Car-thytreg cells, compositions and uses thereof in immunotherapy

Abstract

The invention provides a thymus T regulatory cell (ThyTreg cell) which codes for, or alternatively expresses on its surface, a chimeric antigen receptor (CAR) comprising an extracellular domain, a hinge region, a transmembrane domain and an intracellular domain, wherein the intracellular domain comprises: a cytoplasmatic co-stimulation domain with a sequence having an identity of at least 85% with respect to SEQ ID NO: 1; and a cytoplasmatic stimulation domain. The invention also provides compositions and uses in immunotherapy.

Description

[0001] CAR-ThyTreg CELLS, COMPOSITIONS AND USES THEREOF IN IMMUNOTHERAPY

[0002] FIELD OF THE INVENTION

[0003] The present invention falls within clinical immunology and cell-advanced immunotherapy. In particular, the present invention provides CAR-ThyTreg cells, compositions comprising thereof and uses thereof in inducing a specific immune tolerance.

[0004] BACKGROUND

[0005] The immune system's main function is to defend the organism from pathogenic agents, and it’s also in charge of eliminating tumour cells and preventing cancer development. However, it can also give rise to inadequate responses through the appearance of autoimmune processes, allergies, or transplant rejections. The proper functioning of the immune system is only possible if there is a balance or homeostasis adequate thereto. An excessive response will give rise to pathologies such as allergies or rejection, and a defective response will enable the progression of infections and cancer.

[0006] Autoimmune diseases, hyper-inflammatory processes, and immune rejection are currently treated with immunosuppressive drugs. Despite improvements, they still do not offer a definitive solution to these diseases and continue to cause side effects affecting the patient's clinical evolution. Specifically, long-term immunosuppression causes chronic toxicity, which, in addition to significantly influencing the patient’s quality of life, affects the fulfilment of the treatment. Since most immunosuppressors act in a non- selective way, the entire system is repressed and / or deregulated, which can interfere with normal immune system function, which may have lifelong consequences.

[0007] To this end, achieving immune tolerance that will indefinitely avoid graft rejection or autoimmune disease symptoms that affect millions of people has become the major challenge of modern medicine. The current opinion of the science community is that just one immune tolerance induction that involves re-educating the recipient’s immune response will allow the indefinite survival of the graft, the limitation of the autoimmune symptoms, or the restoration of homeostasis in hyperinflammation processes. However, no therapies have been developed so far and found at the commercial level with the ability to induce immune tolerance, to prevent or cure autoimmune diseases, allogeneic graft rejection, and suppressing hyper-inflammation pathologies. One of the most promising alternatives is to induce immune tolerance through cell immunotherapy (Sicard et al., 2015).

[0008] The discovery of a subset of lymphocytes with a suppressive capacity capable of inducing this tolerance has generated widespread enthusiasm in the clinical sphere. These cells, called regulatory T cells (Treg), constitute an essential part of the immune system, and could play a crucial role in maintaining immune homeostasis beneficial to patients. Tregs can suppress the effector function of many cells, including T CD4+ and CD8+ lymphocytes, Natural Killer cells (NK cells), B cells, macrophages, and dendritic cells (DC) (Sakaguchi et al., 2008).

[0009] The primary role of Tregs in transplants has been confirmed by various studies in animal models of skin and heart transplants, demonstrating that the Tregs present in the receptacle at the time of the transplant are critical to the induction and maintenance of tolerance to the graft (Juneja et al., 2022). These Treg cells will impede the activation and expansion of effector T cells, which are responsible for cellular rejection. Tregs can also induce the death of B cells, preventing humoral rejection, as already demonstrated in a cardiac xenotransplantation model (Ma et al., 2008). Cell therapy with Tregs is therefore postulated to become the great hope in the treatment of diseases mediated by an excessive or inadequate response of the immune system, such as autoimmune processes (Bluestone et al., 2015), graft-versus-host disease in bone marrow transplant patients (Brunstein et al., 2016), or transplant rejection (Safinia et al., 2016). It is assumed that the mechanism of this therapy is based on that a greater number of circulating functional Tregs will be capable of preventing the activation and proliferation of effector cells that trigger these diseases. Treg cell transfer would substantially increase their number in circulation and thus potentiate the recipient’s intrinsic tolerance mechanisms in the receptacle to the transplanted organ or own tissues.

[0010] The safety and potential effectiveness of Treg therapy in humans are reflected in the first Phase-l / ll trials already performed. Most clinical trials with Tregs have been performed within the context of bone marrow transplants in patients with haematological neoplasia, showing that the infusion of Tregs in these patients reduces or prevents graft-versus- host disease (GvHD) (Trzonkowski et al., 2009). Treg-based therapies were also tested with good results in type-1 diabetes children (Marek-Trzonkowska et al., 2014) with a partial response after 1-3 years of follow-up (Gliwihski et al., 2020) or in multiple sclerosis (Chwojnicki et al., 2021).

[0011] Previous data have shown that antigen-specific Treg cells are more effective than nonspecific polyclonal Treg obtained by ex vivo expansion in reducing graft rejection (Sagoo et al., 2011). Up to now, antigen-specific Treg (Ag-specific) are obtained with the coculture of Treg cells and allogeneic antigen-presenting cells (APCs) such as dendritic cells or B cells, making it possible to enrich alloreactive Treg cells in vitro. The main limitation of these strategies is the reduced number of T reg and APC that can be isolated. The CAR (chimeric antigen receptor) technology may be suitable for generating Ag- specific ThyTreg to overcome these limitations (Beheshti et al., 2022). A CAR is a synthetic construct mimicking T-cell receptor activation and redirects specificity and effector function toward a specified antigen when expressed in T cells. CARs comprise an extracellular region (scFv) responsible for binding to a particular antigen and an intracellular domain promoting T cell activity and proliferation.

[0012] Efforts have also been made in designing modified structures to enhance the innate properties of the Treg cells. In this regard, Levings M.K. and colleagues (Dawson et al., 2020) showed that the incorporation of the CD28 domain in the cytoplasmatic region of the CAR significantly enhanced the properties of the Treg cells, contrary to other constructs incorporating other fragments such as CD137 (also known as 4-1 BB).

[0013] Despite the efforts made, however, there remains still the need of further Treg-cell based therapeutic approaches suitable in the management of immune-related conditions.

[0014] SUMMARY OF THE INVENTION

[0015] Prior to the present invention it was well-established the design of CAR-Treg cells based on the incorporation of CD28 domains in the cytoplasmatic region to potentiate their innate properties.

[0016] The inventors found, however, that in the case of thymus Treg cells (hereinafter also referred as ThyTreg cells), the incorporation of the CD28 domain within the cytoplasmatic domain of the CAR, provided a CAR-ThyTreg cell (hereinafter also referred as “CARCD28”) with a low ability to suppress CD8+ cell populations. In FIG. 7, one can see that CARCD28 provided a CD8+-cell suppression effect of around 50%.

[0017] The present inventors have surprisingly found that when CAR-ThyTreg cells incorporated a 4-1 BB domain (SEQ ID NO: 1) within the cytoplasmatic region of the CAR (hereinafter also referred as CAR41 BB-ThyTreg cells), the ability to reduce CD8+ cell population was remarkably improved, reaching more than 90% of the population (FIG. 7). In addition, it was also found that not only the proliferation was down-regulated, but also the viability of CD8+-cells was also significantly reduced (FIG. 5). Therefore, the administration of the ThyTreg cells with this particular CAR construct, based on incorporating 4-1 BB sequence within the cytoplasmatic domain, provides a dual negative effect on the target CD8 population, acting at the level of viability, but also at the level of proliferation.

[0018] The inventors have also found that CAR41 BB-ThyTreg significantly affected the suppression of CD4 cell population (see FIG. 7).

[0019] These experimental data support the immunotolerance-enhancer effect provided by the incorporation of the 4-1 BB domain within CAR’s cytoplasmatic domain on ThyTreg population.

[0020] The above is surprising in view of the prior art (Dawson et al., 2020) wherein it was reported that the incorporation of a 4-1 BB domain provided a reduced activity and that the significant activity was achieved when CD28 was included instead. Not only that, but also, this prior art did not provide any point at all to consider the incorporation of 4-1 BB when designing a CAR-Treg cell with enhance immunotolerance.

[0021] Altogether, the data provided below show that the incorporation of 4-1 BB domain remarkably potentiates the innate properties of the ThyTreg cells, being suitable in the management of diseases and conditions induced or related to an over-activity of the immune system.

[0022] Therefore, in a first aspect the present invention provides a thymus T regulation cell (hereinafter referred as “ThyTreg cell”) which codes for, or alternatively expresses on its surface, a chimeric antigen receptor (CAR) comprising an extracellular domain, a hinge region, a transmembrane domain, and an intracellular domain, wherein the intracellular domain comprises: - a cytoplasmatic co-stimulation domain with a sequence having an identity of at least 85% with respect to SEQ ID NO: 1

[0023] (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL); and

[0024] - a cytoplasmatic stimulation domain, herein also referred as CAR ThyTreg cell; or a Thy Treg cell population comprising or consisting thereof.

[0025] Without being bound to the theory, the present inventors believe that the remarkably different behaviour of ThyTreg cells expressing a CAR including 4-1 BB, is due to the particular marker profile expressed on their surface, CD4+CD8+FOXP3.

[0026] In a second aspect, the present invention provides a process for preparing a CAR- ThyTreg cell population of the invention, the process comprising the step of transfecting or transducing a ThyTreg cell with an expression construct coding for the CAR as defined in the first aspect of the invention.

[0027] In a third aspect, the present invention provides the CAR-ThyTreg cell or cell population obtainable by the process of the second aspect of the invention.

[0028] In a fourth aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the ThyTreg cells object of the invention, together with one or more pharmaceutically acceptable excipients or carriers.

[0029] In a fifth aspect the present invention provides a combination comprising (a) a ThyTreg cell population as provided by the present invention functionalized with one member of a pair of specific binder moieties, particularly a member with the ability to bind to biotin, more particularly streptavidin, and (b) a binding moiety specific of an activated immune cell, which is conjugated to the second member of the pair of specific binder moieties, particularly conjugated to biotin or a fragment thereof.

[0030] In a sixth aspect, the present invention provides the CAR-ThyTreg cell or cell population as defined in the first or third aspect, or the combination of the fifth aspect of the invention, for use in therapy or diagnosis.

[0031] In a seventh aspect, the present invention provides the CAR-ThyTreg cell or cell population or the combination of the invention for use in inducing or restoring tolerance to the immune system. This aspect can be formulated as the use of a CAR-ThyTreg cell or the combination of the invention for the manufacture of a medicament in inducing or restoring tolerance to the immune system. This aspect can also be alternatively formulated as a method for inducing or restoring tolerance to the immune system in a subject, the method comprising administering an effective therapeutic amount of the CAR-ThyTreg cell of the invention, pharmaceutical composition, or combination, to a subject in need thereof.

[0032] Antigen-specific Tregs have been shown to be more effective in suppressing immune responses than polyclonal ones. Our study has shown that CAR-ThyTregs hold great promise due to their stable phenotypes and functions, and their ability to provide more potent and specific immunosuppression compared to polyclonal Tregs. Furthermore, we have found that the intracellular 4-1 BB signal is more effective in inducing suppressive function. This is important because 4-1 BB allows for less exhaustion of the cells, ensuring their durability in treated individuals and enabling them to effectively carry out their immunosuppressive function. This is particularly crucial in autoimmune diseases, immune rejection, allergies, and chronic inflammatory processes, where CAR-ThyTregs must combat chronic inflammatory states.

[0033] Thus, the present invention provides in a final aspect the CAR-ThyTreg cells, pharmaceutical composition, or combination of the invention for use in the treatment of a disease selected from: autoimmune disease, inflammatory processes, allergy, graft- versus host disease, or immune rejection to transplant. This aspect can be formulated as the use of CAR-ThyTreg cells, pharmaceutical compositions, or combinations of the invention for the manufacture of a medicament for the treatment of a disease selected from: autoimmune disease, inflammatory processes, allergy, graft-versus host disease, or immune rejection to transplant. This aspect can also be alternatively formulated as a method for the treatment of a disease selected from: autoimmune disease, inflammatory processes, allergy, graft-versus host disease, or immune rejection to transplant, the method comprising the step of administering a therapeutically effective amount of the CAR-ThyTreg cells, pharmaceutical compositions, and combinations of the invention to a subject in need thereof.

[0034] BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1. (A) Scheme of the CAR-41 BB cDNA sequence. (B) Scheme of the CAR-41 BB protein structure. (C) Scheme of the CAR-41 BB-ThyT reg recognizing one biotinylated antibody specific of a cell surface marker. (D) full-length nucleotide sequence coding CAR4-1 BB. (h: hinge; tm: transmembrane; sd: signalling domain).

[0036] FIG. 2. (COMPARATIVE PURPOSE). (A) Scheme of the CARCD28 cDNA sequence. (B) Scheme of the CARCD28 protein structure. (C) full-length nucleotide sequence coding CARCD28. (h: hinge; tm: transmembrane; sd: signalling domain).

[0037] FIG. 3. (A) Scheme of the CAR-thyTreg recognizing target cells specifically through a biotinylated antibody. (B) Scheme of all co-culture conditions to test the CAR-thyTreg phenotype and function.

[0038] FIG. 4. (A) Dot plot of the CD4 and CD8 gated on living CELLTRACE violet positive target cells. (B) Ratio CD8:CD4 of the targets PBMC in the co-culture experiment with thyTreg NT or engineered thyT reg in combination with specific, non-specific, biotinylated and non-biotinylated antibodies.

[0039] FIG. 5. Represents the frequency of viability of target cells. (A) Frequency of CD8 viability, gated on total living target cells. (B) Frequency of CD8 viability, gated on total living target cells.

