Highly functional autologous stem-derived T cell immunotherapy

Abstract

The present invention relates to a novel and specific T cell therapy strategy based on the use of memory stem T cells (Tscm), in particular highly functional memory stem T cells (Tscm) negatively selected based on inhibitory receptor expression. This novel cellular immunotherapy can be applied to PML patients, but also to other infections or cancers where specific memory T cell responses are functionally impaired.

Description

[Technical field]

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

[0002] Progressive multifocal leukoencephalitis (PML) is a demyelinating opportunistic disease with poor prognosis, which is associated with the replication of polyomavirus JC (JCV) in the central nervous system. PML is observed exclusively during prolonged and severe cellular immunosuppression, mainly in AIDS patients or patients with malignant hematological diseases, or after immunosuppressive therapy, including novel potent immunosuppressive biotherapy. This devastating disease is associated with high mortality and major neurological sequelae in survivors.

[0003] PML is associated with impaired immune control of viral replication in the brain by cytotoxic memory CD8 T lymphocytes, which require functional memory CD4 T cells for optimal functionality. Impairment of anti-JC virus CD8 T cell responses 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 therapeutic approach that has shown some efficacy is functional restoration of anti-JCV T cell responses, if possible, for example by initiating effective antiretroviral therapy in HIV-infected patients or by ceasing immunosuppressive therapy. However, such immune restoration may require long periods of time during which JCV continues its replication, expanding neuropathology, and compromising survival and neurological prognosis. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Szabolcs et al. (1995) J. Immunol. 154:5851~5861 [Non-Patent Document 2] Remington: The Science and Practice of Pharmacy (20th ed.), edited by A. R. Gennaro, Lippincott Williams & Wilkins, 2000 [Non-Patent Document 3] Encyclopedia of Pharmaceutical Technology, edited by J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there is a strong need for novel therapeutic options that can efficiently generate an effective and sustained immune response against JCV in PML patients. [Means for solving the problem]

[0007] The present inventors have developed a novel specific T cell therapy strategy based on the use of memory stem T cells (Tscm), especially highly functional Tscm negatively selected based on inhibitory receptor expression. This approach 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 may remain highly functional in terms of expansion and differentiation, and can generate specific cytotoxic effectors effective ex vivo against virus or tumor antigens; whereas more differentiated memory T cell subsets, such as effector memory (Tem) or central memory (Tcm) or effector (Teff), are poorly functional against virus or tumor antigens. This novel cellular immunotherapy can be applied to PML patients, but also to other infections or cancers in which specific memory T cell responses are functionally impaired.

[0008] Thus, in a first aspect, the present invention provides an in vitro method for obtaining a population of cells comprising antigen-specific T cells, comprising the steps of: a) sorting a population of Tscm cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, and CD95+ from a cell sample from a subject suffering from cancer or a disease caused by a pathogen, in particular a cancer or a disease caused by a pathogen in which specific memory T cell responses are functionally impaired; b) culturing said population of Tscm cells in the presence of antigen-presenting cells loaded with at least one antigen of interest or at least one peptide derived from at least one antigen of interest, and optionally in the presence of IL-7 and IL-15 or other stimulatory cytokines, and optionally c) recovering the cells obtained in step b), in particular the CD8+ and / or CD4+ cells, preferably the CD8+ and CD4+ cells. The present invention relates to a method comprising the steps of:

[0009] The population of Tscm cells sorted in step a) may have a cell surface phenotype further comprising PD1-, TIGIT-, LAG3-, TIM3-, CTLA4- and / or CD160-, preferably PD1-, TIGIT-, LAG3- and / or TIM3-. In particular, the population of Tscm cells sorted in step a) may have a cell surface phenotype further comprising PD1- and TIGIT-, and optionally LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-, more preferably LAG3- and TIM3-.

[0010] Alternatively, the method may comprise: a) sorting a population of Tscm cells from a cell sample from a subject, the Tscm cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1-, and TIGIT-; b) culturing said population of Tscm cells in the presence of antigen-presenting cells loaded with at least one immunogenic peptide derived from at least one antigen of interest, and optionally in the presence of IL-7 and IL-15 or other stimulatory cytokines; and c) sorting CD8+ cells and optionally CD4+ cells from the population of cells obtained in step b), preferably sorting CD8+ cells and CD4+ cells from the population of cells obtained in step b). may include.

[0011] Optionally, the population of Tscm cells sorted in step a) has a cell surface phenotype further comprising LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-.

[0012] The population of Tscm cells sorted in step a) may have a cell surface phenotype further comprising CD3+, CD45RO-, CXCR3+ and / or CD122+, preferably CD3+ and CD45RO-.

[0013] In particular, the population of Tscm cells sorted in step a) may include cells having a cell surface phenotype including CD4+, CD8-, CD45RA+, CD95+, CCR7+, PD1- and TIGIT-, as well as cells having a cell surface phenotype including CD4-, CD8+, CD45RA+, CD95+, CCR7+, PD1- and TIGIT-.

[0014] More particularly, the population of Tscm cells sorted in step a) may comprise cells having a cell surface phenotype comprising: CD4+, CD8-, CD45RA+, CD95+, CCR7+, PD1-, TIGIT-, LAG3 and TIM3-, preferably CD3+, CD45RO-, CD4+, CD8-, CD45RA+, CD95+, CCR7+, PD1-, TIGIT-, LAG3 and TIM3-, as well as cells having a cell surface phenotype comprising: CD4-, CD8+, CD45RA+, CD95+, CCR7+, PD1-, TIGIT-, LAG3- and TIM3-, preferably CD3+, CD45RO-, CD4-, CD8+, CD45RA+, CD95+, CCR7+, PD1-, TIGIT-, LAG3- and TIM3-.

[0015] The antigen presenting cells can be dendritic cells, monocytes, peripheral blood mononuclear cells (PBMCs), Epstein-Barr virus transformed B lymphoblastoid cell line cells (EBV-BLCL cells) or artificial antigen presenting cells (AAPCs).

[0016] Preferably, the antigen presenting cells are autologous to the subject.

[0017] Preferably, the antigen presenting cells are monocytes or dendritic cells, more preferably monocytes or dendritic cells autologous to the subject.

[0018] Said at least one antigen of interest may be a pathogen antigen, preferably a viral, bacterial or fungal antigen, or an antigen expressed by a tumor cell, such as a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA).

[0019] The subject may be suffering from cancer.

[0020] The at least one antigen of interest may be an antigen expressed by a tumor cell, such as a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA).

[0021] The subject may be suffering from a disease caused by human polyomavirus, preferably progressive multifocal leukoencephalitis, Merkel cell carcinoma or BK virus associated nephropathy.

[0022] Preferably, said at least one antigen of interest is an antigen of a human polyomavirus, in particular an antigen selected from the group consisting of Polyomavirus JC, Polyomavirus MPCyV or Polyomavirus BK. More preferably, said at least one antigen of interest is an antigen of Polyomavirus JC or MPCyV, in particular an antigen of Polyomavirus JC.

[0023] In step b), the population of Tscm cells may be cultured in the presence of IL-7 and IL-15, and / or may be cultured for 8 to 20 days, preferably 10 to 18 days, more preferably 12 to 16 days.

[0024] The cell sample can be a bone marrow cell sample, a blood cell sample, a fractionated or unfractionated whole blood sample, a fractionated or unfractionated apheresis collection, tumor infiltrating lymphocytes, PBMCs, or a T cell-enriched population from a blood sample or PBMCs.

[0025] The present invention also relates to 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 population of cells comprising antigen-specific T cells. The isolated population may comprise or consist of Tscm cells, T effector (Teff) cells, T central memory (Tcm) cells, and T effector memory (Tem) cells.

[0026] In an isolated population of cells of the present invention, Tscm, Tcm and Tem cells may constitute up to 90% of the total cells, preferably 50%-90%, and Teff cells may constitute 10%-50%, preferably 10%-20% of the total cells.

[0027] The present invention also relates to an in vitro method for obtaining a population of memory stem T cells (Tscm cells), comprising the step of sorting, from a cell sample from a subject, a population of Tscm cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1- and TIGIT-, and optionally LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-.

[0028] The subject may have cancer or a disease caused by a pathogen, particularly a cancer or a disease caused by a pathogen in which the specific memory T cell response is functionally impaired.

[0029] Preferably, the subject is suffering from a disease caused by a human polyomavirus.

[0030] More preferably, the subject is suffering from progressive multifocal leukoencephalitis, Merkel cell carcinoma or BK virus associated nephropathy.

[0031] The present invention further relates to a population of isolated Tscm cells having a cell surface phenotype comprising: (i) CD4+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1-, TIGIT-, and optionally LAG3- and / or TIM3-; and / or (ii) CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1-, TIGIT-, and optionally LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-, more preferably LAG3- and TIM3-.

[0032] The present invention also relates to a population of isolated cells comprising antigen-specific CD8+ T cells of the present invention and optionally antigen-specific CD4+ T cells, or a population of isolated Tscm cells of the present invention, as a cell therapy medicine, and to a pharmaceutical composition comprising said population of isolated cells comprising antigen-specific CD8+ T cells of the present invention, or said population of isolated Tscm cells of the present invention, and a pharma- ceutical acceptable carrier and / or excipient.

[0033] The present invention further relates to said population of isolated cells comprising antigen-specific CD8+ T cells of the invention and optionally antigen-specific CD4+ T cells, said population of isolated Tscm cells of the invention or said pharmaceutical composition for use in the treatment of cancer or a disease caused by a pathogen, in particular a cancer or a disease caused by a pathogen in which the specific memory T cell response is functionally impaired.

[0034] 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 leukoencephalitis, Merkel cell carcinoma or BK virus-associated nephropathy.

[0035] Preferably, the disease to be treated is a disease caused by a pathogen, where the pathogen is polyomavirus JC and the disease is progressive multifocal leukoencephalitis (PML). Alternatively, the disease to be treated is a disease caused by a pathogen, where the pathogen is polyomavirus MCPyV and the disease is Merkel cell carcinoma, or the disease to be treated is a disease caused by a pathogen, where the pathogen is polyomavirus BKV and the disease is BK virus-associated nephropathy.

[0036] Preferably, the cells used in the treatment are autologous to the subject being treated.

[0037] The dose of the population of isolated cells or pharmaceutical composition administered may comprise 1000-10,000,000 antigen-specific CD8+ T cells per kg of subject body weight. The dose may further comprise 1000-10,000,000 antigen-specific CD4+ T cells per kg of subject body weight.

[0038] The present invention further relates to the use of an isolated population of cells of the invention, or a pharmaceutical composition of the invention, for the preparation of a medicament for the treatment of cancer or a disease caused by a pathogen, in particular a cancer or a disease caused by a pathogen in which the specific memory T cell response is functionally impaired.

