WO2025210263A1

WO2025210263A1 - T cell immunotherapy of cutaneous diseases

Info

Publication number: WO2025210263A1
Application number: PCT/EP2025/059351
Authority: WO
Inventors: Yassine Taoufik; Marie-Ghislaine DE GOËR DE HERVE
Original assignee: Institut Gustave Roussy (IGR); Assistance Publique Hopitaux de Paris APHP; Institut National de la Sante et de la Recherche Medicale INSERM; Universite Paris Saclay
Current assignee: Institut Gustave Roussy (IGR); Assistance Publique Hopitaux de Paris APHP; Institut National de la Sante et de la Recherche Medicale INSERM; Universite Paris Saclay
Priority date: 2024-04-05
Filing date: 2025-04-04
Publication date: 2025-10-09
Anticipated expiration: 2026-10-05

Abstract

The present invention relates to a newly identified skin-tropic subset of Tscm that expresses the cutaneous homing marker CLA+. The identification of this new cell population of skin homing Tscm opens up new cell therapy strategies to treat cutaneous diseases, in particular cutaneous cancers such as Merkel cell carcinoma.

Description

T CELL IMMUNOTHERAPY OF CUTANEOUS DISEASES

FIELD OF THE INVENTION

The present invention relates to the field of medicine, in particular to the treatment of cutaneous diseases such as Merkel cell carcinoma, using a T cell immunotherapy.

BACKGROUND OF THE INVENTION

Merkel cell carcinoma (MCC) is a rare, highly aggressive cutaneous carcinoma of neuroendocrine origin, often already metastatic at diagnosis. Its incidence has been steadily increasing since the 1990s, linked to increased exposure to the sun (in the United States, 0.44 and 0.66 cases per 100,000 in 2010 and 2016, respectively; in Australia, 1.6 and 2.5 cases per 100,000 in 1993 and 2010, respectively). The annual incidence in 2022 is around 7000 new cases for the United States and Europe and 5200 cases for Australia. Overall survival at five years is less than 40%, and at the metastatic stage, less than 14%.

Around 80% of MCCs are directly linked to the human polyomavirus MCPyV with the expression of viral proteins in tumor cells (Feng et al., Science. 2008 Feb 22;319(5866):1096-100). MCPyV is a ubiquitous virus with a seroprevalence of around 70% in adults (Kean et al. PLoS Pathog. 2009 Mar;5(3):el000363). It infects a wide range of cell types and can transform neuroendocrine cells in the skin. The risk factors are age (over 65), fair skin, a history of significant sun exposure, and immunosuppression (HIV infection, hematological malignancies, transplantation).

The MCPyV genome includes an early region coding for two potentially oncogenic proteins, LTA and STA (Large and Small T antigens). There is no viral production in the MCC cells but an intracellular accumulation of LTA and STA proteins (Liu et al. Cell Host Microbe. 2016 Jun 8;19(6):775-87.). The discrepancy between the high seroprevalence of the virus and the rare incidence of Merkel's carcinoma is possibly related to the need for a cascade of rare molecular events, including viral integration into the cellular genome and mutations resulting in a premature stop codon leading to a truncated form of the LT antigen (Harms et al. Nat Rev Clin Oncol. 2018 Dec;15(12):763-776). This truncated form does not allow viral production (Pastrana et al. PLoS Pathog. 2009 Sep;5(9):el000578). Exposure to UV promotes the appearance of mutations in the viral genome (Shuda et al. Proc Natl Acad Sci U S A. 2008 Oct 21;105(42):16272- 7.). The truncated form of LTA binds, among other things, with high affinity to the Rb protein and inhibits its activation. Inhibition of Rb protein promotes cell proliferation. The immunogenicity of MCPyV+ MCCs is linked to the expression of LT and ST proteins whose peptides are presented in MHC class I restriction, resulting in tumor infiltration by CD8 T cells directed against these viral epitopes (Jing et al. Cancer Immunol Res. 2020 May;8(5):648-659).

The first-line treatment combines surgical excision and radiotherapy, and possibly chemotherapy. The relapse rate is high, and drug resistance rapidly develops. The expression of inhibitory receptors by anti-tumor T cells (PD-1, CTLA-4) has motivated the use of immune checkpoint inhibitors (ICI) (Pembrolizumab, Nivolumab, Avelumab, Ipilimumab) in order to improve the effectiveness of anti-tumor lymphocytes. First-line use of ICI is associated with improved survival in 50% of patients. However, 50% of MCCs do not respond to ICI treatments. Therefore, there is a strong need for new therapeutic approaches.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to an in vitro method for obtaining a population of cells comprising skin homing antigen-specific T cells comprising a) sorting from a cell sample from a subject, a population of T cells having a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+ and (iv) CLA+, and b) culturing said population of T 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 cells obtained in step b), in particular CD8+ and/or CD4+ cells, preferably CD8+ and CD4+ cells.

The population of T cells sorted in step a) may have a cell surface phenotype further comprising CD95+.

The antigen-presenting cells may 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).

Said at least one antigen of interest may be an antigen of a cutaneous pathogen, preferably a viral, bacterial or fungal antigen, or an antigen expressed by cutaneous tumor cells such as tumor-specific antigens (TSA) or tumor-associated antigens (TAA). The subject may be suffering from a cutaneous cancer or cutaneous pathogen-caused disease, preferably from a cutaneous cancer, in particular a cutaneous cancer selected from the group consisting of Merkel cell carcinoma, melanoma, squamous cell carcinoma, basal cell carcinoma, angiosarcoma, cutaneous B-cell lymphoma, cutaneous T-cell lymphoma, dermatofibrosarcoma protuberans and sebaceous carcinoma. More preferably, the subject is suffering from Merkel cell carcinoma and said at least one antigen of interest is an antigen of the polyomavirus MCPyV.

The cell sample may 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 population enriched in T cells from a blood sample or PBMCs.

In a second 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 invention. Preferably, said population comprises memory stem T (Tscm) cells having a cell surface phenotype comprising (i) CD45RA+ (ii) CCR7+ and/or CD62L+, and (iii) CLA+; T effector (Teff) cells having a cell surface phenotype comprising (i) CD45RA+ (ii) CCR7- and/or CD62L-, and (iii) CLA+; T central memory (Tern) cells having a cell surface phenotype comprising (i) CD45RA- (ii) CCR7+ and/or CD62L+, and (iii) CLA+; and T effector memory (Tern) cells having a cell surface phenotype comprising (i) CD45RA- (ii) CCR7- and/or CD62L-, and (iii) CLA+. Preferably, CLA+ cells represent more than 90 % of the total cells of said population. Preferably, Tscm, Tern and Tern cells represent from 50% to 90% of the total cells and Teff cells represent from 10% to 50% of the total cells of said population.

In a third aspect, the present invention also relates to an in vitro method for obtaining a population of skin homing memory stem T-cells (Tscm cells), said method comprising sorting from a cell sample from a subject a population of cells having a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+ and (v) CLA+.

In a further aspect, the present invention relates to an isolated population of cells comprising CLA+ Tscm, wherein CLA+ Tscm cells represent more than 30% of the total cells in said population.

In another aspect, the present invention relates to a method for preparing a pharmaceutical composition, said method comprising obtaining a population of skin homing Tscm cells by the method of the invention or a population of cells comprising antigen-specific CD8+ T cells, and optionally antigen-specific CD4+ T cells, by the method of the invention, and optionally mixing said population of cells with CLA- cells, in particular CLA- Tscm cells and/or CLA- T effector (Teff) cells and/or CLA- T central memory (Tern) cells and/or CLA- T effector memory (Tern) cells.

Alternatively, the method may comprise a) obtaining a population of skin homing Tscm cells by the method of the invention or providing an isolated population of cells comprising CLA+ Tscm of the invention, or b) obtaining a population of cells comprising antigen-specific CD8+ T cells, and optionally antigen-specific CD4+ T cells, by the method of the invention, or providing 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 invention, and, after a) or b), optionally mixing said population of cells with CLA- cells, in particular CLA- Tscm cells and/or CLA- T effector (Teff) cells and/or CLA- T central memory (Tern) cells and/or CLA- T effector memory (Tern) cells.

It also relates to a pharmaceutical composition obtained or obtainable by said method.