[0040] FIG. 6. Represents the viability and CAR expression after 8 days post-transduction. (A) Dot plots from flow cytometry analysis showing the size (FSC) and 7AAD (viability) of the cells, non-treated (NT) or genetically modified by viral vectors. (B) Dot plots from flow cytometry analysis showing the GFP and CAR expression (labelled with Atto665-biotin) of the cells, non-treated (NT) or genetically modified by viral vectors.

[0041] FIG. 7. Represents the frequency of the suppression of CD4+ T (dark grey) and CD8+ T (light grey) cell proliferation gated on total living target cells.

[0042] FIG. 8. Obtention and characterization of thymocytes from human thymuses. (A) Representative flow cytometry plot and summary data of thymocytes viability after mechanical dissociation of thymic tissue. (B) Representative flow cytometry plot and summary of the CD4 / CD8 phenotype of thymocytes. (C) Representative flow cytometry plot showing the frequency of CD25+ thymocytes and the expression of FOXP3 within CD25+ thymocytes. Graphs show mean ± SEM. FIG. 9. Comparison of freshly isolated thyTreg with or without previous CD8 depletion. We compared thyTreg cells obtained using the standard protocol (without previous CD8 depletion, w / o depletion, n=17; orange) or with an extra step of CD8 depletion employing the complement-mediated lysis technique with anti-CD8a (OKT-8) and rabbit complement HLA-ABC before CD25+ selection (n=3, green). (A) Yield of thyTreg obtained at day 0. (B) Representative flow cytometry plots and (C) summary of thyTreg CD4 / CD8 phenotype with or without CD8 depletion. (D) Representative flow cytometry histograms and summary data of FOXP3 expression frequencies within isolated thyTreg on day 0. Graphs show mean ± SEM. Comparison between strategies was made using unpaired Mann-Whitney test; *, P < 0.05; and **, P < 0.01. Triangle symbol represents the thyTreg cell products obtained in parallel with both strategies from the same thymic tissue.

[0043] FIG. 10. Characteristics of manufactured thyTreg. (A) Isolation and culture protocol for thyTreg obtention. (B) Representative flow cytometry dot plots showing the viability, purity and CD4 / CD8 phenotype of thyTreg right after isolation (day 0) or after culture (day 7). (C) Summary of the cell viability, purity and CD4 / CD8 phenotype of n=16 thyT reg at days 0 (blue) and 7 (orange). The graph shows min-median-max. **,P < 0.01 and ***,P < 0.001 (paired Wilcoxon test). (D) Representative flow cytometry histograms showing CD25 (left) and FOXP3 (right) expression in thyTreg CD4 / CD8 subsets. To determine the background signal, the fluorescence minus one (FMO) of FOXP3 is shown. (E) Correlation between the frequency of CD4+CD8+DP and the purity of thyTreg product (Pearson correlation analysis).

[0044] FIG. 11. Optimization of thyTreg culture conditions. Freshly isolated thyTreg were cultured in parallel under the standard conditions (orange) or under the test condition (green), and their phenotype was evaluated at day 7. (A) Purity, (B) phenotype, and (C) fold expansion of thyTreg cultured without or with rapamycin at 50 nM final concentration (n=4). (D) Purity, (E) phenotype, and (F) fold expansion of thyTreg cultured without or with human 5% AB serum (n=3). (G) Representative flow cytometry dot plots and (H) summary of purity, (I) phenotype and (J) fold expansion of thyTreg stimulated with TransAct or with Dynabeads at ratio 1 :1 (n=3). Graphs show mean ± SEM. We determined no significant differences between cultured conditions by paired Wilcoxon test.

[0045] FIG. 12. Additional thyTreg characterization. (A) Fold expansion of thyTreg cells over the manufacturing protocol, n=16 (mean ± SEM). (B, C) Summary of the evolution in phenotypic and functionality markers expression within thyTreg cells between day 0 (blue) and day 7 (orange). Graphs show min-median-max. *, P < 0.05; **, P < 0.01 ; and ***, P < 0.001 (unpaired Mann-Whitney test). (D) Global demethylation level of 27 genome regions located in 20 genes (calculated as the mean of demethylation of the CpGs contained in the region) within n=4 thyTreg cell products and n=2 thyTconv cultured in parallel for 7 days. ID13 and ID14 are female donors. The left panel depicts regions with different demethylation patterns within thyTreg and thyTconv. The right panel shows regions with similar demethylation patterns within thyTreg and thyTconv.

[0046] FIG. 13. (A) Frequency of phenotypic and functionality markers within thyTreg cells (day 7). (B) Frequency of homing markers within thyTreg cells (day 7). (C) Quantitation of molecules secreted in day 7 thyTreg culture supernatants. Anti-inflammatory molecules in blue; proinflammatory molecules in red. (D) Representative flow cytometry histograms showing CD4 (green) and CD8 T (purple) cell proliferation as CellTrace Violet lost. C-, negative control of proliferation, PBMC cultured alone without stimulation; C+, positive control of proliferation, PBMC cultured alone with anti-CD3 / anti-CD28 stimulation; 1 :1 to 1 :8, stimulated PBMC cultured with thyTreg cells at different thyTreg:PBMC ratios. (E) Summary of the suppressive capacity of thyTreg cells defined as % inhibition of CD4 (green) and CD8 T (purple) cell proliferation at the indicated ratios. Graphs show mean ± SEM.

[0047] FIG. 14. Stability of thyTreg cell product. (A-D, G) thyTreg cell product was restimulated under control conditions (CT, blue), or under Th1 (orange) or Th17 (green) polarizing conditions and evaluated after 3 days of culture. PBMC were cultured in parallel under the same conditions. (A) Representative flow cytometry histogram showing FOXP3 expression. To determine the background signal, the fluorescence minus one (FMO) of FOXP3 is shown. (B) Frequency of FOXP3, CTLA-4, CD39 and HLA-DR within thyTreg under different culture conditions. Paired Wilcoxon test showed no significant differences between conditions. (C) Quantitation of secreted IFN-y and IL-17A by thyTreg or PBMC under different culture conditions. Comparison between culture conditions within the same cell type was performed using paired Wilcoxon test, and comparison within the same condition between thyTreg and PBMC were performed using unpaired Man- Whitney test (#, P < 0.05). (D) Summary (n=4) of the suppressive capacity of thyTreg cells cultured under different polarizing conditions defined as % inhibition of CD4 (upper panel) and CD8 T (lower panel) cell proliferation at the indicated ratios. Graphs show mean ± SEM. Paired Wilcoxon test showed no significant differences between conditions. (E) Demethylation level of 11 conserved CpGs at the TSDR region of FOXP3 in n=4 thyTreg cell products and n=2 thyTconv cultured in parallel for 7 days. ID13 and I D14 are female donors. (F) Global TSDR demethylation level (calculated as the mean of demethylation of the 11 CpGs) of thyTreg and ThyTconv right after cell isolation (day 0, blue) or after 7 days of culture (day 7, orange). Triangles represent female donors, and circles represent male donors. (G) Global TSDR demethylation level of thyTreg cultured under different polarizing conditions.

[0048] FIG. 15. TSDR methylation pattern of total thyTreg, sorted CD4+SP and sorted CD4+CD8+DP. (A) Flow cytometry plots show the CD4 / CD8 phenotype of one thyTreg product before (input) and after sorting of CD4+SP and CD4+CD8+DP subpopulations. The purity of sorted fractions is above 90%. (B) Percentage of methylated and demethylated CpG islands within the FOXP3 TSDR of the input and sorted fractions.

[0049] FIG. 16. ThyT reg’s marker expression on the thyTreg genetically modified or not. Histograms represent the frequency of living CD25+FOXP3+ thyTreg cells expressing the surface and intracellular markers in non-transduced thyTreg (NT) and genetically modified thyTreg with the second-generation CAR with the 41 -BB intracellular domain (CAR41 BB-thyTreg). ThyTreg were labeled for surface (CD73, LAG-3, HLA-DR, CTLA- 4, ICOS, GITR, Helios, LAP, CCR7, CCR4, CD62L, CXCR3, CD27, and CD103) and intracellular (IL-2, IFNgamma, IL-10, and Granzyme B) markers after activation and cell sorting 8 days post-transduction.

[0050] DETAILED DESCRIPTION OF THE INVENTION

[0051] Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood. Any methods and materials similar or equivalent to those described herein can be used to test the present invention. However, the preferred materials and methods are described herein. It is also to be understood that the terminology herein describes particular embodiments only and is not intended to be limiting.

[0052] Throughout the present specification and the accompanying clauses, the words "comprise" and variations such as "comprises", "comprising" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows. The word “comprise” also includes the term “consists of”.

[0053] In the context of the invention, “T regulation cell”, “T regulatory cell” and “Treg cell” are considered synonyms. “Thymus T regulation cell”, “thymus T regulatory cell” and “ThyTreg cell” are understood as synonyms.

[0054] A “cell population” is understood as more than one cell, preferably a group of cells.

[0055] In a first aspect the present invention provides a CAR-ThyTreg cell as defined herein above or a ThyTreg cell population comprising or consisting thereof. Typically, the thyTreg cell population will comprise at least 40%, preferably at least 45%, at least 50%, at least 55%, at least 60%, at least 65% or at least 70% of CAR-ThyTreg cells. In some embodiments, the thy Treg cell population comprises from 40% to 80% of CAR-ThyTreg cells, such as from 50% to 70% of CAR-ThyTreg cells.

[0056] The CARs of the invention can redirect the ThyTreg specificity and reactivity towards a cell associated with hyper-activation, autoimmune pathology or transplant rejection. Those cells express the antigen that will be recognized by the tagged biomolecules (e.g. monoclonal or polyclonal antibodies, aptamers, nanoparticles, proteins or peptides, and alike).

[0057] In the context of the invention, “Chimeric Antigen Receptor (CAR)” refers to a chimeric construct comprising an extracellular domain joined to a hinge, a transmembrane, and one or more intracellular signalling domains.

[0058] In some embodiments, the cytoplasmatic co-stimulation domain with a sequence having an identity of at least 85%, preferably at least 90%, at least 95%, 96%, 97%, 98%, 99% ore even 100% with SEQ ID NO: 1 , is further characterized by providing a signal mediating a Treg cell specific response; and a cytoplasmatic stimulation domain; or a Thy Treg cell population comprising or consisting thereof.

[0059] The ThyTreg cell activation or ThyTreg cell specific response can be assessed by measuring the expression of activation markers such as, but not limited to, CD71 , CD69, HLA-DR, PD-1 , or ICOS, by flow cytometry or other techniques. In addition, the ThyTreg cell activation or ThyTreg cell specific response can be determined by measuring the cells capacity of inhibiting or blocking effector T cell activation or proliferation, such as described herein in the Examples.

[0060] In one embodiment of the first aspect, the CAR-ThyTreg cell comprises the recombinant nucleic acid coding for the CAR4-1 BB of SEQ ID NO: 7 or 8, or one having at least 85%, preferably at least 90%, at least 95%, 96%, 97%, 98%, 99% or even 100% of identity with SEQ ID NO: 7 or 8. In an alternative embodiment, the ThyTreg cell expresses on the surface a CAR amino acid sequence SEQ ID NO: 6 (CAR4-1 BB) or one having at least 85%, preferably at least 90%, at least 95%, 96%, 97%, 98%, 99% or even 100% of identity with SEQ ID NO: 6. In the present invention, the terms “4-1 BB”, “41-BB” and “41 BB” are considered as synonyms.

[0061] The term "identity" as used herein refers to an exact nucleotide-to-nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity. The “percent identity” of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequence and multiplied by 100. Suitable programs for calculating the percent identity or similarity between sequences are well known in the art, such as the NCBI BLAST program, used for example with default parameters (http: / / www. ncbi. nlm. gov / cgi-bin / BLAST).

[0062] In some embodiments, the CAR is a universal CAR. Universal CAR-T cells can be allogeneic cells that are taken from healthy donors. The T cell can be transduced with several gene-editing methods, such as but without being limited to, zinc-finger nucleases (ZFN), transcription activator-like nucleases (TALENs) and CRISPR / Cas9. Universal CAR-T therapy can also be produced without gene editing.

[0063] The structure of universal CARs can be modular. The CAR is split into two parts: (i) the signaling module on T cells, consisting of the extracellular domain that specifically binds to the switching module and the intracellular domain that transmits the activation signals; (ii) the switching module, usually a bispecific antibody or small molecule recognized by the signaling module on T cells and binding to the targets on the target cells (e.g., an activated immune cell). This split, universal, and programmable (SUPRA) CAR system currently adopts a variety of recognition modes including but not limited to neo-epitopes, SpyTag, biotin, and fluorescein isothiocyanate (FITC) and leucine zippers (Lin et al., 2021 , Sutherland, Owens and Geyer, 2020). In one embodiment, the extracellular domain comprises a molecule from a pair of complementary affinity molecules. In some embodiments, a pair of complementary affinity molecules is selected from the group consisting of: biotin / biotin-binding moiety, antibody / antigen, enzyme / substrate, receptor / ligand, metal / metal-binding protein, carbohydrate / carbohydrate-binding protein, lipid / a lipid-binding protein; and a His- tag / His-tag-binding molecule. In some embodiments, the extracellular domain comprises leucine zippers, anti- fluorescein isothiocyanate (FITC) system, bacterial toxin-antitoxin system, FITC-folate adapter, peptide neoepitope (PNE)-mAb system or a photolyzable molecule adapter system. In preferred embodiments, the pair of complementary affinity molecules is biotin / biotin-binding moiety. In this embodiment, the CAR-ThyTreg cells of the invention will not directly bind to the target but will require the interaction with the other member of the pair of specific binding moieties (in the examples below, a biotinylated intermediate) to stimulate the specific function of the CAR-ThyTreg cells.