[0039] The present invention further relates to a method for treating a subject suffering from cancer or a disease caused by a pathogen, in particular a cancer or a disease caused by a pathogen in which the specific memory T cell response is functionally impaired, comprising the step of administering to said subject a therapeutically effective amount of an isolated population of cells of the invention or a pharmaceutical composition of the invention. [Brief description of the drawings]

[0040] [Figure 1] FIG. 1 shows the 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. [Diagram 2] FIG. 1 shows that inhibitory receptors mainly include PD1 and / or TIGIT. The black part of the pie chart represents cells positive for PD1 and / or TIGIT, alone or in combination with other inhibitory receptors. The grey part represents cells positive for other inhibitory receptors than PD1 and / or TIGIT. The white part represents cells negative for inhibitory receptors. Each row represents one patient. [Diagram 3] Gating strategy for isolation of highly functional Tscm. The same gating strategy was applied to CD4 and CD8 T cells. [Figure 4]Figure 1. Proliferative potential of different sorted T cell subsets. Left: Total Tscm (pooled CD4 and CD8 Tscm) versus more differentiated CD45RO+ memory cells (pooled CD4 and CD8 Tcm and Tems). Right: Tscm negative for PD1, TIGIT, TIM3 and LAG3 versus Tscm positive for PD1 and / or TIGIT and / or TIM3 and / or LAG3. Expansion rate was calculated as follows: [number of cells in culture on day 14] / [number of cells on day 0]. Statistical significance: *p<0.05, Wilcoxon test. [Diagram 5] Figure 1 shows the specific cytotoxic potential of cells obtained from the different sorted T cell subsets after 14 days of culture. Statistical significance: *p<0.05, Wilcoxon test. Cytotoxic potential was assessed by granzyme b and perforin expression after restimulation with autologous cells (CD14 and CD3 depleted PBMCs) loaded with JCV peptides. [Figure 6] FIG. 1 shows differentiation of PD1-TIGIT-Tscm CD8 T cells after 14 days of in vitro culture. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] The present inventors developed a novel autologous specific T cell therapy strategy to circumvent the inhibition of anti-JCV T cell function in PML patients. The approach is based on the use of memory stem T cells (Tscm), preferably highly functional Tscm negatively selected based on inhibitory receptor expression. Indeed, they observed that in patients with severe and long-term immunosuppression, such as PML patients, this rare memory T cell subset may remain highly functional in terms of expansion and differentiation, and can generate specific cytotoxic effectors effective ex vivo against viral or tumor antigens. They also demonstrated that in PML patients from various immunological backgrounds, including HIV infection, hematological malignancies, and treatment with immunosuppressive biotherapy, Tscm cells express lower amounts of inhibitory receptors than more differentiated memory cells. They further demonstrated that selection based on PD1 and TIGIT exclusion allows depleting the majority of inhibitory receptor expressing Tscm. They also showed that these PD1-TIGIT-Tscm cells exhibited better proliferation and cytotoxicity compared to PD1+ and / or TIGIT+Tscm and more differentiated memory cells. These cells efficiently differentiate in vitro into more differentiated memory cells, including effector cells, but the majority retain the Tscm phenotype, allowing further cycles of differentiation in vivo after administration and therefore long-term therapeutic effects. Thus, after in vitro activation, differentiation and expansion, these cells can provide a population of cells that includes antigen-specific T cells and can generate an effective and sustained immune response against JCV in PML patients. This novel cell therapy-based personalized medicine can also be applied to other chronic viral infections or cancers in which specific antiviral or antitumor memory T cell responses are functionally impaired.

[0042] In a first aspect, the present invention provides an in vitro method for obtaining a population of cells comprising antigen-specific T cells, comprising the steps of: a) sorting a population of Tscm cells from a cell sample from a subject, i.e., a population of Tscm cells having a cell surface phenotype comprising: (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CD95+, and (iv) CCR7+ and / or CD62L+; b) culturing said population of Tscm cells in the presence of antigen-presenting cells loaded with at least one antigen of interest or one or more peptides derived from said at least one antigen of interest, and optionally in the presence of IL-7 and IL-15 or other stimulatory cytokines, and optionally c) recovering the cells obtained in step b), in particular the CD8+ and / or CD4+ cells, preferably the CD8+ and CD4+ cells. The present invention relates to a method comprising the steps of:

[0043] Preferably, in step a), the population of Tscm cells has a cell surface phenotype further comprising PD1-, TIGIT-, LAG3-, TIM3-, CTLA4- and / or CD160-, more preferably PD1-, TIGIT-, LAG3- and / or TIM3-.

[0044] In a preferred embodiment, the method comprises: a) sorting a population of highly functional Tscm cells from a cell sample from a subject, i.e., a population of Tscm cells having a cell surface phenotype comprising: (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CD95+, (iv) CCR7+ and / or CD62L+, (v) PD1-, and (vi) TIGIT-; b) culturing said population of Tscm cells in the presence of antigen-presenting cells loaded with at least one antigen of interest or one or more peptides derived from said at least one antigen of interest, and optionally in the presence of IL-7 and IL-15 or other stimulatory cytokines, and optionally c) recovering the cells obtained in step b), in particular the CD8+ and / or CD4+ cells, preferably the CD8+ and CD4+ cells. Includes.

[0045] In particular, in step c), CD8+ cells, and optionally CD4+ cells, may be sorted from the population of cells obtained in step b).

[0046] Optionally, step a) may further comprise depleting cells expressing one or several other inhibitory receptors, such as LAG3, TIM3, CTLA4 or CD160. In particular, step a) may further comprise depleting cells expressing LAG3, TIM3, CTLA4 and / or CD160. In this case, the population of cells to be sorted may have a cell surface phenotype comprising: (i) CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1- and TIGIT-, and (ii) LAG3-, TIM3-, CTLA4- and / or CD160-.

[0047] Preferably, step a) further comprises depleting cells expressing LAG3 and / or TIM3, in which case the population of cells to be sorted may have a cell surface phenotype comprising: (i) CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1- and TIGIT-, and (ii) LAG3- and / or TIM3-, preferably a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1-, TIGIT-, LAG3- and TIM3-.

[0048] Optionally, step a) may further comprise depleting cells expressing CD45RO and / or selecting cells expressing CD3. In this case, the population of cells to be sorted may have a cell surface phenotype comprising: (i) CD4+ or CD8+, CD3+, CD45RA+, CD45RO-, CD95+, CCR7+ and / or CD62L+, preferably CD4+ or CD8+, CD3+, CD45RA+, CD45RO-, CD95+, CCR7+ and / or CD62L+, PD1- and / or TIGIT-, more preferably CD4+ or CD8+, CD3+, CD45RA+, CD45RO-, CD95+, CCR7+ and / or CD62L+, PD1- and TIGIT-, and optionally (ii) LAG3-, TIM3-, CTLA4- and / or CD160-. Preferably, step a) further comprises depleting cells expressing LAG3 and / or TIM3, preferably depleting cells expressing LAG3 and TIM3, in which case the population of cells to be sorted may have a cell surface phenotype comprising: (i) CD4+ or CD8+, CD3+, CD45RA+, CD45RO-, CD95+, CCR7+ and / or CD62L+, preferably CD4+ or CD8+, CD3+, CD45RA+, CD45RO-, CD95+, CCR7+ and / or CD62L+, PD1- and / or TIGIT-, more preferably CD4+ or CD8+, CD3+, CD45RA+, CD45RO-, CD95+, CCR7+ and / or CD62L+, PD1- and TIGIT-, and (ii) LAG3- and / or TIM3-, preferably LAG3- and TIM3-.

[0049] As used herein, the term "CD4" refers to the T cell surface glycoprotein CD4, a glycoprotein that serves as a co-receptor for the T cell receptor (TCR). In humans, the CD4 protein is encoded by the CD4 gene.

[0050] As used herein, the term "CD8" refers to a transmembrane glycoprotein that serves as a coreceptor for the T cell receptor (TCR). There are two isoforms of the protein, 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, which in humans is encoded by the CD8A gene.

[0051] As used herein, the term "CD3" refers to a protein complex and a T cell coreceptor. In mammals, this complex contains the CD3γ chain, the 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 bound to major histocompatibility complex classes I and II during the immune response. As used herein, the term "CD3" refers to the CD3γ chain, which in humans is encoded by the CD3G gene, the CD3δ chain, which in humans is encoded by the CD3D gene, or the CD3ε chain, which in humans is encoded by the CD3E gene.

[0052] As used herein, the term "CD45RA" refers to the 200-220 kDa isoform of receptor tyrosine-protein phosphatase C, also named CD45. In humans, the CD45 protein is encoded by the PTPRC gene. This tyrosine phosphatase is required for T cell activation through the antigen receptor. The CD45RA isoform contains only the A protein region.

[0053] As used herein, the term "CD45RO" refers to the 180 kDa isoform of receptor tyrosine-protein phosphatase C, also named CD45. This isoform is the shortest CD45 isoform and lacks all three of the A, B and C regions.

[0054] 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.

[0055] As used herein, the term "CCR7" refers to CC chemokine receptor type 7, also known as CD197, which is a member of the G protein-linked receptor family. In humans, the CCR7 protein is encoded by the CCR7 gene.

[0056] As used herein, the term "CD62L" refers to L-selectin, a calcium-dependent lectin that mediates cell adhesion by binding to glycoproteins on neighboring cells. In particular, CD62L mediates the adhesion of lymphocytes to endothelial cells of high endothelial venules in peripheral lymph nodes. In humans, CD62L is encoded by the SELL gene.

[0057] As used herein, the term "PD1" refers to programmed cellular D protein 1, also known as CD279. PD1 is a cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on the surface of T and B cells. In humans, the PD-1 protein is encoded by the PDCD1 gene.

[0058] As used herein, the term "TIGIT" refers to an immune receptor, also known as T cell immune receptor with Ig and ITIM domains, WUCAM or Vstm3. In humans, the TIGIT protein is encoded by the TIGIT gene.

[0059] As used herein, the term "LAG3" refers to lymphocyte activation gene 3, also known as CD223. LAG3 is a cell surface molecule that has diverse biological effects on T cell function. In humans, the LAG3 protein is encoded by the LAG3 gene.

[0060] As used herein, the term "TIM3" refers to T-cell immunoglobulin and mucin domain-containing protein 3, also known as Hepatitis A Virus Cellular Receptor 2 (HAVCR2). TIM3 is a surface receptor implicated in modulating innate and adaptive immune responses. In humans, the TIM3 protein is encoded by the HAVCR2 gene.

[0061] 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 and downregulates immune responses. In humans, CTLA4 protein is encoded by the CTLA4 gene.

[0062] As used herein, the term "CD160" refers to a glycoprotein receptor on immune cells that can deliver stimulatory or inhibitory signals that regulate cell activation and differentiation. In humans, the CD160 protein is encoded by the CD160 gene.