In another aspect, the present invention relates (i) an isolated population of cells comprising antigen-specific CD8+ T cells, and optionally antigen-specific CD4+ T cells, of the invention, (ii) an isolated population of cells comprising CLA+ Tscm of the invention or (ii) a pharmaceutical composition of the invention, for use as a cell therapy medicament, in particular in the treatment of a cutaneous cancer or a cutaneous pathogen-caused disease. The population of cells may be used in combination with CLA- cells, in particular CLA- Tscm cells and/or CLA- T effector (Teff) cells and/or CLA- T central memory (Tern) cells and/or CLA- T effector memory (Tern) cells. Preferably, the isolated population or pharmaceutical composition is for use in the treatment of a cutaneous cancer, more preferably selected from the group consisting of Merkel cell carcinoma, melanoma, squamous cell carcinoma, basal cell carcinoma, angiosarcoma, cutaneous B-cell lymphoma, cutaneous T-cell lymphoma, dermatofibrosarcoma protuberans, sebaceous carcinoma and Kaposi's sarcoma, event more preferably being Merkel cell carcinoma.

Preferably, the population of cells is autologous to the subject to be treated. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Gating strategy for isolation of a population of CLA+ Tscm. After CD3+ CD4+ (upper panel) or CD3+ CD8+ (lower panel) gating, CLA expression was analyzed in CD45RO- CD45RA+ CCR7+ CD95+ cells. CLA+ staining is also shown in CD45RO+ CD45RA- cells.

Figure 2 : percentages of CLA+ cells in the CD4+ and CD8+ Tscm subsets. Data are median, 25^e and 75^e percentiles.

Figure 3: CLA+ cells within the CD45RA+ CD62L+ CLA+ are mainly Tscm cells. Boxes represent median, 25th and 75th percentiles, whiskers represent 10th and 90th percentiles.

Figure 4 : CLA expression was determined by flow cytometry in Tscm, naive T cells, Tern, Tern and Teff from a series of 24 healthy donors. Results are expressed as the percentage of positive cells.

Figure 5 : CLA+ CD4 and CD8 Tscm express higher levels of CCR4 and CCR10. CCR4 and CCR10 expression was examined in T cells from healthy controls. The results of CCR4 and CCR10 are expressed in mean fluorescence intensity (MFI). The statistical significance was calculated by the Wilcoxon test (* p< 0.05; ** p< 0.01) n=24. TN: naive T cells ; TSCM: stem-cell-like memory T cells.

Figure 6 : Tscm CLA+ robustly expand after culture in the presence of MCPyV peptides: highly purified CLA+ Tscm, CLA- Tscm, CLA+ CD45RO+ T cells and/or CLA- CD45RO+ T cells (CD45RO+ T cells corresponding to conventional memory T cells that include central memory T and effector memory T cells) were sorted and cultured in vitro with autologous CD14+ monocytes loaded with a MCPyV peptides pool covering ST and LT antigens sequences, in the presence of IL-7 and IL-15 in Grex 24-well plates for 14 days. Cell expansion was determined on day 14. Results represent the fold expansion (live T cells number at day 14 / live T cells seeded per well on day 1). The statistical significance was calculated by the Wilcoxon test (** p< 0.01).

Figure 7 : Tscm CLA+ and CLA- differentiate into other memory subsets, and maintain their initial phenotype. Highly purified CLA+ and CLA- Tscm, and CLA+ and CLA- CD45RO+ memory T cells (that include Tern and Tern) were sorted and cultured in vitro with autologous CD14+ monocytes loaded with MCPyV peptide pools (covering large and small T antigens of MCPyV), in the presence of IL-7 and IL-15 in Grex 24-well plates for 14 days. Cells were analyzed by flow cytometry and the proportions of Tscm, Tern, Tern and terminally differentiated effectors (Teff) were determined. DETAILED DESCRIPTION OF THE INVENTION

In International patent application WO 2023/073062, the inventors described a new autologous and specific T-cell therapy strategy to bypass T-cell functional inhibition in patients with a medical condition of immunosuppression. The approach was based on the use of memory stem T-cells (Tscm). Indeed, they observed that, in patients with severe and prolonged immunosuppression, this rare memory T cell subset may maintain high functionality in terms of expansion and differentiation, and may generate ex vivo effective specific cytotoxic effectors against viral or tumoral antigens.

In the unpublished European patent application n° 23 170212.7, the inventors also demonstrated that similar results can be obtained using a population comprising Tscm and naive T cells instead of a population comprising only Tscm thereby simplifying the protocol to select the initial population of the method and thus improving its feasibility on a large clinical scale. Indeed, they observed that when culturing Tscm and naive T cells with antigen-peptides coated on autologous PBMCs, only memory cells, i.e. Tscm, were activated thereby leading to selective amplification and differentiation of Tscm cells.

In the present application, the inventors have now identified, for the first time, a new skintropic subset of Tscm that expresses the cutaneous homing marker, i.e. CLA (Cutaneous Lymphocyte-associated Antigen). The identification of this new cell population of skin homing Tscm opens up new cell therapy strategies, in particular to treat the cutaneous diseases such as MCC.

In a first aspect, the present invention relates to an in vitro method for obtaining a population of cells comprising skin homing antigen-specific T cells comprising a) sorting from a cell sample from a subject a population of skin homing Tscm cells, and b) culturing said population of T 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 cells obtained in step b), in particular CD8+ and/or CD4+ cells, preferably CD8+ and CD4+ cells.

In particular, in step c), CD8+ cells, and optionally CD4+ cells, may be sorted from the population of cells obtained in step b). The population of skin homing Tscm cells is a population of Tscm cells exhibiting a skin homing receptor selected from the group consisting of CLA, CCR4 or CCR10, preferably the skin homing receptor CLA. Thus, the population of skin homing Tscm cells is a population of T cells having a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+, preferably CD62L+, and (iv) CLA+ and/or CCR4+ and/or CCR10+, preferably CLA+.

In preferred embodiment, the population of skin homing Tscm cells sorted in step a) is a population of T cells having a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+, preferably CD62L+, and (iv) CLA+.

Preferably, in step a), the population of T cells sorted in step a) has a cell surface phenotype further comprising CD95+. Thus, in a particular embodiment, step a) comprises sorting from a cell sample from a subject a population of T cells having a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+, preferably CD62L+, (iv) CLA+ and/or CCR4+ and/or CCR10+, preferably CLA+, and (v) CD95+.

Optionally, step a) may further comprise depleting cells expressing one or several other inhibitory receptors such as PD1, TIGIT, LAG3, TIM3, CTLA4 and CD160. In particular, step a) may further comprise depleting cells expressing PD1, TIGIT, LAG3, TIM3, CTLA4, and/or CD160, preferably expressing PD1 and/or TIGIT, more preferably expressing PD1. In this case, the population of sorted cells may have a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+, preferably CD62L+, (iv) CLA+ and/or CCR4+ and/or CCR10+, preferably CLA+, and (v) PD1-, TIGIT-, LAG3-, TIM3-, CTLA4- and/or CD160-, preferably PD1- and/orTIGIT-, more preferably PD1-, and optionally (vi) CD95+. Thus, in a particular embodiment, step a) comprises sorting from a cell sample from a subject a population of T cells having a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+, preferably CD62L+, (iv) CLA+ and/or CCR4+ and/or CCR10+, preferably CLA+, and, (v) PD1-, TIG IT- , LAG3-, TIM3-, CTLA4- and/or CD160-, preferably PD1- and/or TIGIT-, more preferably PD1-, and optionally (vi) CD95+.

Optionally, step a) may further comprise depleting cells expressing CD45RO. In this case, the population of sorted cells may have a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+, preferably CD62L+, (iv) CLA+ and/or CCR4+ and/or CCR10+, preferably CLA+, and (v) CD45RO-. Thus, in a particular embodiment, step a) comprises sorting from a cell sample from a subject a population of T cells having a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+, preferably CD62L+, (iv) CLA+ and/or CCR4+ and/or CCR10+, preferably CLA+, and, (v) CD45RO-, and optionally CD95+, PD1-, TIGIT-, LAG3-, TIM3-, CTLA4- and/or CD160-, preferably CD95+.

Optionally, step a) may further comprise selecting cells expressing CD3. In this case, the population of sorted cells may have a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+, preferably CD62L+, (iv) CLA+ and/or CCR4+ and/or CCR10+, preferably CLA+, and, (v) CD3+. Thus, in a particular embodiment, step a) comprises sorting from a cell sample from a subject a population of T cells having a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+, preferably CD62L+, (iv) CLA+ and/or CCR4+ and/or CCR10+, preferably CLA+, and, (v) CD3+, and optionally CD95+, CD45RO-, PD1-, TIGIT-, LAG3-, TIM3-, CTLA4- and/or CD160-, preferably CD95+ and CD45RO-, more preferably CD95+.