[0064] In another embodiment, the extracellular domain includes a biotin-binding moiety. Illustrative non-limitative examples of biotin-binding moieties suitable in the context of the invention are rizavidin, avidin, streptavidin, bradavidin, tamavidin, lentiavidin, zebavidin, neutravidin, captavidin™, combinations or functional fragments thereof. By “functional fragment” it is understood a fragment of the biotin-binding moiety which retains part or all the binding ability of the full-length moiety. In this embodiment, the CAR-ThyTreg cells of the invention will not bind to the target, but to biotinylated moieties (such as an antibody or fragment thereof, or aptamer, for instance) to stimulate the specific function of the CAR-ThyTreg cells.

[0065] In one embodiment, the extracellular domain comprises or consists of streptavidin of a fragment thereof.

[0066] Streptavidin is a 52.8 kDa protein from the bacterium Streptomyces avidinii. Streptavidin exists in nature as a homo-tetramer. The secondary structure of a streptavidin monomer is composed of eight antiparallel p-strands, which fold to give an antiparallel beta barrel tertiary structure. A biotin binding-site is located at one end of each p-barrel. Four identical streptavidin monomers (i.e. four identical p-barrels) associate to give streptavidin's tetrameric quaternary structure. The biotin binding-site in each barrel consists of residues from the interior of the barrel, together with a conserved Trp120 from neighbouring subunit. In this way, each subunit contributes to the binding site on the neighbouring subunit, and so the tetramer can also be considered a dimer of functional dimers. The streptavidin domain of the CAR system of the present invention may consist essentially of a streptavidin monomer, dimer, or tetramer. Particularly, it is a monomeric streptavidin having a sequence with an identity of at least 85%, at least 90% or at least 95% with respect to SEQ ID NO: 2 (high affinity monomeric streptavidin domain, known as mSA2, already disclosed in Lohmueller, J. J., et al., 2017):

[0067] SEQ ID NO: 2

[0068] METDTLLLWVLLLWVPGSTGGAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRA QGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQ WNLTYEGGSGPATEQGQDTFTKVKPSAASGS

[0069] In another embodiment of the first aspect of the invention, the extracellular domain does not include neither is fused to an antibody or fragment thereof.

[0070] In one embodiment, the Chimeric Antigen Receptor (CAR) of the invention comprises an extracellular domain having a monomeric high-affinity streptavidin recognition domain, a hinge region, a transmembrane domain, and one or two cytoplasmic domains. In this embodiment, the extracellular domain does not include neither is fused to an antibody or fragment thereof.

[0071] The hinge region in CARs functions as a flexible spacer for the extracellular domain and improves the recognition / function of CARs for antigens closer located to the surface of target cells. For large number of constructs, the hinge region may consist of the CH2CH3 domain of a human IgG antibody, which can bind to Fc-receptors on macrophages and other cells, thereby resulting in cross-activation and activation-induced cell death in vivo independent of recognition of the target antigen. In one embodiment of the invention, the hinge region originates from CD8a (SEQ ID NO: 3):

[0072] SEQ ID NO: 3

[0073] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0074] The CAR must include a transmembrane domain fused to the hinge and intracellular domains. The transmembrane domain can be derived from a natural or a synthetic source. When the transmembrane domain is from a natural source, the domain can be derived from any membrane-bound or transmembrane protein. For example, transmembrane domain from CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD154. Preferably, the transmembrane domain is the CD28 transmembrane domain having SEQ ID NO: 4:

[0075] M FWVLVWGGVLACYSLLVTVAFI I FWV.

[0076] In the context of the present invention, the term “Intracellular Domain” is the signaltransmission portion of a classical CAR. In the signalling system of the present invention the intracellular signalling domain (signalling domain) is located in the signalling component.

[0077] In the context of the present invention, the term “co-stimulation domain” refers to a signalling moiety that provides to T cells a signal which, in addition to the primary signal provided by, for instance, the CD3 chain of the TCR / CD3 complex, mediates a T cell response, including, but not limited to, activation, proliferation, differentiation, cytokine secretion, and the like. A co-stimulatory domain can include, in addition to 4-1 BB (SEQ ID NO: 1), all or a portion of CD27, CD28, 0X40 (CD134), CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83. In some embodiments, the co-stimulatory signalling domain is an intracellular signalling domain that interacts with other intracellular mediators to mediate a cell response including activation, proliferation, differentiation and cytokine secretion, and the like. In one embodiment of the present invention, the cytoplasmatic co-stimulation domain is CD3zeta (SEQ ID NO: 5): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R

[0078] In one embodiment, the co-stimulatory domain, in addition to 4-1 BB, is CD28. In one preferred embodiment, the intracellular domain comprises the fused CD28-4-1 BB sequence referred as SEQ ID NO: 12 (SEQ ID NO: 12): RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGGGKRGRKKLLYIFK QPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0079] In an alternative embodiment, the intracellular domain consists of 4-1 BB sequence SEQ ID NO: 1.

[0080] In another embodiment of the first aspect of the invention, the CAR comprises (i) mSA2, (ii) CD8a hinge region, (iii) CD28 transmembrane region, (iv) SEQ ID NO: 1 or 7, and (v) CD3zeta, wherein (i) to (v) are provided in N-terminal to C-terminal order. In one embodiment, the CAR sequence is at least 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100 % identical to SEQ ID NO: 6:

[0081] SEQ ID NO: 6:

[0082] METDTLLLWVLLLWVPGSTGGAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRA QGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQ WNLTYEGGSGPATEQGQDTFTKVKPSAASGSTTTPAPRPPTPAPTIASQPLSLRPEA CRPAAGGAVHTRGLDFACDMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIF

[0083] KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0084] The nucleic acid coding to the CAR of the invention is a sequence with at least 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100 % of identity with SEQ ID NO: 7:

[0085] GGCGCAGAGGCGGGTATCACCGGGACATGGTACAACCAACACGGAAGCACATTT ACAGTCACCGCTGGAGCAGACGGGAATCTGACCGGACAGTACGAGAACAGGGCT CAGGGGACAGGTTGTCAGAACAGTCCGTATACTCTGACTGGGAGGTACAATGGC ACGAAGCTGGAGTGGCGAGTCGAGTGGAATAATTCCACGGAAAACTGTCACAGTA

[0086] GAACAGAGTGGAGGGGACAGTACCAGGGGGGAGCAGAGGCCCGGATCAACACC CAATGGAACTTGACATATGAAGGCGGGTCAGGCCCCGCGACAGAGCAAGGACAG GATACATTCACGAAGGTCAAGCCAAGCGCAGCCTCTGGCTCTACCACAACTCCAG CTCCCCGGCCCCCTACTCCTGCTCCAACCATTGCCTCACAGCCACTGAGCCTGC

[0087] GGCCCGAAGCTTGTAGACCTGCTGCTGGAGGAGCTGTGCATACCAGAGGCCTGG ACTTCGCCTGCGATATGTTCTGGGTGCTGGTGGTGGTGGGCGGGGTGCTGGCCT GCTACAGCCTGCTGGTGACAGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAG AAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCA

[0088] AGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGA ACTGCGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCA GAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCT GGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGA

[0089] ACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCT ACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGG CCTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACAT GCAGGCCCTGCCCCCAAGG

[0090] Before expansion and genetic modification of the regulatory T cells of the invention, a source of ThyTreg cells is obtained from a subject.

[0091] Preferably, the Treg cells suitable in the context of the invention are characterized by comprising CD4+CD8+FOXP3. A population with these suitable Treg cells can be obtained following the method disclosed in EP3759215A1 , whose content is incorporated herein by reference. The resulting cells have a great capacity to suppress effector cells of the immune system, an undifferentiated (naive) phenotype, high viability, and a stable expression of FOXP3 even under pro-inflammatory conditions and functional markers such as CTLA-4 and CD39.

[0092] In some embodiments, the ThyTreg cell population of the invention comprises at least 60% of CD25+FOXP3+ cells, preferably at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, 96%, 97%, 98%, 99% or even 100% of CD25+FOXP3+ cells.

[0093] In some embodiments, optionally in combination with any of the features or embodiments described herein, the ThyTreg cell population of the preceding aspects wherein at least 10% of CD4+CD8+ cells, preferably at least 15%, at least 20%, at least 25%, at least 30%, or at least 35%, even more preferably at least 40% of CD4+CD8+ cells.

[0094] In some embodiments, optionally in combination with any of the features or embodiments described herein, at least 60%, preferably at least 65%, more preferably at least 70%, even more preferably at least 75%, 80%, 85%, 90%, 95% or even 100% of the cells of the ThyTreg cell population express one or more of the markers selected from the group consisting of cytotoxic T-lymphocyte associated protein (CTLA-4), inducible T-cell costimulator (ICOS), HELIOS, latency-associated peptide (LAP), CD27, CCR4 and CD62L. In a preferred embodiment, at least 60%, preferably at least 65%, more preferably at least 70%, even more preferably at least 75%, 80%, 85%, 90%, 95% or even 100% of the cells of the ThyTreg cell population express one or more of the markers selected from the group consisting of cytotoxic T-lymphocyte associated protein (CTLA-4), inducible T-cell co-stimulator (ICOS), HELIOS, latency-associated peptide (LAP), CCR4 and CD62L.

[0095] In another embodiment, optionally in combination with any of the features or embodiments described herein, at least 25%, preferably at least 30%, 35%, 40%, 45% or at least 50% of the cells of the ThyTreg cell population express one or more of the markers selected from the group consisting of T cell immunoreceptor with Ig and ITIM domains (TIGIT), glucocorticoid-induced tumor necrosis factor receptor (GITR), latency- associated peptide (LAP), HLA-DR and CD45RA. In a preferred embodiment, at least 25%, preferably at least 30%, 35%, 40%, 45% or at least 50% of the cells of the ThyTreg cell population express one or more of the markers selected from the group consisting of glucocorticoid-induced tumor necrosis factor receptor (GITR) and HLA-DR. In preferred embodiments of any thereof, the levels of expression are from 25% to 60%, preferably from 30% to 50%.

[0096] In a further embodiment, optionally in combination with any of the features or embodiments described herein, 20% or less, preferably 15% or less, 10% or less, 5% or less, 2% or less, 1 % or less, 0.5% or less, 0.25% or less, or even 0.1% or less of the cells of the ThyTreg cell population express one or more of the markers selected from the group consisting of CD39, CD73, lymphocyte activation gene 3 (LAG-3) and CXCR3. In a preferred embodiment, 20% or less, preferably 15% or less, 10% or less, 5% or less, 2% or less, 1% or less, 0.5% or less, 0.25% or less, or even 0.1% or less of the cells of the ThyTreg cell population express one or more of the markers selected from the group consisting of CD73, lymphocyte activation gene 3 (LAG-3) and CXCR3.

[0097] Preferably, the ThyTreg cell population is characterized by: a) at least 60%, preferably at least 65%, more preferably at least 70%, even more preferably at least 75%, 80%, 85%, 90%, 95% or even 100% of the cells of the ThyTreg cell population express CD27 and / or CCR4; b) at least 25%, preferably at least 30%, 35%, 40%, 45% or at least 50% of the cells of the ThyTreg cell population express HLA-DR and / or CD45RA; and c) 20% or less, preferably 15% or less, 10% or less, 5% or less, 2% or less, 1 % or less, 0.5% or less, 0.25% or less, or even 0.1 % or less of the cells of the ThyT reg cell population express CD73, LAG-3 and / or CXCR3. In one embodiment, the ThyTreg cell population as described herein, is characterized by a high level of expression of anti-inflammatory cytokines, such as IL-10 and / or TGF-p, and / or other inhibitory molecules associated to Treg functionality, such as granzyme B, soluble LAG-3 and / or TIM-3, preferably wherein the expression levels are of at least 50 pg / ml, more preferably at least 100 pg / ml, wherein the expression levels are determined as specified in the Examples with regards to cytokine production analysis.

[0098] In one embodiment, the ThyTreg cell population as described herein, is characterized by expression levels of at least 100 pg / ml, preferably at least 150 pg / ml of IL-10 and / or TGF- P, wherein the expression levels are determined as specified in the Examples with regards to cytokine production analysis.

[0099] In preferred embodiments, the ThyTreg cell population as described herein is characterized by: a) expression levels of at least 100 pg / ml of granzyme B, soluble LAG-3 and / or TIM- 3; and / or b) expression levels of less than 10 pg / ml of IL-17-A and / or PD-L1 , wherein the expression levels are determined as specified in in the Examples with regards to cytokine production analysis.

[0100] In one embodiment, the ThyTreg cell population as described herein is characterized by having a stable FOXP3 expression. In some embodiments, the ThyTreg cell population derives from a male subject and has at least 70%, preferably at least 75%, at least 80%, at least 85% or more preferably at least 90% of demethylation in the Treg-specific demethylated region (TSDR) of the FOXP3 gene. In other embodiments, the ThyTreg cell population derives from a female subject and has at least 30%, preferably at least 35%, at least 40%, or more preferably at least 45% of demethylation in the Treg-specific demethylated region (TSDR) of the FOXP3 gene.

[0101] In another embodiment, the ThyTreg cell population as described herein has a mean inhibitory capacity of CD4+ and / or CD8+ T cells proliferation of at least 60%, preferably at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or more preferably at least 90% at a thyTreg : responder cells ratio of 1 :1 ; and / or a mean inhibitory capacity of CD4+ and / or CD8+ T cells proliferation of at least 25%, preferably at least 30%, at least 35% or more preferably at least 40% at a thyTreg : responder cells ratio of 1 :4.

[0102] In one embodiment, the ThyTreg cell population as described herein does not comprise effector T cells.

[0103] In one embodiment, the thymic Treg cells are obtained by a method comprising or consisting of the following steps: a. mechanically disaggregating an isolated thymic tissue; b. filtering the product obtained after stage (a), and resuspending the precipitate comprising thymocytes in a culture medium in the presence of IL-2; c. isolating CD25+ cells from the product obtained after stage (b); d. culturing the cell population obtained after stage (c) in a culture medium in the presence of a T cell activator and IL-2, wherein said T cell activator comprises at least CD3 and CD28 agonists; and e. removing the T cell activator from the culture medium of stage (d); f. optionally, further culturing the regulatory T cells in a culture medium in the presence of IL-2; with the proviso that prior to step (d) the cell population has not been depleted from CD8+ cells.

