may have cancer or pathogen-induced diseases, particularly cancer or pathogen-induced diseases in which specific memory T cell responses are functionally impaired.

[0033] Preferably, the subjects suffer from a disease caused by human polyomavirus.

[0034] More preferably, the subjects have progressive multifocal leukoencephalopathy, Merkel cell carcinoma, or BK virus-associated nephropathy.

[0035] The present invention further relates to isolated populations of Tscm cells and naive T cells comprising (i) CD4+, CD45RA+, CCR7+ and / or CD62L+, PD1-, TIGIT- and optionally LAG3- and / or TIM3-, and / or (ii) CD8+, CD45RA+, CCR7+ and / or CD62L+, PD1-, TIGIT- and optionally LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-, more preferably LAG3- and TIM3-, wherein the population comprises both CD95- and CD95+ cells. Preferably, the population comprises at least 10%, preferably at least 30%, and more preferably at least 50% naive T cells relative to the total number of cells in the population.

[0036] The present invention also relates to an isolated cell population containing the antigen-specific CD8+ T cells of the present invention and optionally antigen-specific CD4+ T cells, or an isolated population of Tscm cells and naive T cells of the present invention, as a cell therapy. The present invention also relates to a pharmaceutical composition comprising an isolated cell population containing the antigen-specific CD8+ T cells of the present invention or an isolated population of Tscm cells and naive T cells of the present invention, as well as a carrier and / or excipient.

[0037] The present invention further relates to an isolated cell population comprising antigen-specific CD8+ T cells and optionally antigen-specific CD4+ T cells, an isolated population of Tscm cells and naive T cells, or a pharmaceutical composition, for use in the treatment of diseases caused by cancer or pathogens, particularly cancer or pathogen-induced diseases in which a specific memory T cell response is functionally impaired.

[0038] Preferably, the disease to be treated is a disease caused by a polyomavirus, more preferably a disease caused by a human polyomavirus. In particular, the disease to be treated may be progressive multifocal leukoencephalopathy, Merkel cell carcinoma, or BK virus-associated nephropathy.

[0039] Preferably, the disease to be treated is a disease caused by a pathogen, the pathogen being polyomavirus JC, and the disease being progressive multifocal leukoencephalopathy (PML). Alternatively, the disease to be treated is a disease caused by a pathogen, the pathogen being polyomavirus MCPyV, and the disease being Merkel cell carcinoma, or the disease to be treated is a disease caused by a pathogen, the pathogen being polyomavirus BKV, and the disease being BK virus-associated nephropathy.

[0040] Preferably, the cells used in the treatment are of the same origin as the patient being treated.

[0041] The dose of the isolated cell population or pharmaceutical composition administered may contain 1,000 to 10,000,000 antigen-specific CD8+ T cells / kg of target body weight. The dose may further contain 1,000 to 10,000,000 antigen-specific CD4+ T cells / kg of target body weight.

[0042] The present invention further relates to the use of isolated cell populations or pharmaceutical compositions of the present invention for preparing pharmaceuticals for the treatment of diseases caused by cancer or pathogens, particularly diseases caused by cancer or pathogens in which specific memory T cell responses are functionally impaired.

[0043] The present invention further relates to a method for treating patients suffering from cancer or pathogen-induced diseases, particularly cancer or pathogen-induced diseases in which a specific memory T cell response is functionally impaired, comprising administering a therapeutically effective amount of an isolated cell population or a pharmaceutical composition of the present invention to the subject. [Brief explanation of the drawing]

[0044] [Figure 1] Expression of any combination of inhibitory receptors (PD1, TIGIT, LAG3, TIM3) on Tscm, Tcm, Tem CD4, or CD8 T cells. Each row represents one patient. [Figure 2] The inhibitory receptors primarily include PD1 and / or TIGIT. The black areas in the pie chart represent cells positive for PD1 and / or TIGIT, either alone or in combination with other inhibitory receptors. The gray areas represent cells positive for other inhibitory receptors than for PD1 and / or TIGIT. The white areas represent cells negative for the inhibitory receptors. Each row represents one patient. [Figure 3] A gating strategy for isolating T cell populations, including Tscm cells and naive T cells, according to the present invention. The same gating strategy was also applied to CD4 and CD8 T cells. [Figure 4]Proliferative capacity of various selected T cell subsets. Left: Total Tscm (pooled CD4+ and CD8 Tscm) vs. more differentiated CD45RO+ memory cells (pooled CD4 and CD8 Tcm and Tem). Right: PD1, TIGIT, TIM3, and LAG3-negative Tscm vs. PD1 and / or TIGIT and / or TIM3 and / or LAG3-positive Tscm. Proliferation rates were calculated as follows: [number of cultured cells on day 14] / [number of cells on day 0]. Statistical significance: *p<0.05, Wilcoxon test. [Figure 5] Specific cytotoxic activity of cells obtained after culturing different selected T cell subsets for 14 days. Statistical significance: *p<0.05, Wilcoxon test. Cytotoxic activity was assessed by the expression of granzyme b and perforin after restimulation with autologous cells (CD4 and CD3 deficient PBMCs) loaded with JCV peptide. [Figure 6] Differentiation of PD1-TIGIT-Tscm CD8 T cells after 14 days of in vitro culture. [Figure 7] The proportion of naive T cells (CD95-) within a selected T cell subset (CD45RA+ CD62L+) of CD4 and CD8 T cells. Data were obtained from 23 different patients. [Figure 8] PBMCs were stained with CD3, CD4, CD8, CD45RA, CD62L, and CD95 antibodies. (A) The percentage of Tscm (CD45RA+ CD62L+ CD95+) was measured in total CD4+ and CD8+ CD3+ cells (top) and CD45RA+ CD62L+ CD3+ CD4+ (or CD8+) cells (bottom). Data are from 25 PML patients. Boxes and whiskers indicate the median, 10th, and 90th percentiles. + indicates the mean. (B) This figure shows the percentage of total CD4+ and CD8+ CD3+ cells (top), and the percentage of Tscm and naive T cells (Tn) in CD4+ and CD8+ CD45RA+ CD62L+ CD3+ cells (bottom). [Figure 9]PBMCs were stained with CD3, CD4, CD8, CD45RA, CD62L, CD95, PD1, and TIGIT antibodies. The percentage of cells negative for the inhibitory receptors PD1 and TIGIT was analyzed within (A) CD45RA+ CD62L+ cells (CD3+ CD4+ / CD8+ CD45RA+ CD62L+) and (B) Tscm cells (CD3+ CD4+ / CD8+ CD45RA+ CD62L+ CD95+). Data are from 10 patients. Boxes and whiskers indicate the median, 10th, and 90th percentiles. + indicates the mean. [Modes for carrying out the invention]

[0045] In International Publication No. 2023 / 073062, the inventors developed a novel autologous and specific T-cell therapy method to avoid functional inhibition of anti-JCV T cells in PML patients. This method is based on the use of stem cell memory T cells (Tscm). In fact, the inventors observed that in patients with severe and prolonged immunosuppression, such as PML patients, this rare memory T cell subset maintains high functionality in terms of proliferation and differentiation, and can generate effective specific cytotoxic effectors against viral or tumor antigens ex vivo. The inventors also demonstrated that Tscm cells express less inhibitory receptors than more differentiated memory cells in PML patients with various immunological backgrounds, including HIV infection, malignant hematological disorders, and treatment with immunosuppressive biotherapy. The inventors further demonstrated that sorting based on the elimination of PD1 and TIGIT allows for the depletion of the majority of Tscm expressing inhibitory receptors. The inventors also demonstrated that these PD1-TIGIT-Tscm cells exhibit superior proliferative capacity and superior cytotoxicity compared to PD1+ and / or TIGIT+ Tscm and more differentiated memory cells. These cells efficiently differentiate into more differentiated memory cells, including effector cells, in vitro, but the majority retain the Tscm phenotype after administration, enabling further differentiation cycles and thus long-lasting therapeutic effects in vivo. After in vivo activation, differentiation, and proliferation, these cells can thus provide a cell population containing antigen-specific T cells that can generate an effective and sustained immune response against JCV in PML patients. Personalized medicine based on this novel cell therapy may also be applicable to other chronic viral infections or cancers in which specific antiviral or antitumor memory T cell responses are functionally impaired.

[0046] In this specification, the inventors now provide an alternative to the method that improves its feasibility on a large clinical scale. This alternative is based on the use of a population containing Tscm and naive T cells instead of a population containing only Tscm, thereby simplifying the protocol for selecting the initial population in the method. In fact, the inventors observed that when {CD45RA+ CCR7+ and / or CD62L+} sorted cells (Tscm and naive T cells) coated with autologous PBMCs and JCV peptide are cultured, only memory cells, i.e., stem cell memory T cells, are activated, since naive T cell activation requires dendritic cell priming in vivo. Thus, in vitro activation with JCV peptide in the presence of autologous monocytes does not activate naive T cells. In contrast, memory T cells, i.e., Tscm cells, within the sorted gate {CD45RA+ CCR7+ and / or CD62L+} are activated. Therefore, activation of cells sorted based on the {CD45RA+ CCR7+ and / or CD62L+} gate leads to the selective amplification and differentiation of Tscm cells, thereby simplifying the protocol for selecting the T cell population used in the aforementioned method.

[0047] In its first form, the present invention is a) A step of selecting from the target cell sample Tscm cells and naive T cell populations, i.e., T cell populations having cell surface phenotypes including (i) CD4+ or CD8+, (ii) CD45RA+, and (iii) CCR7+ and / or CD62L+, b) A step of culturing the T cell population in the presence of antigen-presenting cells carrying at least one target antigen or one or more peptides derived from the at least one target antigen, and optionally in the presence of IL-7 and IL-15 or other stimulating cytokinins, and optionally c) A step of collecting the cells obtained in step b), particularly CD8+ and / or CD4+ cells, preferably CD8+ and CD4+ cells, This relates to an in vitro method for obtaining a cell population containing antigen-specific T cells.

[0048] The method of the present invention does not include a sorting step based on a CD95 marker. The T cell population sorted in step a) therefore includes both naive T cells (CD95-) and Tscm cells (CD95+). Naive T cells have a cell surface phenotype including (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and / or CD62L+, and (iv) CD95-. Tscm cells have a cell surface phenotype including (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and / or CD62L+, and (iv) CD95+. Preferably, the T cell population sorted in step a) contains at least 10%, preferably at least 30%, and more preferably at least 50% naive T cells relative to the total number of cells in the population.

[0049] In step a), the T cell population may have a cell surface phenotype further comprising PD1-, TIGIT-, LAG3-, TIM3-, CTLA4- and / or CD60-, more preferably PD1-, TIGIT-, LAG3- and / or TIM3-.

[0050] In particular, the above method is a) A step of selecting a population of T cells from a target cell sample that have cell surface phenotypes including (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and / or CD62L+, (iv) PD1-, and (v) TIGIT-, b) A step of culturing the T cell population in the presence of antigen-presenting cells carrying at least one target antigen or one or more peptides derived from the at least one target antigen, and optionally in the presence of IL-7 and IL-15 or other stimulating cytokinins, and optionally c) A step of recovering the cells obtained in step b), particularly CD8+ cells and / or CD4+ cells, preferably CD8+ cells and CD4+ cells, It may include.