As used herein, the term "CLA" or "cutaneous lymphocyte-associated antigen" refers to a fucose-containing carbohydrate that can decorate P-selectin glycoprotein ligand-1 on T cells (Fuhlbrigge et al. Nature 1997. 389:978-981). This carbohydrate epitope is recognized by the monoclonal antibody HECA-452 (Duijavestijn et al., Am J Pathol, 1988, vol. 130, 147-55) and acts as a ligand for E-selectin.

As used herein, the term "CCR4" or "C-C chemokine receptor type 4" refers to a cell surface protein, also designated CD194, and belonging to the G protein-coupled receptor family. CCR4 is a receptor for the following CC chemokines: CCL2, CCL4, CCL5, CCL17 and CCL22. In humans, the CCR4 protein is encoded by the CCR4 gene.

As used herein, the term "CCR10" or "C-C chemokine receptor type 10" refers to a cell surface protein belonging to the G protein-coupled receptor family. CCR10 is a receptor for the CC chemokines CCL27 and CCL28. In humans, the CCR10 protein is encoded by the CCR10 gene.

As used herein, the term "CD4" refers to 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.

As used herein, the term "CD8" refers to a transmembrane glycoprotein that serves as a co-receptor 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-a chain encoded, in humans, by the CD8A gene.

As used herein, the term "CD3" refers to a protein complex and T cell co-receptor. In mammals, the complex contains a CD3y chain, a CD36 chain, and two CD3E chains. The CD3 is part of a bigger complex which includes the T Cell Receptor (TCR). CD3 complex associated with the TCR is involved in the recognition of peptides bound to the major histocompatibility complex class I and II during the immune response. As used herein, the term "CD3" refers to the CD3y chain encoded, in humans, by the CD3G gene, to the CD36 chain encoded, in humans, by the CD3D gene or the CD3E chain encoded, in humans, by the CD3E gene.

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

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

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.

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

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 adherence of lymphocytes to endothelial cells of high endothelial venules in peripheral lymph nodes In humans, the CD62L is encoded by the SELL gene.

As used herein, the term "PD1" refers to Programmed cel ID 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.

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

As used herein, the term "LAG3" refers to Lymphocyte-activation gene 3 also known as CD223. LAG3 is a cell surface molecule with diverse biologic effects on T cell function. In humans, the LAG3 protein is encoded by the LAG3 gene. As used herein, the term "TIM3" refers to T cell immunoglobulin and mucin domaincontaining 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.

As used herein, the term "CTLA4" refers to cytotoxic T-lymphocyte-associated protein 4 also known as CD152. CTLA4 is protein receptor that functions as an immune checkpoint and downregulates immune responses. In humans, the CTLA4 protein is encoded by the CTLA4 gene.

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

As used herein, the term "cell surface phenotype" refers to the presence or absence of a combination of specific cell surface markers at the surface of the cells. By "cell surface marker" is intended a molecule expressed on the surface of a cell that can be detected, for example, using labeled antibodies or other means known in the art. A cell surface marker can comprise a protein, glycoprotein, or carbohydrate, or group of proteins and/or glycoproteins and/or carbohydrates. In the present case, the population of skin homing Tscm cells sorted/selected in step a) may be identified by expression of a particular combination of markers comprising (i) CD4 or CD8, (ii) CD45RA, (iii) CCR7 and/or CD62L, preferably CD62L, and (iv) CLA, CCR4 and/or CCR10, preferably CLA, and optionally CD3 and/or CD95. Optionally, this population may be further identified by the lack of expression of a particular combination of markers comprising CD45RO, PD1, TIGIT, LAG3, TIM3, CTLA4, CD160, preferably CD45RO.

The population of T cells obtained in step a) is enriched in T cells having a particular cell surface phenotype. In particular, the population of T cells obtained in step a) may be enriched in T cells having a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+, preferably CD62L+, and (iv) CLA+ and/or CCR4+ and/or CCR10+, preferably CLA+, and optionally CD3+, CD95+, CD45RO-, PD1-, TIGIT-, LAG3-, TIM3-, CTLA4- and/or CD160-, preferably CD3+, CD95+ and CD45RO-, more preferably CD3+ and CD95+.

By "enriched" is meant a composition comprising cells present in a greater percentage of total cells than is found in another composition. In particular, in the population obtained in step a), T cells having a particular cell surface phenotype as defined above are present in a higher percentage of total cells as compared to their percentage in the cell sample. In the population obtained in step a), T cells having said particular cell surface phenotype, e.g. a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+, preferably CD62L+, and (iv) CLA+ and/or CCR4+ and/or CCR10+, preferably CLA+, and optionally CD3+, CD95+, CD45RO-, PD1-, TIG IT-, LAG3-, TIM3-, CTLA4- and/or CD160-, preferably CD3+, CD95+ and CD45RO-, more preferably CD3+ and CD95+, represent more than 70%, preferably represent more than 80%, 90%, 95%, 96%, 97%, 98% or 99% of the total cells in said population, even more preferably represent more than 95%, 96%, 97%, 98% or 99% of the total cells in said population. Preferably, in the population obtained in step a), T cells having a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+, preferably CD62L+,and (iv) CLA+, and optionally CD95+, represent more than 70%, preferably represent more than 80%, 90%, 95%, 96%, 97%, 98% or 99% of the total cells in said population, even more preferably represent more than 95%, 96%, 97%, 98% or 99% of the total cells in said population.

Conversely, the population of T cells obtained in step a) may be depleted in cells which do not express any skin homing receptor, preferably CLA, CCR4 or CCR10. By "depleted" is meant a composition comprising cells present in a lower percentage of total cells than is found in another composition, in particular than is found in the cell sample. In preferred embodiments, in the population obtained in step a), T cells having a cell surface phenotype comprising CLA-, CCR4- or CCR10-, preferably CLA-, are present in a lower percentage of total cells as compared to their percentage in the cell sample. In particular, in the population obtained in step a), T cells which do not express any skin homing receptor, preferably which do not express CLA, CCR4 or CCR10, more preferably which do not express CLA, may represent less than 5%, 2% or 1% of the total cells in said population.

In a particular embodiment, the population obtained in step a) consists of T cells having a cell surface phenotype as defined above, preferably a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+, preferably CD62L+, and (iv) CLA+, CCR4+ and/or CCR10+, preferably CLA+, and optionally CD3+, CD95+, CD45RO-, PD1-, TIGIT-, LAG3-, TIM3-, CTLA4- and/or CD160-, preferably CD95+ and CD45RO-, more preferably CD95+.

In another particular embodiment, the population obtained in step a) consists of T cells having a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+, preferably CCR7+, (iv) CLA+, and (v) CD95+.

Preferably, the population obtained in step a) exhibits a ratio CD4+ / CD8+ of at least 0.2.

In embodiments wherein the population obtained in step a) exhibits a ratio CD4+ / CD8+ lower than 0.2, anti-CD40 antibodies may be added to the population in order to compensate for the lack of CD4 T cell helping signals.

In step a), cells having a specific cell surface phenotype are sorted and recovered from a cell sample. Sorting of the cells having a specific cell surface phenotype may be carried out using any method known in the art. Positive and/or negative selection can be readily accomplished using materials and techniques known in the art. For example, cells expressing a particular cell surface marker(s) can be separated from other cells using monoclonal antibodies that bind to the marker and are coupled to columns or magnetic beads; the separation is readily performed according to standard techniques and/or manufacturer or provider directions. In particular, in step a), cells may be sorted by fluorescence-activated cell sorting (FACS) or by magnetic separation.

The cell sample may be any sample containing T cells and in particular Tscm cells, or cells that can be induced in culture to become Tscm cells. Preferably, the sample is a sample containing Tscm cells. Examples of suitable samples include, but are not limited to, a bone marrow cell sample, a blood cell sample, a fractionated or unfractionated whole blood sample, a fractionated or unfractionated apheresis collection (e.g., a leukapheresis collection), tumor infiltrating lymphocytes, PBMCs, or a T cell population (e.g., a population enriched in T cells from a blood sample or PBMCs).

In a particular embodiment, the cell sample is PBMCs. PBMCs can be isolated from a blood sample by any method known in the art such as by ficoll density gradient centrifugation.

As used herein, the term "isolated" means separated from constituents with which the cells are normally associated with in nature.

In another particular embodiment, the cell sample is a population enriched in T cells from PBMCs or from a blood sample, preferably a population enriched in 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 selection of CD3+ cells.

The method may further comprise providing said cell sample from the subject.

As used herein, the term "subject" or "patient" relates to an animal, preferably a mammal, more preferably a human being. As described below, the population of cells obtained by the method of the invention may be used to provide adoptive cell therapy, in particular autologous therapy (by infusing cells derived from said T cells back into the same patient) or allogeneic therapy (by infusing cells derived from said T cells into another patient). The cell sample may be thus obtained from a healthy subject, in particular for allogeneic therapy, or from a subject having a disease to be treated with said adoptive cell therapy.