[0104] The thyTreg cells resulting from the above isolation method are typically characterized by a higher level of CD27, CD45RA, and CCR4; as well as a lower amount of activated HLA-DR, the latter reducing their immunogenicity. In preferred embodiments, the thyTreg cells obtained by the above isolation method are characterized as described herein above.

[0105] In the context of the invention, the “thymic tissue” is any tissue sample from the thymus, which is the gland of the lymphatic system where T cells or lymphocytes mature, located in front of the heart and behind the breastbone. The thymic tissue can be removed by any method known in the art that serves such purpose, such as for example by means of a thymectomy, which can be transsternal, transcervical or videoscopic. Preferably, the thymic tissue is removed, prior to carrying out the method, during a surgical intervention, more preferably intended for treating a heart disease, such as for example congenital heart disease, or during a heart transplant. Even more preferably, the thymic tissue of the invention is removed during a paediatric heart transplant. The thymic tissue can come from a human or a non-human mammal such as, for example, but not limited to, rodents, pigs, primates, ruminants, felines or canines. In a preferred embodiment of the method of the invention, the thymic tissue comes from a human, more preferably a human aged between 0 (newborn) and 16 years, even more preferably between 0 and 10 years, particularly between 0 and 24 months.

[0106] In another preferred embodiment, the thymic tissue comes from the same or different individual to whom the ThyTreg cells obtained at the end of the method of the invention are going to be subsequently administered for cell immunotherapy.

[0107] In the present invention, “paediatric patient” or “child” is understood to be a human aged between 0 and 16 years, preferably between 0 and 10 years, even more preferably between 0 and 24 months.

[0108] Any tissue dissociator from among those commercially available in the state of the art could be used in stage (a) of the method of the invention. Examples of these dissociators are, but not limited to, the gentleMACS Dissociator or the gentleMACS Octo Dissociator from Miltenyi Biotec, the TissueLyser LT from Qiagen or tissue dissociators from Worthington Biochemical, Sigma-Aldrich or Roche Diagnostics. Preferably, the tissue dissociator used in the present invention is the gentleMACS Octo Dissociator from Miltenyi Biotec in one embodiment, step a) comprises mechanically disaggregating the thymic tissue in the presence of a culture medium and without using enzymes.

[0109] Whether before or after genetic modification of the T reg cells to express a desirable CAR, the T cells can be activated and expanded. Generally, the T cells of the invention are expanded by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. In the invention, once ThyTreg are freshly isolated from the thymus, and after verification of their purity by flow cytometry (CD25+FOXP3+), cells can be activated and expanded with anti-CD3 / anti-CD28 magnetic beads (Dynabeads™) at a ratio of 1 :1 (bead : cell). In one embodiment, the ratio of CD3:CD28 antibody bound to the beads ranges from 100:1 to 1 :100 and all integer values there between. In one embodiment, the cells can be re-activated and re-stimulated for expansion after 7 days after the first activation and expansion. In this embodiment the re-activation and re-stimulation can be done with anti-CD3 / anti-CD28 magnetic beads (Dynabeads™) or anti-CD3 / anti-CD28 matrix (Miltenyi Biotec™). The ratio between the matrix and the cells ranges from 1 :50 to 1 :1000, with all integer values between. In one embodiment, the cells (104to 109ThyTreg cells) and matrices or beads are combined in culture medium, preferably X-vivo15 or PBS (without divalent cations such as calcium and magnesium). Those of ordinary skill in the art can readily appreciate any cell concentration that may be used. For example, a concentration of about 2 million cells / ml is used in one embodiment. In another embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, 50 or 100 million cells / ml is used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion.

[0110] In one embodiment, the mixture may be cultured for several hours (about 18 hours) to about 72 hours or any hourly integer value in between. Several cycles of stimulation / expansion may also be desired such that the culture time of T cells can be 28 days or more.

[0111] Conditions appropriate for T cell culture include an appropriate media, preferably X- Vivo 15 (Lonza), but also Minimal Essential Media or RPMI Media1640 (Lonza) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), IL-7, or IL-15.

[0112] Other additives for the growth of cells include, but are not limited to, reducing agents such as 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, C- MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or Supplemented with an appropriate amount of serum (or plasma), and / or an amount of cytokine(s) sufficient for the growth and expansion of T cells. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37°C.) and atmosphere (e.g., air plus 5% CO).

[0113] 24h after activation and without removing activation beads, viral vectors coding for our CAR are added to the cell mixture. 24h after lentivector treatment (in one embodiment, 48 hours after lentivector treatment), magnetic beads are removed with a magnet. Cells are then washed with a warm culture medium, preferably X-vivo15 complemented with 5% human serum, and tested for viability with the 7-Aminoactinomycin D (7-AAD, Figure 6A) and GFP expression by flow cytometry. The transduction frequency is around 40% (from 20 to 70%, Figure 6B). If cells presented less than 30% of transduction, GFP+ cells are sorted by the Tyto sorter (Miltenyi Biotec). Cells are then let to proliferate for 5 days adding fresh medium RPMI 1640 complemented with 5% human serum (Lonza) and recombinant human IL-2 (600 U / ml, preferably Immunotools). After 8 days, cells are labelled with a biotinylated marker (preferably Atto665-biotin, Sigma-Aldrich) for CAR detection (Figure 6B). Since the CAR plasmids encode mSA2 and GFP, the doublepositive cells were well-transduced and expressed GFP and CAR.

[0114] In one embodiment, the isolation of CD25+ cells in step c) comprises the use of magnetic beads conjugated to antibodies against CD25.

[0115] In one embodiment, the T cell activator of stage d) is a colloidal polymer nanomatrix (magnetic beads) conjugated with humanised CD3 and CD28 agonists. These magnetic beads facilitate the efficient activation of T cells while maintaining the viability thereof.

[0116] In one embodiment, the T-cell activator of stage d) is used in a T-cell activator: cell ratio range between 1 :10 and 1 : 100.

[0117] In one embodiment, the cells are cultured in step d) for at least 2 or 3 days, preferably for at least 3 days.

[0118] In one embodiment, the cells are cultured in step d) in the absence of rapamycin.

[0119] In one embodiment, the removal of stage e) is carried out by means of centrifugation or magnet.

[0120] In one embodiment, in step f) the regulatory T cells are cultured for another one to seven days, preferably for another four days in the presence of IL-2.

[0121] In one embodiment, the cells are cultured in step f) in the absence of rapamycin.

[0122] In one embodiment, said culture medium is a GMP culture medium.

[0123] In one embodiment, the culture medium further comprises antibiotics, preferably 5% of antibiotics.

[0124] The CAR-ThyTreg cells of the invention can be generated by introducing an expression construct comprising the CAR as defined in any of the embodiments provided above. In one embodiment, the expression construct comprises a sequence with at least 85%, at least 90% or at least 95% of identity with respect to sequence SEQ ID NO: 7.

[0125] The nucleic acid CAR sequence can be cloned into many types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and cosmid. Further, the expression vector may be provided to a cell as a viral vector. Viral vector technology is well-known in the art. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers.

[0126] The method of the present invention uses a DNA construct comprising a nucleic acid coding for the CAR as defined in any of the above embodiments. In one embodiment, the expression vector derives from a lentivirus. Lentivirus are suitable tools for long-term gene transfer since they allow long-term, stable transgene integration and its propagation in daughter cells. Lentiviral vectors have the advantage over other vector sources: they can transduce non-proliferating cells and have low immunogenicity.

[0127] The expression of the nucleic acid coding for the CAR object of the invention is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide or portions thereof to a promoter and incorporating the construct into an expression vector. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters helpful in regulating the expression of the desired nucleic acid sequence.

[0128] Illustrative non-limitative examples of promoter sequences are the cytomegalovirus (CMV), simian virus 40 (SV40) early promoter, mouse mammary tumour virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMul V promoter, an avian leukaemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as but not limited to, the actin promoter, the myosin promoter, the haemoglobin promoter, human elongation factor 1 alpha (EF1a), and the creatine kinase promoter. Further, the invention should not be limited to constitutive promoters. Inducible promoters are also contemplated as part of the invention. Using an inducible promoter provides a molecular switch capable of turning on the expression of the polynucleotide sequence, which is operatively linked when such expression is desired or turning off the expression when the expression is not desired.

[0129] In the context of the present invention, the terms “operably linked”, “operably linking”, refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in the expression of the later. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions in the same reading frame.

[0130] T o assess the expression of a CAR polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate the identification and selection of expressing cells from the population of cells sought to be transfected or transduced through viral vectors. Both, selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.

[0131] Reporter genes identify potentially transduced cells and evaluate regulatory sequences' functionality. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. The reporter gene can be coded in the same reading frame as the CAR sequences, separated by a T2A sequence, the 2A peptide from Thosea asigna virus capsid protein; or, alternatively, it can be under a promoter that can be different from the CAR promoter.

[0132] In another embodiment, the expression construct consists of a sequence with at least 85%, at least 90%, or at least 95% of identity with respect to sequence SEQ ID NO: 8.

[0133] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell, by any method in the art. Biological methods for introducing a polynucleotide of interest into a host cell include DNA and RNA vectors. Viral vectors, especially retroviral vectors, have become one of the most widely used methods for inserting genes into mammals, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno- associated viruses, and the like. Regardless of the method used to introduce exogenous nucleic acids into a host cell, various assays may be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include, for example, “molecular biological' assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISA, flow cytometry and Western blots) or by assays described herein to identify agents falling within the scope of the invention.

[0134] In a further aspect the present invention provides CAR-ThyTreg cells obtainable by the process of the invention.

[0135] The cells resulting from the above process are characterized by a high expression, among others, of perforin, granzyme B or both. Preferably, the cells show overexpression of both perforin and granzyme B.

[0136] For the purposes of the invention the expressions "obtainable", "obtained" and equivalent expressions are used interchangeably, and in any case, the expression "obtainable" encompasses the expression "obtained".

[0137] The present invention is based on improving the immunosuppressive ThyTreg ability in modulating immune system status in hyper-inflammation processes, autoimmune diseases, or transplant rejection, among others.

[0138] Accordingly, the invention provides CAR-ThyTreg cells and methods of their use for adoptive therapy. In one embodiment, the CAR-ThyTreg cells of the invention can be generated by introducing a lentiviral vector comprising a desired CAR, for example, a CAR comprising anti-high affinity monomeric streptavidin, CD8alpha hinge, a CD28 transmembrane domain, and human 4-1 BB and the CD3zeta signalling domains, into the cells. The CAR-ThyTreg cells of the invention can replicate in vivo resulting in longterm persistence that can lead to sustained immunosuppressive control.

[0139] In one embodiment, the invention relates to administering a genetically modified ThyTreg cell expressing a CAR for treating a patient with hyper-inflammation processes, autoimmune diseases, or transplant rejection. The CAR-ThyTreg could be autologous or allogeneic. The ThyTreg can be isolated from human thymus from pediatric patients undergoing cardiac surgery.

[0140] In a further aspect the present invention provides a combination comprising: a ThyTreg cell population as defined in any of the previous embodiments, incorporating (e.g., as extracellular domain of a CAR) a first molecule from a pair of complementary affinity molecules, such as a biotin-binding moiety; and an activated immune cell specific-binding moiety, which is conjugated to the second molecule from the pair of complementary affinity molecules, such as biotin, a biotin analog or a fragment of any thereof. A biotin analog can be, but is not limited to, a- dehydrobiotin, niocytin, norbiotin, norbiotin sulfoxide, norbiotin sulfone, norbiotin carbamate, norbiotin carbonate, homobiotin, oxybiotin, iminobiotin, biotin sulfone, biotin carbonate, noroxybiotin, noriminobiotin, nordesthiobiotin, nordiaminobiotin, homooxybiotin, homoiminobiotin, homodesthiobiotin, homodiaminobiotin, homobiotin sulfoxide, homobiotin sulfone, homobiotin carbamate, homobiotin carbonate, 2- imidazolidinone-4-butanoic acid, 2-imidazolidinone-4-propanoic acid, y imidazolidinone- 4-hexanoic acid.

[0141] All the embodiments provided under the first aspect of the invention, related to the biotinbinding moieties and CAR sequences included thereof, are also embodiments of the combination of the invention.

[0142] The “activated immune cell specific-binding moiety” can be, in the context of the invention, any molecule with the ability to bind to an immune cell. The term “immune cell” as used herein includes any cell that is involved in the generation, regulation, or effect of the acquired or innate immune system. Immune cells include T cells such as CD4+ cells, CD8+ cells and various other T cell subsets, B cells, natural killer cells, macrophages, monocytes and dendritic cells, and neutrophils. Thus, the “activated immune cell specificbinding moiety” can be an antibody, aptamer, cytokine, any nucleic or peptide sequence, or fragment thereof with the ability to bind to a particular component (marker) related to the activation of the immune cell. Illustrative non-limitative examples of surface markers already known in the state of the art as being specific of activated immune cells are CD19, CD30, CD8, and the interleukin 23 receptor, among others.

[0143] In the context of the invention the term “antibody”, refers to an immunoglobulin molecule which specifically binds with an antigen. Illustrative non-limitative examples of “antigen” in the context of the invention are such as, but not limited to, CD30, HLA-A2, CD19, Insulin, 2,4,6-trinitrophenol (TNP), Carcinoembryonic antigen (CEA), Myelinoligodendrocyte glycoprotein (MOG), Myelin basic protein (MBP), B cell maturation antigen (BCMA), or Desmosomal core glycoprotein-3 (DSG3).