[0051] In particular, in step c), CD8+ cells and optionally CD4+ cells can be selected from the cell population obtained in step b).

[0052] Optionally, step a) may further include depleting one or more other inhibitory receptors such as LAG3, TIM3, CTLA4, or CD160. In particular, step a) may further include depleting cells expressing LAG3, TIM3, CTLA4, and / or CD160. In this case, the selected cell population may have cell surface phenotypes comprising (i) CD4+ or CD8+, CD45RA+, CCR7+, and / or CD62L+, PD1-, and TIGIT- and (ii) LAG3-, TIM3-, CTLA4-, and / or CD160-.

[0053] In particular, step a) may further include depletion of cells expressing LAG3 and / or TIM3. In this case, the selected cell population may have a cell surface phenotype comprising (i) CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, PD1- and TIGIT-, and (ii) LAG3- and / or TIM3-, preferably a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, PD1-, TIGIT-, LAG3- and TIM3-.

[0054] Optionally, step a) may further include depletion of cells expressing CD45RO and / or sorting of cells expressing CD3. In this case, the sorted population of cells may have a cell surface phenotype comprising (i) CD4+ or CD8+, CD3+, CD45RA+, CD45RO-, CCR7+ and / or CD62L+, preferably CD4+ or CD8+, CD3+, CD45RA+, CD45RO-, CCR7+ and / or CD62L+, PD1- and / or TIGIT-, more preferably CD4+ or CD8+, CD3+, CD45RA+, CD45RO-, CCR7+ and / or CD62L+, PD1- and TIGIT-, and optionally, (ii) LAG3-, TIM3-, CTLA4- and / or CD160-. Preferably, step a) further comprises depletion of cells expressing LAG3 and / or TIM3, preferably depletion of cells expressing LAG3 and TIM3. In this case, the selected cell population may have a cell surface phenotype comprising (i) CD4+ or CD8+, CD3+, CD45RA+, CD45RO-, CCR7+ and / or CD62L+, preferably CD4+ or CD8+, CD3+, CD45RA+, CD45RO-, CCR7+ and / or CD62L+, PD1- and / or TIGIT-, more preferably CD4+ or CD8+, CD3+, CD45RA+, CD45RO-, CCR7+ and / or CD62L+, PD1- and TIGIT-, and (ii) LAG3- and / or TIM3-, preferably LAG3- and TIM3-.

[0055] As used herein, the term "CD4" refers to the T cell surface glycoprotein CD4, a glycoprotein that functions as a co-receptor for the T cell receptor (TCR). In humans, the CD4 protein is encoded by the CD4 gene.

[0056] As used herein, the term "CD8" refers to a transmembrane glycoprotein that functions as a co-receptor for the T cell receptor (TCR). This protein has two isoforms, alpha and beta, each encoded by a different gene. CD8 forms a dimer consisting of a pair of CD8 chains. As used herein, the term "CD8" refers to the CD8-α chain, encoded by the CD8A gene in humans.

[0057] As used herein, the term “CD3” refers to the protein complex and the T cell coreceptor. In mammals, the complex comprises one CD3γ chain, one CD3δ chain, and two CD3ε chains. CD3 is part of a larger complex that includes the T cell receptor (TCR). The CD3 complex associated with the TCR is involved in the recognition of peptides that bind to major histocompatibility complexes class I and II during the immune response. As used herein, the term “CD3” refers in humans to the CD3γ chain encoded by the CD3G gene, the CD3δ chain encoded by the CD3D gene, or the CD3ε chain encoded by the CD3E gene.

[0058] As used herein, the term "CD45RA" refers to the 200- to 220-kDa isoform of receptor tyrosine protein phosphatase C, also known as CD45. In humans, the CD45 protein is encoded by the PTPRC gene. This tyrosine phosphatase is required for T cell activation via antigen receptors. The CD45RA isoform contains only the A protein region.

[0059] As used herein, the term "CD45RO" refers to the 180-kDa isoform of receptor tyrosine protein phosphatase C, also known as CD45. This isoform is the shortest CD45 isoform and lacks all three regions: A, B, and C.

[0060] As used herein, the term "CD95" refers to the Fas receptor, also known as Fas, FasR, apoptosis antigen 1, or tumor necrosis factor receptor superfamily member 6 (TNFRSF6). In humans, the CD95 protein is encoded by the FAS gene. Specifically, Tscm cells are CD95+, while naive T cells are CD95-.

[0061] As used herein, the term "CCR7" refers to CC chemokine receptor type 7, also known as CD197, and is a member of the G protein-binding receptor family. In humans, the CCR7 protein is encoded by the CCR7 gene.

[0062] As used herein, the term "CD62L" refers to an L-selectin, a calcium-dependent lectin that mediates cell adhesion by binding to glycoproteins of adjacent cells. In particular, CD62L mediates the adhesion of lymphocytes to endothelial cells in the high endothelial venules of peripheral lymph nodes. In humans, CD62L is encoded by the SELL gene.

[0063] As used herein, the term "PD1" refers to programmed cell death protein 1, also known as CD279. PD1 belongs to the immunoglobulin superfamily and is a cell surface receptor expressed on the surface of T and B cells. In humans, the PD-1 protein is encoded by the PDCD1 gene.

[0064] As used herein, the term "TIGIT" refers to an immune receptor also known as a T cell immune receptor, which has Ig and ITIM domains, WUCAM, or Vstm3. In humans, the TIGIT protein is encoded by the TIGIT gene.

[0065] As used herein, the term "LAG3" refers to lymphocyte activation gene 3, also known as CD223. LAG3 is a cell surface molecule with diverse biological effects on T cell function. In humans, the LAG3 protein is encoded by the LAG3 gene.

[0066] As used herein, the term "TIM3" refers to T-cell immunoglobulin and mucin domain-containing protein 3, also known as hepatitis A virus cell receptor 2 (HAVCR2). TIM3 is a surface receptor involved in the regulation of innate and adaptive immune responses. In humans, the TIM3 protein is encoded by the HAVCR2 gene.

[0067] As used herein, the term "CTLA4" refers to cytotoxic T lymphocyte-associated protein 4, also known as CD152. CTLA4 is a protein receptor that functions as an immune checkpoint, reducing the immune response. In humans, the CTLA4 protein is encoded by the CTLA4 gene.

[0068] As used herein, the term "CD160" refers to a glycoprotein receptor on immune cells that can transmit signals to regulate, promote, or inhibit cell activation and differentiation. In humans, the CD160 protein is encoded by the CD160 gene.

[0069] As used herein, the term “cell surface phenotype” refers to the presence or absence of a particular combination of cell surface markers on the surface of a cell. “Cell surface marker” means a molecule expressed on the surface of a cell that can be detected, for example, by the use of a labeled antibody or by other means known in the Art. Cell surface markers may include proteins, glycoproteins, or groups of proteins and / or glycoproteins. In this specification, the T cell populations selected in step a) may be identified by the expression of a particular combination of markers including CD4 or CD8, CD45RA, CCR7 and / or CD62L, and preferably CD3, and by the absence of expression of a particular combination of markers including PD1 and / or TIGIT, preferably PD1 and TIGIT, and optionally LAG3, TIM3, CTLA4 and / or CD160, preferably LAG3 and / or TIM3, more preferably LAG3 and TIM3. Optionally, the T cell populations selected in step a) may be further identified by the absence of CD45RO expression. In this invention, the T cell population sorted / selected in step a) is not selected based on the presence or absence of the CD95 marker. The selected population therefore includes both CD95+ and CD95- cells.

[0070] The T cell population obtained in step a) is rich in T cells having a specific cell surface phenotype.

[0071] In a particular embodiment, the T cell population obtained in step a) is rich in T cells having a cell surface phenotype including CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, and optionally CD3+ and / or CD45RO-, preferably T cells having a cell surface phenotype including CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, CD3+ and CD45RO-. The cell surface phenotype may further include PD1-, TIGIT-, LAG3-, TIM3-, CTLA4- and / or CD160-, preferably PD1- and / or TIGIT-, and optionally LAG3- and / or TIM3-, more preferably PD1- and TIGIT-, and optionally LAG3- and / or TIM3-, and even more preferably PD1-, TIGIT-, LAG3- and TIM3-.

[0072] In particular, the T cell population obtained in step a) may be rich in T cells having a cell surface phenotype that includes CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, PD1- and TIGIT-, and optionally LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-, more preferably LAG3- and TIM3-. Preferably, the T cells have a cell surface phenotype that further includes CD3+ and CD45RO-.

[0073] "Abundantly present" means a composition that contains cells that are present in a higher proportion to the total cells than in other compositions. In particular, in the population obtained in step a), T cells having specific cell surface phenotypes, such as (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and / or CD62L+, and optionally (iv) cell surface phenotypes including PD1-, TIGIT-, LAG3-, TIM3-, CTLA4-, and / or CD160-, are present in a higher proportion to the total cells compared to their proportion in the cell sample. In the population obtained in step a), T cells having the specific cell surface phenotypes, for example (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and / or CD62L+, and optionally (iv) cell surface phenotypes including PD1-, TIGIT-, LAG3-, TIM3-, CTLA4-, and / or CD160-, account for 70% or more of the total cells in the population, preferably 80%, 90%, 95%, or 99% or more, and more preferably 95% or 99% or more of the total cells in the population.

[0074] Conversely, the T cell population obtained in step a) may deplete cells expressing the inhibitory receptors PD1, TIGIT, LAG3, TIM3, CTLA4 and / or CD160, preferably PD1 and TIGIT, and optionally cells expressing LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3 and / or TIM3. Preferably, the T cell population obtained in step a) is further depleted of cells expressing CD45RO. "Depleted" means a composition containing cells that are present in a smaller proportion of the total cells than they are in other compositions, and in particular, than they are in the cell sample. In particular, in the T cell population obtained in step a), T cells having a cell surface phenotype including PD1+ and TIGIT+, and optionally LAG3+, TIM3+, CTLA4+ and / or CD160+, preferably LAG3+ and / or TIM3+, are present in a smaller proportion of the total cells compared to the proportion in the cell sample. In particular, in the population obtained in step a), T cells expressing the inhibitory receptors PD1 and TIGIT, and optionally LAG3, TIM3, CTLA4 and / or CD160, preferably LAG3 and / or TIM3, may constitute 5%, 2%, or 1% or less of the total cells in the population.

[0075] Preferably, in the population obtained in step a), T cells having a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, and (iii) CCR7+ and / or CD62L+, preferably (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and / or CD62L+, and (iv) CD3+, more preferably (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and / or CD62L+, (iv) CD3+, and (v) CD45RO- constitute 95%, 96%, 97%, 98%, or 99% or more of the total cells in the population.