In particular, the subject may have an infection or a cancer for which the specific memory T cell responses are functionally impaired. Preferably, this impaired functionality involves a T cell anergy, in particular an anergy of T cell responses against an antigen of interest, i.e. a tumoral or pathogen antigen, and/orT cell exhaustion characterized in particular by high levels of expression of inhibitory receptors such as PD-1 or TIGIT, and/or any other mechanisms of T-cell functional negative regulation. Thus, in preferred embodiments, the subject has an infection or a cancer and exhibits a T cell anergy, in particular an anergy of T cell responses against an antigen of interest, and/or T cell exhaustion, and/or any other mechanisms of T-cell functional negative regulation. In some particular embodiments, the subject has an infection or a cancer and exhibits a T cell anergy, in particular an anergy of T cell responses against an antigen of interest, and/or T cell exhaustion.

Preferably, the subject has a cutaneous cancer or a cutaneous pathogen-caused disease as described below.

In some embodiments, the subject is suffering from a cutaneous pathogen-caused disease. The cutaneous pathogen-caused disease may be a cutaneous disease caused by a bacterium, a fungus or a virus, preferably caused by a bacterium, a fungus or a virus. The cutaneous pathogen- caused disease may be selected from the group consisting of bacterial infections such as cellulitis, impetigo or Staphylococcus infections, virus infections such as Merkel cell polyomavirus (MCPyV or MCV), herpes simplex viruses (HHV1, HHV2, HHV8), varicella-zoster virus (VZV), human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), or human papilloma virus (HPV) infection, fungal infection such as Candida infection or dermatophyte infection.

In preferred embodiments, the subject is suffering from a cutaneous cancer, preferably selected from the group consisting of Merkel cell carcinoma, melanoma, squamous cell carcinoma, basal cell carcinoma, angiosarcoma, cutaneous B-cell lymphoma, cutaneous T-cell lymphoma, dermatofibrosarcoma protuberans, sebaceous carcinoma, Kaposi's sarcoma. The cutaneous cancer may be a nonmetastatic cancer or a metastatic cancer.

In step b) of the method of the invention, the population of T cells obtained in step a) are 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 with at least one immunogenic peptide derived from at least one antigen of interest.

Herein, the terms "peptide", and "protein" are employed interchangeably and refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming said chain.

The antigen presenting cells (APCs) used in this step can be any antigen presenting cells suitable for presenting said at least one antigen of interest or at least one immunogenic peptide and activating T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with a TCR on the surface of a 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 are monocytes.

APCs used in step b) may be autologous (i.e. obtained from the same subject providing the cell sample, and preferably from the subject to be treated) or may be allogeneic (i.e. obtained from another subject than the subject providing the cell sample, and preferably from another subject than the subject to be treated).

In a preferred embodiment, APCs used in step b) are autologous. The skilled person may use a wide range of known procedures to generate autologous APCs using distinct sources, such as peripheral blood monocytes, naturally occurring DCs or CD34+ hematopoietic precursor cells mobilized from the bone marrow. Preferably, autologous APCs are obtained from peripheral blood monocytes or naturally occurring DCs, more preferably from peripheral blood monocytes.

CD34+ stem cells can be differentiated into dendritic cells by incubating the cells with appropriate cytokines, as is 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-a (see, e.g., Szabolcs, et al. (1995) J. Immunol. 154: 5851-5861). Dendritic cells can be then isolated by fluorescence activated cell sorting (FACS) based on expression of cell surface markers or by any other standard methods. In particular, the method may further comprise before step b) 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 autologous APCs may be identical or different from the cell sample used in step a) but both are obtained from the same subject.

Preferably, the method further comprises before step b) sorting from a cell sample from the subject a population of monocytes cells 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.

Alternatively, before or after antigen-loading, monocytes obtained from the sample, e.g. using CD14+ positive selection, may be cultured in the presence of GM-CSF and IL-4 in order to induce differentiation into dendritic cells. Optionally, between day 5 and day 10 of culture, preferably at day 6, IL-6, IL-ip and TNF-a are added to the culture medium during about 24h in order to induce optimal maturation of dendritic cells.

APCs can be loaded by any antigen-loading methods known by the skilled person. For example, APCs, in particular dendritic cells, monocytes or PBMCs may be loaded by pulsing or incubating APCs with one or more antigens of interest and/or one or more peptides, in particular one or more immunogenic peptides, derived from said one or more antigens of interest, or delivering one or more antigens and/or one or more peptides, in particular one or more immunogenic peptides, derived from said one or more antigens of interest into APCs using viral vectors or mRNA transfection.

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

An antigen of interest may be any antigen which may be targeted by the immune system to provide a therapeutic effect. The antigen(s) of interest is(are) easily selected by the skilled person depending on the disease to be treated. In preferred embodiments, the antigen(s) of interest is(are) selected depending on the disease to be treated in the subject providing the cell sample, i.e. a cancer or an infection.

In particular, the antigen(s) of interest may be selected from pathogen antigens or antigens expressed by tumor cells such as tumor-specific antigens (TSA) (i.e. antigens found on tumor cells only and not on healthy cells) or tumor-associated antigens (TAA) (i.e. antigens which have elevated levels on tumor cells but are also expressed at lower levels on healthy cells).

In an embodiment, the antigen(s) of interest are selected from one or more antigens of a cutaneous cancer. The term "cancer" or "tumor", as used herein, refers to the presence of cells possessing typical features of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. This term refers to any type of malignancy (primary or metastasis) and refers to solid or hematopoietic cancers.

In another embodiment, the antigen(s) of interest are selected from one or more antigens of a pathogen, in particular a virus, bacterium or fungus, preferably a virus, bacterium or fungus.

In a preferred embodiment, the antigen(s) of interest are selected from one or more viral antigens, preferably one or more antigens from a human virus. Preferably, the virus is selected from the group consisting of polyomaviruses, varicella-zoster virus (VZV), human immunodeficiency viruses (HIV), human T-lymphotropic virus (HTLV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), Herpes viruses and Papillomaviruses. More preferably, the virus is a human polyomavirus, even more preferably, the Merkel cell polyomavirus (MCPyV or MCV).

In a particular embodiment, the antigen(s) of interest are selected from antigens of a polyomavirus. For example, peptides presented by APCs may include one or more peptides of a polyomavirus, 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, peptides presented by APCs may be overlapping peptides covering one or several of these proteins. More particularly, peptides presented by APCs, preferably immunogenic peptides, may include overlapping peptide pools covering Large T protein, small T protein, or the VP1, VP2, and/or VP3 regions of said polyomavirus.

In a preferred embodiment, the antigen(s) of interest are selected from antigens of the polyomavirus MCPyV. For example, peptides presented by APCs may include 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, peptides presented by APCs may be overlapping peptides covering one or several of these proteins. More particularly, peptides presented by APCs, preferably immunogenic peptides, may include overlapping peptide pools covering the entirety of Large T and small T proteins of MCPyV.

Overlapping peptides can range from 10 to 20 amino acids in length, preferably from 12 to 18 amino acids in length and/or may overlap by 5 to 15 amino acids, preferably by 8 to 12 amino acids.

Antigens used to load APCs can be prepared by any method known by the skilled person depending on the nature of said antigens. For example, said antigens may be prepared by chemical synthesis, recombinant expression, from a sample from the subject, in particular from subject's own cancer cells, e.g. using whole tumor lysate, or from cancer cell line lysate.

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

Methods to obtain antigen-specific T cells from a population comprising Tscm cells using APCs are well known in the art and the skilled person may use any of these known methods.

Typically, the culture is carried out in the presence of IL-15, IL-7 and/or other stimulatory cytokines, preferably recombinant cytokines, such as IL-21. Preferably, the culture step comprises culture supplementation with IL-15 and IL-7, and optionally IL-21. Said supplementation starts preferably within the first seven days of culturing, more preferably between day 2 and day 4 of culture. IL-15 and IL-7 may help to maintain stem cell-like phenotype of the Tscm cells

At the beginning of the culture, the ratio of Tscm cells to APCs may be adjusted in order to be set from 1/1 (number of Tscm/number of APC) to 1/20, preferably from 1/5 to 1/15 and more preferably from 1/9 to 1/11.

The culture of T cells in the presence of APCs may last between 8 to 20 days, preferably between 10 to 18 days, more preferably between 12 to 16 days. In a particular embodiment, the culture of T cells in the presence of APCs lasts 14 days.