[0144] Antibodies can be intact immunoglobulins derived from natural or recombinant sources and immunoreactive portions of intact immunoglobulins. Antibodies may exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, multispecific antibodies such as bispecific antibodies, humanised antibodies, and fragment antibodies, as long as they exhibit the desired target-binding activity. Non-limiting examples of antibody fragments are Fv, Fab, Fab, Fab-SH, and F(ab)2, diabodies, linear antibodies, single chain antibodies and multispecific antibodies formed from antibody fragments.

[0145] In one embodiment, the biotin binding moiety-CAR protein (e.g. mSA2-CAR protein) can be designed to target biotinylated aptamers.

[0146] In the context of the invention, the term “aptamer” refers to an oligonucleotide (nucleic acid aptamer) or peptide molecule that binds to a specific target molecule. Nucleic acid aptamers are sequences engineered through repeated selection rounds to bind to various molecular targets such as small molecules, proteins, nucleic acids, cells, or tissues.

[0147] In one embodiment, the biotin binding moiety-CAR protein (e.g. mSA2-CAR) can be designed to target biotinylated cytokines.

[0148] In one embodiment, the biotin binding moiety-CAR protein (e.g. mSA2-CAR) can be designed to target biotinylated receptor ligands.

[0149] In one embodiment, the biotin binding moiety-CAR protein (e.g. mSA2-CAR) can be designed to target a biotinylated nucleic acid sequence.

[0150] By “biotinylation” in biochemistry is the process of covalently attaching biotin to a protein, nucleic acid, or another biomolecule. There are well-known protocols in the state of the art to biotinylate the molecule of interest.

[0151] However, the invention should not be construed as limited solely to the targets and diseases disclosed herein. Instead, the invention should be construed to include any antigenic target associated with a disease where a CAR can be suitable to be used.

[0152] The CAR-modified T cells of the present invention may be administered either alone or as a pharmaceutical composition in combination with one or more pharmaceutical acceptable excipients or carriers.

[0153] As used herein, "pharmaceutically acceptable excipient", or "pharmaceutically acceptable carrier" means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and, without limiting the scope of the present invention, include: additional buffering agents; preservatives; co-solvents; antioxidants, including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers, such as polyesters; salt-forming counterions, such as sodium, polyhydric sugar alcohols; amino acids, such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactitol, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [alpha]- monothioglycerol, and sodium thio sulfate; low molecular weight proteins, such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; and hydrophilic polymers, such as polyvinylpyrrolidone.

[0154] In the context of the invention, the term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject being sought by the researcher, veterinarian, medical doctor, or other clinicians. The term “therapeutically effective amount” includes the amount of a compound that, when administered, is sufficient to prevent the development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated. The dose for obtaining a therapeutically effective number depends on a variety of factors such as example, age, weight, sex, pathological condition, or tolerance of the individual to whom the composition of the invention is going to be administered. Preferably, the therapeutically effective amount of the invention comprised in the composition of the invention is between of 104to 109cells / kg body weight, preferably 105to 107cells / kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages.

[0155] The compositions described herein may be administered to a patient subcutaneously, intradermally, intracranial, intranodally, intramedullary, by intravenous (i.v.) injection, or intraperitoneally. The CAR-ThyTreg cell compositions of the present invention are preferably administered by i.v. injection.

[0156] In certain embodiments of the present invention, cells activated, expanded, and engineered using the methods described herein, or other methods known in the art where T cells are expanded to therapeutic levels, are administered to a patient in conjunction with (e.g., before, simultaneously or following) any biomolecules biotinylated that correspond to the therapeutic necessity. The medicament of the invention can be used both alone and in combination with other drugs or compositions for immune tolerance induction or for treating and / or preventing a pathological condition selected from the list consisting of autoimmune disease, inflammatory processes, allergy, graft- versus-host disease and / or immune rejection in transplanted individuals. These other medicaments or compositions to be administered as therapy combined with the medicament of the invention can form part of the same composition or can be administered utilizing different compositions and administered simultaneously with the medicament of the invention or at different times. The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition and the treatment recipient. The scaling of dosages for human administration can be performed according to art-accepted practices.

[0157] In a more preferred embodiment, the medicament of the invention is administered once a previous therapy with immunosuppressive drugs has been completed. At the moment of administering the medicament of the invention, the administration of immunosuppressive drugs can be eliminated to give way to the exclusive treatment with the medicament of the invention, or the dose of said immunosuppressive drugs can be gradually decreased until wholly eliminated. In the context of the invention, "combination therapy", “in combination with” or “in conjunction with” as used herein denotes any form of concurrent, parallel, simultaneous, sequential, or intermittent treatment with at least two distinct treatment modalities (i.e., compounds, components, targeted agents or therapeutic agents). As such, the terms refer to administration of one treatment modality before, during, or after administration of the other treatment modality to the subject. The modalities in combination can be administered in any order. The therapeutically active modalities are administered together (e.g., simultaneously in the same or separate compositions, formulations, or unit dosage forms) or separately (e.g., on the same day or on different days and in any order as according to an appropriate dosing protocol for the separate compositions, formulations, or unit dosage forms) in a manner and dosing regimen prescribed by a medical caretaker or according to a regulatory agency. In general, each treatment modality will be administered at a dose and / or on a time schedule determined for that treatment modality. Optionally, three or more modalities may be used in a combination therapy. Additionally, the combination therapies provided herein may be used in conjunction with other types of treatment. For example, other anti-cancer treatment may be selected from the group consisting of chemotherapy, surgery, radiotherapy (radiation) and / or hormone therapy, amongst other treatments associated with the current standard of care for the subject.

[0158] Another aspect of the invention relates to a method for inducing or restoring immune tolerance in an individual, more preferably in a transplanted or grafted individual, or who has an autoimmune disease, or in an inflammatory process or an allergy or graft-versus- host disease, wherein said method comprises the administration of the CAR-ThyTreg cell or cell population, or the pharmaceutical composition or the combination, to said individual.

[0159] The term "subject" or “individual” as used herein refers to a mammalian subject. Preferably, it is selected from a human, companion animal, non-domestic livestock or zoo animal. For example, the subject may be selected from a human, mouse, rat, dog, cat, cow, pig, sheep, horse, bear, and so on. In a preferred embodiment, said mammalian subject is a human subject

[0160] Another aspect of the invention relates to a method for treating and / or preventing a pathological condition selected from the list consisting of autoimmune disease, inflammatory process, allergy, graft-versus-host disease and / or immune rejection to a transplant, wherein said method comprises the administration of the CAR-ThyTreg cell or cell population of the invention or the pharmaceutical composition of the invention to an individual suffering from a said pathological condition.

[0161] “Autoimmune disease" is a disorder resulting from an autoimmune response which results from an inappropriate and excessive response to a self-antigen or autoantigen. Examples of “autoimmune diseases” that could be treated and / or prevented with the medicament of the invention are, but not limited to, type I diabetes, arthritis (such as, for example, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis or juvenile idiopathic arthritis), multiple sclerosis, intestinal inflammatory affection of autoimmune origin (such as Chron’s Disease or ulcerative colitis), vasculitis (such as Wegener’s Disease or atherosclerosis), asthma, inflammatory autoimmune affection of the bile duct (such as primary biliary cirrhosis or primary sclerosing cholangitis), autoimmune thyroiditis (Hashimoto’s Disease), hyperthyroidism (Graves’s Disease), autoimmune adrenal insufficiency (Addison’s Disease), autoimmune oophoritis, autoimmune orchitis, autoimmune hepatitis, autoimmune haemolytic anaemia, paroxysmal cold haemoglobinuria, autoimmune thrombocytopenia, autoimmune neutropenia, pernicious anaemia, pure red cell aplasia, autoimmune coagulopathies, myasthenia gravis, autoimmune polyneuritis, pemphigus and other blistering disorders, rheumatic heart disease, Goodpasture Syndrome, postcardiotomy syndrome, lupus erythematosus, Sjogren Syndrome, polymyositis, dermatomyositis, sclerodermia, chronic obstructive pulmonary diseases, chronic inflammatory diseases, celiac disease, Churg-Strauss Syndrome, cardiovascular disease, polydermatomyositis, septic shock, rhinitis, psoriasis, cancer-associated cachexia, eczema, Vitiligo, Reiter Syndrome, Kawasaki’s Disease, idiopathic thrombocytopenic purpura, Guillain-Barre Syndrome, antiphospholipid antibody syndrome (APS) or narcolepsy.

[0162] In one embodiment, the CAR-ThyTreg cell or cell population of the invention, when they include a member of a pair of members (such as mSA2), any of the therapeutic uses provided in the present invention will include administering an antibody, peptides or aptamer which is tagged with a member of the pair of specific binding moieties (such as biotin); and administering the CAR-ThyTreg cell or cell population or pharmaceutical composition of the invention, both steps being performed in any order (simultaneously or sequentially). It is contemplated that any embodiment discussed in this specification can be implemented with respect to any ThyTreg cell or cell population, method of obtaining thereof, combination, pharmaceutical composition, medical or diagnostic use, method of treatment, or method of manufacturing a medicament described herein. It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

[0163] All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0164] The use of the word "a" or "an" may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one". The use of the term “another” may also refer to one or more. The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0165] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. The term “comprises” also encompasses and expressly discloses the terms “consists of” and “consists essentially of”. As used herein, the phrase "consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. As used herein, the phrase "consisting of excludes any element, step, or ingredient not specified in the claim except for, e.g., impurities ordinarily associated with the element or limitation. As used herein, words of approximation such as, without limitation, "about", "around”, “approximately” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as "about" may vary from the stated value by ±1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15%. Preferably the term “about” means exactly the indicated value (± 0%).

[0166] The following examples serve to illustrate the present invention and should not be construed as limiting the scope thereof.

[0167] EXAMPLES

[0168] EXAMPLE 1 - CAR-ThyTreg generation, characterization and suppressive function determination

[0169] The invention is further described in detail by reference to the following experimental example. This section is provided for illustration only and is not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following example but, rather, should be construed to encompass any variations that become evident because of the instruction provided herein. Without further description, it is believed that using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples, therefore, specifically point out the preferred embodiments of the present invention and are not to be construed as limiting in any way the remainder of the disclosure.

[0170] New CAR-ThyTreg were generated with potent activity composed of the highly functional and naive ThyTreg engineered with high-affinity monomeric streptavidin (mSA2) protein for biotin. Higher affinity can lead to greater ThyTreg activity and response. It was determined that biotinylated antibodies and the presence of target cells efficiently stimulate mSA2-ThyTreg cells. Moreover, they were capable of potent suppressive function associated with direct target cell lysis.

[0171] Materials and methods

[0172] Lentivector constructs and lentivector production: lentiviral vector encoding for CAR (mSA2, SEQ ID NO: 2) coding regions were listed in FIG. 1 (CAR4-1 BB-ThyTreg), and FIG. 2 (CARCD28, for comparative purpose), and were synthesized (Creative Biolabs) and cloned into Lenti-EF1a (Creative Biolabs). The viral vector was generated using the psPAX2 and pMD2.G (both plasmids from Addgene, gift from Didier Trono Addgene plasmid # 12260 y # 12259, respectively). All plasmids were transfected into 293T cells (ref # CRL-3216 from ATCC) using the lentivector production protocol (Salmon and Trono, 2006). Lentiviral vectors were produced by the co-transfection of 293T cells (ATCC, LGC Standards S.L.U., Barcelona, Spain) with pCAR, psPAX2, and pMD2.G using a calcium phosphate transfection kit (Sigma-Aldrich, St. Louis, MO, USA). The physical titers of the vectors were evaluated after 0.45 pm filtration (Corning, Corning, NY, USA) by quantifying HIV-1-p24gag with an ELISA kit (Abeam, Cambridge, UK). In brief, on day one, one million 293T cells were plated in a 10 cm dish. On day two, the cells were transfected with three plasmids using a calcium phosphate-DNA precipitate method. The plasmids were mixed in sterile water to a final concentration of 1-10 pg / pl and added dropwise to 2.5 M CaCI2 solution mixed with HBS. After incubating for 20-30 minutes, the DNA-CaCI2-HBS mixture was added dropwise to the cells and incubated for 18 hours at 37°C. The cells were then rinsed and cultured for 24-72 hours before proceeding to lentivector filtration and storage at -80°C.

[0173] Biotinylated Antibodies: anti-CD8 (#REA734, Miltenyi Biotec), Isotype control, rh-lgG1 (#REA293, Miltenyi Biotec), and anti-CD8alpha (#OKT8, Creative Biolabs) were all purchased already biotinylated. The antibody anti-CD8 (#REA734, Miltenyi Biotec) nonbiotinylated was also purchased as control of the experiments.

[0174] Cell lines: Human cell lines 293T for lentivector production (# CRL-3216) were cultured at 37°C. 293T cells were cultured in RPMI medium supplemented with 1% mix of antibiotics (125 pg / mL ampicillin, 125 pg / mL cioxacillin, and 40 pg / mL gentamicin) and 5% of fetal bovine serum (FBS). Primary peripheral blood mononuclear cells (PBMC). PBMC were isolated from deidentified human buffy coat samples obtained from the Madrid Blood Center under a research agreement. PBMC were cultured at 37°C in supplemented RPMI media with 1 % mix of antibiotics (125 pg / mL ampicillin, 125 pg / mL cioxacillin, and 40 pg / mL gentamicin) and 5% FBS.

[0175] Primary ThyTreg cells and lentiviral transduction: All experiments in primary cells were performed on ThyTreg isolated from de-identified thymus obtained from pediatric cardiac surgery (infants without syndrome affecting the thymus (such as Down syndrome or DiGeorge syndrome) as described in PCT / EP2019 / 055221 , Example 1. ThyTreg were cultured at 37°C in X-vivo15 supplemented with 5% human serum (Sigma-Aldrich) instead of FBS. And the media was supplemented with 600 ll / rnl human recombinant IL- 2 (Immunotools). Briefly, thymus was then disaggregated mechanically (gentleMACS™ Dissociators) and ThyTreg from thymocytes were selected using CD25 MicroBeads II (human, miltenyi Biotec). ThyTreg were stimulated and expanded using antiCD3 / CD28 beads (Dynabeads). For transduction, 24 hours after stimulation, lentivirus, as obtained above, was added to cells at an MOI of 1-50. After 48 hours, cells were washed and resuspended in fresh ThyTreg media containing 600 ll / rnl IL-2. After an additional 2 days of stimulation and expansion, the resulting cells were flow-sorted by EGFP expression. Sorted CAR-ThyTreg then underwent additional 2 days of culture prior to being assayed.