[0063] In addition, the population of Tscm cells sorted in step a) may also express CXCR3 and / or CD122 and may be selected based on these additional markers. In this case, the population of cells sorted is (i) CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, CXCR3+ and / or CD122+, preferably CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, CXCR3+ and / or CD122+, PD1- and / or TIGIT-, more preferably CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, CXCR3+ and / or CD122+, PD1- and TIGIT-, or (ii) CD4+ or CD8+ , CD3+, CD45RA+, CD45RO-, CD95+, CCR7+ and / or CD62L+, CXCR3+ and / or CD122+, preferably CD4+ or CD8+, CD3+, CD45RA+, CD45RO-, CD95+, CCR7+ and / or CD62L+, CXCR3+ and / or CD122+, PD1- and / or TIGIT-, more preferably CD4+ or CD8+, CD3+, CD45RA+, CD45RO-, CD95+, CCR7+ and / or CD62L+, CXCR3+ and / or CD122+, PD1- and TIGIT-. Optionally, these sorted cell populations may have a cell surface phenotype further comprising LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-, more preferably LAG3- and TIM3-.

[0064] As used herein, the term "CXCR3" refers to the chemokine receptor CXCR3, also known as G protein-linked receptor 9 (GPR9) and CD183. In humans, CXCR3 is encoded by the CXCR3 gene. As used herein, the term "CD122" refers to the interleukin-2 receptor subunit beta, also known as IL15RB. CD122 is a receptor for interleukin-2. This beta subunit is involved in receptor-mediated endocytosis and transmits the mitogenic signal of IL2. In humans, CD122 is encoded by the IL2RB gene.

[0065] As used herein, the term "cell surface phenotype" refers to the presence or absence of a combination of specific cell surface markers on the surface of a cell. By "cell surface marker" is intended a molecule expressed on the surface of a cell that can be detected, for example, using a labeled antibody or other means known in the art. A cell surface marker may include a protein, a glycoprotein, or a group of proteins and / or glycoproteins. In this case, the population of Tscm cells sorted / selected in step a) may be identified by the expression of a specific combination of markers including CD4 or CD8, CD45RA, CD95, CCR7 and / or CD62L, and preferably CD3, and the lack of expression of a specific 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 population of Tscm cells sorted / selected in step a) may be further identified by the lack of expression of CD45RO.

[0066] The population of Tscm cells obtained in step a) is enriched for Tscm cells having a particular cell surface phenotype.

[0067] In certain embodiments, the population of Tscm cells obtained in step a) is enriched for Tscm cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, and optionally CD3+, CD45RO-, CXCR3+ and / or CD122+, preferably Tscm cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, CD3+ and CD45RO-. Preferably, the cell surface phenotype further comprises 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-.

[0068] In a preferred embodiment, the population of Tscm cells obtained in step a) is enriched for Tscm cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CD95+, 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 Tscm cells have a cell surface phenotype further comprising CD3+ and CD45RO-.

[0069] By "enriched" is meant a composition comprising cells that are present in a greater proportion of total cells than found in another composition. In particular, in the population obtained in step a), Tscm cells having a particular cell surface phenotype as defined above, e.g., cell surface phenotypes including CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1- and TIGIT-, and optionally LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-, are present in a higher proportion of total cells compared to their proportion in the cell sample. In the population obtained in step a), Tscm cells having a particular said cell surface phenotype, for example a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1- and TIGIT-, and optionally LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-, constitute more than 70% of the total cells in said population, preferably constitute more than 80%, 90%, 95% or 99%, and even more preferably constitute more than 95% or 99% of the total cells in said population.

[0070] Conversely, the population of Tscm cells obtained in step a) may be depleted for cells expressing the inhibitory receptors PD1, TIGIT, LAG3, TIM3, CTLA4 and / or CD160, preferably for cells expressing the inhibitory receptors PD1 and TIGIT, and optionally LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3 and / or TIM3. Preferably, the population of Tscm cells obtained in step a) is further depleted for cells expressing CD45RO. By "depleted" is meant a composition comprising cells present in a lower proportion of total cells than found in another composition, in particular than found in a cell sample. In a preferred embodiment, in the population obtained in step a), Tscm cells having a cell surface phenotype comprising PD1+ and TIGIT+, and optionally LAG3+, TIM3+, CTLA4+ and / or CD160+, preferably LAG3+ and / or TIM3+, are present in a lower proportion of total cells compared to their proportion in the cell sample.In particular, in the population obtained in step a), Tscm cells expressing inhibitory receptors PD1 and TIGIT, and optionally LAG3, TIM3, CTLA4 and / or CD160, preferably LAG3 and / or TIM3, may constitute less than 5%, 2% or 1% of total cells in said population.

[0071] Preferably, in the population obtained in step a), Tscm cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, preferably comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+ and CD3+, more preferably comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, CD3+ and CD45RO- constitute more than 95%, 96%, 97%, 98% or 99% of all cells in said population.

[0072] More preferably, in the population obtained in step a), Tscm cells having a cell surface phenotype comprising: (i) CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1- and TIGIT-, preferably CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, CD3+, PD1- and TIGIT-, more preferably CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, CD3+, CD45RO-, PD1- and TIGIT-, and (ii) optionally LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-, constitute more than 95%, 96%, 97%, 98% or 99% of all cells in said population. In this case, Tscm cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1+ and / or TIGIT+, preferably CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, CD3+, PD1+ and / or TIGIT+, more preferably CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, CD3+, CD45RO-, PD1+ and TIGIT+, and optionally LAG3+, TIM3+, CTLA4+ and / or CD160+, preferably LAG3+ and / or TIM3+, may comprise less than 5% of the total cells in the population, more preferably less than 2% or 1% of the total cells in the population.

[0073] In a particular embodiment, the population obtained in step a) consists of Tscm cells having a cell surface phenotype as defined above, preferably comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, preferably comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+ and CD3+, more preferably comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, CD3+ and CD45RO-.

[0074] In another particular embodiment, the population obtained in step a) consists of Tscm cells having a cell surface phenotype as defined above, preferably (i) comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1- and TIGIT-, preferably comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, CD3+, PD1- and TIGIT-, more preferably comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, CD3+, CD45RO-, PD1- and TIGIT-, and (ii) optionally comprising LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-.

[0075] 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, anti-CD40 antibodies may be added to the population to compensate for the lack of CD4 T cells helping signals.

[0076] In step a), cells with a particular cell surface phenotype are sorted and recovered from the cell sample. Sorting of cells with a particular cell surface phenotype can be performed using any method known in the art. Positive and / or negative selection can be easily achieved using materials and techniques known in the art. For example, cells expressing a particular cell surface marker can be separated from other cells using monoclonal antibodies that bind to the marker and are coupled to columns or magnetic beads; separation is easily performed according to standard techniques and / or manufacturer's or distributor's instructions. In particular, in step a), cells can be sorted by fluorescence-activated cell sorting (FACS) or by magnetic separation.

[0077] The cell sample can be any sample that contains T cells and in particular Tscm cells, or cells that can be induced to become Tscm cells in culture.Preferably, the sample is a sample that contains Tscm 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 collections (e.g., leukapheresis collections), tumor infiltrating lymphocytes, PBMCs, or T cell populations (e.g., T cell-enriched populations from blood samples or PBMCs).

[0078] In certain embodiments, the cell sample is PBMCs. PBMCs can be isolated from blood samples by any method known in the art, for example, by Ficoll density gradient centrifugation.

[0079] As used herein, the term "isolated" means that the cell has been separated from components with which it is normally associated in nature.

[0080] In another particular embodiment, the cell sample is a population enriched with T cells from PBMCs or from a blood sample, preferably a population enriched with T cells from PBMCs. T cells can be enriched from PBMCs or from a blood sample by any method known in the art. For example, T cells can be enriched from PBMCs or from a blood sample by depletion of CD14+ cells and / or by sorting using an anti-CD3 antibody and retaining CD3+ cells. Preferably, T cells are enriched from PBMCs or from a blood sample by depletion of CD14+ cells and selection of CD3+ cells.

[0081] The method may further comprise the step of providing said cell sample from a subject.

[0082] As used herein, the term "subject" or "patient" relates to animals, preferably mammals, and more preferably humans.

[0083] As described below, the population of cells obtained by the method of the present invention can be used to provide adoptive cell therapy, particularly autologous therapy (by injecting cells derived from the Tscm cells back into the same patient) or allogeneic therapy (by injecting cells derived from the Tscm cells into another patient). Cell samples can thus be obtained from healthy subjects, particularly for allogeneic therapy, or from subjects with a disease to be treated with the adoptive cell therapy.

[0084] In particular, the subject may have an infection or cancer in which the specific memory T cell response is functionally impaired. Preferably, this impaired functionality comprises T cell anergy, in particular anergy of the T cell response to an antigen of interest, i.e. a tumor antigen or a pathogen antigen, and / or T cell exhaustion, characterized in particular by high levels of expression of inhibitory receptors such as PD-1 or TIGIT, and / or any other mechanism of negative regulation of T cell function. Thus, in a preferred embodiment, the subject has an infection or cancer and exhibits T cell anergy, in particular anergy of the T cell response to an antigen of interest, and / or T cell exhaustion, and / or any other mechanism of negative regulation of T cell function. In some particular embodiments, the subject has an infection or cancer and exhibits T cell anergy, in particular anergy of the T cell response to an antigen of interest, and / or T cell exhaustion.

[0085] Preferably, the subject has cancer or a disease caused by a pathogen as described below, more preferably a disease caused by a polyomavirus, even more preferably a disease caused by a human polyomavirus.

[0086] In a preferred embodiment, the subject has progressive multifocal leukoencephalitis (PML), Merkel cell carcinoma or BK virus-associated nephropathy, preferably progressive multifocal leukoencephalitis (PML) or Merkel cell carcinoma, more preferably progressive multifocal leukoencephalitis.

[0087] In step b) of the method of the present invention, the population of Tscm cells obtained in step a) is cultured in the presence of antigen-presenting cells loaded with at least one antigen of interest or at least one immunogenic peptide derived from at least one antigen of interest, preferably at least one immunogenic peptide derived from at least one antigen of interest.

[0088] As used herein, the terms "peptide" and "protein" are used interchangeably and refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming the chain.

[0089] The antigen presenting cells (APCs) used in this step can be any antigen presenting cells suitable for presenting at least one of said antigens of interest or at least one of said immunogenic peptides and activating T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC complexed with the peptide interacts with a TCR on the surface of the T cell. Examples of APCs include, but are not limited to, dendritic cells, monocytes, peripheral blood mononuclear cells (PBMCs), Epstein-Barr virus transformed B lymphoblastoid cell line cells (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 and PBMCs, and combinations thereof. More preferably, the APCs used in step b) are monocytes or dendritic cells, preferably monocytes.

[0090] The APCs used in step b) may be autologous (i.e. obtained from the same patient providing the cell sample, preferably from the subject being treated) or allogeneic (i.e. obtained from a subject other than the subject providing the cell sample, preferably from a subject other than the subject being treated).

[0091] In a preferred embodiment, the APC used in step b) is autologous.Those skilled in the art can use a wide range of known techniques to generate autologous APC using separate sources, such as peripheral blood monocytes, naturally occurring DCs, or CD34+ hematopoietic progenitor cells mobilized from bone marrow.Preferably, autologous APC is obtained from peripheral blood monocytes or naturally occurring DCs, more preferably from peripheral blood monocytes.