Optionally, the cells may be cultured for a longer period of time, preferably in the absence of antigen-loaded APCs. In particular, 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), preferably in the absence of antigen loaded APCs.

In some particular embodiments wherein the subject is affected with a retrovirus such as HIV, the culture may be conducted in the presence of one or several antiretroviral compounds.

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

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

In particular embodiments, in step c) of the method of the invention, the population of cells obtained in step b) is sorted in order to select/ re cover CD8+ cells and CD4+ cells. CD8+ cells and CD4+ cells may be recovered separately or together. In some embodiments, CD8+ cells and CD4+ cells are recovered separately, preferably before to being subsequently mixed. This separation allows to adjust the ratio CD8+/CD4+ in the obtained population of cells.

In preferred embodiments, in step c), the population of cells obtained in step b), i.e. all cells of the culture, is recovered and includes CD8+ cells and CD4+ cells. Recovered cells may also include other cell types, in particular APCs such as monocytes or dendritic cells.

Cells may be recovered by any method known by the skilled person including filtration methods or cell sorting methods as described above.

In particular, the population selected/recovered in step c) may comprise Tscm cells (with a cell surface phenotype comprising (i) CD45RA+ (ii) CCR7+ and/or CD62L+, preferably CD62L+, (iii) CLA+), T effector (Teff) cells (with a cell surface phenotype comprising (i) CD45RA+ (ii) CCR7- and/or CD62L-, preferably CD62L-, (iii) CLA+), T central memory (Tern) cells (with a cell surface phenotype comprising (i) CD45RA- (ii) CCR7+ and/or CD62L+, preferably CD62L+, (iii) CLA+), and T effector memory (Tern) cells (with a cell surface phenotype comprising (i) CD45RA- (ii) CCR7- and/or CD62L-, preferably CD62L-, (iii) CLA+).

Preferably, CLA+ cells represent more than 90 % of the total cells in the selected/recovered population.

In preferred embodiments, Tscm, Tern and Tern cells represent up to 90 %, preferably from 50% to 90%, of the total cells in the selected/recovered population, allowing further cycles of differentiation in vivo and therefore a prolonged therapeutic effect. Typically, Teff cells may represent from 10% to 50%, preferably from 10% to 20%, of the total cells in the selected/recovered population.

Optionally, before step b), a population of T cells having a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+ and (iv) CLA-, and optionally CD95+, is mixed with the population of T cells sorted in step a). This population may be obtained as described above for the population of CLA+ cells with the exception of the selection of CLA- ce I Is. In this case, steps b) and c) are carried out in the presence of these two populations of cells including CLA+ and CLA- cells. Recovering CLA+ and CLA- cells before to being subsequently mixed allows to adjust the ratio CLA+/CLA- in the obtained population of cells. In this case, in step c), CLA+ cells and CLA- cells may also be recovered separately before being subsequently mixed, e.g. at a specific ratio. Preferably, the population of CLA+ T cells sorted in step a) is not mixed with a population of CLA- T cells before step b) or step c).

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 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.

All embodiments disclosed above and relating to the method for obtaining a population of cells comprising antigen-specific T cells, are also encompassed in this aspect.

In particular, said population may comprise, or consist of, Tscm cells (with a cell surface phenotype comprising (i) CD45RA+ (ii) CCR7+ and/or CD62L+, preferably CD62L+, (iii) CLA+), T effector (Teff) cells (with a cell surface phenotype comprising (i) CD45RA+ (ii) CCR7- and/or CD62L-, preferably CD62L-, (ii) CLA+), T central memory (Tern) cells (with a cell surface phenotype comprising (i) CD45RA- (ii) CCR7+ and/or CD62L+, preferably CD62L+, (iii) CLA+), and T effector memory (Tern) cells (with a cell surface phenotype comprising (i) CD45RA- (ii) CCR7- and/or CD62L-, preferably CD62L-, (iii) CLA+).

Preferably, CLA+ cells represent more than 90 % of the total cells in the isolated population of the invention.

Preferably, Tscm, Tern and Tern cells represent up to 90 %, preferably from 50% to 90%, of the total cells in the isolated population of the invention. Typically, Teff cells may represent from 10% to 50%, preferably from 10% to 20%, of the total cells in the isolated population of the invention.

Preferably, antigen-specific CD8+ T cells represent from 10% to 90% of the total cells and antigen-specific CD4+T cells represent from l% to 90% of the total cells in the isolated population of the invention. In particular, antigen-specific CD8+ T cells may represent from 50% to 90% of the total cells in the isolated population of the invention, and antigen-specific CD4+ T cells represent from 1% to 50% of the total cells in the isolated population of the invention.

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 obtained or obtainable by a method comprising a) sorting from a cell sample from a subject suffering from a cutaneous cancer or a cutaneous pathogen-caused disease, a population of T cells having a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+, preferably CD62+ and (iv) CLA+, CCR4+ and/or CCR10+, preferably CLA+, and optionally CD3+, CD95+, CD45RO-, PD1-, TIGIT-, LAG3-, TIM3-, CTLA4- and/or CD160-, preferably CD95+ and CD45RO-, more preferably CD95+, b) culturing said population of T 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, in particular at least one immunogenic peptide, 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 cells obtained in step b), in particular CD8+ and/or CD4+ cells, preferably CD8+ and CD4+ cells.

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

The subject may suffer from a cutaneous cancer. In this case, said at least one antigen of interest may be selected from antigens expressed by tumor cells such as tumor-specific antigens (TSA) or tumor-associated antigens (TAA).

The subject may suffer from a cutaneous pathogen-caused disease. In this case, said at least one antigen of interest may be selected from antigens of said pathogen.

In a preferred embodiment, the subject suffers from Merkel cell carcinoma. Preferably, said at least one antigen of interest is selected from antigens of the Merkel cell polyomavirus (MCPyV or MCV), in particular from VPl, VP2, VP3, Large T, small T proteins and/or from any other protein of MCPyV.

In another aspect, the present invention relates to an in vitro method for obtaining a population of skin homing memory stem T-cells (Tscm cells), comprising sorting from a cell sample from a subject a population of cells having a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+, preferably CD62+ and (iv) CLA+, CCR4+ and/or CCR10+, preferably CLA+, and optionally CD3+, CD95+, CD45RO-, preferably CD95+ and CD45RO- , more preferably CD95+.

Optionally, the method may further comprises depleting cells expressing one or several other inhibitory receptors such as PD1, TIGIT, LAG3, TIM3, CTLA4 and/or CD160.

Optionally, the method further comprises amplifying the selected population of Tscm. This step may be carried out by any method well-known by the skilled person such as the culture of said Tscm cells in the presence of feeders such as monocytes (non-loaded monocytes), and suitable cytokines.

The cell sample may be obtained from a subject suffering from a cutaneous cancer or a cutaneous pathogen-caused disease as described above. Preferably, the subject has Merkel cell carcinoma.

Alternatively, the cell sample may be obtained from a healthy subject, preferably from a subject who is not suffering from a cutaneous infection or cancer and/or does not exhibit T cell anergy.

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.

In a further aspect, the present invention relates to an isolated population of skin homing Tscm cells, in particular an isolated population of skin homing Tscm cells obtained or obtainable by the method of the invention for obtaining a population of skin homing Tscm cells.

This population may comprise Tscm cells having a cell surface phenotype comprising

1) CD4+ , CD45RA+, CD95+, CCR7+, CLA+; and/or

2) CD8+, CD45RA+, CD95+, CCR7+, CLA+; and/or,

3) CD4+, CD45RA+, CD95+, CD62L+, CLA+; and/or, 4) CD8+, CD45RA+, CD95+, CD62L+, CLA+; and/or,

5) CD4+ , CD45RA+, CD95+, CCR7+, CCR4+; and/or

6) CD8+, CD45RA+, CD95+, CCR7+, CCR4+; and/or,

7) CD4+, CD45RA+, CD95+, CD62L+, CCR4+; and/or,

8) CD8+, CD45RA+, CD95+, CD62L+, CCR4+; and/or,

9) CD4+ , CD45RA+, CD95+, CCR7+, CCR10+; and/or

10) CD8+, CD45RA+, CD95+, CCR7+, CCR10+; and/or,

11) CD4+, CD45RA+, CD95+, CD62L+, CCR10+; and/or,

12) CD8+, CD45RA+, CD95+, CD62L+, CCR10+.

Preferably, this population may comprise Tscm cells having a cell surface phenotype comprising

(i) CD4+ , CD45RA+, CD95+, CCR7+, CLA+; and/or

(ii) CD8+, CD45RA+, CD95+, CCR7+, CLA+; and/or,

(iii) CD4+, CD45RA+, CD95+, CD62L+, CLA+; and/or,

(iv) CD8+, CD45RA+, CD95+, CD62L+, CLA+.