[0176] Flow cytometry staining: ThyTreg and CAR-ThyTreg (CARCD28 or CAR4-1 BB) were stained using the indicated antibodies in staining buffer (PBS + 2% FBS), for 30 min at 4°C. Alternatively, CAR expression on ThyTreg cells was determined by using the biomolecules Atto 665 Biotin in PBS (Merck Sigma-Aldrich), for 60 min at room temperature. After two washes with PBS, cells were stained with Fixable lity Dye (Miltenyi Biotec) 30 min at 4°C in PBS and washed twice with staining buffer. Live cells were gated based on forward (FSC) and the signal negative for Fixable Viability Dye (Fig. 6A). CAR- ThyTreg were gated on double positive cells EGFP and Atto 665 Biotin (Fig. 6B). 50,000 total events were recorded per sample. The flow cytometry parameters are adjusted using isotypic controls and utilizing fluorescence minus one (FMO) methodology.

[0177] Suppressive function assay (Fig. 4-5-7): the co-culture experiments were based on a series of controls, target cells cultured alone, cultured with ThyT reg or CAR-ThyT reg with or without biotinylated antibody. Moreover, a specific biotinylated antibody and several other antibodies were used as negative controls: the specific non-biotinylated antibody and an unspecific biotinylated antibody. The target cells were PBMC. CD8 T cells from PBMC were considered the real target cells since the biotinylated antibody was anti- CD8. The non-CD8 T cells were used as off-target controls. The day before co-cultures, target PBMCs were stained with CELLTRACE™ Violet (ThermoFisher) following the manufacturer’s instructions. 1 million were then activated with 1 million antiCD3 / CD28 magnetic beads (Dynabeads™) for 18 hours in a 24-well plate, in 2 ml of X-vivo15 complemented with 5% Human Serum and 60 U / ml IL-2. 1 million effector cells ThyTreg or CAR-ThyTreg) were then activated with 1 million antiCD3 / CD28 magnetic beads (Dynabeads™) for 18 hours in a 24-well plate, in 2 ml of X-vivo15 complemented with 5% Human Serum and 600 U / ml IL-2. After 18 hours of stimulation, beads were removed, and effector and target cells were washed with pre-warm X-vivo15 complemented with 5% Human Serum and 600 U / ml IL-2. 50,000 CellTrace-violet-PBMC were cultured alone or incubated for 24 or 72 hours with 100,000 ThyTreg or CAR-ThyTreg in 250 pl in a 96 well-U-bottom plate (ratio ThyTreg: target ratio of 2:1) in X-vivo15 complemented with 5% Human Serum and 600 U / ml IL-2 without antibodies or with 1 ,5 pg / ml of anti- CD8-biotin, anti-IgG-biotin or anti-CD8 pure. Cells alone and co-cultured cells were incubated 24 or 72 hours at 37°C in X-vivo15 complemented with 5% Human Serum and 600 U / ml IL-2. After 72 hours post-coculture, cells were harvested, and effector cells (ThyTreg or CAR-ThyTreg) were differentiated from target PBMC since PBMC were labelled CELLTRACE violet by flow cytometry. Harvested cells were labelled in different cytometry panels for superficial markers: CD8-APC / Cy5 (Beckman Coulter), and CD4- ECD (Miltenyi Biotec). The target cell proliferation was followed by the loss of the CELLTRACE violet signal using flow cytometry analysis. The viability of target cells was monitored using 7-Aminoactinomycin D (7-AAD), which produces a positive signal in cells undergoing death, as measured by flow cytometry.

[0178] The percentage of suppression was calculated by comparing the percentage of target cell proliferation co-cultured with the effector cells against the percentage of target cell proliferation cultured alone.

[0179] Results

[0180] Lentivectors encoding the two mSA2-CARs with two intracellular signalling domains (FIG. 1 and FIG. 2), mSA2-CD28-CD3 (also referred as “CARCD28”, comparative purpose) and mSA2-41 BB-CD3 (also referred as “CAR4-1 BB”, embodiment of the invention) were constructed. Driven by the EF1 alpha promoter, the CAR-coding region is formed by the murine igK leader sequence, the codon-optimized mSA2, the CD8alpha hinge domain, the CD28 transmembrane domain, either 41 BB or CD28 signaling domain and the CD3 cytoplasmic domain. Additionally, the EGFP gene was added via a T2A co-translation peptide. All the vectors were packaged into lentivirus and transduced into primary human ThyTreg cells.

[0181] After three days of stimulation and two days of transduction, transduced ThyTreg were evaluated for their viability (FIG 6A, analysed with 7AAD) and for the frequency of transduction using ATTO665-biotin labeling and GFP expression followed by flow cytometry (Fig 6B). It was found that the staining with ATTO665-biotin was specific for CAR expression since ATTO665-biotin and EGFP expression correlated. Therefore, by flow cytometry, mSA2-CAR was found to be expressed on the ThyTreg cell surface. Then, for the cells that expressed less than 30% of CAR, cells were sorted.

[0182] Next, it was tested if CAR-ThyT reg could be combined with a specific antibody to mediate specific ThyTreg functions. For these studies, ThyTreg (isolated from thymus tissue), CARCD28 and CAR4-1 BB ThyTreg cells were co-cultured with primary allogeneic human PBMC (FIG. 3) without (w / o) antibody, with a non-biotinylated specific antibody (anti-CD8 pure), with biotinylated non-specific antibody (anti-IgG-Biotin) or biotinylated specific antibody (anti-CD8-Biotin). Following three days of co-cultures of effectors cells (ThyTreg) and target cells (PBMC-CELLTRACE violet), the cells were harvested and labelled for determining viability, CD4 and CD8 subsets in the target cells determined as CELLTRACE violet positive cells.

[0183] It was found that the CD8 population disappeared when co-culture with engineered CAR4-1 BB ThyTreg (FIG. 4A). Interestingly, analyzing all the culture conditions, it was also found that the ratio of CD8:CD4 in the target cells was modified only when the coculture was PBMC + CAR4-1 BB-ThyTreg + anti-CD8-Biotin conditions, with an evident diminution of CD8 subset frequency compared to CD4 subset. Moreover, this diminution in the ratio was not detected when the co-culture was done with ThyTreg non-treated or when PBMC were cultured alone with antibodies (FIG. 4B). Therefore, and importantly, the presence of the specific biotinylated antibody and target cells (in this case, CD8) was required for all the genetically modified ThyTreg to be activated. Cell death is one mechanism used to suppress effector cells. Therefore, in this co-culture experiment (FIG 4), we investigated whether the disappearance of CD8 was due to the induction of cell death. Analyzing the frequencies of 7AAD+ cells, we showed that the suppressive function induced by the genetically modified ThyTreg compared to the nonmodified ThyTreg (ThyTreg-NT) was due to specific cell death (Fig 5). Interestingly, analyzing all the culture conditions, it was found that the lowest viability of the CD8 subset into the total PBMC target cells was achieved when the co-culture was PBMC + CAR4- 1 BB-ThyTreg + anti-CD8-Biotin conditions (FIG. 5A), with a clear low CD8 subset viability compared to the maintain viability of the CD4 subset (FIG. 5B). Moreover, this diminution in the frequency was not detected when the co-culture was done with nontreated ThyTreg or when PBMC were cultured alone with antibodies. The specific biotinylated antibody and target cells (in this case, CD8) were required for all the genetically modified ThyTreg to be activated and functional. It is important to note that cell death was not induced by polyclonal ThyTreg. This point makes a novel difference in the function of the CAR-ThyTreg compared with the initial polyclonal and non-modified ThyT reg product.

[0184] On the other hand, the suppressive function may be induced by a suppression of the cellular proliferation. We observed that the genetically modified thyTreg compared to the non-modified ThyTreg (ThyTreg-NT) were able to induce a suppression of proliferation. Indeed, the disappearance of the CD8 target subset observed in FIG. 4, compared to the CD4 subset, was also due to a suppression of cell proliferation. Analyzing all the culture conditions, it was found that the proliferation ability of the CD8 subset into the total PBMC target cells was diminished when the co-culture was PBMC + CAR4-1 BB- ThyTreg + anti-CD8-Biotin conditions (FIG. 7, light grey) compared to the suppression of the cellular proliferation of the CD4 subset (dark grey). Moreover, the PBMC + CAR4- 1 BB-ThyTreg + anti-CD8-Biotin condition show the higher suppressive ability (82% of suppression on CD8+ T cells (+ / - SEM: 7.1%) and 53.7% of suppression for CD4+ T cells (+ / - SEM 18.8%)) and killing ability (FIG 5). This suppression was higher than that observed for the ThyTreg NT with 47.9% of suppression on CD8+T cells (+ / - SEM 6.2%) and 30.6% of suppression on CD4+T cells (+ / - SEM 22.1 %).

[0185] The cells CAR-ThyTreg based on mSA2 has a high potential specific suppressive capacity that improves those found for polyclonal ThyTreg. Besides, the CAR-ThyTreg of the invention were found to suppress target cells through a different mechanism than polyclonal ThyTreg, inducing target cell death. These cells can be used with the antibodies already approved by the FDA or DEA or currently in clinical development. Therefore, the cells can be used as an off-the-shelf reagent for preselecting in vitro the best candidate antibodies for antigen-binding domains of traditional CAR before proceeding with their construction.

[0186] EXAMPLE 2 - ThyTreg cell population isolation and characterization

[0187] Materials and Methods

[0188] Thymic tissue obtention. Human thymuses used for this research were excised and discarded in pediatric cardiac surgeries at the Pediatric Cardiac Surgery Unit of Gregorio Maranon Hospital (HGUGM). Thymic tissue was collected in sterile containers with TexMACS GMP medium (Miltenyi Biotec) supplemented with 1% antifungal antibiotic (Penicillin-streptomycin-amphotericin B; Sigma-Aldrich) and kept at 4° C until processing. The study was conducted after the HGUGM ethics committee's approval and according to the principles expressed in the Declaration of Helsinki. Informed written consent from the legal guardians was obtained before the patient's enrolment.

[0189] ThyTreg production in the research laboratory. Thymic tissue fragments were mechanically disaggregated in TexMACS GMP medium (Miltenyi Biotec) with the gentleMACS Dissociator (Miltenyi Biotec). Total thymocytes obtained were filtered through a 40 pm pore, and CD25+ cells were immunomagnetically selected using human CD25 Microbeads II and LS columns (Miltenyi Biotec). After isolation, CD25+ (thyTreg day 0) and CD25- (thyTconv day 0) were cultured in TexMACS GMP medium supplemented with 600 U / ml IL-2 (Miltenyi Biotec) at 106cells / ml at 37° C and 5% CO2. Cells were stimulated with T Cell TransAct (Miltenyi Biotec), a polymeric nanomatrix, to activate and expand human T cells via CD3 and CD28 following the manufacturer's instructions. On day 3, half of the medium was removed and replaced with fresh TexMACS GMP medium supplemented with 600 U / ml IL-2. Cells were monitored on days 4, 5 and 6 and passage was performed when required. On day 7, cells were harvested, and their phenotype, functionality and stability were analyzed (Figure 10A). Additionally, dry cell pellets and culture supernatants were stored at -80° C for further analysis.

[0190] Flow cytometry and cell sorting. We evaluated the cell viability and phenotype in the different stages of the procedure by flow cytometry. Briefly, cell surface markers staining was followed by staining with Fixable Viability Dye-eFluor450 (eBioscience). Then, the cells were fixed and permeabilized using the FOXP3 transcription factor staining kit (eBioscience) for intracellular staining. All the antibodies are listed in Table 1. Flow cytometry analysis of labeled cells was performed with a MACSQuant16 cytometer (Miltenyi Biotec), acquiring at least 100,000 events, and the data were analyzed using Kaluza software (Beckman Coulter).

[0191] Table 1. Antibodies used for flow cytometric analysis.

[0192] Marker Fluorochrome Clone

[0193] Surface

[0194] CD4 APC / Cy7 13B8.2

[0195] CD25 PC7 M-A251

[0196] CD45RA ECD 2H4 Immu357

[0197] CD73 BV605 AD2 11C3C65

[0198] CD39 FITC MZ1823C8 C398.4A

[0199] TIGIT VioBright 515 REA 1004 REA 1007

[0200] LAP APC FNLAP L291 H4

[0201] CD62L BV650 DREG-56 REA232

[0202] Intracellular BNI3

[0203] FOXP3 PE PCH101 22F6

[0204] Others To isolate the CD4+ single-positive (SP) and CD4+CD8+ double-positive (DP) thyTreg cells, 50x106of total thyTreg cells were labeled with anti-CD4-VioBlue (Miltenyi Biotec) and anti-CD8-FITC (Beckman Coulter). Cells were washed and resuspended at 5x106cells / ml in MACSQuant Tyto Running Buffer (Miltenyi Biotec) and were subjected to two consecutive rounds of sorting with High-Speed MACSQuant Tyto Cartridges (MACSQuant Tyto cell sorter, Miltenyi Biotec). After the first round, CD4+SP cells were collected from the positive fraction. The negative fraction was loaded into a second cartridge, and CD4+CD8+ DP cells were collected from the positive fraction.