[0076] Preferably, in the population obtained in step a), T cells having a cell surface phenotype including (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and / or CD62L+, and (iv) CD95+ account for 4% to 70% of the total cells in the population, preferably 20% to 70%, and T cells having a cell surface phenotype including (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and / or CD62L+, and (iv) CD95- account for 30% to 96% of the total cells in the population, preferably 30% to 80%.

[0077] In some embodiments, in the population obtained in step a), T cells having a cell surface phenotype comprising (i) CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, PD1- and TIGIT-, preferably CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, CD3+, PD1- and TIGIT-, more preferably CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, CD3+, CD45RO-, PD1- and TIGIT-, and (ii) LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3- constitute 95%, 96%, 97%, 98%, or 99% or more of the total cells in the population. In this case, T cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, PD1+ and / or TIGIT+, preferably CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, CD3+, PD1+ and / or TIGIT+, more preferably CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, CD3+, CD45RO-, PD1+ and TIGIT+, and optionally LAG3+, TIM3+, CTLA4+ and / or CD160+, preferably LAG3+ and / or TIM3+, constitute 5% or less of the total cells in the population, more preferably 2% or 1% or less of the total cells in the population.

[0078] In a particular embodiment, the population obtained in step a) consists of T cells having the above-mentioned cell surface phenotypes, preferably including (i) CD4+ or CD8+, (ii) CD45RA+, and (iii) CCR7+ and / or CD62L+, preferably including (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and / or CD62L+, and (iv) CD3+, more preferably including (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and / or CD62L+, (iv) CD3+, and (v) CD45RO-.

[0079] In another specific embodiment, the population obtained in step a) consists of T cells having the above-mentioned cell surface phenotypes, preferably (i) containing CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, PD1- and TIGIT-, more preferably containing CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, CD3+, PD1- and TIGIT-, more preferably CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, CD3+, CD45RO-, PD1- and TIGIT-, and (ii) optionally containing LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-.

[0080] Preferably, the population obtained in step a) exhibits a CD4+ / CD8+ ratio of at least 0.2. In embodiments in which the population obtained in step a) exhibits a CD4+ / CD8+ ratio lower than 0.2, an anti-CD40 antibody may be added to the population to compensate for the lack of signaling CD4 T cells.

[0081] In step a), cells having a specific cell surface phenotype are sorted and recovered from the cell sample. Sorting of cells having a specific cell surface phenotype can be performed using any method known in the art. Positive and / or negative sorting can be readily achieved using materials and techniques known in the art. For example, cells expressing a specific cell surface marker can be separated from other cells using a monoclonal antibody bound to the marker and linked to a column or magnetic beads, and such separation can be readily performed according to standard techniques and / or instructions from the manufacturer or provider. In particular, in step a), cells can be sorted by fluorescence-activated cell sorting (FACS) or by magnetic separation.

[0082] The cell sample may be any sample containing T cells, particularly Tscm cells and naive T cells, or cells that can be induced to become Tscm cells and naive T cells during culture. Preferably, the sample is a sample containing Tscm cells and naive T cells. Examples of suitable samples include, but are not limited to, bone marrow cell samples, blood cell samples, fractionated or unfractionated whole blood samples, fractionated or unfractionated apheresis recoveries (e.g., leukocyte apheresis recoveries), tumor-infiltrating lymphocytes, PBMCs, or T cell populations (e.g., populations rich in T cells from blood samples or PBMCs).

[0083] In certain embodiments, the cell sample is PBMCs. PBMCs can be isolated from a blood sample by any method known in the art, such as Ficoll density gradient centrifugation.

[0084] As used herein, the term “isolated” means separated from the components that a cell would normally be surrounded by in nature.

[0085] In another specific embodiment, the cell sample is a population rich in T cells from PBMCs or a blood sample, preferably a population rich in T cells from PBMCs. T cells can be abundantly present from PBMCs or a blood sample by any method known in the art. For example, T cells can be abundantly present from a blood sample or PBMC by depletion of CD14+ cells and / or by sorting and retaining CD3+ cells using an anti-CD3 antibody.

[0086] The method may further include providing the cell sample from the subject.

[0087] As used herein, the terms “subject” or “patient” refer to animals, preferably mammals, and more preferably humans.

[0088] As described below, the cell populations obtained by the method of the present invention can be used to provide adoptive cell therapy, in particular autologous therapy (by reinjecting the T cell-derived cells into the same patient), or allogeneic therapy (by injecting the T cell-derived cells into another patient). Cell samples can therefore be obtained from healthy subjects, particularly in allogeneic therapy, or from subjects with the disease to be treated by adoptive cell therapy.

[0089] In particular, the subject may have an infection or cancer in which a specific memory T cell response is functionally impaired. Preferably, this dysfunction includes mechanisms of negative regulation of T cell function, particularly T cell anergy, i.e., T cell response to the antigen of interest, i.e., tumor or pathogen antigen, and / or T cell exhaustion, characterized in particular by high levels of expression of inhibitory receptors such as PD-1 or TIGIT. Thus, in preferred embodiments, the subject has an infection or cancer and exhibits mechanisms of negative regulation of T cell function, particularly T cell anergy, particularly T cell response to the antigen of interest, and / or T cell exhaustion. In some specific embodiments, the subject has an infection or cancer and exhibits T cell anergy, particularly T cell response to the antigen of interest, and / or T cell exhaustion.

[0090] Preferably, the subject is cancer or a disease caused by the following pathogens, more preferably a disease caused by a polyomavirus, and even more preferably a disease caused by a human polyomavirus.

[0091] In a preferred embodiment, the subject has progressive multifocal leukoencephalopathy (PML), Merkel cell carcinoma, or BK virus-associated nephropathy, preferably progressive multifocal leukoencephalopathy (PML) or Merkel cell carcinoma, and more preferably progressive multifocal leukoencephalopathy.

[0092] In step b) of the method of the present invention, the T cell population obtained in step a) is cultured in the presence of antigen-presenting cells carrying at least one target antigen or at least one immunogenic peptide derived from at least one target antigen, preferably at least one immunogenic peptide derived from at least one target antigen.

[0093] Here, the terms "peptide" and "protein" are used interchangeably and refer to amino acid chains linked by peptide bonds, regardless of the number of amino acids forming the chain.

[0094] The antigen-presenting cells (APCs) used in this process can be any antigen-presenting cells suitable for activating T cells by presenting at least one target antigen or at least one immunogenic peptide when the major histocompatibility complex (MHC) receptor on the surface of the APC, when complexed with a peptide, interacts with the TCR on the surface of the T cell. Examples of APCs include, but are not limited to, dendritic cells, monocytes, monocyte-derived dendritic cells, peripheral blood mononuclear cells (PBMCs), Epstein-Barr virus-transformed B lymphoblastic cell lines (EBV-BLCL cells), or artificial antigen-presenting cells (AAPCs). Preferably, the APCs used in step b) are selected from the group consisting of dendritic cells, monocytes, peripheral blood mononuclear cells (PBMCs), Epstein-Barr virus-transformed B lymphoblastic cell lines (EBV-BLCL cells), or artificial antigen-presenting cells (AAPCs). More preferably, the APCs used in step b) are selected from the group consisting of dendritic cells, monocytes, and PBMCs, and combinations thereof. More preferably, the APC used in step b) is a monocyte or dendritic cell, and is preferably a monocyte.

[0095] The APC used in step b) may be self-derived (i.e., obtained from the same subject as the subject providing the cell sample, preferably from the subject being treated) or allogeneic (i.e., obtained from a different subject than the subject providing the cell sample, preferably from a different subject than the subject being treated).

[0096] In a preferred embodiment, the APC used in step b) is autologous. Those skilled in the art may use a wide range of known procedures to generate autologous APCs using different sources such as peripheral blood monocytes, spontaneously generated DCs, or CD34+ hematopoietic progenitor cells returned from bone marrow. Preferably, autologous APCs are obtained from peripheral blood monocytes or spontaneously generated DCs, more preferably from peripheral blood monocytes.

[0097] As is known in the art, CD34+ stem cells can be differentiated into dendritic cells by incubation with appropriate cytokines. For example, human CD34+ hematopoietic stem cells can be differentiated in vitro by culturing them with human GM-CSF and TNF-α (see, e.g., Szabolcs, et al. (1995) J. Immunol. 154: 5851-5861). Dendritic cells can then be isolated by fluorescence-activated cell sorting (FACS) based on the expression of cell surface markers, or by other standard methods.

[0098] In particular, the method may further include, before step b), obtaining the autologous APC from a cell sample derived from the subject, and loading the autologous APC with at least one target antigen or at least one immunogenic peptide derived from at least one target antigen, preferably at least one immunogenic peptide derived from at least one target antigen. The cell sample used to obtain the autologous APC may be the same as or different from the cell sample used in step a), but both are obtained from the same subject.

[0099] Preferably, the method further comprises, prior to step b), sorting a population of monocyte cells from a cell sample derived from a subject using CD14+ positive selection and loading the monocytes with at least one target antigen or at least one immunogenic peptide derived from at least one target antigen, preferably at least one immunogenic peptide derived from at least one target antigen. Preferably, the monocytes are obtained from a PBMC sample derived from a subject.

[0100] Alternatively, to induce differentiation into dendritic cells before or after antigen loading, monocytes obtained from the sample, for example using CD14+ positive screening, may be cultured in the presence of GM-CSF and IL-4. Optionally, to induce optimal maturation of dendritic cells, IL-6, IL-1β, and TNF-α are added to the culture medium for approximately 24 hours between day 5 and day 10 of culture, preferably on day 6.

[0101] APCs can be loaded by any antigen loading method known to those skilled in the art. For example, APCs, in particular dendritic cells, monocytes, or PBMCs, can be loaded by pulsing or incubating the APCs with one or more target antigens and / or one or more peptides, in particular one or more immunogenic peptides derived from the one or more target antigens, or by delivering one or more antigens and / or one or more peptides, in particular one or more immunogenic peptides derived from the one or more target antigens, to the APCs using a viral vector or mRNA transfection.

[0102] In a preferred embodiment, the APC contains one or more peptides, particularly one or more immunogenic peptides derived from the one or more target antigens, preferably an overlapping peptide pool, particularly an overlapping immunogenic peptide pool derived from the one or more target antigens.

[0103] The target antigen may be any antigen that can be targeted by the immune system to provide a therapeutic effect. The target antigen can be easily selected by those skilled in the art depending on the disease being treated. In a preferred embodiment, the target antigen is selected depending on the disease being treated of the subject of the cell sample, i.e., cancer or infection.

[0104] In particular, the target antigen can be selected from pathogen antigens or antigens expressed by tumor cells, such as tumor-specific antigens (TSAs) (i.e., antigens found only in tumor cells and not in healthy cells) or tumor-associated antigens (TAAs) (i.e., antigens that are elevated in tumor cells but expressed at low levels in healthy cells).

[0105] In some embodiments, the target antigen is selected from one or more cancer antigens. As used herein, the terms “cancer” or “tumor” refer to the presence of cells that possess typical characteristics of cancerous cells, such as unrestrained growth, immortality, metastatic ability, rapid growth and proliferation rates, and certain characteristic morphological features. The term refers to any type of malignant tumor (primary or metastatic) and refers to solid or hematopoietic carcinoma.