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

Optionally, these Tscm cells may have a cell surface phenotype further comprising PD1-, TIGIT-, LAG3-, TIM3-, CTLA4 and/or CD160.

Preferably, the population of Tscm cells of the invention comprises at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 or at least 90% (of the total cells of the population) of skin homing Tscm cells, CLA+, CCR4+ or CCR10+ Tscm cells, preferably CLA+ Tscm cells.

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.

In a further aspect, the present invention also relates to

- 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 the 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 skin homing Tscm cells of the invention (i.e. obtained or obtainable by the method of the invention for obtaining a population of skin homing Tscm cells), as a cell therapy medicament.

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

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

The present invention also relates to a method for preparing a pharmaceutical composition, said method comprising obtaining a population of skin homing Tscm cells by the method of the invention or a population of cells comprising antigen-specific CD8+ T cells, and optionally antigen-specific CD4+ T cells, by the method of the invention, and optionally mixing said population of cells with CLA- cells, in particular CLA- Tscm cells and/or CLA- T effector (Teff) cells and/or CLA- T central memory (Tern) cells and/or CLA- T effector memory (Tern) cells.

CLA- cells to be mixed with CLA+ cells may be obtained by a method similar to the method of the invention for obtaining a population of cells comprising skin homing antigen-specific T cells, with the exception of the CLA- phenotype, i.e. a method comprising a) sorting from a cell sample from a subject, a population of T cells having a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+ and (iv) CLA-, and b) culturing said population of T 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 cells obtained in step b), in particular CD8+ and/or CD4+ cells, preferably CD8+ and CD4+ cells.

Preferably, the population of CLA- T cells is cultured in the presence of antigen-presenting cells loaded with the same antigen(s) of interest or the same peptide(s) derived from antigen(s) of interest than the CLA+ cells.

Preferably, the sample used in step a) is the same or is obtained from the same subject as for CLA+ cells and CLA- cells.

Preferably, CLA- cells and CLA+ cells are mixed at a cell number ratio CLA-/CLA+ from 0.2 to 5. This ratio may be adjusted according to the clinical parameters of the patient.

In a further aspect, the present invention relates to a pharmaceutical composition comprising

- an isolated population of skin homing Tscm cells of the invention (i.e. obtained or obtainable by the method of the invention for obtaining a population of skin homing Tscm cells).

Optionally, the pharmaceutical composition further comprises CLA- cells such as CLA- Tscm cells and/or CLA- T effector (Teff) cells and/or CLA- T central memory (Tern) cells and/or CLA- T effector memory (Tern) cells.

In preferred embodiments, the pharmaceutical composition comprises 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 the 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.

The pharmaceutical composition is formulated in a pharmaceutically acceptable carrier and/or excipient according to the route of administration.

Preferably, the pharmaceutical composition is formulated in order to be suitable for use in a cell based therapy in a subject in need thereof. The pharmaceutical composition may be formulated in accordance with standard pharmaceutical practice (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) known by a person skilled in the art.

Preferably, the pharmaceutical composition is suitable for parenteral administration, preferably intravenous infusion.

Pharmaceutical compositions suitable for such administration may comprise the population of cells of the invention, in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions (e.g., balanced salt solution (BSS)), dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes or suspending or thickening agents.

Optionally, the composition comprising cells may be frozen for storage at any temperature appropriate for storage of the cells. For example, the cells may be frozen at about -150°C or - 196°C. Cryogenically frozen cells may be stored in appropriate containers and prepared for storage to reduce rick of cell damage and maximize the likelihood that the cells will survive thawing.

The amount of cells to be administered may be determined by standard procedure well known by those of ordinary skill in the art. Physiological data of the patient (e.g. age, size, and weight) and type and severity of the disease being treated have to be taken into account to determine the appropriate dosage.

The pharmaceutical composition of the invention may be administered as a single dose or in multiple doses. Each unit dosage may contain, for example, from 10⁵ to 7.10⁸ cells, preferably from 7.10⁶ to 7.10⁸ cells.

The pharmaceutical composition of the invention may further comprise additional active compounds such as therapeutic monoclonal antibodies to deplete a lymphocyte subset or to block a receptor involved in immune function such as anti-PD-1 or anti-TIGIT.

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

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

As used herein, the term "treatment", "treat" or "treating" refers to any act intended to ameliorate the health status of patients such as therapy, prevention, prophylaxis and retardation of the disease. In certain embodiments, such term refers to the amelioration or eradication of a disease or symptoms associated with a disease. In other embodiments, this term refers to minimizing the spread or worsening of the disease resulting from the administration of one or more therapeutic agents to a subject with such a disease.

The effective amount may be a therapeutically or prophylactically effective amount. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. In particular, this term refers to an amount of the pharmaceutical composition of the invention administered to a patient that is sufficient to provide an immune response against the targeted pathogen or tumor cells. The therapeutically effective amount may vary according to various factors such as the disease to be treated, the physiological condition of the subject to be treated, the severity of the affliction and the administration route. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount would be less than the therapeutically effective amount. Suitable means and measures to determine the therapeutically or prophylactically effective amount are available to the person skilled in the art.

Preferably, the pharmaceutical composition is administered via parenteral route, more preferably intravenous infusion. In some embodiments, in particular for the treatment of localized disease, the administration may be targeted in order to deliver cells in the organ or tissue affected by said disease.

In a particular embodiment, the method of the invention comprises administering from 10³ to 10⁸ cells / kg of body weight, preferably 10⁴ to 10⁸ cells / kg of body weight, more preferably from 10⁵ to 10⁷ cells / kg of body weight, of 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, of the invention to said subject.

More particularly, the method of the invention may comprise administering an isolated population of cells comprising antigen-specific CD8+ T cells, and optionally antigen-specific CD4+ T cells, of the invention, and in particular from 1000 to 10,000,000 antigen-specific CD8+ T cells / kg of body weight, preferably from 5000 to 1,000,000 antigen-specific CD8+ T cells / kg of body weight, more preferably from 5000 to 100,000 antigen-specific CD8+ T cells / kg of body weight. In this case, the dose to be administered may be obtained by quantifying CD8+ T cells present in the population or pharmaceutical composition of the invention.

In some embodiments, the method of the invention may comprise administering an isolated population of cells comprising antigen-specific CD8+ T cells and antigen-specific CD4+ T cells, of the invention. Preferably, from 1000 to 10,000,000 antigen-specific CD8+ T cells / kg of body weight, preferably from 5000 to 1,000,000 antigen-specific CD8+ T cells / kg of body weight, more preferably from 5000 to 100,000 antigen-specific CD8+ T cells / kg of body weight are to be administered, and from 1000 to 10,000,000 antigen-specific CD4+ T cells / kg of body weight of the subject, preferably from 5000 to 1,000,000 antigen-specific CD4+ T cells / kg of body weight of the subject, more preferably from 5000 to 100,000 antigen-specific CD4+ T cells / kg of body weight of the subject, are to be administered. In this case, the dose to be administered may be obtained by quantifying CD8+ T cells and optionally quantifying CD4+ T cells present in the population or pharmaceutical composition of the invention.

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. In embodiments wherein the subject to be treated is suffering from a metastatic cutaneous cancer, it may be advantageous to use not only skin homing cells, e.g. CLA+ cells, but also nonskin homing cells, e.g. CLA- cells. In this case, the pharmaceutical composition used for the treatment may be prepared by the method of the invention comprising obtaining a population of skin homing Tscm cells by the method of the invention or a population of cells comprising antigen-specific CD8+ T cells, and optionally antigen-specific CD4+ T cells, by the method of the invention, and mixing said population of cells with CLA- cells, in particular CLA- Tscm cells and/or CLA- T effector (Teff) cells and/or CLA- T central memory (Tern) cells and/or CLA- T effector memory (Tern) cells.

Alternatively, the isolated population of cells or pharmaceutical population of the invention may be used in combination with CLA- cells, in particular CLA- Tscm cells and/or CLA- T effector (Teff) cells and/or CLA- T central memory (Tern) cells and/or CLA- T effector memory (Tern) cells. In this case, CLA- cells may be administered simultaneously or sequentially.

CLA- cells to be used in combination or to be mixed with CLA+ cells may be obtained by a method similar to the method of the invention for obtaining a population of cells comprising skin homing antigen-specific T cells, with the exception of the CLA- phenotype, i.e. a method comprising a) sorting from a cell sample from a subject, a population of T cells having a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+ and (iv) CLA-, and b) culturing said population of T 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 cells obtained in step b), in particular CD8+ and/or CD4+ cells, preferably CD8+ and CD4+ cells.