[0092] CD34+ stem cells can be differentiated into dendritic cells by incubating the cells with appropriate cytokines, as known in the art. For example, human CD34+ hematopoietic stem cells can be differentiated in vitro by culturing the cells 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 any other standard method.

[0093] In particular, the method may further comprise, prior to step b), the step of obtaining said autologous APCs from a cell sample from the subject and loading said autologous APCs with at least one antigen of interest or at least one immunogenic peptide derived from at least one antigen of interest, preferably with at least one immunogenic peptide derived from at least one antigen of interest. The cell sample used to obtain the autologous APCs may be the same as or different from the cell sample used in step a), but both are obtained from the same subject.

[0094] Preferably, the method further comprises, prior to step b), the step of sorting a population of monocyte cells from a cell sample from the subject using CD14+ positive selection and loading said monocytes with at least one antigen of interest or at least one immunogenic peptide derived from at least one antigen of interest, preferably with at least one immunogenic peptide derived from at least one antigen of interest. Preferably, the monocytes are obtained from a PBMC sample from the subject.

[0095] Alternatively, monocytes obtained from the sample, before or after antigen loading, for example using CD14+ positive selection, can be cultured in the presence of GM-CSF and IL-4 to induce differentiation into dendritic cells. Optionally, IL-6, IL-1β and TNF-α are added to the culture medium for about 24 hours between days 5 and 10 of culture, preferably on day 6, to induce optimal maturation of dendritic cells.

[0096] 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 APCs with one or more antigens of interest and / or one or more peptides derived from said antigen(s) of interest, in particular one or more immunogenic peptides, or by using viral vectors or mRNA transfection to deliver one or more antigens and / or one or more peptides derived from said antigen(s) of interest, in particular one or more immunogenic peptides, to APCs.

[0097] In a preferred embodiment, APCs are loaded with one or more peptides derived from said one or more antigens of interest, in particular one or more immunogenic peptides, preferably a pool of overlapping peptides derived from said one or more antigens of interest, in particular a pool of overlapping immunogenic peptides.

[0098] The antigen of interest may be any antigen that can be targeted by the immune system to provide a therapeutic effect. The antigen of interest may be easily selected by a person skilled in the art depending on the disease to be treated. In a preferred embodiment, the antigen of interest is selected depending on the disease, i.e., cancer or infection, to be treated in the subject providing the cell sample.

[0099] In particular, the antigen of interest may be selected from pathogen antigens, or antigens expressed by tumor cells, such as tumor-specific antigens (TSAs) (i.e., antigens that are found only on tumor cells and not on healthy cells) or tumor-associated antigens (TAAs) (i.e., antigens that are expressed at high levels on tumor cells, but at lower levels on healthy cells).

[0100] In one embodiment, the antigen of interest is selected from one or more cancer antigens. The term "cancer" or "tumor" as used herein refers to the presence of cells that possess typical characteristics of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid proliferation and proliferation rate, and certain characteristic morphological characteristics. This term refers to any type of malignant tumor (primary or metastatic), and refers to solid cancer or hematopoietic cancer.

[0101] In another embodiment, the antigen of interest is selected from one or more antigens of a pathogen, in particular a virus, a bacteria or a fungus.

[0102] In a preferred embodiment, the antigen of interest is selected from one or more viral antigens, preferably one or more antigens from human viruses. Preferably, the virus is selected from the group consisting of polyomavirus, human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes virus and papilloma virus. More preferably, the virus is selected from the group consisting of human polyomavirus, in particular polyomavirus John Cunningham (JC), BK virus (BKV) and Merkel cell polyomavirus (MCPyV or MCV).

[0103] In a particular embodiment, the antigen of interest is selected from the antigens of polyomavirus. For example, the peptides presented by APCs may comprise one or more polyomavirus peptides, in particular one or more immunogenic peptides, from VP1, VP2, VP3, large T, small T proteins and / or from any other protein of said polyomavirus. In particular, the peptides presented by APCs may be overlapping peptides that encompass one or several of these proteins. More particularly, the peptides presented by APCs, preferably immunogenic peptides, may comprise an overlapping peptide pool that encompasses the VP1, VP2 and VP3 regions of said polyomavirus.

[0104] In a more particular embodiment, the antigen of interest is selected from the antigens of polyomavirus MCPyV. For example, the peptides presented by APCs may comprise one or more MCPyV peptides, in particular one or more MCPyV immunogenic peptides, from VP1, VP2, VP3, large T, small T proteins and / or from any other protein of MCPyV. In particular, the peptides presented by APCs may be overlapping peptides that encompass one or several of these proteins. More particularly, the peptides presented by APCs, preferably immunogenic peptides, may comprise an overlapping peptide pool that encompasses the VP1, VP2 and VP3 regions of MCPyV.

[0105] In another more specific embodiment, the antigen of interest is selected from the antigens of polyomavirus BKV. For example, the peptides presented by APCs may comprise one or more BKV peptides, particularly one or more BKV immunogenic peptides, from VP1, VP2, VP3, large T, small T proteins and / or from any other protein of BKV. In particular, the peptides presented by APCs may be overlapping peptides that encompass one or several of these proteins. More particularly, the peptides presented by APCs, preferably immunogenic peptides, may comprise an overlapping peptide pool that encompasses the VP1, VP2 and VP3 regions of BKV.

[0106] In another more specific embodiment, the antigen of interest is selected from the antigens of polyomavirus JC. For example, the peptides presented by the APCs may comprise one or more JCV peptides, in particular one or more JCV immunogenic peptides, from the VP1, VP2, VP3, large T, small T proteins and / or from any other protein of JCV. In particular, the peptides presented by the APCs may be overlapping peptides encompassing one or several of these proteins. More particularly, the peptides presented by the APCs, preferably immunogenic peptides, may comprise an overlapping peptide pool encompassing the VP1, VP2 and VP3 regions of JCV.

[0107] The antigens used to load the APCs can be prepared by any method known to those skilled in the art depending on the nature of the antigen, for example, they can be prepared by chemical synthesis, recombinant expression, from a sample from the subject, in particular from the subject's own cancer cells, for example using whole tumor lysates, or from cancer cell line lysates.

[0108] In step b) of the method of the present invention, the Tscm cells are cultured in the presence of APCs as described above, thereby expanding and differentiating into a population comprising T cells reactive to a specific antigen or set of antigens.

[0109] Methods for obtaining antigen-specific T cells from a population of Tscm cells using APCs are well known in the art, and one of skill in the art may use any of these known methods.

[0110] Typically, the culture is performed in the presence of IL-15, IL-7 and / or other stimulatory cytokines, preferably recombinant cytokines such as IL-21. Preferably, the culture process includes culture supplementation with IL-15 and IL-7, and optionally IL-21. Said supplementation is preferably initiated 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.

[0111] At the beginning of the culture, the ratio of Tscm cells to APCs can be adjusted to be set at 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.

[0112] The culture of Tscm cells in the presence of APCs can continue for 8 to 20 days, preferably 10 to 18 days, more preferably 12 to 16 days. In a specific embodiment, the culture of Tscm cells in the presence of APCs continues for 14 days.

[0113] Optionally, the cells may be cultured for a longer period, preferably in the absence of antigen-loaded APCs. In particular, the cells may be cultured after step a) and before step b) in the absence of antigen-loaded APCs and / or after step b) and before step c) in the absence of antigen-loaded APCs.

[0114] In certain embodiments, where the subject is affected by a retrovirus, such as HIV, the culturing may be performed in the presence of one or several anti-retroviral compounds.

[0115] Optionally, the method of the invention may further comprise a step c) of recovering the cells obtained in step b), in particular the CD8+ and / or CD4+ cells, preferably the CD8+ and CD4+ cells.

[0116] In particular, in step c), the population of cells obtained in step b) may be sorted to select for CD8+ cells and optionally CD4+ cells.

[0117] In a particular embodiment, in step c) of the method of the invention, the population of cells obtained in step b) is sorted to select / recover CD8+ and CD4+ cells. CD8+ and CD4+ cells can be recovered separately or together. In some embodiments, CD8+ and CD4+ cells are recovered separately and preferably then subsequently mixed. This separation makes it possible to modulate the CD8+ / CD4+ ratio in the population of cells obtained.

[0118] In a preferred embodiment, in step c), the population of cells obtained in step b), i.e. all the cells of the culture, is recovered and comprises CD8+ and CD4+ cells. The recovered cells may also comprise other cell types, in particular APCs, such as monocytes or dendritic cells.

[0119] The cells may be recovered by any method known to those of skill in the art, including filtration or cell sorting methods described above.

[0120] In particular, the population selected / recovered in step c) may comprise Tscm cells (having a cell surface phenotype comprising CD45RA+ CCR7+), T effector (Teff) cells (having a cell surface phenotype comprising CD45RA+ CCR7-), T central memory (Tcm) cells (having a cell surface phenotype comprising CD45RA- CCR7+), and T effector memory (Tem) cells (having a cell surface phenotype comprising CD45RA- CCR7).

[0121] In a preferred embodiment, Tscm, Tcm and Tem cells comprise up to 90%, preferably 50%-90%, of the total cells in the selected / recovered population, allowing further cycles of differentiation in vivo and therefore long-term therapeutic effect.Typically, Teff cells may comprise 10%-50%, preferably 10%-20%, of the total cells in the selected / recovered population.

[0122] 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, obtained or obtainable by the method of the present invention for obtaining a population of cells comprising antigen-specific T cells. Preferably, the population comprises antigen-specific CD8+ T cells and antigen-specific CD4+ T cells.

[0123] All embodiments disclosed above and relating to methods for obtaining a population of cells comprising antigen-specific T cells are also encompassed in this aspect.

[0124] In particular, the population may include Tscm cells (having a cell surface phenotype comprising CD45RA+ CCR7+), T effector (Teff) cells (having a cell surface phenotype comprising CD45RA+ CCR7-), T central memory (Tcm) cells (having a cell surface phenotype comprising CD45RA- CCR7+), and T effector memory (Tem) cells (having a cell surface phenotype comprising CD45RA- CCR7).

[0125] Preferably, Tscm, Tcm and Tem cells comprise up to 90%, preferably 50% to 90%, of all cells in the isolated population of the present invention.Typically, Teff cells may comprise 10% to 50%, preferably 10% to 20%, of all cells in the isolated population of the present invention.

[0126] Preferably, antigen-specific CD8+ T cells make up 10% to 90% of all cells in the isolated population of the present invention, and antigen-specific CD4+ T cells make up 1% to 90% of all cells. In particular, antigen-specific CD8+ T cells may make up 50% to 90% of all cells in the isolated population of the present invention, and antigen-specific CD4+ T cells make up 1% to 50% of all cells in the isolated population of the present invention.