[0205] In vitro suppression assay. Peripheral blood mononuclear cells (PBMC) were obtained from buffy coats of healthy donors from the Madrid Transfusion Center and cryopreserved until further use. Thawed PBMC were cultured overnight in RPMI 1640 (Biochrome) supplemented with 5% serum fetal bovine serum (FBS, Biowest) and 60 ll / rnl of IL-2 (ImmunoTools). The following day, the PBMC were stained with 1 pM of CellTrace Violet (CTVio, Life Technologies). 1x105CTVio-labeled allogeneic PBMC were co-cultured with thyTreg at different thyTreg: PBMC ratios (1 :1 , 1 :2, 1 :4 and 1 : 8) in the presence of anti-CD3 / anti-CD28 coated-beads (Dynabeads; Gibco) at a bead: PBMC ratio of 0.5:1 in X-VI O 15 (Lonza) supplemented with 5% serum human AB (Sigma- Aldrich) and 600 U / ml of IL-2 (ImmunoTools) in round bottom 96 well culture plate. PBMC cultured alone in the presence or absence of Dynabeads were used as positive (C+) and negative (C-) control of proliferation, respectively. After 3 days in culture, the cells were labeled with anti-CD4-PC7 (Beckman Coulter), anti-CD8-FITC (Beckman Coulter) and 0.5 pg / mL of 7AAD (Sigma-Aldrich) to differentiate living and dead cells. Cells were acquired in a MACSQuant16 cytometer (Miltenyi Biotec), and data analysis was performed using Kaluza software (Beckman Coulter). The percentage of suppression of proliferation was calculated according to the "Division index method" (McMurchy and Levings, 2012) within CD4+ and CD8+ T cells.

[0206] Cytokine production analysis. The levels of different secreted cytokines or soluble proteins were measured using ELLA Protein-Simple (Biotechne) immunoassay technology in the culture supernatant of the thyTreg product (day 7). The supernatants were thawed at room temperature and centrifuged to remove cell debris. Samples were pre-treated (in case of TGF- [3 detection) and diluted according to the manufacturer (Simple Plex, Protein Simple). Samples were then loaded along with the necessary controls into SimplePlex cartridges, following the kit instructions for their quantification by triplicate. Graphs show the concentration of each molecule in pg / ml; each point represents the mean of the replicate measurements. The limit of detection (LD) and the quantification range for each of the evaluated molecules are: IL-10, 0.14 (0.46-5530 pg / ml); TGF-p, 5.29 (20.8-12684 pg / ml); Granzyme-B, 0.385 (1.31-5000 pg / ml); LAG-3, 15 (39.6-151050 pg / ml); TIM-3, 0.33 (2.04-7780 pg / ml); IFN-y, 0.05 (0.17-4000 pg / ml); IL-17A, 0.38 (0.82-8490 pg / ml); IL-4, 0.05 (0.319-1290 pg / ml); and PD-L1, 0.741 (3.45- 13172 pg / ml). Values above the limit of quantification are shown as the maximum limit of quantification. Values below the limit LD are shown as 0.

[0207] Stability assay under proinflammatory conditions. The thyTreg cell product (day 7) was cultured at 1x106cells / ml in TexMACS GMP medium supplemented with 600 U / ml IL-2 and restimulated with TransAct alone or together with the following cytokines: 10 ng / ml of IL-12 (polarizing condition to Th1); and 10 ng / ml I L-1 p, 10 ng / ml IL-6, 10 ng / ml of IL- 23 and 20 ng / ml of TNF-a (polarizing condition to Th17). All cytokines were purchased from ImmunoTools. PBMC were also cultured in parallel under the same conditions. Cells were cultured for 3 days, removing excess TransAct matrix on day 2. On day 3, thyTreg and PBMC culture supernatants were frozen at -80°C for cytokine analysis, and thyTreg were assessed for cell viability, phenotype, and suppressive capacity as described above. The remaining cells were saved as dry pellets at -80°C for TSDR methylation studies.

[0208] Methylation analysis. DNA was isolated from cell pellets using DNeasy Blood & Tissue Kit (Qiagen). The methylation status of 141 CpG sites located in 29 different genome regions comprising 20 different genes, including the Treg-specific demethylated region (TSDR), was analyzed by targeted Next-Gen bisulfite sequencing (NGS070V3 assay) performed by EpigenDx Inc (Hopkinton, MA, USA).

[0209] Statistical analysis. The results are expressed as the mean ± SEM (Standard Error of the Mean) or min-median-max. Continuous data were tested for normality using the Shapiro-Wilk test. Comparisons were based on the unpaired Mann-Whitney U test and the paired Wilcoxon test for nonparametric data. The statistical test used to evaluate each experiment is specified within the respective figure legend. The statistical associations between variables were calculated by linear regression and Pearson correlation analysis, p-values < 0.05 were considered to be statistically significant. The following criteria to distinguish significance levels was used: * = < 0.05, ** = < 0.01 and *** = < 0.001. Results

[0210] ThyTreg isolation and phenotype

[0211] Thymocytes obtained by mechanical disaggregation from freshly removed pediatric thymuses (n=20; age range 0-48 months; Table 2) presented high viability (96.33% ± 0.99%) (Figure 8A). Most of them (76.68% ± 2.03%) exhibited a CD4+CD8+ doublepositive (DP) phenotype, while 12.57% ± 1.23% were CD4+ single-positive (SP) cells, and 6.98% ± 1.26% were CD8+SP cells (Figure 8B). Because it has been shown that CD4+CD8+DP thymic Treg cells significantly contribute to the Treg pool in the human thymus (Nunes-Cabago et al., 2011 ; Martin-Gayo et al., 2010; Vanhanen et al., 2020), we decided to directly isolate CD25+ thymocytes (2.36% ± 0.34%) without previous depletion of CD8+ cells. The average frequency of FOXP3+ cells on isolated CD25+ thymocytes was 67.08% ± 2.22% (representative plot in Figure 8C). The benefits of preserving DP thyTreg were supported by comparing the thyTreg cells obtained with or without CD8+ depletion. The thyTreg yield was significantly higher without CD8+ depletion (p=0.04; Figure 9A), as well as the proportion of DP cells (p=0.003; Figure 9B) while maintaining cell viability and percentages of CD8+SP and FOXP3+ cells (Figure 9B-D). Following this strategy, we obtained 6.54 x 106thyTreg per 109thymocytes (range 2.44 x 106- 11.65 x 106) after CD25+ immunomagnetic selection (Table 2), with cell viability over 85%. Therefore, the estimated thyTreg number per gram of thymus was around 9.96 x 106(range 1.32 x 106- 21.59 x 106), which corresponds to 200.3 x 106highly pure thyTreg for an average thymus weight of 20.10 grams.

[0212] Table 2. Characteristics of processed thymuses and thyTreg obtention. Individual 9 5 34.67 2.11 5.04 14.36

[0213] 11 3 12.33 0.88 3.38 4.64

[0214] 13 0.4 5.90 1.21 6.80 23.97

[0215] 15 0.2 3.30 0.95 7.60 30.40

[0216] 17* 4 35.20 1.80 9.20 39.65

[0217] 19* 28 47.00 0.54 2.44 20.95

[0218] Mean 7.65 20.10 1.49 6.54 52.28

[0219] Range 0-48 3.3-47 0.54-3.37 2.44-11.65 4.64-209.47

[0220] ThyTreg culture and product characterization

[0221] Following thyTreg isolation, cells were activated for 3 days and cultured for an additional 4 days, as depicted in Figure 10A. It is to note that the culture conditions were kept as simple as possible with the idea of maintaining the immature nature of the thyTreg, avoiding extra compounds usually employed during Treg expansion such as rapamycin and human AB serum, which showed no advantage in terms of thyT reg purity, phenotype or fold expansion (Figure 11A-F). These cell characteristics were also maintained using TransAct instead of Dynabeads for cell activation to avoid the cell loss associated with the Dynabeads removal (Figure 11G-J). Cell phenotype on day 0 and day 7 is shown in Figure 10B, C (n=16). The thyTreg cells harvested at day 7 presented very high viability (92.41% ± 1.02%) and purity in terms of CD25+FOXP3+ (95.2% ± 0.74%); being both parameters higher compared to day 0. During this short-time culture period, thyTreg proliferated 6.9 ± 1.42-fold (Figure 12A). Considering 200.3 x 106thyTreg isolated at day 0 and the average fold expansion, the theoretical number of thyTreg that could be obtained from a single thymus is around 1 ,500 x 106, reaching a yield of 13,649 x106thyTreg cells from a single thymus in the best case.

[0222] We observed that the proportion of CD4+SP thyTreg decreased during the cell culture, being offset by the increased proportion of CD4+CD8+DP thyTreg (Figure 10B, C; bottom panels). Remarkably, at day 7, these CD4+CD8+DP thyTreg presented a similar phenotype to the CD4+SP thyTreg, characterized by a high expression of CD25 and F0XP3 (Figure 10D). Indeed, there was a positive correlation between the proportion of CD4+CD8+DP thyTreg and the frequency of CD25+FOXP3+ thyTreg (Figure 10E).

[0223] To further characterize the thyTreg product, we analyzed a series of cellular markers related to Treg phenotype and functionality (Figure 13A). In summary, thyTreg product was characterized by high expression of the cytotoxic T-lymphocyte associated protein (CTLA-4), inducible T-cell co-stimulator (ICOS), thymic origin marker HELIOS, and CD27; intermediate expression of T cell immunoreceptor with Ig and ITIM domains (TIGIT), glucocorticoid-induced tumor necrosis factor receptor (GITR), latency- associated peptide (LAP), HLA-DR, and CD45RA; and low expression of CD39, CD73, and lymphocyte activation gene 3 (LAG-3). In addition, to evaluate the homing capacity of thyTreg cells, we determined the expression of the chemokine receptors CCR4, CXCR3, and the CD62L selectin (Figure 13B). ThyTreg cells showed high expression of CCR4, indicating their putative ability to migrate to organs with large epithelial surfaces (such as skin, gut or lungs) (Sather et al., 2007), and CD62L, favoring their location in lymph nodes (Lamarche and Levings, 2018). As previously shown (Dijke et al., 2016), the expression of several functionality markers, including CTLA-4, CD73, ICOS, GITR and LAP, significantly increased during the cell culture; whereas CD39 expression decreased (Figure 12B). Moreover, CD45RA expression increased, which could reflect the last switch from CD45RO to CD45RA occurring as a final step of maturation in the thymus (Fujii et al., 1992) (Figure 12B). Regarding the expression of chemokine receptors, CCR4 and CD62L significantly increased after 7 days of culture; whereas CXCR3 expression decreased (Figure 12C), indicative of an undifferentiated phenotype (Groom and Luster, 2011).

[0224] We then analyzed the profile of secreted molecules by thyTreg in culture supernatants (Figure 13C). We detected high levels of the anti-inflammatory cytokines IL-10 and transforming growth factor [3 (TGF-[3) (188.03 ± 36.04 and 237.73 ± 50.83 pg / ml, respectively); as well as high levels of other inhibitory molecules associated with Treg functionality, such as Granzyme B, soluble LAG-3 and soluble T-cell immunoglobulin mucin 3 (TIM3). On the contrary, we detected very low expression of proinflammatory cytokines such as IFN-y, IL-4, IL-17A; and PD-L1. Finally, we evaluated in vitro the capacity of thyTreg cells to suppress the proliferation of CD4+, and CD8+ stimulated T cells (Figure 13D, E). We found that thyTreg exhibited a very high suppressive capacity, with more than 80% mean inhibition at a thyT reg: responder cells ratio of 1 :1 and more than 40% at 1 :4 ratio.

[0225] To determine the stability of the thyT reg product, we restimulated thyT reg cells exposed to a cocktail of cytokines polarizing to Th1 (IL-2, IL-12) or polarizing to Th17 (IL-2, IL-1 p, IL-6, IL-23, TNF-a). We observed that thyT reg cell phenotype in terms of FOXP3, CTLA- 4, CD39, and HLA-DR expression remains imperturbable (Figure 14A, B). Furthermore, thyT reg cells were not prompted to produce IFN-y or IL-17A under polarizing conditions (Figure 14C) and conserved their suppressive function (Figure 14D). To support these findings, we determined the stability of F0XP3 expression by analyzing the methylation profile of the TSDR (Figure 14E-F). We observed an intermediate level of TSDR demethylation in thyT reg cells at day 0 (62.93% ± 4.3% for males and 22.74% ± 8.03% for females), which increased after culture to 89.03% ± 2.57% for males and 51.26% ± 0.36% for females. Differences in demethylation levels between gender is due to the methylation-mediated inactivation of one X-chromosome in females. In contrast, the TSDR demethylation of freshly isolated (day 0) or cultured (day 7) thymic CD25- (thyTconv) was around 5%. The differential methylation pattern between thyT reg and thyT conv was observed not only in the F0XP3 gene but in other 8 out of 19 genes related to Treg, including CTLA-4, IKZF2 or ILR2A (Figure 12D). Notably, the TSDR demethylation status in thyT reg cells was maintained under proinflammatory conditions (Figure 14G).

[0226] Since one of the hallmarks of our thyT reg product is the existence of a CD25+FOXP3+CD4+CD8+ DP population, we decided to evaluate their commitment to a T reg phenotype by analyzing the methylation status of the TSDR. For that, after 7 days of thyT reg culture, we sorted the CD4+SP and the CD4+CD8+DP populations and analyzed their TSDR demethylation status compared with the whole thyT reg cell product (Figure 15). We indeed confirmed that the proportion of CD4+CD8+DP cells with a demethylated TSDR (94.1 %) was similar to that observed in the CD4+SP or the total thyT reg population (92.8% and 91.6%, respectively), confirming the stability of F0XP3 expression in this cell subset. EXAMPLE 3 - Immunophenotypic characterization of the CAR-ThyTreg population

[0227] Materials and Methods

[0228] After isolation, activation, transduction, sorting, and culture for a total of 8 days, thyTreg (NT) and CAR-thyTreg cells were stained using the specified antibodies for surface markers in staining buffer (PBS + 2% FBS), incubated for 30 minutes at 4°C. After two washes with PBS, cells were stained with Fixable Viability Dye (Miltenyi Biotec) for 30 minutes at 4°C in PBS. Excess dye was removed by washing the cells with staining buffer.