Preferably, the sample used in step a) is the same or is obtained from the same subject, preferably the subject to be treated, as for CLA+ cells and CLA- cells. The treatment may be an autologous therapy (by administering cells derived from a subject back into the same subject) or allogeneic therapy (by administering cells derived from a subject into another subject). Preferably, the treatment is an autologous therapy.

As mentioned above, the subject to be treated, preferably a human being, is suffering from a cutaneous cancer or cutaneous pathogen-caused disease.

The cancer or pathogen-caused disease to be treated may be any cutaneous infection or cancer.

The cutaneous pathogen-caused disease may be a cutaneous disease caused by a bacterium, a fungus or a virus, preferably caused by a virus.

To treat cutaneous pathogen-caused diseases, the pharmaceutical composition may comprise an isolated population of skin homing Tscm cells of the invention (i.e. obtained or obtainable by the method of the invention for obtaining a population of skin homing Tscm cells). These Tscm cells are administered in order to increase the pool of Tscm cells and allow the in vivo activation of said cells by contacting in vivo APCs.

Preferably, to treat cutaneous pathogen-caused diseases, the pharmaceutical composition comprises an isolated population of cells comprising antigen-specific CD8+ T cells, and optionally antigen-specific CD4+ T cells, of the invention, preferably an isolated population of cells comprising antigen-specific CD8+ T cells and antigen-specific CD4+ T cells of the invention. To treat this category of disease, the population of cells comprising antigen-specific CD8+ T cells, and optionally antigen-specific CD4+ T cells, of the invention has been obtained by culturing T cells in the presence of APCs loaded with at least one antigen of the pathogen to be targeted or at least one peptide, in particular immunogenic peptide, derived from said at least one antigen, in order to obtain a population of T cells that are activated to recognize target cells bearing said at least one antigen.

The cutaneous pathogen-caused disease may be selected from the group consisting of bacterial infections such as cellulitis, impetigo or Staphylococcus infection, virus infections such as Merkel cell polyomavirus (MCPyV or MCV), herpes simplex virus (HSV), varicella-zoster virus (VZV), human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), or human papilloma virus (HPV) infection, fungal infection such as Candida infection. In an embodiment, the cutaneous pathogen-caused disease is a chronic or acute viral infection caused by a virus selected from the group consisting of Merkel cell polyomavirus (MCPyV or MCV), herpes simplex virus (HSV), varicella-zoster virus (VZV), human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), and human papilloma virus (HPV) infection. Preferably, the virus is the Merkel cell polyomavirus (MCPyV or MCV).

In a particular embodiment, the pathogen is the Merkel cell polyomavirus and the cutaneous pathogen-caused disease is the Merkel carcinoma.

To treat a pathogen-caused disease, the cell-based therapy of the invention may be used alone or in combination with other treatment(s) such as antibiotic treatment, antiviral (or antiretroviral) treatment or antifungal treatment.

In other embodiments, the disease to be treated is a cutaneous cancer. To treat this category of diseases, the pharmaceutical composition may comprise an isolated population of cells comprising antigen-specific CD8+ T cells, and optionally antigen-specific CD4+ T cells, of the invention that has been obtained by culturing T cells in the presence of APCs loaded with at least one antigen expressed by tumor cells such as tumor-specific antigens (TSA) or tumor-associated antigens (TAA), or at least one peptide, in particular immunogenic peptide, derived from said at least one antigen, in order to obtain a population of T cells that are activated to recognize target cells bearing said at least one antigen.

Alternatively, and in particular for disease for which there is no identified specific antigen, the pharmaceutical composition may comprise an isolated population of skin homing Tscm cells of the invention (i.e. obtained or obtainable by the method of the invention for obtaining a population of skin homing Tscm cells). These skin homing Tscm cells are administered in order to increase the pool of skin homing Tscm cells and allow the in vivo activation of said cells by contacting in vivo APCs.

To treat a cancer, the cell-based therapy of the invention may be used alone or in combination with other treatment(s) such as chemotherapeutic treatment, surgical treatment and/or radiotherapeutic treatment.

In a particular embodiment, the disease to be treated is Merkel cell carcinoma and the pathogen is the Merkel cell polyomavirus (MCPyV or MCV). In this embodiment, the population of cells comprising antigen-specific T cells to be administered may be obtained by the method comprising a) sorting from a cell sample from the subject suffering from Merkel cell carcinoma a population of T cells having a cell surface phenotype comprising CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+ preferably or CD62L+, and (iv) CLA+, CCR4+ and/or CCR10+, preferably CLA+, and optionally CD3+, CD95+, CD45RO-, PD1-, TIGIT-, LAG3-, TIM3-, CTLA4- and/or CD160-, preferably CD95+ and CD45RO-, more preferably CD95+, b) culturing said population of T cells in the presence of antigen-presenting cells loaded with at least one antigen of the polyomavirus MCV or at least one peptide, in particular immunogenic peptide, derived from said at least one antigen , and, optionally, in the presence of IL-7 and IL-15 or other stimulatory cytokines, and, optionally c) recovering cells obtained in step b), in particular CD8+ and/or CD4+ cells, preferably CD8+ and CD4+ cells.

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

Preferably, peptides presented by APCs may include one or more MCV peptides, in particular one or more MCV immunogenic peptides, from VP1, VP2, VP3, Large T, small T proteins and/or from any other protein of MCV. In particular, peptides presented by APCs may be overlapping peptides covering one or several of these proteins. More particularly, peptides presented by APCs, preferably immunogenic peptides, may include overlapping peptide pools covering Large T and small T proteins of MCV.

If the subject is further affected with a retrovirus such as HIV, the culture of step b) may be conducted in the presence of an antiretroviral compound.

All the references cited in this description are incorporated by reference in the present application. Others features and advantages of the invention will become clearer in the following examples which are given for purposes of illustration and not by way of limitation.

EXAMPLES EXEMPLE 1

Materials and methods

PBMC isolation

100 mL of heparinized blood were used. Blood was obtained from healthy donors or MCC patients with or without a medical condition of immunosuppression. Blood was diluted with Nacl 0,9% (v/v) and PBMCs were isolated by Ficoll density gradient centrifugation (Ficoll Hypaque).

T-cell subsets

One million PBMCs were stained with the following combination : anti-CD3, anti-CD4, anti- CD8, anti-CD45RA, anti-CD45RO, anti-CCR7, anti-CD62L, anti-CD95, anti-CLA. Cells were fixed in PBS IX containing 1% PFA and analyzed by flow cytometry (BD LSR Fortessa). data were analyzed by means of the FlowJo software.

Cell sorting

T cell subset isolation

PBMCs were washed in Buffer (PBS IX, EDTA 2 mM SVF 0,5%). Monocytes were isolated by means of anti-CD14 coated magnetic beads. T cells were subsequently isolated on the negatively selected fraction, by means of antibodies-coated magnetic beads allowing depletion of non-CD3+ cells.

T cells were then washed in PBS containing 0,5% SVF. Cell concentration was adjusted at 20 million cells per mL then cells were stained with the following antibodies : anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, anti-CD62L, anti-CCR7, anti-CD95, anti-CLA, for 15 minutes at 4°C. Cells were washed, filtered on a 0.22pm filter to remove cell clumps, and processed for cell sorting.

Gating strategy

Cells were first gated on forward and side scatters, then on FSC-A and FSC-H to exclude doublets. Cell subsets were defined as follows, in both CD3+ CD4+ T-cells and CD3+ CD8+ T cells

- Stem cell memory (Tscm) : CD45RA+ CD45RO- CCR7+ CD95+ or CD45RA+ CD45RO- CD62L+

CD95+ - CLA+ Tscm : CD45RA+ CD45RO- CCR7+ CD95+ CLA+ or CD45RA+ CD45RO- CD62L+ CD95+

CLA+

- CLA+ memory cells : CLA+ CD45RO+-

Cells were sorted on an SVF-coated tube. Cells were then resuspended in a culture medium.

CD4 and CD8 cell count

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

Analysis

Results were analyzed with the FlowJo Software. Statistical analysis and graphs were performed by means of GraphPad Prism software.

Results

Derm-specific blood T-cells were characterized by the expression of the cutaneous leukocyte antigen CLA. CLA-positive cells were found in both CD4+ and CD8+ T cell subsets and were detectable within the Tscm compartment and the memory CD45RO+ compartment (Figure 1). These CLA-positive cells represented a mean percentage of 15 % of CD4+ Tscm cells and 15% of CD8+ Tscm cells (Figure 2).