[0127] In a particular embodiment, the isolated population of cells comprising antigen-specific CD8+ T cells, and optionally antigen-specific CD4+ T cells, preferably comprising antigen-specific CD8+ T cells and antigen-specific CD4+ T cells, is a) sorting a population of Tscm cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+ from a cell sample from a subject suffering from cancer or a disease caused by a pathogen, in particular a cancer or a disease caused by a pathogen in which specific memory T cell responses are functionally impaired; b) culturing said population of Tscm cells in the presence of antigen-presenting cells, preferably autologous to the subject, loaded with at least one antigen of interest or at least one peptide derived from said at least one antigen of interest, in particular at least one immunogenic peptide, and optionally in the presence of IL-7 and IL-15 or other stimulatory cytokines, and optionally c) recovering the cells obtained in step b), in particular the CD8+ and / or CD4+ cells, preferably the CD8+ and CD4+ cells. The method is obtained or obtainable by a process comprising:

[0128] In step c), CD8+ cells, and optionally CD4+ cells, may be sorted from the population of cells obtained in step b).

[0129] The subject may be suffering from cancer, in which case the at least one antigen of interest may be selected from antigens expressed by tumor cells, such as tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs).

[0130] The subject may be suffering from a disease caused by a pathogen, in which case the at least one antigen of interest may be selected from antigens of the pathogen.

[0131] Preferably, the subject suffers from progressive multifocal leukoencephalitis (PML), Merkel cell carcinoma or BK virus-associated nephropathy. For subjects suffering from Merkel cell carcinoma, at least one of the antigens of interest may be selected from antigens of Merkel cell polyomavirus (MCPyV or MCV), in particular from VP1, VP2, VP3, large T, small T protein and / or from any other protein of MCPyV. For subjects suffering from BK virus-associated nephropathy, at least one of the antigens of interest may be selected from antigens of BK virus (BKV), in particular from VP1, VP2, VP3, large T, small T protein and / or from any other protein of BKV.

[0132] More preferably, the subject suffers from progressive multifocal leukoencephalitis (PML) and said at least one antigen of interest is selected from antigens of polyomavirus JC, preferably from VP1, VP2, VP3, large T, small T proteins and / or from any other protein of BKV. For example, peptides presented by APCs, preferably immunogenic peptides, may comprise an overlapping pool of peptides of JCV encompassing the VP1, VP2 and VP3 regions of JCV.

[0133] Preferably, the population of Tscm cells sorted in step a) has a cell surface phenotype that further comprises 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-, even more preferably PD1-, TIGIT-, LAG3- and TIM3-.

[0134] Preferably, the population of Tscm cells sorted in step a) has a cell surface phenotype further comprising CD3+, CD45RO-, CXCR3+ and / or CD122+, preferably CD3+ and CD45RO-.

[0135] In another aspect, the present invention relates to an in vitro method for obtaining a population of memory stem T cells (Tscm cells), i.e., a population of highly functional Tscm cells, comprising the step of sorting a population of Tscm cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1- and TIGIT- from a cell sample from a subject.

[0136] Preferably, the population of Tscm cells has a cell surface phenotype further comprising CD3+, CD45RO-, CXCR3+ and / or CD122+, preferably CD3+ and CD45RO-.

[0137] Optionally, the method may further comprise the step of depleting cells expressing one or several other inhibitory receptors, such as LAG3, TIM3, CTLA4 or CD160. In particular, the method may further comprise the step of depleting cells expressing LAG3, TIM3, CTLA4 and / or CD160, preferably LAG3 and / or TIM3, more preferably LAG3 and TIM3. In this case, the population of cells to be sorted may have a cell surface phenotype comprising: (i) CD4+ or CD8+, CD45RA+, CD95+, 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 population of cells to be sorted has a cell surface phenotype comprising: (i) CD4+ or CD8+, CD45RA+, CD95+, 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-.

[0138] Optionally, the method further comprises a step of amplifying the selected population of Tscm, which can be performed by any method known to those skilled in the art, for example, by culturing said Tscm cells in the presence of feeders, such as monocytes (unloaded monocytes), and suitable cytokines.

[0139] All embodiments disclosed above and relating to step a) of the method of the invention for obtaining a population of cells comprising antigen-specific T cells are also encompassed in this aspect.

[0140] In another aspect, the present invention relates to an in vitro method for obtaining a population of memory stem T cells (Tscm cells), i.e., a population of Tscm cells, comprising the step of sorting a population of Tscm cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+ from a cell sample from a subject, wherein the subject is suffering from cancer or a disease caused by a pathogen, particularly a cancer or a disease caused by a pathogen in which specific memory T cell responses are functionally impaired.

[0141] Preferably, the population of Tscm cells has a cell surface phenotype further comprising CD3+, CD45RO-, CXCR3+ and / or CD122+, preferably CD3+ and CD45RO-.

[0142] Preferably, the subject has a disease caused by the human polyomavirus.

[0143] More preferably, the subject has progressive multifocal leukoencephalitis (PML), Merkel cell carcinoma or BK virus associated nephropathy.

[0144] In a preferred embodiment, the subject has progressive multifocal leukoencephalitis (PML).

[0145] Optionally, the method may further comprise the step of depleting cells expressing one or several inhibitory receptors, such as PD1, TIGIT, LAG3, TIM3, CTLA4 or CD160.

[0146] In particular, the method may further comprise the step of depleting 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 population of cells to be sorted may have a cell surface phenotype comprising: (i) CD4+ or CD8+, CD45RA+, CD95+, 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 population of cells to be sorted has a cell surface phenotype comprising: (i) CD4+ or CD8+, CD45RA+, CD95+, 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-.

[0147] More preferably, the method may further comprise the step of depleting cells expressing PD1, TIGIT LAG3 and TIM3. In this case, the population of cells to be sorted may have a cell surface phenotype comprising: (i) CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, and (ii) PD1-, TIGIT-, LAG3- and TIM3-. Preferably, the population of cells to be sorted has a cell surface phenotype comprising: (i) CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, CD3+ and CD45RO-, and (ii) PD1-, TIGIT-, LAG3- and TIM3-.

[0148] Optionally, the method further comprises a step of amplifying the selected population of Tscm, which can be performed by any method known to those skilled in the art, for example, by culturing said Tscm cells in the presence of feeders, such as monocytes (unloaded monocytes), and suitable cytokines.

[0149] All embodiments disclosed above and relating to step a) of the method of the invention for obtaining a population of cells comprising antigen-specific T cells are also encompassed in this aspect.

[0150] In a further aspect, the present invention relates to an isolated population of Tscm cells obtained or obtainable by the method of the present invention for obtaining a population of Tscm cells.

[0151] This group: (i) CD4+, CD45RA+, CD95+, CCR7+, PD1- and TIGIT-; and / or (ii) CD8+, CD45RA+, CD95+, CCR7+, PD1- and TIGIT-; and / or (iii) CD4+, CD45RA+, CD95+, CD62L+, PD1- and TIGIT-; and / or (iv) CD8+, CD45RA+, CD95+, CD62L+, PD1- and TIGIT-; and / or (v) CD4+, CD45RA+, CD95+, CCR7+, CD62L+, PD1- and TIGIT-; and / or (vi) CD8+, CD45RA+, CD95+, CCR7+, CD62L+, PD1-, and TIGIT- The Tscm cells may include a Tscm cell having a cell surface phenotype comprising:

[0152] Preferably, these Tscm cells have a cell surface phenotype further comprising CD3+ and / or CD45RO-, preferably CD3+ and CD45RO-.

[0153] Optionally, these Tscm cells may have a cell surface phenotype further comprising LAG3-, TIM3-, CTLA4 and / or CD160, preferably LAG3- and / or TIM3-, more preferably LAG3- and TIM3-.

[0154] Preferably, the population of Tscm cells comprises at least 95%, 96%, 97%, 98% or 99% (of all cells) Tscm cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1- and TIGIT-, preferably CD4+ or CD8+, CD45RA+, CD95+, 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-.

[0155] More preferably, the population of Tscm cells comprises at least 95% or at least 99% (of all cells) Tscm cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1- and TIGIT-, preferably CD4+ or CD8+, CD45RA+, CD95+, 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-.

[0156] In a preferred embodiment, Tscm cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1+ and / or TIGIT+, and optionally LAG3+, TIM3+, CTLA4+ and / or CD160+, preferably LAG3+ and / or TIM3+-, comprise less than 5% of the total cells in the population, preferably less than 2% or 1% of the total cells in the population.

[0157] In certain embodiments, the population of Tscm cells consists of Tscm cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1- and TIGIT-, preferably CD4+ or CD8+, CD45RA+, CD95+, 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-.

[0158] All embodiments disclosed above and relating to step a) of the method of the invention for obtaining a population of cells comprising antigen-specific T cells or relating to the method of the invention for obtaining a population of Tscm cells are also encompassed in this aspect.

[0159] In a further aspect, the present invention also relates to a cell therapy medicament, - an isolated population of cells comprising antigen-specific CD8+ T cells and optionally antigen-specific CD4+ T cells of the invention (i.e. obtained or obtainable by a method of the invention for obtaining a population of cells comprising antigen-specific T cells), preferably an isolated population of cells comprising antigen-specific CD8+ T cells and antigen-specific CD4+ T cells of the invention, or - an isolated population of Tscm cells of the invention (i.e. obtained or obtainable by the method of the invention for obtaining a population of Tscm cells); Regarding.

[0160] The present invention also relates to said population for use in cell-based therapy.

[0161] All embodiments disclosed above relating to the population of cells comprising antigen-specific T cells, the method of the invention for obtaining an isolated population of cells comprising antigen-specific CD8+ T cells of the invention and optionally antigen-specific CD4+ T cells, the population of Tscm cells and the method of the invention for obtaining an isolated population of Tscm cells of the invention are also encompassed in this aspect.

[0162] In a further aspect, the present invention provides a method for producing a composition comprising the steps of: - an isolated population of cells comprising antigen-specific CD8+ T cells and optionally antigen-specific CD4+ T cells of the invention (i.e. obtained or obtainable by a method of the invention for obtaining a population of cells comprising antigen-specific T cells), preferably an isolated population of cells comprising antigen-specific CD8+ T cells and antigen-specific CD4+ T cells of the invention, or - an isolated population of Tscm cells of the invention (i.e. obtained or obtainable by the method of the invention for obtaining a population of Tscm cells); The present invention relates to a pharmaceutical composition comprising:

[0163] In a preferred embodiment, the pharmaceutical composition comprises an isolated population of cells comprising antigen-specific CD8+ T cells of the invention and optionally antigen-specific CD4+ T cells (i.e. obtained or obtainable by a method of the invention for obtaining a population of cells 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 of the invention.

[0164] Pharmaceutical compositions are formulated in pharma- ceutically acceptable carriers and / or excipients depending on the route of administration.

[0165] Preferably, the pharmaceutical composition is formulated so as to be suitable for use in a cell-based therapy in a subject in need thereof.

[0166] Pharmaceutical compositions may be formulated according to standard pharmaceutical practice known to those skilled in the art (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

[0167] Preferably, the pharmaceutical composition is suitable for parenteral administration, preferably intravenous injection.