[0106] In another embodiment, the target antigen is selected from one or more antigens of a pathogen, particularly a virus, bacteria, or fungus.

[0107] In a preferred embodiment, the target antigen is selected from one or more viral antigens, preferably from one or more human viruses. Preferably, the virus is selected from the group consisting of polyomaviruses, human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpesviruses, and papillomaviruses. More preferably, the virus is selected from the group consisting of human polyomaviruses, particularly from John Cunningham (JC) polyomavirus, BK virus (BK), and Merkel cell polyomavirus (MCPyV or MCV).

[0108] In certain embodiments, the target antigen is selected from polyomavirus antigens. For example, the peptide presented by the APC may include one or more polyomavirus peptides, particularly one or more immunogenic peptides, from the VP1, VP2, VP3, large T, small T proteins of the polyomavirus and / or other proteins of the polyomavirus. In particular, the peptide presented by the APC may be an overlapping peptide covering one or more of these proteins. More preferably, the peptide presented by the APC, preferably an immunogenic peptide, may include an overlapping peptide pool covering the VP1, VP2, and VP3 regions of the polyomavirus.

[0109] In more specific embodiments, the target antigen is selected from the antigens of the polyomavirus MCPyV. For example, the peptide presented by the APC may include one or more MCPyV peptides, particularly one or more MCPyV immunogenic peptides, from the VP1, VP2, VP3, large T, small T proteins of MCPyV, and / or other MCPyV proteins. In particular, the peptide presented by the APC may be an overlapping peptide covering one or more of these proteins. More preferably, the peptide presented by the APC, preferably an immunogenic peptide, may include an overlapping peptide pool covering the VP1, VP2, and VP3 regions of MCPyV.

[0110] In other, more specific embodiments, the target antigen is selected from the antigens of polyomavirus BKV. For example, the peptide presented by the APC may include one or more BKV peptides, particularly one or more BKV immunogenic peptides, from the VP1, VP2, VP3, large T, small T proteins of BKV, and / or other BKV proteins. In particular, the peptide presented by the APC may be an overlapping peptide covering one or more of these proteins. More specifically, the peptide presented by the APC, preferably an immunogenic peptide, may include an overlapping peptide pool covering the VP1, VP2, and VP3 regions of BKV.

[0111] In other, more specific embodiments, the target antigen is selected from the antigens of polyomavirus JC. For example, the peptide presented by the APC may include one or more JCV peptides, particularly one or more JCV peptides, from the VP1, VP2, VP3, large T, small T proteins of JCV, and / or other JCV proteins. In particular, the peptide presented by the APC may be an overlapping peptide covering one or more of these proteins. More specifically, the peptide presented by the APC, preferably an immunogenic peptide, may include an overlapping peptide pool covering the VP1, VP2, and VP3 regions of JCV.

[0112] The antigens used to mount the APC can be prepared by any method known to those skilled in the art, depending on the properties of the antigen. For example, the antigens can be prepared by chemical synthesis, recombinant expression, from a sample derived from the subject, particularly from the subject's own cancer cells (e.g., using whole tumor lysates), or from cancer cell line lysates.

[0113] In step b) of the method of the present invention, T cells are cultured in the presence of APC as described above, thereby proliferating and differentiating into a population containing T cells that are responsive to a specific antigen or set of antigens.

[0114] Since the activation of naive T cells requires in vivo priming by dendritic cells, in step b), the Tscm cells of the T cell population obtained in step a) are activated.

[0115] Methods for obtaining antigen-specific T cells from a population containing Tscm cells using APCs are well known in the art, and those skilled in the art may use any of these known methods.

[0116] Generally, the culture is carried out in the presence of IL-15, IL-7, and / or other stimulating cytokinins, preferably recombinant cytokinins such as IL-21. Preferably, the culture step includes culture supplementation with IL-15 and IL-7, and optionally IL-21. The supplementation preferably begins within the first 7 days of culture, more preferably between days 2 and 4 of culture. IL-15 and IL-7 may help maintain the stem cell-like phenotype of Tscm cells.

[0117] At the start of culture, the ratio of Tscm cells to APCs can be adjusted to 1 / 1 (number of Tscm cells / number of APCs) to 1 / 20, preferably 1 / 5 to 1 / 15, and more preferably 1 / 9 to 1 / 11.

[0118] T cell culture in the presence of APC may continue for 8 to 20 days, preferably 10 to 18 days, and more preferably 12 to 16 days. In certain embodiments, T cell culture in the presence of APC may continue for 14 days.

[0119] Optionally, cells may be cultured for longer periods, preferably in the absence of antigen-presenting APCs. In particular, cells may be cultured in the absence of antigen-presenting APCs after step a) and before step b), and / or after step b) and before step c), preferably in the absence of antigen-presenting APCs.

[0120] In some specific embodiments in which the subjects are infected with retroviruses such as HIV, the culture may be carried out in the presence of one or more antiretroviral compounds.

[0121] Optionally, the method of the present invention may further include step c), which involves recovering the cells obtained in step b), particularly CD8+ and / or CD4+ cells, preferably CD8+ and CD4+ cells.

[0122] In particular, in step c), the cell population obtained in step b) can be sorted to select CD8+ cells and, optionally, CD4+ cells.

[0123] In certain embodiments of the method of the present invention, in step c), the cell population obtained in step b) is sorted to select / recover CD8+ cells and CD4+ cells. CD8+ cells and CD4+ cells may be recovered separately or together. In some embodiments, CD8+ cells and CD4+ cells are preferably recovered separately before being mixed. This separation allows for adjustment of the CD8+ / CD4+ ratio of the resulting cell population.

[0124] In a preferred embodiment, in step c), the cell population obtained in step b), i.e., all cells of the culture, are harvested and include CD8+ cells and CD4+ cells. The harvested cells may also include other cell types, particularly APCs such as monocytes or dendritic cells.

[0125] The cells can be recovered by any method known to those skilled in the art, including a filtration method or the cell sorting method described above.

[0126] In particular, the population selected / recovered in step c) may include Tscm cells (having a cell surface phenotype including CD45RA+ CCR7+), T effector (Teff) cells (having a cell surface phenotype including CD45RA+ CCR7-), T central memory (Tcm) cells (having a cell surface phenotype including CD45RA- CCR7+), and T effector memory (Tem) cells (having a cell surface phenotype including CD45RA- CCR7).

[0127] In preferred embodiments, Tscm, Tcm, and Tem cells constitute up to 90%, preferably 50% to 90%, of the total cells in the selected / recovered population, enabling further differentiation cycles in vivo and thus long-term therapeutic effects. Generally, Teff cells may constitute 10% to 50%, preferably 10% to 20%, of the total cells in the selected / recovered population.

[0128] In another aspect, the present invention relates to an isolated population of cells comprising antigen-specific CD8+ T cells and optionally antigen-specific CD4+ T cells, which can be obtained or obtained by the method of the present invention for obtaining a cell population comprising antigen-specific T cells. Preferably, the population comprises antigen-specific CD8+ T cells and antigen-specific CD4+ T cells.

[0129] All embodiments of the method disclosed above for obtaining a cell population containing antigen-specific T cells also encompass this embodiment.

[0130] In particular, the population may include Tscm cells (having a cell surface phenotype including CD45RA+ CCR7+), T effector (Teff) cells (having a cell surface phenotype including CD45RA+ CCR7-), T central memory (Tcm) cells (having a cell surface phenotype including CD45RA- CCR7+), and T effector memory (Tem) cells (having a cell surface phenotype including CD45RA- CCR7).

[0131] Preferably, Tscm, Tcm, and Tem cells make up to 90% of the total cells in the isolated population of the present invention, preferably 50% to 90%. Generally, Teff cells may make up 10% to 50% of the total cells in the isolated population of the present invention, preferably 10% to 20%.

[0132] Preferably, antigen-specific CD8+ T cells constitute 10% to 90% of the total cells in the isolated population of the present invention, and antigen-specific CD4+ T cells constitute 1% to 90% of the total cells. In particular, antigen-specific CD8+ T cells may constitute 50% to 90% of the total cells in the isolated population of the present invention, and antigen-specific CD4+ T cells may constitute 1% to 50% of the total cells in the isolated population of the present invention.

[0133] In certain embodiments, an isolated population of cells containing antigen-specific CD8+ T cells and optionally antigen-specific CD4+ T cells, preferably antigen-specific CD8+ T cells and antigen-specific CD4+ T cells, a) A step of selecting a population of T cells having a cell surface phenotype including (i) CD4+ or CD8+, (ii) CD45RA+, and (iii) CCR7+ and / or CD62L+ from a cell sample derived from a subject suffering from a disease caused by cancer or a pathogen, in particular a disease caused by cancer or a pathogen in which a specific memory T cell response is functionally impaired; b) A step of culturing the T cell population, preferably self-derived from the target, carrying at least one target antigen or at least one peptide, particularly at least one immunogenic peptide, in the presence of antigen-presenting cells derived from the at least one target antigen, and optionally in the presence of IL-7 and IL-15 or other stimulating cytokinins, c) A step of collecting the cells obtained in step b), particularly CD8+ and / or CD4+ cells, preferably CD8+ and CD4+ cells, It can be obtained or obtained by a method that includes [the specified element].

[0134] The method does not include a sorting step based on the CD95 marker. The T cell population sorted in step a) therefore includes both naive T cells (CD95-) and Tscm cells (CD95+). Preferably, the population contains at least 10%, preferably at least 30%, and more preferably at least 50% naive T cells of the total number of cells in the population.

[0135] In step c), CD8+ cells and optionally CD4+ cells can be selected from the cell population obtained in step b).

[0136] The subjects may have cancer. In this case, the at least one target antigen may be selected from antigens expressed by tumor cells, such as tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs).

[0137] The subject may suffer from a disease caused by a pathogen. In this case, the at least one target antigen may be selected from the antigens of the pathogen.

[0138] Preferably, the subjects have progressive multifocal leukoencephalopathy (PML), Merkel cell carcinoma, or BK virus-associated nephropathy. For subjects with Merkel cell carcinoma, the at least one target antigen may be selected from the antigens of Merkel cell polyomavirus (MCPyV or MCV), particularly from the VP1, VP2, VP3, large T, small T proteins, and / or other MCPyV proteins of MCPyV. For subjects with BK virus-associated nephropathy, the at least one target antigen may be selected from the antigens of BK virus (BKV), particularly from the VP1, VP2, VP3, large T, small T proteins, and / or other BKV proteins of BKV.

[0139] More preferably, the subject suffers from progressive multifocal leukoencephalopathy (PML), and the at least one target antigen is selected from the antigens of polyomavirus JC, preferably from the VP1, VP2, VP3, large T, and small T proteins of BKV, and / or other BKV proteins. For example, the peptide presented by the APC, preferably an immunogenic peptide, may include a JCV overlapping peptide pool covering the VP1, VP2, and VP3 regions of JCV.