The percentages of Tscm cells (CD95+) within the CD4+ or CD8+ CD45RA+ CD62L+ CLA+ cell gates were also analyzed. The results show that more than half of the CD45RA+ CD62L+ CLA+ cells express CD95 and are Tscm (Figure 3). Remaining cells harbor a naive phenotype. Thus, the sorted cell population defined according to the following markers : CD3+ CD4+ [or CD8+] CD45RA+ CD62L+ CLA+ exclusively includes Tscm and naive cells.

With these results, the inventors have identified a new skin-tropic subset of Tscm that expresses the cutaneous homing marker CLA+. This subset and other skin-tropic memory T cells can be used to treat the cutaneous diseases such as cutaneous location of MCC (primitive Tumor). One can consider using personalized cell therapy products that combine at different ratios (and according to clinical situations) CLA+ and CLA- Tscm to better treat cutaneous and metastatic locations of cutaneous cancer such as MCC.

EXEMPLE 2: Identification in peripheral blood mononuclear cells (PBMC) of a new subset of stem cell memory T cells (Tscm) with cutaneous tropism.

As shown in Example 1, in peripheral blood mononuclear cells, the inventors have identified a subset of Tscm that expresses the cutaneous domiciliation receptor CLA (cutaneous-lymphocyte- associated antigen). Tscm corresponds to the least differentiated memory T cell subset. This subset is known to traffic between blood and lymphoid tissues, like naive T cells. This finding of CLA+ Tscm shows that some of those cells may enter the skin to sustain local immune responses. By contrast, as shown in Figure 4, naive T cells do not significantly express CLA.

CLA+ Tscm also express higher levels of CCR4 and CCR10, two chemokine receptors involved in skin domiciliation of T cells (Figure 5), confirming their cutaneous tropism.

As shown in Figure 6, the functionality of highly purified CLA+ Tscm specific against MCPyV was tested and compared to CLA- Tscm and to other circulating memory T cells sorted based on CD45RO expression (called conventional memory T cells). Conventional memory T cells include central memory T cells (Tern) and effector memory T cells (Tern). Cells were activated for 2 weeks in the presence of monocytes loaded with MCPyV peptide pools covering large T and small T proteins of MCPyv.

CLA+ Tscm showed the strongest anti-MCPyV expansion among blood memory T cells. CLA+ Tscm expanded 67 fold (mean) (Figure 6). This high functional capacity against MCPyV is related to the higher functionality of Tscm as compared to CLA+ conventional memory T cells. Moreover, as MCPyV is mainly located in the skin in healthy donors, the proportion of MCPyV-specific cells may be higher in CLA+ Tscm than in CLA-Tscm, which may explain their higher functionality against MCPyV.

As shown in Figure 7, besides their high functionality, CLA+ Tscm have strong differentiation capacities. CLA+ Tscm generate following 14-day-specific activation, a continuum of differentiation to terminally differentiated effector T cells (Teff - CD45RA+ CD62L-) that include Tern (central memory T cells - CD45RO+ CD62L+) and Teff (memory effector T cells - CD450+ CD62L-). The results demonstrate that CLA+ Tscm strongly proliferate and differentiate in response to MCPyV. This 14-day expansion and differentiation process leads to MCPyV-specific cells with effector functions (terminally differentiated effectors and Tem) and cells capable of subsequent expansion and differentiation in vivo including Tscm and Tcm (Figure 7). Administration of a cell preparation generated from MCPyV-activated CLA+Tscm following 14 days may therefore have an immediate therapeutic effect owing to the presence of a pool of MCPyV-specific terminally differentiated effectors and Tem, and a prolonged effect due to the presence of MCPyV-specific Tscm and Tcm.

Claims

1. An in vitro method for obtaining a population of cells comprising skin homing antigenspecific T cells comprising a) sorting from a cell sample from a subject, a population of T cells having a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+ and (iv) CLA+, and b) culturing said population of T 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 cells obtained in step b), in particular CD8+ and/or CD4+ cells, preferably CD8+ and CD4+ cells.

2. The method of claim 1, wherein the population of T cells sorted in step a) has a cell surface phenotype further comprising CD95+.

3. The method of claim 1 or 2, 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).

4. The method of any of claims 1 to 3, wherein said at least one antigen of interest is an antigen of a cutaneous pathogen, preferably a viral, bacterial or fungal antigen, or an antigen expressed by cutaneous tumor cells such as tumor-specific antigens (TSA) or tumor-associated antigens (TAA).

5. The method of any of claims 1 to 4, wherein the subject is suffering from a cutaneous cancer or cutaneous pathogen-caused disease.

6. The method of any of claims 1 to 5, wherein the subject is suffering from a cutaneous cancer, preferably selected from the group consisting of Merkel cell carcinoma, melanoma, squamous cell carcinoma, basal cell carcinoma, angiosarcoma, cutaneous B-cell lymphoma, cutaneous T-cell lymphoma, dermatofibrosarcoma protuberans and sebaceous carcinoma.

7. The method of any of claims 1 to 6, wherein the subject is suffering from Merkel cell carcinoma and said at least one antigen of interest is an antigen of the polyomavirus MCPyV.

8. The method of any of claims I to 7, 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 population enriched in T cells from a blood sample or PBMCs.

9. 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 any of claims 1 to 8.

10. The isolated population of cells of claim 9, wherein said population comprises memory stem T (Tscm) cells having a cell surface phenotype comprising (i) CD45RA+ (ii) CCR7+ and/or CD62L+, and (iii) CLA+); T effector (Teff) cells having a cell surface phenotype comprising (i) CD45RA+ (ii) CCR7- and/or CD62L-, and (iii) CLA+; T central memory (Tern) cells having a cell surface phenotype comprising (i) CD45RA- (ii) CCR7+ and/or CD62L+, and (iii) CLA+; and T effector memory (Tern) cells having a cell surface phenotype comprising (i) CD45RA- (ii) CCR7- and/or CD62L-, and (iii) CLA+.

11. The isolated population of cells of claim 9 or 10, wherein CLA+ cells represent more than 90 % of the total cells.

12. The isolated population of cells of any of claims 9 to 11, wherein Tscm, Tern and Tern cells represent from 50% to 90% of the total cells and Teff cells represent from 10% to 50% of the total cells.

13. An in vitro method for obtaining a population of skin homing memory stem T-cells (Tscm cells), said method comprising sorting from a cell sample from a subject a population of cells having a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+ and (iv) CLA+.

14. An isolated population of cells comprising CLA+ Tscm, wherein CLA+ Tscm cells represent more than 30% of the total cells in said population, and wherein said CLA+ Tscm cells have a cell surface phenotype comprising (i) CD4+ or CD8+, (ii) CD45RA+, (iii) CCR7+ and/or CD62L+, (iv) CLA+, and (v) CD95+.

15. A method for preparing a pharmaceutical composition, said method comprising obtaining a population of skin homing Tscm cells by the method of claim 13 or providing a population of cells of claim 14, or b) obtaining a population of cells comprising antigen-specific CD8+ T cells, and optionally antigen-specific CD4+ T cells, by the method of any of claims 1 to 8, or providing a population of cells of any of claims 9 to 12, and, after a) or b), optionally mixing said population of cells with CLA- cells, in particular CLA- Tscm cells and/or CLA- T effector (Teff) cells and/or CLA- T central memory (Tern) cells and/or CLA- T effector memory (Tern) cells.

16. A pharmaceutical composition obtained or obtainable by the method of claim 15.

17. An isolated population of cells of any of claims 9 to 12 or 14 or the pharmaceutical composition of claim 16, for use as a cell therapy medicament, in particular in the treatment of a cutaneous cancer or a cutaneous pathogen-caused disease.

18. The isolated population of cells for use of claim 17 or the pharmaceutical composition for use of claim 17, wherein the population of cells is used in combination with CLA- cells, in particular CLA- Tscm cells and/or CLA- T effector (Teff) cells and/or CLA- T central memory (Tern) cells and/or CLA- T effector memory (Tern) cells.

19. The isolated population of cells for use of claim 17 or 18 or the pharmaceutical composition for use of claim 17 or 18, wherein the isolated population of cells is for use in the treatment of a cutaneous cancer, preferably selected from the group consisting of Merkel cell carcinoma, melanoma, squamous cell carcinoma, basal cell carcinoma, angiosarcoma, cutaneous B-cell lymphoma, cutaneous T-cell lymphoma, dermatofibrosarcoma protuberans, sebaceous carcinoma and Kaposi's sarcoma.

20. The isolated population of cells for use of any of claims 17 to 19 or the pharmaceutical composition for use of any of claims 17 to 19, wherein said population is autologous to the subject to be treated.