[0168] Pharmaceutical compositions suitable for such administration may comprise a population of cells of the invention in combination with one or more pharma- ceutically acceptable sterile isotonic aqueous or non-aqueous solutions (e.g., balanced salt solutions (BSS)), dispersions, suspensions or emulsions, which may contain antioxidants, buffers, bacteriostats, solutes, or suspending or thickening agents, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately prior to use.

[0169] In some cases, the composition containing the cells can be frozen for storage at any temperature suitable for storing the cells. For example, the cells can be frozen at about -150°C or -196°C. The deep-frozen cells can be stored and prepared in a suitable container for storage that reduces the risk of cell damage and maximizes the chances that the cells will survive thawing.

[0170] The amount of cells to be administered can be determined by standard techniques known to those skilled in the art. The physiological data of the patient (e.g., age, size and weight) and the type and severity of the disease to be treated must be taken into account to determine the appropriate dosage.

[0171] The pharmaceutical composition of the present invention may be administered as a single dose or in multiple doses. Each unit dose may contain, for example, 10 5 ~7.10 8cells, preferably 7.10 6 ~7.10 8 The cell may contain cells.

[0172] The pharmaceutical compositions of the present invention may further comprise additional active compounds, for example therapeutic monoclonal antibodies for depleting lymphocyte subsets or for blocking receptors involved in immune function, such as anti-PD-1 or anti-TIGIT.

[0173] The present invention also relates to a pharmaceutical composition of the present invention for use in cell-based therapy in a subject in need thereof.The present invention also relates to a pharmaceutical composition of the present invention for use in the treatment of cancer or a disease caused by a pathogen.The present invention also relates to a method for treating a subject suffering from cancer or a disease caused by a pathogen, comprising administering to said subject a therapeutically effective amount of a pharmaceutical composition of the present invention.The present invention also relates to the use of a pharmaceutical composition of the present invention for preparing a medicament for treating cancer or a disease caused by a pathogen.

[0174] All embodiments disclosed above relating to the methods of the invention for obtaining a population of cells comprising antigen-specific T cells, an isolated population of cells comprising antigen-specific CD8+ T cells of the invention and optionally antigen-specific CD4+ T cells, a population of Tscm cells, an isolated population of Tscm cells of the invention and a pharmaceutical composition of the invention are also encompassed in this aspect.

[0175] As used herein, the terms "treatment," "treat," or "treating" refer to any action intended to improve the health status of a patient, such as the treatment, prevention, prophylaxis, and delay of disease. In certain embodiments, such terms refer to the amelioration or eradication 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 a disease.

[0176] The effective amount may be a therapeutically effective amount or a prophylactically effective amount. "Therapeutically effective amount" refers to an amount effective to achieve a desired therapeutic or prophylactic result at the required dosage and for the required period of time. In particular, this term refers to an amount of the pharmaceutical composition of the present invention administered to a patient sufficient to provide an immune response against the targeted pathogen or tumor cell. The therapeutically effective amount may vary depending on various factors such as the disease being treated, the physiological state of the subject being treated, the severity of the affliction, and the route of administration. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects are outweighed by the therapeutically beneficial effects. "Prophylactically effective amount" refers to an amount effective to achieve a desired prophylactic result at the required dosage and for the required period of time. Typically, but not necessarily, a prophylactic dose is used in subjects prior to or at an early stage of disease, so that the prophylactically effective amount may be less than the therapeutically effective amount. Suitable means and methods for determining a therapeutically effective amount or a prophylactically effective amount are available to those skilled in the art.

[0177] Preferably, the pharmaceutical composition is administered via a parenteral route, more preferably via intravenous infusion. In some embodiments, particularly for the treatment of localized diseases, administration can be targeted to deliver cells to the organ or tissue affected by the disease.

[0178] In a particular embodiment, the method of the present invention comprises administering to the subject cells 10 3 ~10 8 10 cells / kg body weight, preferably 10 cells 4 ~10 8 cells / kg body weight, more preferably 10 cells 5 ~10 7 The method includes administering to the patient an isolated population of cells of the invention, preferably an isolated population of cells comprising antigen-specific CD8+ T cells and optionally antigen-specific CD4+ T cells, at 100 cells / kg of body weight.

[0179] More particularly, the method of the present invention may comprise a step of administering a population of isolated cells comprising the antigen-specific CD8+ T cells of the present invention and optionally antigen-specific CD4+ T cells, in particular 1000-10,000,000 antigen-specific CD8+ T cells / kg body weight, preferably 5000-1,000,000 antigen-specific CD8+ T cells / kg body weight, more preferably 5000-100,000 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.

[0180] In some embodiments, the method of the present invention may include a step of administering a population of isolated cells including antigen-specific CD8+ T cells and antigen-specific CD4+ T cells of the present invention. Preferably, 1000 to 10,000,000 antigen-specific CD8+ T cells / kg body weight, preferably 5000 to 1,000,000 antigen-specific CD8+ T cells / kg body weight, more preferably 5000 to 100,000 antigen-specific CD8+ T cells / kg body weight, and 1000 to 10,000,000 antigen-specific CD4+ T cells / kg body weight of the subject, preferably 5000 to 1,000,000 antigen-specific CD4+ T cells / kg body weight of the subject, more preferably 5000 to 100,000 antigen-specific CD4+ T cells / kg body weight of the subject are administered. 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 and, optionally, by quantifying the CD4+ T cells.

[0181] The pharmaceutical composition may be administered as a bolus or repeatedly. The frequency of administration may be, for example, every two weeks, every month, every three months, or every six months.

[0182] The treatment can be an autologous therapy (by administering cells derived from a subject back to the same subject) or an allogeneic therapy (by administering cells derived from a subject to another subject). Preferably, the treatment is an autologous therapy.

[0183] As mentioned above, the subject to be treated, preferably a human, suffers from cancer or a disease caused by a pathogen.

[0184] The cancer or pathogen-caused disease to be treated may be any infection or cancer, particularly any infection or cancer in which the specific memory T cell response is functionally impaired.

[0185] A disease caused by a pathogen can be a viral, bacterial or fungal infection.

[0186] For treating diseases caused by pathogens, pharmaceutical compositions can include a population of isolated Tscm cells of the invention (i.e., obtained or obtainable by the methods of the invention for obtaining a population of Tscm cells), which are administered to increase the pool of Tscm cells and allow for in vivo activation of said cells by contacting them with APCs in vivo.

[0187] Preferably, for treating diseases caused by pathogens, the pharmaceutical composition comprises a population of isolated cells comprising antigen-specific CD8+ T cells and optionally antigen-specific CD4+ T cells of the present invention, preferably a population of isolated cells comprising antigen-specific CD8+ T cells and antigen-specific CD4+ T cells of the present invention. For treating diseases of this category, the population of cells comprising antigen-specific CD8+ T cells and optionally antigen-specific CD4+ T cells of the present invention is obtained by culturing Tscm cells in the presence of APCs loaded with at least one antigen of the pathogen to be targeted or at least one peptide derived from at least one said antigen, in particular an immunogenic peptide, thus obtaining a population of T cells that are activated to recognize target cells bearing at least one said antigen.

[0188] 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 polyomaviruses John Cunningham (JC), BK virus (BKV) and Merkel cell polyomavirus (MCPyV or MCV).

[0189] In certain embodiments, the pathogen is polyomavirus JC and the disease caused by the pathogen is progressive multifocal leukoencephalitis (PML).

[0190] In another specific embodiment, the pathogen is BK virus and the disease caused by the pathogen is BK virus associated nephropathy.

[0191] In another specific embodiment, the pathogen is Merkel cell polyomavirus and the disease caused by the pathogen is Merkel cell carcinoma.

[0192] 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), herpes virus and papilloma virus, and the disease caused by the pathogen is a chronic or acute viral infection.

[0193] 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, antiviral, or antiretroviral therapies.

[0194] The disease to be treated may also be a solid cancer or a hematopoietic cancer, whether or not associated with an oncogenic virus.

[0195] The term "cancer" or "tumor" as used herein refers to the presence of cells that possess typical characteristics of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid proliferation and proliferation rate, and certain characteristic morphological properties. The term refers to any type of malignant tumor (primary or metastatic).

[0196] Examples of solid cancers 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 ORL cancer, and childhood tumors (neuroblastoma, glioblastoma multiforme).

[0197] Examples of hematopoietic cancers include, but are not limited to, lymphoma, leukemia, myeloma, seminoma, Hodgkin's, and hematological malignancies.

[0198] For treating diseases of this category, the pharmaceutical composition may comprise a population of isolated cells comprising antigen-specific CD8+ T cells and optionally antigen-specific CD4+ T cells of the invention, obtained by culturing Tscm cells in the presence of APCs loaded with 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 at least one said antigen, in particular an immunogenic peptide, thus obtaining a population of T cells that are activated to recognize target cells bearing at least one said antigen.

[0199] Alternatively, and particularly for diseases for which there is no identified specific antigen, the pharmaceutical composition may comprise an isolated population of Tscm cells of the invention (i.e., obtained or obtainable by the methods of the invention for obtaining a population of Tscm cells), which are administered to expand the pool of Tscm cells and allow for in vivo activation of said cells by contacting them with APCs in vivo.

[0200] To treat cancer, the cell-based therapies of the present invention can be used alone or in combination with other treatments, such as chemotherapy, surgery and / or radiation therapy treatments.

[0201] In a particular embodiment, the disease to be treated is a disease caused by a pathogen, where the pathogen is polyomavirus JC and the disease is progressive multifocal leukoencephalitis (PML). In this embodiment, the population of cells comprising antigen-specific T cells administered is a) sorting a population of Tscm cells from a cell sample from a subject suffering from PML, the Tscm cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1- and TIGIT-, optionally LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-, more preferably LAG3- and TIM3-; b) culturing said population of Tscm cells in the presence of antigen-presenting cells loaded with at least one antigen of the polyomavirus JC or at least one peptide derived from said antigen, in particular an immunogenic peptide, and optionally in the presence of IL-7 and IL-15 or other stimulatory cytokines, and optionally c) recovering the cells obtained in step b), in particular the CD8+ and / or CD4+ cells, preferably the CD8+ and CD4+ cells. The method can be obtained by a method comprising the steps of:

[0202] Alternatively, the population of cells comprising antigen-specific T cells to be administered is a) sorting a population of Tscm cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+ from a cell sample from a subject suffering from PML; b) culturing said population of Tscm cells in the presence of antigen-presenting cells loaded with at least one antigen of the polyomavirus JC or at least one peptide derived from said antigen, in particular an immunogenic peptide, and optionally in the presence of IL-7 and IL-15 or other stimulatory cytokines, and optionally c) recovering the cells obtained in step b), in particular the CD8+ and / or CD4+ cells, preferably the CD8+ and CD4+ cells. The method can be obtained by a method comprising the steps of:

[0203] Preferably, in step a), the population of Tscm cells has a cell surface phenotype further comprising PD1-, TIGIT-, LAG3-, TIM3-, CTLA4- and / or CD160-, more preferably PD1-, TIGIT-, LAG3- and / or TIM3-.