[0140] In particular, the T cell population selected in step a) may have a cell surface phenotype comprising PD1-, TIGIT-, LAG3-, TIM3-, CTLA4- and / or CD160-, preferably comprising PD1-, TIGIT-, LAG3- and / or TIM3-, more preferably comprising PD1- and TIGIT-, and optionally LAG3- and / or TIM3-, and even more preferably comprising PD1-, TIGIT-, LAG3- and TIM3-.

[0141] Preferably, the T cell population selected in step a) has a cell surface phenotype that further includes CD3+ and CD45RO-.

[0142] In another aspect, the present invention relates to an in vitro method for obtaining a T cell population, comprising the step of selecting a T cell population having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, PD1-, and TIGIT- from a cell sample derived from a subject. The method does not include a CD95 marker-based selection step. The T cell population therefore includes both naive T cells (CD95-) and Tscm cells (CD95+). Preferably, the population contains at least 10%, preferably at least 30%, and more preferably at least 50% naive T cells of the total number of cells in the population.

[0143] Preferably, the T cell population has a cell surface phenotype further comprising CD3+ and / or CD45RO-, preferably CD3+ and CD45RO-.

[0144] Optionally, the method may further include depletion of cells expressing one or more other inhibitory receptors, such as LAG3, TIM3, CTLA4, or CD160. In particular, the method may further include depletion of cells expressing LAG3, TIM3, CTLA4, and / or CD160, preferably LAG3 and / or TIM3, more preferably LAG3 and TIM3. In this case, the selected population of cells may have a cell surface phenotype comprising (i) CD4+ or CD8+, CD45RA+, CCR7+, and / or CD62L+, PD1-, and TIGIT-, and (ii) LAG3-, TIM3-, CTLA4, and / or CD160, preferably LAG3- and / or TIM3-, more preferably LAG3- and TIM3-. Preferably, the selected cell population may have a cell surface phenotype comprising (i) CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, CD3+, CD45RO-, PD1-, and TIGIT-, and (ii) LAG3-, TIM3-, CTLA4 and / or CD160, preferably LAG3- and / or TIM3-, more preferably LAG3- and TIM3-.

[0145] Optionally, the method further includes amplification of a selected population of T cells. This step may be carried out by any method well known to those skilled in the art, such as culturing the T cells in the presence of a feeder such as monocytes (uncarcinomatous monocytes) and appropriate cytokines.

[0146] All embodiments relating to step a) of the method of the present invention for obtaining a cell population containing antigen-specific T cells, as disclosed above, are also included in this embodiment.

[0147] In another aspect, the present invention relates to an in vitro method for obtaining a T cell population, comprising the step of selecting a T cell population having a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, and (ii) CCR7+ and / or CD62L+, from a cell sample derived from a subject, wherein the subject suffers from a disease caused by cancer or a pathogen, in particular a disease caused by cancer or a pathogen in which a specific memory T cell response is functionally impaired. The method does not include a selection step based on a CD95 marker. The T cell population therefore comprises both naive T cells (CD95-) and Tscm cells (CD95+). Preferably, the population comprises at least 10%, preferably at least 30%, and more preferably at least 50% naive T cells relative to the total number of cells in the population.

[0148] Preferably, the T cell population has a cell surface phenotype further comprising CD3+ and / or CD45RO-, preferably CD3+ and CD45RO-.

[0149] Preferably, the subject has a disease caused by a human polyomavirus.

[0150] More preferably, the subject has progressive multifocal leukoencephalopathy (PML), Merkel cell carcinoma, or BK virus-associated nephropathy.

[0151] In a preferred embodiment, the subject has progressive multifocal leukoencephalopathy (PML).

[0152] Optionally, the method may further include depletion of cells expressing one or more inhibitory receptors, such as PD1, TIGIT, LAG3, TIM3, CTLA4, or CD160.

[0153] In particular, the method may further include the depletion of cells expressing PD1 and / or TIGIT, and optionally cells expressing LAG3, TIM3, CTLA4 and / or CD160, preferably cells expressing PD1 and TIGIT, and optionally cells expressing LAG3 and / or TIM3. In this case, the selected population of cells may have a cell surface phenotype comprising (i) CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, and (ii) PD1-, TIGIT-, LAG3-, TIM3-, CTLA4 and / or CD160, preferably PD1-, TIGIT-, LAG3- and / or TIM3-, more preferably PD1- and TIGIT-, and optionally LAG3- and / or TIM3-. Preferably, the selected cell population may have a cell surface phenotype comprising (i) CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, CD3+ and CD45RO-, and (ii) PD1-, TIGIT-, LAG3-, TIM3-, CTLA4 and / or CD160, preferably PD1-, TIGIT-, LAG3- and / or TIM3-, more preferably PD1- and TIGIT-, and optionally LAG3- and / or TIM3-.

[0154] More preferably, the method may further include depletion of cells expressing PD1, TIGIT, LAG3, and TIM3. In this case, the selected cell population may have a cell surface phenotype comprising (i) CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+ and (ii) PD1-, TIGIT-, LAG3-, and TIM3-. Preferably, the selected cell population has a cell surface phenotype comprising (i) CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, CD3+, and CD45RO- and (ii) PD1-, TIGIT-, LAG3-, and TIM3-.

[0155] Optionally, the method further includes amplification of a selected population of T cells. This step can be carried out in any way well known to those skilled in the art, such as culturing the T cells in the presence of a feeder such as monocytes (uncarcinomatous monocytes) and appropriate cytokines.

[0156] All embodiments relating to step a) of the method of the present invention for obtaining a population of cells including antigen-specific T cells, as disclosed above, are also included in this embodiment.

[0157] In a further aspect, the present invention relates to an isolated population of T cells that can be obtained or obtained by the method of the present invention for obtaining a T cell population.

[0158] This group, (i) CD4+, CD45RA+, CCR7+, PD1-, and TIGIT-; and / or (ii) CD8+, CD45RA+, CCR7+, PD1-, and TIGIT-; and / or (iii) CD4+, CD45RA+, CD62L+, PD1- and TIGIT-; and / or (iv) CD8+, CD45RA+, CD62L+, PD1-, and TIGIT-; and / or (v) CD4+, CD45RA+, CCR7+, CD62L+, PD1-, and TIGIT-; and / or (vi) CD8+, CD45RA+, CCR7+, CD62L+, PD1- and TIGIT- It may include T cells having a cell surface phenotype that includes the following.

[0159] The T cell population includes both naive T cells (CD95-) and Tscm cells (CD95+).

[0160] Preferably, the population comprises at least 10%, preferably at least 30%, and more preferably at least 50% of the total number of cells in the population as naive T cells.

[0161] More preferably, in the population, T cells having a cell surface phenotype including (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and / or CD62L+, and (iv) CD95+ account for 4% to 70% of the total cells in the population, preferably 20% to 70%, and T cells having a cell surface phenotype including (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and / or CD62L+, and (iv) CD95- account for 30% to 96% of the total cells in the population, preferably 30% to 80%.

[0162] Preferably, these T cells have a cell surface phenotype further comprising CD3+ and / or CD45RO-, preferably CD3+ and CD45RO-.

[0163] Selectively, these T cells may have a cell surface phenotype further comprising LAG3-, TIM3-, CTLA4 and / or CD160, preferably LAG3- and / or TIM3-, and more preferably LAG3- and TIM3-.

[0164] In particular, the T cell population may contain at least 95%, 96%, 97%, 98%, or 99% (of the total cells) T cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, CD3+, CD45RO-, PD1-, and TIGIT-, preferably CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, CD3+, CD45RO-, PD1-, and TIGIT-, and optionally LAG3-, TIM3-, CTLA4 and / or CD160, preferably LAG3- and / or TIM3-, more preferably LAG3- and TIM3-.

[0165] More specifically, the T cell population may comprise at least 95% or at least 99% (of the total cells) of T cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, PD1- and TIGIT-, preferably CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, CD3+, CD45RO-, PD1- and TIGIT-, and optionally LAG3-, TIM3-, CTLA4 and / or CD160, preferably LAG3- and / or TIM3-, more preferably LAG3- and TIM3-.

[0166] In some embodiments, T cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, PD1+ and / or TIGIT+, and optionally LAG3+, TIM3+, CTLA4+ and / or CD160+, preferably LAG3+ and / or TIM3+-, constitute less than 5% of the total cells in the population, preferably less than 2% or 1% of the total cells in the population.

[0167] In a particular embodiment, the T cell population consists of T cells having cell surface phenotypes including (i) CD4+ or CD8+, (ii) CD45RA+, and (iii) CCR7+ and / or CD62L+.

[0168] In a more specific embodiment, the T cell population consists of T cells having a cell surface phenotype that includes CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, PD1- and TIGIT-, preferably CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, CD3+, CD45RO-, PD1- and TIGIT-, and optionally LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-, more preferably LAG3- and TIM3-.

[0169] All embodiments of the method of the present invention for obtaining a cell population containing antigen-specific T cells, as disclosed above, or of the method of the present invention for obtaining a T cell population, are also included in this embodiment.

[0170] In a further aspect, the present invention relates to a cell therapy drug, -An isolated population of cells comprising antigen-specific CD8+ T cells and optionally antigen-specific CD4+ T cells, preferably an isolated population of cells comprising antigen-specific CD8+ T cells and antigen-specific CD4+ T cells, obtained or obtainable by the method of the present invention for obtaining a cell population comprising antigen-specific T cells, or -An isolated population of T cells obtained or obtainable by the method of the present invention for obtaining a T cell population, This also relates to that.

[0171] The present invention also relates to the aforementioned population for use in cell-based therapies.

[0172] All embodiments of the present invention relating to the method for obtaining a cell population containing antigen-specific T cells, the isolated cell population containing antigen-specific CD8+ T cells and optionally antigen-specific CD4+ T cells, the method for obtaining a T cell population, and the isolated T cell population of the present invention are also included in this embodiment.

[0173] In a further manner, the present invention is -An isolated population of cells comprising antigen-specific CD8+ T cells and optionally antigen-specific CD4+ T cells, preferably an isolated population of cells comprising antigen-specific CD8+ T cells and antigen-specific CD4+ T cells, obtained or obtainable by the method of the present invention for obtaining a cell population comprising antigen-specific T cells, or -An isolated population of T cells obtained or obtainable by the method of the present invention for obtaining a T cell population, This relates to a pharmaceutical composition containing [a specific ingredient / component].

[0174] In a preferred embodiment, the pharmaceutical composition comprises an isolated population of cells comprising antigen-specific CD8+ T cells and optionally antigen-specific CD4+ T cells, obtained or obtainable by the method of the present invention for obtaining a cell population comprising antigen-specific T cells (i.e., the method of the present invention for obtaining a cell population comprising antigen-specific T cells). Preferably, the pharmaceutical composition comprises an isolated population of cells comprising antigen-specific CD8+ T cells and antigen-specific CD4+ T cells according to the present invention.

[0175] The pharmaceutical composition is formulated in a pharmaceutically acceptable carrier and / or excipient depending on the route of administration.

[0176] Preferably, the pharmaceutical composition is formulated to be suitable for use in cell-based therapy in a target that requires it.

[0177] Pharmaceutical compositions may be formulated in accordance with standard pharmaceutical practices known to those skilled in the art (see, for example, Remington: The Science and Practice of Pharmacy (20th ed.), ed. AR Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and JC Boylan, 1988–1999, Marcel Dekker, New York).

[0178] Preferably, the pharmaceutical composition is suitable for parenteral administration, and more preferably for intravenous injection.

[0179] Pharmaceutical compositions suitable for such administration may contain the cell population of the present invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions (e.g., equilibrium salt solutions (BSS)), dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injection solutions or dispersions immediately before use and may contain antioxidants, buffers, bactericides, solutes or suspensions or thickeners.

[0180] Optionally, compositions containing cells may be frozen for storage at any temperature suitable for cell storage. For example, cells may be frozen at approximately -150°C or -196°C. To reduce the risk of cell damage and maximize the chances of cells surviving thawing, cryogenically frozen cells may be stored in appropriate containers and prepared for storage.

[0181] The amount of cells to be administered can be determined by standard procedures well known to those skilled in the art. The patient's physiological data (e.g., age, size, and weight), as well as the type and severity of the disease being treated, must be taken into consideration when determining the appropriate dosage.

[0182] The pharmaceutical composition of the present invention can be administered as a single dose or in multiple doses. Each dose unit is, for example, 10 5 ~7.10 8 Cells, preferably 7.10 6 ~7.10 8 It may contain cells.

[0183] The pharmaceutical composition of the present invention may further comprise additional active compounds, such as therapeutic monoclonal antibodies, for the purpose of depleting lymphocyte subsets or inhibiting receptors involved in immune function, such as anti-PD-1 or anti-TIGIT.

[0184] The present invention also relates to the pharmaceutical composition of the present invention for use in cell-based therapy in subjects requiring such treatment. The present invention also relates to the pharmaceutical composition of the present invention for use in the treatment of cancer or diseases caused by pathogens. The present invention also relates to a method of treating a subject suffering from cancer or a disease caused by a pathogen, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention to the subject. The present invention also relates to the use of the pharmaceutical composition of the present invention for preparing a medicament for the treatment of cancer or diseases caused by pathogens.

[0185] All embodiments of the present invention relating to the method for obtaining a cell population containing antigen-specific T cells, the isolated cell population containing antigen-specific CD8+ T cells and optionally antigen-specific CD4+ T cells, the method for obtaining a T cell population, the isolated T cell population, and the pharmaceutical composition of the present invention, as disclosed above, are also included in this embodiment.

[0186] As used herein, the terms “treatment” or “treat or treating” refer to any action aimed at improving a patient’s health condition, such as the treatment, prevention, prophylaxis, and retardation of a disease. In certain embodiments, such terms refer to the improvement or elimination of a disease or symptoms associated with a disease. In other embodiments, the terms refer to the minimization of the spread or worsening of a disease resulting from the administration of one or more therapeutic agents to a subject with such a disease.

[0187] An effective dose can be a therapeutic or prophylactic effective dose. “Therapeutic effective dose” refers to the amount effective in the required dosage and duration to achieve a desired therapeutic or prophylactic outcome. In particular, this term refers to the amount of the pharmaceutical composition of the present invention administered to a patient sufficient to provide an immune response against the target pathogen or tumor cells. The therapeutic effective dose can vary depending on various factors, such as the disease being treated, the physiological state of the subject being treated, the severity of the disease, and the route of administration. The therapeutic effective dose encompasses the amount in which the therapeutically beneficial effect outweighs any toxic or adverse effects. “Prophylactic effective dose” refers to the amount effective in the required dosage and duration to achieve a desired prophylactic outcome. Generally, but not necessarily, a prophylactic effective dose will be less than a therapeutic effective dose, as prophylactic doses are used for subjects before or earlier in the disease. Appropriate means and measurements for determining therapeutic or prophylactic effective doses are available to those skilled in the art.

[0188] Preferably, the pharmaceutical composition is administered via a parenteral route, more preferably by intravenous injection. In some embodiments, particularly in the treatment of local diseases, the administration can be targeted to deliver to the cells of the organ or tissue affected by the disease.

[0189] In certain embodiments, the method of the present invention comprises administering to the subject a purified population of the cells of the present invention, preferably a purified population of cells comprising the antigen-specific CD8+ T cells of the present invention and optionally antigen-specific CD4+ T cells, at a dose of 10 3 ~10 8 cells / kg body weight, preferably 10 4 ~10 8 cells / kg body weight, more preferably 10 5 ~10 7 cells / kg body weight.

[0190] More specifically, the method of the present invention may comprise administering a purified population of cells comprising the antigen-specific CD8+ T cells of the present invention and optionally antigen-specific CD4+ T cells, particularly 1000 to 10000000 antigen-specific CD8+ T cells / kg body weight, preferably 5000 to 1000000 antigen-specific CD8+ T cells / kg body weight, more preferably 5000 to 100000 antigen-specific CD8+ T cells / kg body weight. In this case, the dose to be administered can be obtained by quantifying the CD8+ T cells present in the population or pharmaceutical composition of the present invention.

[0191] In some embodiments, the method of the present invention may involve administering an isolated population of cells comprising the antigen-specific CD8+ T cells and antigen-specific CD4+ T cells of the present invention. Preferably, 1,000 to 1,000,000 antigen-specific CD8+ T cells / kg body weight, preferably 5,000 to 1,000,000 antigen-specific CD8+ T cells / kg body weight, more preferably 5,000 to 1,000,000 antigen-specific CD8+ T cells / kg body weight, and 1,000 to 1,000,000 antigen-specific CD4+ T cells / kg target body weight, preferably 5,000 to 1,000,000 antigen-specific CD4+ T cells / kg target body weight, more preferably 5,000 to 1,000,000 antigen-specific CD4+ T cells / kg target body weight. In this case, the dose administered may be obtained by quantifying the CD8+ T cells and optionally quantifying the CD4+ T cells present in the population or pharmaceutical composition of the present invention.

[0192] The pharmaceutical composition may be administered as a bolus or in repeated doses. The frequency of administration may be, for example, every other week, monthly, every three months, or every six months.

[0193] The treatment may be autologous cell therapy (by reinjecting cells derived from the target into the same target) or allogeneic cell therapy (by injecting cells derived from the target into another target). Preferably, the treatment is autologous cell therapy.

[0194] As described above, the subject being treated, preferably a human, suffers from cancer or a disease caused by a pathogen.

[0195] The cancer or disease caused by the pathogen being treated may be any infection or cancer, in particular any infection or cancer in which a specific memory T cell response is functionally impaired.

[0196] Diseases caused by pathogens can be infections caused by viruses, bacteria, or fungi.

[0197] To treat diseases caused by pathogens, a pharmaceutical composition may comprise an isolated population of T cells obtained or obtainable by the present invention (i.e., by the method of the present invention for obtaining a T cell population). These T cells are administered to increase the pool of Tscm cells and to enable in vivo activation of the cells by contacting APCs in vivo.

[0198] Preferably, to treat a disease caused by a pathogen, the pharmaceutical composition comprises an isolated population of cells comprising the antigen-specific CD8+ T cells of the present invention and optionally antigen-specific CD4+ T cells, preferably an isolated population comprising the antigen-specific CD8+ T cells and antigen-specific CD4+ T cells of the present invention. To treat this class of diseases, the cell population comprising the antigen-specific CD8+ T cells and optionally antigen-specific CD4+ T cells of the present invention is obtained by culturing a T cell population of the present invention, comprising Tscm and naive T cells, in the presence of an APC carrying at least one antigen of a targeted pathogen or at least one peptide, particularly an immunogenic peptide, derived from the at least one antigen, to obtain an activated T cell population that recognizes target cells having the at least one antigen.

[0199] Preferably, the disease caused by the pathogen is a viral infection, particularly a chronic viral infection. In one embodiment, the disease caused by the pathogen is a viral infection caused by a virus selected from the group consisting of polyomaviruses, preferably human polyomaviruses, human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpesviruses, and papillomaviruses. Preferably, the virus is selected from the group consisting of human polyomaviruses, particularly John Cunningham (JC) polyomavirus, BK virus (BKV), and Merkel cell polyomavirus (MCPyV or MCV).

[0200] In certain embodiments, the pathogen is polyomavirus JC, and the disease caused by the pathogen is progressive multifocal leukoencephalopathy (PML).

[0201] In another specific embodiment, the pathogen is the BK virus, and the disease caused by the pathogen is BK virus-associated nephropathy.

[0202] In another specific embodiment, the pathogen is Merkel cell polyomavirus, and the disease caused by the pathogen is Merkel cell carcinoma.

[0203] In another specific embodiment, the pathogen is selected from the group consisting of human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpesviruses, and papillomaviruses, and the disease caused by the pathogen is a chronic or acute viral infection.

[0204] To treat diseases caused by pathogens, the cell-based therapies of the present invention may be used alone or in combination with other therapies, such as antibiotic therapy, antiviral therapy, or antiretroviral therapy.

[0205] The diseases being treated may include solid tumors or hematopoietic cancers, whether associated with or unrelated to cancer viruses.

[0206] As used herein, the terms “cancer” or “tumor” refer to the presence of cells that possess the typical characteristics of cancerous cells, such as unrestrained growth, immortality, metastatic ability, rapid growth and proliferation rates, and certain characteristic morphological features. The term refers to any type of malignant tumor (primary or metastatic).

[0207] Examples of solid tumors include, but are not limited to, breast cancer, gastric cancer, esophageal cancer, sarcoma, ovarian cancer, endometrial cancer, bladder cancer, cervical cancer, rectal cancer, colon cancer, lung cancer, or oral mucosal cancer, and pediatric tumors (neuroblastoma, glioblastoma multiforme).

[0208] Examples of hematopoietic cancers include, but are not limited to, lymphoma, leukemia, myeloma, seminomas, Hodgkin lymphoma, and malignant hematological disorders.

[0209] To treat this type of disease, the pharmaceutical composition may include an isolated population of cells containing antigen-specific CD8+ T cells and optionally antigen-specific CD4+ T cells, obtained by culturing a T cell population of the present invention, including Tscm and naive T cells, in the presence of an APC carrying at least one antigen expressed by tumor cells, such as a tumor-specific antigen (TSA) or tumor-associated antigen (TAA), or at least one peptide derived from the said at least one antigen, particularly an immunogenic peptide, in order to obtain a T cell population that is activated and recognizes target cells having the said at least one antigen.

[0210] Alternatively, particularly in diseases lacking a specific specific antigen, the pharmaceutical composition may comprise an isolated population of T cells obtained or obtainable by the present invention (i.e., by the method of the present invention for obtaining a T cell population). These T cells are administered to increase the pool of Tscm cells and enable their in vivo activation by contact with in vivo APCs.

[0211] To treat cancer, the cell-based therapies of the present invention may be used alone or in combination with other therapies such as chemotherapy, surgery, and / or radiotherapy.