[0204] Preferably, in step a), the population of Tscm cells has a cell surface phenotype further comprising CD3+ and / or CD45RO-, preferably CD3+ and CD45RO-.

[0205] In step c), CD8+ cells and optionally CD4+ cells can be sorted from the population of cells obtained in step b).

[0206] Preferably, the peptides presented by the APCs may comprise one or more JCV peptides, in particular one or more JCV immunogenic peptides, from the VP1, VP2, VP3, large T, small T proteins and / or from any other protein of JCV. In particular, the peptides presented by the APCs may be overlapping peptides encompassing one or several of these proteins. More particularly, the peptides presented by the APCs, preferably immunogenic peptides, may comprise an overlapping pool of peptides encompassing the VP1, VP2 and VP3 regions of JCV.

[0207] If the subject is additionally affected by a retrovirus such as HIV, the culturing in step b) may be performed in the presence of an anti-retroviral compound.

[0208] Aspects of the invention Various aspects and embodiments of the present invention are also described in clauses 1-15 listed below:

[0209] Clause 1. An in vitro method for obtaining a population of cells comprising antigen-specific T cells, comprising: a) sorting a population of Tscm cells from a cell sample from a subject, the Tscm cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1- and TIGIT-; b) culturing said population of Tscm cells in the presence of antigen-presenting cells loaded with at least one immunogenic peptide derived from at least one antigen of interest, and optionally in the presence of IL-7 and IL-15 or other stimulatory cytokines; and c) sorting CD8+ cells and optionally CD4+ cells from the population of cells obtained in step b). The method includes:

[0210] Clause 2. The method of clause 1, wherein the population of Tscm cells sorted in step a) has a cell surface phenotype further comprising LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-.

[0211] Clause 3. The method of clause 1 or 2, wherein the antigen presenting cell is a dendritic cell, a monocyte, a peripheral blood mononuclear cell (PBMC), an Epstein-Barr virus transformed B lymphoblastoid cell line cell (EBV-BLCL cell) or an artificial antigen presenting cell (AAPC).

[0212] Clause 4. The method of any one of clauses 1 to 3, wherein at least one said antigen of interest is a pathogen antigen, preferably a viral, bacterial or fungal antigen, or an antigen expressed by a tumor cell, such as a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA).

[0213] Clause 5. The method of any one of clauses 1 to 4, wherein at least one said antigen of interest is an antigen of a human polyomavirus, preferably an antigen selected from the group consisting of Polyomavirus JC, Polyomavirus MPCyV or Polyomavirus BK.

[0214] Clause 6. The method of any one of clauses 1 to 5, wherein at least one said antigen of interest is an antigen of Polyomavirus JC.

[0215] Clause 7. An isolated population of cells comprising antigen-specific CD8+ T cells and optionally antigen-specific CD4+ T cells, obtained or obtainable by a method according to any one of clauses 1 to 6.

[0216] Clause 8. The population of isolated cells of clause 7, comprising or consisting of Tscm cells, T effector (Teff) cells, T central memory (Tcm) cells, and T effector memory (Tem) cells.

[0217] Clause 9. The population of isolated cells according to clause 7 or 8 as a cell therapy medicament.

[0218] Clause 10. A pharmaceutical composition comprising the population of isolated cells of clause 9 and a pharma- ceutically acceptable carrier and / or excipient.

[0219] Clause 11. The population of isolated cells according to any one of clauses 7 to 9 or the pharmaceutical composition according to clause 10 for use in the treatment of cancer or a disease caused by a pathogen, preferably a disease caused by a human polyomavirus.

[0220] Clause 12. The population of isolated cells or pharmaceutical composition for use according to clause 11, wherein the pathogen is Polyomavirus JC and the disease is Progressive Multifocal Leukoencephalitis (PML).

[0221] Clause 13. The population of isolated cells or pharmaceutical composition for use according to clause 11 or 12, wherein said population is autologous to the subject to be treated.

[0222] Clause 14. An in vitro method for obtaining a population of memory stem T cells (Tscm cells), comprising the step of sorting from a cell sample from a subject a population of Tscm cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1- and TIGIT-, and optionally LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-.

[0223] Clause 15. A population of isolated Tscm cells having a cell surface phenotype comprising: (i) CD4+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1-, TIGIT-, and optionally LAG3- and / or TIM3-; and / or (ii) CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1-, TIGIT-, and optionally LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-.

[0224] All references cited in this description are incorporated by reference in this application. Other characteristics and advantages of the present invention will become more apparent in the following examples, given for purposes of illustration and not limitation. EXAMPLES

[0225] Materials and Methods PBMC isolation 100 mL of heparinized blood was used. Blood was obtained from PML patients with various immunological backgrounds, including HIV infection, hematological malignancies, and treatment with immunosuppressive biotherapy. Blood was diluted with NaCl 0.9% (v / v) and PBMCs were isolated by Ficoll density gradient centrifugation.

[0226] Inhibitory receptor phenotyping 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, 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 by means of FlowJo software.

[0227] Cell subsets were defined for both CD3+ CD4+ and CD3+ CD8+ T cells as follows: - Naive T cells: CD45RA+ CD45RO- CCR7+ CD95- - Stem Cell Memory (Tscm): CD45RA+ CD45RO- CCR7+ CD95+ - Central memory (Tcm): CD45RA- CD45RO+ CCR7+ CD95+ - Effector Memory (Tem): CD45RA- CD45RO+ CCR7- CD95+

[0228] PD1, TIGIT, LAG3, and TIM3 expression was analyzed in each CD4+ and CD8+ T cell subset.

[0229] Cell sorting T cell subset isolation PBMCs were washed in buffer (PBS 1X, EDTA 2mM SVF 0.5%). Monocytes were isolated by means of anti-CD14 coated magnetic beads. T cells were then isolated by means of antibody coated magnetic beads, which allow the depletion of non-CD3+ cells for the negative selection fraction.

[0230] T cells were then washed in PBS containing 0.5% SVF. The cell concentration was adjusted to 20 million cells per mL, and then the cells were stained with the following antibodies: anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, anti-CD62L, anti-CD95, anti-PD1, anti-TIGIT for 15 minutes at 4° C. The cells were washed and filtered through a 0.22 μm filter to remove cell clumps and processed for cell sorting.

[0231] Gating Strategy Cells were first gated on forward and side scatter, then on FSC-A and FSC-H to exclude doublets, and then gated as follows: CD4 Tscm negative for inhibitory receptors: CD3+ CD4+ CD8- CD45RA+ CD45RO- CCR7+ CD95+ PD1- TIGIT- CD4 Tscm positive for inhibitory receptors: CD3+ CD4+ CD8- CD45RA+ CD45RO- CCR7+ CD95+ and NOT (PD1- TIGIT-) CD8 Tscm negative for inhibitory receptors: CD3+ CD4- CD8+ CD45RA+ CD45RO- CCR7+ CD95+ PD1- TIGIT- CD8 Tscm positive for inhibitory receptors: CD3+ CD4- CD8+ CD45RA+ CD45RO- CCR7+ CD95+ and NOT (PD1- TIGIT-)

[0232] The cells were sorted onto SVF-coated tubes and then resuspended in culture medium.

[0233] cell culture Five million monocytes were seeded in 2 mL of culture medium per well. Overlapping 15-mer JCV peptides (final concentration 10 μg / mL for each pool) with an overlap of 11 amino acids spanning the entire sequence of VP1, VP2 and VP3 proteins were added for 2 h at 37° C. Monocytes were then washed twice in culture medium. Purified Tscm were centrifuged and resuspended in culture medium. Tscm were cultured with 5 million peptide-loaded monocytes in a total volume of 6 mL in a 24-well culture plate. This was considered as day 0. On day 2, rIL-7 and rIL-15 were added to the culture (final concentration 10 ng / mL each). 4 mL of medium was removed every 3 days and replaced with fresh medium containing 10 ng / mL of Il-7 and IL-15. On day 14, cells were counted.

[0234] CD4 and CD8 cell count Cells were stained with anti-CD3, anti-CD4 and anti-CD8. The percentage of CD4 and CD8 T cells in the live gate was analyzed. We calculated the number of CD4 and CD8 T cells in each well by multiplying the percentage of CD4 or CD8 T cells by the total number of cells contained in each well. Fold expansion was calculated by dividing the cell count at day 14 by the cell count at day 0.

[0235] Cytotoxicity assay CD14-depleted and CD3-depleted cells were thawed, washed, and incubated for 2 hours with JCV peptide pools spanning the VP1, VP2, and VP3 proteins (final concentration for each pool: 10 μg / mL). After washing, the cells were used as target cells to restimulate cultured Tscm at a ratio of 1 target cell / 10 cultured T cells. The cells were cultured overnight.

[0236] Cells were then washed and stained with anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, anti-CD45RO, anti-CCR7, anti-CD27, and anti-CD95. Cells were then washed and fixed for 20 minutes at +4°C. Cells were washed and permeabilized. Anti-granzyme B and anti-perforin antibodies were added for 30 minutes at +4°C, followed by a final wash and flow cytometric data acquisition.

[0237] analysis Results were analyzed using FlowJo software. Statistical analysis was performed and graphs were generated using GraphPad Prism software.

[0238] result 1- CD4 and CD8 Tscm express significantly fewer inhibitory receptors than Tcm or Tem We analyzed the expression of inhibitory receptors PD1, TIGIT, LAG3 and TIM3 by Tscm, Tcm and Tem subsets among CD4 and CD8 T cells from PML patients. We showed that in nine patients, CD4 and CD8 Tscm expressed fewer inhibitory receptors than Tcm or Tem cells (see FIG. 1 : white areas correspond to cells that do not express inhibitory receptors).

[0239] Among these four inhibitory receptors, PD1 and TIGIT were the most highly expressed (Fig. 2, black). The results suggest that PD1- and TIGIT-based negative selection may be able to deplete the majority of inhibitory receptor-expressing cells. Therefore, we isolated Tscm negative for both PD1 and TIGIT and analyzed their ability to proliferate after in vitro culture.

[0240] 2- Tscm negative for PD1 and TIGIT inhibitory receptors have better cell proliferation We established a gating strategy to purify PD1-TIGIT-Tscm cells (see Figure 3).

[0241] The sorted cells were cultured in the presence of JCV peptide-loaded autologous monocytes for 14 days in the presence of IL-7 and IL-15 to expand JCV-specific cells. We compared the fold expansion of cell numbers at the end of culture on day 14 (see FIG. 4). We compared the proliferation capacity of i) Tscm (see FIG. 4a) versus other memory cells (CD45RO positive cells including Tcm and Tems regardless of inhibitory receptor expression status), and ii) PD1 and TIGIT negative Tscm (see FIG. 4b) versus PD1 and / or TIGIT positive Tscm.

[0242] These data show that Tscm expand more efficiently than other (more differentiated) memory cells (Figure 4A), and that PD1-TIGIT-Tscm expand more efficiently than Tscm expressing PD1 and / or TIGIT (Figure 4B).