[0212] In a particular embodiment, the disease to be treated is a disease caused by a pathogen, the pathogen being polyomavirus JC, and the disease being progressive multifocal leukoencephalopathy (PML). In this embodiment, the cell population to be administered, which includes antigen-specific T cells, a) A step of selecting a population of T cells having a cell surface phenotype containing CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, PD1- and TIGIT-, optionally LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-, more preferably LAG3- and TIM3- from a cell sample derived from a subject suffering from PML, b) A step of culturing the T cell population in the presence of antigen-presenting cells carrying at least one antigen of polyomavirus JC or at least one peptide, particularly an immunogenic peptide, derived from the at least one antigen, and optionally in the presence of IL-7 and IL-15 or other stimulating cytokines, and optionally c) A step of recovering the cells obtained in step b), particularly CD8+ and / or CD4+ cells, preferably CD8+ and CD4+ cells, It can be obtained by a method that includes

[0213] Alternatively, the cell population containing antigen-specific T cells that are administered is a) A step of selecting a population of T cells having cell surface phenotypes including (i) CD4+ or CD8+, (ii) CD45RA+, and (iii) CCR7+ and / or CD62L+ from a cell sample derived from a subject suffering from PML, b) A step of culturing the T cell population in the presence of antigen-presenting cells carrying at least one antigen of polyomavirus JC or at least one peptide, particularly an immunogenic peptide, derived from the at least one antigen, and optionally in the presence of IL-7 and IL-15 or other stimulating cytokines, and optionally c) A step of recovering the cells obtained in step b), particularly CD8+ and / or CD4+ cells, preferably CD8+ and CD4+ cells, It can be obtained by a method that includes

[0214] The method does not include a sorting step based on the CD95 marker. The T cell population sorted in step a) therefore includes both naive T cells (CD95-) and Tscm cells (CD95+). Preferably, the population contains at least 10%, preferably at least 30%, and more preferably at least 50% naive T cells of the total number of cells in the population. More preferably, in the population, T cells having a cell surface phenotype including (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and / or CD62L+, and (iv) CD95+ account for 4% to 70% of the total cells in the population, preferably 20% to 70%, and T cells having a cell surface phenotype including (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and / or CD62L+, and (iv) CD95- account for 30% to 96% of the total cells in the population, preferably 30% to 80%.

[0215] Preferably, in step a), the T cell population has a cell surface phenotype further comprising PD1-, TIGIT-, LAG3-, TIM3-, CTLA4- and / or CD160-, more preferably PD1-, TIGIT-, LAG3- and / or TIM3-.

[0216] Preferably, in step a), the T cell population has a cell surface phenotype further comprising CD3+ and / or CD45RO-, preferably CD3+ and CD45RO-.

[0217] In step c), CD8+ cells and optionally CD4+ cells can be selected from the cell population obtained in step b).

[0218] Preferably, the peptides presented by the APC may include one or more JCV peptides, particularly one or more JCV immunogenic peptides, from VP1, VP2, VP3, large T, small T proteins and / or other JCV proteins. In particular, the peptides presented by the APC may be overlapping peptides covering one or more of these proteins. More specifically, the peptides presented by the APC, preferably immunogenic peptides, may include an overlapping peptide pool covering the VP1, VP2, and VP3 regions of the JCV.

[0219] If the subject is also infected with a retrovirus such as HIV, the culture in step b) may be carried out in the presence of an antiretroviral compound.

[0220] Preferably, in all aspects of the present invention, T cells (including T cells used in or obtained from the methods of the present invention) are not recombinant T cells, and in particular are not TCR-T (TCR-transformed T) cells or CAR-T (chimeric antigen receptor-T) cells.

[0221] All references cited in this description are incorporated herein by reference. Other features and advantages of the present invention will become more apparent in the following examples, which are provided for illustrative purposes only and not for limitation. [Examples]

[0222] (Example 1) material and method Isolation of PBMCs 100 mL of heparinized blood was used. The blood was obtained from PML patients with various immunosuppressive causes, including AIDS, malignant hematological disorders, and treatments involving immunosuppressive therapy and biotherapy. The blood was diluted with 0.9% (v / v) NaCl, and PBMCs were isolated by Ficoll density gradient centrifugation.

[0223] Phenotypic testing of inhibitory receptors One million PBMCs were stained with the following combinations: anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, anti-CD45RO, anti-CCR7, anti-CD95, anti-PD1, anti-TIGIT, anti-LAG-3, and anti-TIM-3. CD62L may be used instead of CCR7 if necessary. Cells were fixed in PBS 1X containing 1% PFA and analyzed by flow cytometry (BD LSR Fortessa). Data were analyzed using FlowJo software.

[0224] In both CD3+ CD4+ T cells and CD3+ CD8+ T cells, cell subsets were defined as follows: -Naive T cells: CD45RA+ CD45RO- CCR7+ CD95- -Stem cell memory (Tscm): CD45RA+ CD45RO- CCR7+ CD95+ -Central Memory (Tcm): CD45RA- CD45RO+ CCR7+ CD95+ -Effects Memory (Tem): CD45RA- CD45RO+ CCR7- CD95+ -Cell subset including Tscm and naive T cells: CD45RA+, CD45RO-, CD62L+

[0225] The expression levels of PD1, TIGIT, LAG3, and TIM3 were analyzed in CD4+ and CD8+ T cell subsets, respectively.

[0226] Cell sorting Isolation of T cell subsets PBMCs were washed in buffer (PBS 1X, EDTA 2mM, SVF 0.5%). Monocytes were isolated by magnetic beads coated with anti-CD14. T cells were subsequently isolated in a fraction negatively selected by antibody-coated magnetic beads, allowing for the depletion of non-CD3+ cells.

[0227] The T cells were then washed in PBS containing 0.5% SVF. The cell concentration was adjusted to 20 million cells / mL, and the cells were then stained with the following antibodies: anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, anti-CD62L, anti-CD95, anti-PD1, and anti-TIGIT for 15 minutes at 4°C. The cells were washed, filtered through a 0.22 μm filter to remove cell clumps, and processed for cell sorting.

[0228] Gating Strategy The cells were first gated with forward and side scatter light, then with FSC-A and FSC-H to remove doublets, and subsequently gated as follows: Inhibitor receptor negative CD4 Tscm:CD3+ CD4+ CD8- CD45RA+ CD45RO- CCR7+ CD95+ PD1- TIGIT- Inhibitor receptor positive CD4 Tscm:CD3+ CD4+ CD8- CD45RA+ CD45RO- CCR7+ CD95+ and not (PD1- TIGIT-) Inhibitor receptor negative CD8 Tscm:CD3+ CD4- CD8+ CD45RA+ CD45RO- CCR7+ CD95+ PD1- TIGIT- Inhibitor receptor positive CD8 Tscm:CD3+ CD4- CD8+ CD45RA+ CD45RO- CCR7+ CD95+ and not (PD1- TIGIT-)

[0229] The cells were sorted in SVF-coated tubes. The cells were then resuspended in culture medium.

[0230] cell culture Five million monocytes were seeded per well in 2 mL of culture medium. Overlapping 15-mer JCV peptides (final concentration of 10 μg / mL in each pool), with 11 amino acid duplications across the entire sequences of VP1, VP2, and VP3 proteins, were added at 37°C for 2 hours. The monocytes were then washed twice with culture medium. The purified Tscm was centrifuged in culture medium and resuspended. Tscm was cultured in a total volume of 6 mL in a 24-well culture plate together with 5 million peptide-loaded monocytes. This was considered day 0. On day 2, rIL-7 and rIL-15 were added to the medium (final concentration, 10 ng / mL each). Every 3 days, 4 mL of medium was removed and replaced with fresh medium containing 10 ng / mL of Il-7 and IL-15. On day 14, the cells were counted.

[0231] CD4 and CD8 cell counting Cells were stained with anti-CD3, anti-CD4, and anti-CD8. The proportion of CD4 and CD8 T cells in the live gate was analyzed. The number of CD4 and CD8 T cells in each well was calculated by multiplying the proportion of CD4 or CD8 T cells by the total number of cells in each well. The proliferation rate was calculated by dividing the cell count on day 14 by the cell count on day 0.

[0232] Cytotoxicity assay CD14- and CD3- depleted cells were thawed, washed, and incubated for 2 hours with a JCV peptide pool containing VP1, VP2, and VP3 proteins (final concentration: 10 μg / mL per pool). After washing, the cells were used as target cells for restimulating cultured Tscm at a ratio of 1 target cell / 10 cultured T cells. The cells were cultured overnight.

[0233] The cells were then washed and stained with anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, anti-CD45RO, anti-CCR7, anti-CD27, and anti-CD95. The cells were then washed and fixed at +4°C for 20 minutes. The cells were washed and permeabilized. Before the final wash and acquisition by flow cytometry, anti-granzyme B and anti-perforin antibodies were added at +4°C for 30 minutes.

[0234] analysis The results were analyzed using FlowJo Software. Statistical analysis and graphing were performed using GraphPad Prism software.

[0235] result 1- CD4 and CD8 Tscm express significantly fewer inhibitory receptors than Tcm or Tem. The expression of inhibitory receptors PD1, TIGIT, LAG3, and TIM3 in CD4 and CD8 T cells from PML patients was analyzed by Tscm, Tcm, and Tem subsets. In nine patients, CD4 and CD8 Tscm cells expressed fewer inhibitory receptors than Tcm or Tem cells (see Figure 1: white areas correspond to cells that do not express inhibitory receptors).

[0236] Among these four inhibitory receptors, PD1 and TIGIT were most expressed (Figure 2, black). The results suggest that negative selection based on PD1 and TIGIT can deplete the majority of cells expressing these inhibitory receptors. Therefore, we isolated Tscm cells that were negative for both PD1 and TIGIT and analyzed their proliferative capacity after in vitro culture.

[0237] 2-PD1 and TIGIT inhibitor receptor-negative Tscm cells exhibit good cell proliferation. We established a gating strategy for purifying PD1-TIGIT-Tscm cells (see Figure 3).

[0238] The selected cells were cultured for 14 days in the presence of IL-7 and IL-15, and in the presence of JCV peptide carrying autologous monocytes, to proliferate JCV-specific cells. On day 14, the proliferation rate of cells at the culture endpoint was compared (see Figure 4). The proliferation capacity of i) Tscm versus other memory cells (CD45RO-positive cells including Tcm and Tem, regardless of inhibitory receptor expression status) (see Figure 4a) and ii) PD1 and TIGIT-negative Tscm versus PD1 and / or TIGIT-positive Tscm (see Figure 4b) was compared.

[0239] These data show that Tscm proliferates more efficiently than other (more differentiated) memory cells (Figure 4A), and that PD1-TIGIT-Tscm proliferates more efficiently than Tscm expressing PD1 and / or TIGIT (Figure 4B).

[0240] 3-PD1 and TIGIT inhibitor receptor-negative Tscm exhibit better cytotoxic activity. Inhibitor receptor-negative Tscm cells were then tested for cytotoxicity after restimulation. At the end of the proliferation phase (day 14), autologous cells carrying the JCV peptide were added to the culture medium overnight. Intracellular perforin and granzyme B staining were performed. The percentage of CD8 T cells expressing granzyme B and perforin among all CD8 T cells was measured (see Figure 5). These data indicate that effector CD8 T cells derived from inhibitor receptor-negative Tscm cells exhibit higher cytotoxic activity than Tscm cells expressing the inhibitor receptor, or other (more differentiated) memory CD8 T cells.

[0241] 4. CD8 T cells obtained from high-functioning Tscm after in vitro culture retain high differentiation potential and can be used in vivo. At the end of the culture period (day 14), PD1 and TIGIT-negative high-functioning stem cell memory cells (T SCM ), or more differentiated memory T cells (T MEM The phenotype of CD8 T cells differentiated from either ) or is as follows: Stem cell memory (Tscm): CD45RA+ CCR7+ Effector cells (Teff): CD45RA+ CCR7- Central Memory (Tcm): CD45RA- CCR7+ Effector Memory (Tem): CD45RA- CCR7- The analysis was based on the following criteria.

[0242] We found that Tscm differentiates into Tcm, Tem, and Teff, but the majority retain the Tscm phenotype (see Figure 6).

[0243] The CD4+ and CD8+ T cell subsets, characterized by the 5-CD45RA+, CD45RO-, and CD62L+ phenotypes, constitute a significant proportion of Tscm cells. We analyzed the proportion of Tscm cells (T cells in CD45RA+ CD45RO- CD62L+ cells) within PBMCs from 25 different PML patients. The results showed that Tscm cells accounted for 21% (median) of CD3+ CD4+ lymphocytes derived from CD45RA+ CD45RO- CD62L+ cells, and 17% (median) of CD3+ CD8+ lymphocytes derived from CD45RA+ CD45RO- CD62L+ cells. In comparison, Tscm cells accounted for only 5% and 2.5% (median) of all CD4+ and CD8+ T cells, respectively (see Figures 8a and 8b).

[0244] 6. High-functioning CD4+ and CD8+ Tscm cells are contained within the CD45RA+, CD45RO-, and CD62L+ cell gates. In 10 PML patients, the expression of the inhibitory receptors PD1 and TIGIT was analyzed in CD45RA+ CD45RO- CD62L+ CD4+ T cells, CD45RA+ CD45RO- CD62L+ CD8+ T cells, and CD4+ and CD8+ Tscm cells.

[0245] The data show that CD45RA+ CD45RO- CD62L+ CD4+ T cells and CD45RA+ CD45RO- CD62L+ CD8+ T cells contain 77% and 87% (median, see Figure 9a), respectively, of PD1-TIGIT- cells. CD4 and CD8 Tscm cells contain 69% and 63% (median) of PD1-TIGIT- cells, respectively (see Figure 9b).

[0246] conclusion In short, these results are i) In PML patients with different immunological backgrounds, Tscm cells express fewer inhibitory receptors than more differentiated memory cells. These inhibitory receptors include mostly PD-1 and TIGIT; ii) Total Tscm proliferates better than conventional memory T cells (including Tcm and Tem); iii) Selection based on the exclusion of PD1 and TIGIT allows for the depletion of the majority of Tscm expressing inhibitory receptors; iv) These PD1-TIGIT-Tscm cells exhibit greater proliferative capacity compared to PD1+ and / or TIGIT+ Tscm cells, and compared to more differentiated memory cells; v) PD1-TIGIT-Tscm exhibits greater cytotoxicity compared to PD1+ and / or TIGIT+Tscm, and compared to more differentiated memory cells; vi) PD1-TIGIT-Tscm efficiently differentiates in vitro into more differentiated memory cells, including effector cells, but the majority retain the Tscm phenotype. This may enable further differentiation cycles in vivo and therefore long-term therapeutic effects; vii) PD1-TIGIT-Tscm is found in CD45RA+ CD45RO- CD62L+ cells, This was demonstrated.

[0247] In short, these data demonstrate that PD1-TIGIT-Tscm can generate an effective and sustained immune response against JCV in PML patients.

[0248] (Example 2) Isolation of T cell subsets PBMCs were washed in buffer (PBS 1X, EDTA 2mM, FCS 0.5%). Monocytes were isolated by magnetic beads coated with anti-CD14. T cells were subsequently isolated in a fraction negatively selected by antibody-coated magnetic beads, allowing for the depletion of non-CD3+ cells.

[0249] The T cells were then washed in PBS containing 0.5% FCS. The cell concentration was adjusted to 20 million cells / mL, and the cells were then stained with the following antibodies: anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, and anti-CD62L for 15 minutes at 4°C. The cells were washed, filtered through a 0.22 μm filter to remove cell clumps, and processed for cell sorting.

[0250] Gating Strategy The cells were first gated with forward and side scatter light, then with FSC-A and FSC-H to remove doublets, and subsequently gated as follows: CD4 Tscm and naive T cells: CD3+ CD4+ CD8- CD45RA+ CD62L+ CD8 Tscm and naive T cells: CD3+ CD4- CD8+ CD45RA+ CD62L+

[0251] The cells were sorted in SVF-coated tubes. The cells were then resuspended in culture medium.

[0252] Quantification of naive T cells PBMCs were washed with PBS containing 0.5% FCS. Two million cells were stained with the following antibodies: anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, anti-CD62L and anti-CD95 at 4°C for 15 minutes. Cells were then washed and analyzed by flow cytometry.

[0253] The first gate was defined by the following phenotype: CD3+ CD4+ CD8− CD45RA+ CD62L+. Within this cell gate, naive CD4+ T cells were defined as CD95− cells and Tscm were defined as CD95+ cells.

[0254] The second gate was defined by the following phenotype: CD3+ CD4− CD8+ CD45RA+ CD62L+. Within this cell gate, naive CD8+ T cells were defined as CD95− cells and Tscm were defined as CD95+ cells.

[0255] Figure 7 shows the percentages of naive CD4+ T cells (left) and naive CD8+ T cells (right) within the CD3+ CD4+ CD8− CD45RA+ CD62L+ and CD3+ CD4− CD8+ CD45RA+ CD62L+ cell gates, respectively. Data are the median, first and third quartiles, 10th and 90th percentiles of 23 different healthy blood donors.

Claims

1. a) A step of selecting a population of T cells having a cell surface phenotype including CD4+ or CD8+, CD45RA+, and CCR7+ and / or CD62L+ from a cell sample derived from a subject suffering from cancer or a disease caused by a pathogen, wherein the population includes CD95+ and CD95- cells, b) A step of culturing the T cell population in the presence of antigen-presenting cells carrying at least one target antigen or at least one peptide derived from at least one target antigen, and optionally in the presence of IL-7 and IL-15 or other stimulating cytokines, c) A step of collecting the cells obtained in step b), particularly CD8+ and / or CD4+ cells, preferably CD8+ and CD4+ cells, An in vitro method for obtaining a cell population containing antigen-specific T cells, including [specific cells].

2. The method according to claim 1, wherein the T cell population selected in step a) contains at least 10%, preferably at least 30%, and more preferably at least 50% naive T cells of the total number of cells in the population.

3. The method according to claim 1 or 2, wherein in the T cell population selected in step a), 4% to 70%, preferably 20% to 70%, of the total cells in the population are T cells having a cell surface phenotype including (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and / or CD62L+, and (iv) CD95+, and 30% to 96%, preferably 30% to 80%, of the total cells in the population.

4. The method according to any one of claims 1 to 3, wherein the T cell population selected in step a) has a cell surface phenotype further comprising PD1-, TIGIT-, LAG3-, TIM3-, CTLA4- and / or CD160-, preferably PD1-, TIGIT-, LAG3-, TIM3- and / or CTLA4-, more preferably PD1-, TIGIT-, LAG3- and / or TIM3-.

5. The method according to any one of claims 1 to 3, wherein the T cell population selected in step a) further has a cell surface phenotype comprising PD1- and TIGIT-.

6. The method according to any one of claims 1 to 5, wherein the antigen-presenting cells are dendritic cells, monocytes, monocyte-derived dendritic cells, peripheral blood mononuclear cells (PBMCs), Epstein-Barr virus-transformed B lymphoblastic cell lines (EBV-BLCL cells), or artificial antigen-presenting cells (AAPCs).

7. The method according to any one of claims 1 to 5, wherein the antigen-presenting cells are dendritic cells, monocytes, peripheral blood mononuclear cells (PBMCs), Epstein-Barr virus-transformed B lymphoblastic cell lines (EBV-BLCL cells), or artificial antigen-presenting cells (AAPCs).

8. The method according to any one of claims 1 to 7, wherein the antigen-presenting cells are of autologous origin to the subject.

9. The method according to any one of claims 1 to 8, wherein the at least one target antigen is a pathogen antigen, preferably a viral, bacterial, or fungal antigen, or an antigen expressed by tumor cells such as a tumor-specific antigen (TSA) or tumor-associated antigen (TAA).

10. The method according to any one of claims 1 to 9, wherein the subject suffers from progressive multifocal leukoencephalopathy, Merkel cell carcinoma, or BK virus-associated nephropathy, and the at least one target antigen is selected from the group consisting of polyomavirus JC, polyomavirus MPCyV, and polyomavirus BK.

11. The method according to any one of claims 1 to 10, wherein the subject suffers from progressive multifocal leukoencephalopathy, and the at least one target antigen is an antigen of polyomavirus JC.

12. The method according to any one of claims 1 to 9, wherein the subject has cancer, and preferably the at least one target antigen is an antigen expressed by tumor cells, such as a tumor-specific antigen (TSA) or tumor-associated antigen (TAA).

13. The method according to any one of claims 1 to 12, wherein the cell sample is a bone marrow cell sample, a blood cell sample, a fractionated or unfractionated whole blood sample, a fractionated or unfractionated apheresis recovery product, tumor-infiltrating lymphocytes, PBMCs, or a population rich in T cells from a blood sample or PBMCs.

14. An isolated cell population comprising antigen-specific CD8+ T cells and optionally antigen-specific CD4+ T cells, obtained or obtainable by the method described in any one of claims 1 to 13.

15. An isolated cell population according to claim 14, as a cell therapy drug.

16. An isolated cell population according to claim 14, for use in the treatment of cancer or a disease caused by a pathogen.

17. An isolated cell population according to claim 16 for use in the treatment of progressive multifocal leukoencephalopathy, Merkel cell carcinoma, or BK virus-associated nephropathy.

18. An isolated cell population according to claim 16 for use in the treatment of progressive multifocal leukoencephalopathy.

19. The isolated cell population according to any one of claims 16 to 18, wherein the population is of autologous origin to the subject being treated.

20. A method for treating a subject suffering from cancer or a disease caused by a pathogen, comprising administering to the subject a therapeutically effective dose of the isolated cell population described in claim 14.

21. The method according to claim 20, wherein the treatment is autologous cell therapy.

22. The method according to claim 20 or 21, wherein the subject is suffering from progressive multifocal leukoencephalopathy, Merkel cell carcinoma, or BK virus-associated nephropathy, preferably progressive multifocal leukoencephalopathy.

23. Use of the isolated cell population according to claim 14 for preparing a pharmacopoeia for the treatment of cancer or a disease caused by a pathogen.

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