[0243] 3- Tscm negative for PD1 and TIGIT inhibitory receptors exhibit superior cytotoxic potential The inhibitory receptor-negative Tscm were then tested for cytotoxic properties after restimulation. At the end of the expansion phase (day 14), JCV peptide-loaded autologous cells were added to the cultures overnight. Intracellular perforin and granzyme B were stained. The percentage of granzyme B and perforin-expressing CD8 T cells among total CD8 T cells was determined (see FIG. 5). These data indicate that effector CD8 T cells derived from inhibitory receptor-negative Tscm exhibit higher cytotoxic potential than inhibitory receptor-expressing Tscm or other (more differentiated) memory CD8 T cells.

[0244] 4- CD8 T cells obtained from highly functional Tscm after in vitro culture retain high differentiation potential that can be used in vivo We identified PD1- and TIGIT-negative highly functional stem cell memory (T SCM ), or more differentiated memory T cells (T MEM The phenotype of CD8 T cells differentiated from either 100% IgG4- or IgG5-specific CD4+ T cells was analyzed based on the following combinations: Stem cell memory (Tscm): CD45RA+ CCR7+ Effector cells (Teff): CD45RA+ CCR7- Central memory (Tcm): CD45RA- CCR7+ Effector memory (Tem): CD45RA- CCR7-

[0245] We found that Tscm differentiate into Tcm, Tems and Teffs, but the majority retain the Tscm phenotype (see FIG. 6).

[0246] conclusion Taken together, these results demonstrated the following: i) In PML patients from various immunological backgrounds, Tscm cells express lower amounts of inhibitory receptors than more differentiated memory cells, most of which include PD-1 and TIGIT; ii) total Tscm expand better than conventional memory T cells (including Tcm and Tem); iii) selection based on PD1 and TIGIT exclusion allows depletion of the majority of Tscm expressing inhibitory receptors; iv) those PD1- TIGIT- Tscm exhibit greater proliferative potential compared to PD1+ and / or TIGIT+ Tscm and compared to more differentiated memory cells; v) PD1-TIGIT-Tscm exhibits superior cytotoxicity compared to PD1+ and / or TIGIT+ Tscm and compared to more differentiated memory cells; and vi) PD1-TIGIT-Tscm differentiate efficiently in vitro into more differentiated memory cells, including effector cells, but mostly retain the Tscm phenotype, which may enable further cycles of differentiation in vivo and therefore long-term therapeutic efficacy.

[0247] Together, these data indicate that PD1-TIGIT-Tscm is able to generate an effective and sustained immune response against JCV in PML patients.

Claims

1. 1. An in vitro method for obtaining a population of cells comprising antigen-specific T cells, comprising: a) sorting a population of Tscm cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CCR7+ and / or CD62L+, and CD95+ from a cell sample from a subject suffering from cancer or a disease caused by a pathogen; b) culturing said population of Tscm cells in the presence of antigen-presenting cells loaded with at least one antigen of interest or at least one peptide derived from at least one antigen of interest, and optionally in the presence of IL-7 and IL-15 or other stimulatory cytokines; and optionally c) recovering the cells obtained in step b), in particular the CD8+ and / or CD4+ cells, preferably the CD8+ and CD4+ cells. A method comprising:

2. 2. The method of claim 1, wherein the population of Tscm cells sorted in step a) has a cell surface phenotype further comprising PD1-, TIGIT-, LAG3-, TIM3-, CTLA4-, and / or CD160-, preferably PD1-, TIGIT-, LAG3-, and / or TIM3-.

3. 2. The method of claim 1, wherein the population of Tscm cells sorted in step a) has a cell surface phenotype comprising PD1- and TIGIT-, and optionally further comprising LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-, more preferably LAG3- and TIM3-.

4. 2. The method of claim 1, wherein the population of Tscm cells sorted in step a) has a cell surface phenotype further comprising CD3+, CD45RO-, CXCR3+ and / or CD122+, preferably CD3+ and CD45RO-.

5. 2. The method of claim 1, wherein the population of Tscm cells sorted in step a) comprises cells having a cell surface phenotype comprising CD4, CD8, CD45RA, CD95, CCR7, PD1, and TIGIT, and cells having a cell surface phenotype comprising CD4, CD8, CD45RA, CD95, CCR7, PD1, and TIGIT.

6. 2. The method of claim 1, wherein the population of Tscm cells sorted in step a) comprises cells having a cell surface phenotype comprising CD4, CD8, CD45RA, CD95, CCR7, PD1, TIGIT, LAG3, and TIM3, preferably CD3, CD45RO, CD4, CD8, CD45RA, CD95, CCR7, PD1, TIGIT, LAG3, and TIM3, and cells having a cell surface phenotype comprising CD4, CD8, CD45RA, CD95, CCR7, PD1, TIGIT, LAG3, and TIM3, preferably CD3, CD45RO, CD4, CD8, CD45RA, CD95, CCR7, PD1, TIGIT, LAG3, and TIM3.

7. The method of claim 1, wherein the antigen-presenting cells are dendritic cells, monocytes, peripheral blood mononuclear cells (PBMCs), Epstein-Barr virus-transformed B lymphoblastoid cell line cells (EBV-BLCL cells), or artificial antigen-presenting cells (AAPCs).

8. The method of claim 1, wherein the antigen-presenting cells are autologous to the subject.

9. 2. The method of claim 1, wherein the antigen-presenting cells are monocytes or dendritic cells, preferably autologous monocytes or dendritic cells for the subject.

10. 2. The method of claim 1, wherein at least one said antigen of interest is a pathogen antigen, preferably a viral, bacterial or fungal antigen, or an antigen expressed by a tumor cell, such as a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA).

11. 11. The method of any one of claims 1 to 10, wherein the subject is suffering from cancer.

12. 12. The method of claim 11, wherein at least one said antigen of interest is an antigen expressed by a tumor cell, such as a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA).

13. 11. The method of any one of claims 1 to 10, wherein the subject is suffering from a disease caused by a human polyomavirus.

14. 14. The method of claim 13, wherein at least one said antigen of interest is an antigen of human polyomavirus.

15. 14. The method of claim 13, wherein the subject is suffering from progressive multifocal leukoencephalitis, Merkel cell carcinoma, or BK virus-associated nephropathy.

16. 14. The method of claim 13, wherein the at least one antigen of interest is selected from the group consisting of Polyomavirus JC, Polyomavirus MPCyV, and Polyomavirus BK.

17. The method of any one of claims 1 to 10, wherein the subject is suffering from Merkel cell carcinoma.

18. 18. The method of claim 17, wherein at least one said antigen of interest is an antigen of the polyomavirus MPCyV.

19. 11. The method of any one of claims 1 to 10, wherein the subject is suffering from progressive multifocal leukoencephalitis.

20. 20. The method of claim 19, wherein at least one said antigen of interest is an antigen of Polyomavirus JC.

21. The method of claim 1, wherein in step b), the population of Tscm cells is cultured in the presence of IL-7 and IL-15.

22. 2. The method of claim 1, wherein in step b), the population of Tscm cells is cultured for 8 to 20 days, preferably 10 to 18 days, more preferably 12 to 16 days.

23. 10. The method of claim 1, 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 collection, tumor infiltrating lymphocytes, PBMCs, or a T cell-enriched population from a blood sample or PBMCs.

24. 2. A population of isolated cells comprising antigen-specific CD8+ T cells, and optionally antigen-specific CD4+ T cells, obtained or obtainable by the method of claim 1.

25. 25. The isolated population of cells of claim 24, comprising or consisting of Tscm cells, T effector (Teff) cells, T central memory (Tcm) cells, and T effector memory (Tem) cells.

26. 25. The population of isolated cells of claim 24, wherein in said population of isolated cells, Tscm, Tcm and Tem cells constitute up to 90%, preferably 50% to 90%, of the total cells, and Teff cells constitute 10% to 50%, preferably 10% to 20% of the total cells.

27. A composition for use as a cell therapy pharmaceutical, comprising a population of isolated cells according to any one of claims 24 to 26.

28. 27. A pharmaceutical composition comprising the population of isolated cells of any one of claims 24 to 26 and a pharmaceutically acceptable carrier and / or excipient.

29. 29. The pharmaceutical composition of claim 28 for use in the treatment of cancer or a disease caused by a pathogen.

30. 30. The pharmaceutical composition of claim 29 for use in the treatment of a disease caused by human polyomavirus.

31. 31. The pharmaceutical composition of claim 30, wherein the disease is progressive multifocal leukoencephalitis, Merkel cell carcinoma, or BK virus-associated nephropathy.

32. 31. The pharmaceutical composition of claim 30, wherein the pathogen is Polyomavirus JC and the disease is progressive multifocal leukoencephalitis.

33. 31. The pharmaceutical composition of claim 30, wherein the pathogen is polyomavirus MCPyV and the disease is Merkel cell carcinoma.

34. 30. The pharmaceutical composition of claim 29, wherein the population is autologous to the subject being treated.

35. 30. The pharmaceutical composition of claim 29, wherein the dose of the population of isolated cells or pharmaceutical composition administered comprises 1,000 to 10,000,000 antigen-specific CD8+ T cells per kg of body weight of the subject.

36. 36. The pharmaceutical composition of claim 35, wherein the dose of the population of isolated cells or pharmaceutical composition to be administered further comprises 1,000 to 10,000,000 antigen-specific CD4+ T cells per kg of body weight of the subject.

37. A pharmaceutical for treating cancer or a disease caused by a pathogen, comprising a population of isolated cells described in any one of claims 24 to 26.

38. 27. A composition for treating a subject suffering from cancer or a disease caused by a pathogen, the composition comprising a population of isolated cells according to any one of claims 24 to 26.

39. 1. An in vitro method for obtaining a population of memory stem T cells (Tscm cells), comprising the step of sorting, from a cell sample from a subject, a population of Tscm cells having a cell surface phenotype comprising CD4+ or CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1- and TIGIT-, and optionally LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-.

40. 40. The method of claim 39, wherein the subject is suffering from cancer or a disease caused by a pathogen.

41. 41. The method of claim 40, wherein the subject is suffering from a disease caused by a human polyomavirus.

42. 42. The method of claim 41, wherein the subject is suffering from progressive multifocal leukoencephalitis, Merkel cell carcinoma, or BK virus-associated nephropathy.

43. 43. The method of claim 42, wherein the subject is suffering from progressive multifocal leukoencephalitis.

44. 43. The method of claim 42, wherein the subject is suffering from Merkel cell carcinoma.

45. A population of isolated Tscm cells having a cell surface phenotype comprising: (i) CD4+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1-, TIGIT-, and optionally LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-; and / or (ii) CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1-, TIGIT-, and optionally LAG3-, TIM3-, CTLA4- and / or CD160-, preferably LAG3- and / or TIM3-.

46. 46. ​​The isolated population of Tscm cells of claim 45, having a cell surface phenotype comprising: (i) CD4+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1-, TIGIT-, LAG3-, and TIM3-; and / or (ii) CD8+, CD45RA+, CD95+, CCR7+ and / or CD62L+, PD1-, TIGIT-, LAG3-, and TIM3-.

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