[0229] In some experiments, intracellular labeling was necessary. For this, cells were treated with ionomycin (Sigma-Aldrich) and phorbol 12-myristate 13-acetate (Sigma-Aldrich) for 5 hours. During the last two hours of stimulation, the cells were treated with GolgiStop (BD Biosciences), which arrests Golgi apparatus-mediated cytokine secretion in stimulated cells. Cells are then harvested and stained using the specified antibodies for surface markers in staining buffer (PBS + 2% FBS) for 30 minutes at 4°C. After two washes with PBS, cells are stained with Fixable Viability Dye (Miltenyi Biotec) for 30 minutes at 4°C in PBS and washed once with PBS. Following viability labeling, cells are treated with the Cytofix / Cytoperm™ Fixation / Permeabilization solution (BD Biosciences). This product is part of the BD Cytofix / Cytoperm™ Plus kit (BD Biosciences), which includes fixation and permeabilization of the cells for 20 minutes at 4°C in the dark. After cell fixation and permeabilization, BD Perm / Wash™ Buffer is used to wash the cells twice and to dilute the antibodies for intracellular staining for 30 minutes at 4°C in the dark. Finally, after another two washes, the cells are analyzed by flow cytometry. 50,000 total events were recorded per sample by flow cytometry.

[0230] Discussion

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[0257] CLAUSES

[0258] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

[0259] Clause 1. A thymus T regulation cell (ThyTreg cell) which codes for, or alternatively expresses on its surface, a chimeric antigen receptor (CAR) comprising an extracellular domain, a hinge region, a transmembrane domain and an intracellular domain, wherein the intracellular domain comprises: a cytoplasmatic co-stimulation domain with a sequence having an identity of at least 85% with respect to SEQ ID NO: 1 ; and a cytoplasmatic stimulation domain.

[0260] Clause 2. The ThyTreg cell of clause 1 , wherein the extracellular domain comprises or consists of a biotin-binding moiety; particularly the biotin-binding moiety is selected from: neutravidin, bradavidin, tamavidin, shwanavidin, zebavidin, streptavidin, streptavidin derivatives, or any functional fragment thereof; particularly the biotin-binding moiety is streptavidin or a functional fragment thereof; particularly the biotin-binding moiety is monomeric streptavidin (mSA2).

[0261] Clause 3. The ThyTreg cell of any one of the clauses 1-2, wherein the cytoplasmatic costimulation domain consists of SEQ ID NO: 1.

[0262] Clause 4. The ThyTreg cell of any one of the preceding clauses, wherein the cytoplasmatic stimulation domain is CD3zeta.

[0263] Clause 5. The ThyTreg cell of any one of the preceding clauses, wherein the transmembrane domain is selected from CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154; particularly is CD28 (SEQ ID NO: 4).

[0264] Clause 6. The ThyTreg cell of any one of the preceding clauses, wherein the hinge region originates from CD8a (SEQ ID NO: 3). Clause 7. The ThyTreg cell of any one of the preceding clauses, wherein the CAR comprises (i) mSA2, (ii) CD8a hinge region, (iii) CD28 transmembrane region, (iv) SEQ ID NO: 1 , and (v) CD3zeta, wherein (i) to (v) are provided in N-terminal to C-terminal order; particularly it has at least 85% of identity with respect to sequences SEQ ID NO: 6.

[0265] Clause 8. The ThyTreg cell of any one of the preceding clauses, wherein the extracellular domain does not include neither is fused to an antibody or fragment thereof.

[0266] Clause 9. The ThyTreg cell of any one of the preceding clauses 1-7, wherein the extracellular domain is fused to a binding moiety specific of an activated immune cell, particularly an antibody or fragment thereof, more particularly an anti-CD8+ antibody or fragment thereof.

[0267] Clause 10. A method for preparing a ThyTreg cell as defined in any one of the preceding clauses 1-9 which comprises the step of transfecting or transducing isolated ThyTreg cells with an expression vector codifying for the CAR as defined in any of the preceding clauses.

[0268] Clause 11. The method of clause 10, wherein the expression vector is a virus, particularly a lentivirus.

[0269] Clause 12. The method of any one of the clauses 10-11 , wherein the expression vector comprises a sequence with at least 85% of identity with SEQ ID NO: 7; particularly, the expression vector comprises a sequence 100% identical to SEQ ID NO: 7.

[0270] Clause 13. The method of any one of the clauses 10-12, wherein the expression vector has sequence with at least 85% of identity with respect to sequence SEQ ID NO: 8; particularly, the expression vector is 100% identical to SEQ ID NO: 8.

[0271] Clause 14. The method of any one of the clauses 10-13, wherein the thymic Treg cells are obtained following the steps: a. mechanically disaggregating an isolated thymic tissue; b. filtering the product obtained after stage (a), and resuspending the precipitate comprising thymocytes in a culture medium; c. isolating CD25+ cells from the product obtained after stage (b); d. culturing the cell population obtained after stage (c) in a culture medium in the presence of a T cell activator and IL-2, wherein said T cell activator comprises at least CD3 and CD28 agonists; and e. removing the T cell activator from the culture medium of stage (d); the method optionally further comprising a step (f) culturing the regulatory T cells in a culture medium in the presence of IL-2; with the proviso that prior to step (d) the cell population has not been depleted from CD8+ cells.

[0272] Clause 15. The method according to clause 14, wherein step (a) comprises mechanically disaggregating the thymic tissue in the presence of a culture medium and without using enzymes.

[0273] Clause 16. The method according to anyone of clauses 14-15, wherein isolation of CD25+ cells in step (c) comprises the use of magnetic beads conjugated to antibodies against CD25.

[0274] Clause 17. The method according to anyone of clauses 14-16, wherein the T cell activator of stage (d) is a colloidal polymer nanomatrix conjugated with humanised CD3 and CD28 agonists.

[0275] Clause 18. The method according to anyone of clauses 14-17, wherein the T cell activator of stage (d) is used in a T cell activator: cell ratio range between 1 :10 and 1 : 100.

[0276] Clause 19. The method according to anyone of clauses 14-18, wherein the cells are cultured in step (d) for at least 2 or 3 days, preferably for at least 3 days.

[0277] Clause 20. The method, according to anyone of clauses 14-19, wherein the cells are cultured in step (d) in the absence of rapamycin.

[0278] Clause 21. The method according to anyone of clauses 14-20, wherein the removal of stage (e) is carried out by a magnet or centrifugation.

[0279] Clause 22. The method according to anyone of clauses 14-21 , wherein in step (f) the regulatory T cells are cultured for another one to seven days, preferably for another four days in the presence of IL-2. Clause 23. The method, according to anyone of clauses 14-22, wherein the cells are cultured in step (f) in the absence of rapamycin.

[0280] Clause 24. The method according to anyone of clauses 14-23, wherein said culture medium is a GMP culture medium.

[0281] Clause 25. The method according to anyone of clauses 14-24, wherein said culture medium further comprises antibiotic, preferably 5% of antibiotic.

[0282] Clause 26. The method, according to anyone of clauses 14-25, wherein the thymic tissue comes from a human.

[0283] Clause 27. The ThyTreg cell obtainable by the method of any one of the clauses 10-26.

[0284] Clause 28. A pharmaceutical composition comprising a therapeutically effective amount of the CAR-ThyTreg cells as defined in anyone of the clauses 1 to 8 or 27, and one or more pharmaceutically acceptable excipients or carriers.

[0285] Clause 29. A combination comprising a Treg cell population of ThyTreg cells as defined in any of the preceding clauses 2-8, and a binding moiety specific of an activated immune cell, this moiety being conjugated to biotin.

[0286] Clause 30. The combination of clause 29, wherein the binding moiety specific of an activated cell is an antibody or fragment thereof.

[0287] Clause 31. The combination of clause 30, wherein the antibody is anti-CD8+.

[0288] Clause 32. The combination of any one of the clauses 29-31 , wherein the ThyTreg cells has an extracellular domain consisting of mSA2.

[0289] Clause 33. A chimeric antigen receptor (CAR) comprising an extracellular domain, a hinge region, a transmembrane domain and an intracellular domain, wherein the extracellular domain comprises an antibody anti-CD8+ or a fragment thereof; and the intracellular domain comprises: a cytoplasmatic co-stimulation domain with a sequence having an identity of at least 85% with respect to SEQ ID NO: 1; and a cytoplasmatic stimulation domain.

[0290] Clause 34. A nucleic acid encoding the CAR as defined in clause 34.

[0291] Clause 35. An expression vector comprising the nucleic acid of clause 35.

[0292] Clause 36. A cell transduced with the vector of clause 36.

[0293] Clause 37. The cell of clause 37, which is a Treg cell, particularly a thyTreg cell.

[0294] Clause 38. A pharmaceutical composition comprising an effective amount of the vector of clause 36 or a therapeutically effective amount of the cells transduced with the vector of clause 36, and one or more pharmaceutically acceptable vehicles or carriers.

[0295] Clause 39. The CAR-ThyTreg cell as defined in anyone of the clauses 1-8, 27 or 36, the pharmaceutical composition of clause 28 or 38, or the combination of any one of the clauses 29-32, for use in therapy or diagnosis.

[0296] Clause 40. A CAR-ThyTreg cell as defined in anyone of the clauses 1-8, 27 or 36-37, the pharmaceutical composition of clause 28 or 39, or the combination of any one of the clauses 29-32 for use in a method for inducing or restoring tolerance to the immune system.

[0297] Clause 41. A CAR-ThyTreg cell as defined in anyone of the clauses 1-8, 27 or 36-37, the pharmaceutical composition of clause 28 or 39, or the combination of any one of the clauses 29-32 for use in a method of treatment of a disease selected from: autoimmune disease, inflammatory processes, allergy, graft-versus host disease and immune rejection to transplant.

[0298] Clause 42. The combination of any one of clauses 29-31 for use according to any of the clauses 40 to 41, wherein the method comprises the separate administration of the ThyTreg cell population and binding moiety specific of an activated immune cell, to the subject.

Claims

CLAIMS1. A thymus T regulatory cell (ThyTreg cell) which codes for, or alternatively expresses on its surface, a chimeric antigen receptor (CAR) comprising an extracellular domain, a hinge region, a transmembrane domain, and an intracellular domain, wherein the intracellular domain comprises: a cytoplasmatic co-stimulation domain with a sequence having an identity of at least 85% with respect to SEQ ID NO: 1 which provides a signal mediating a Treg cell specific response; and a cytoplasmatic stimulation domain; or a Thy Treg cell population comprising or consisting thereof.

2. The ThyTreg cell population of claim 1 , wherein the cell population comprises at least 60% of CD25+FOXP3+ cells, preferably at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, 96%, 97%, 98%, 99% or even 100% of CD25+FOXP3+ cells.

3. The ThyTreg cell population of claims 1 or 2, wherein the cell population comprises at least 10% of CD4+CD8+ cells, preferably at least 15%, at least 20%, at least 25%, at least 30%, or at least 35%, even more preferably at least 40% of CD4+CD8+ cells.

4. The ThyTreg cell population of any one of claims 1 to 3, wherein at least 60% of the cells of the ThyTreg cell population express one or more of the markers selected from the group consisting of cytotoxic T-lymphocyte associated protein (CTLA-4), inducible T- cell co-stimulator (ICOS), HELIOS, latency-associated peptide (LAP), CCR4 and CD62L.

5. The ThyTreg cell population of any one of the preceding claims, wherein at least 25% of the cells of the ThyTreg cell population express one or more of the markers selected from the group consisting of glucocorticoid-induced tumor necrosis factor receptor (GITR) and HLA-DR.

6. The ThyTreg cell population of any one of the preceding claims, wherein 20% or less of the cells of the ThyTreg cell population express one or more of the markers selected from the group consisting of CD73, lymphocyte activation gene 3 (LAG-3) and CXCR3.

7. The ThyTreg cell population of any one of the preceding claims, wherein:d) at least 60% of the cells of the ThyTreg cell population express CCR4; e) at least 25% of the cells of the ThyTreg cell population express HLA-DR and / or CD45RA; and f) 20% or less of the cells of the ThyTreg cell population express CD73, LAG-3 and / or CXCR3.

8. The ThyTreg cell population of any one of the preceding claims, wherein the ThyTreg cell population is characterized by a high level of expression of anti-inflammatory cytokines, such as IL-10 and / or TGF-p, and / or other inhibitory molecules associated to Treg functionality, such as granzyme B, soluble LAG-3 and / or TIM-3, preferably wherein the expression levels are of at least 50 pg / ml, more preferably at least 100 pg / ml, wherein the expression levels are determined as specified in the description.

9. The ThyTreg cell population of any one of the preceding claims, wherein the ThyTreg cell population is characterized by expression levels of at least 100 pg / ml, preferably at least 150 pg / ml of IL-10 and / or TGF-p, wherein the expression levels are determined as specified in the description.

10. The ThyTreg cell population of any one of the preceding claims, wherein the ThyTreg cell population is characterized by low expression levels of anti-inflammatory cytokines, such as IFN-y, IL-4, IL-17A and / or PD-L1 , preferably wherein the expression levels are of 20 pg / ml or less, more preferably of 10 pg / ml or less, wherein the expression levels are determined as specified in the description.11 . The ThyT reg cell population of any one of the preceding claims, wherein the ThyT reg cell population is characterized by: c) expression levels of at least 100 pg / ml of granzyme B, soluble LAG-3 and / or TIM- 3; and / or d) expression levels of less than 10 pg / ml of IL-17-A and / or PD-L1 , wherein the expression levels are determined as specified in the description.

12. The ThyTreg cell population of any one of the preceding claims, wherein the ThyTreg cell population is characterized by having a stable FOXP3 expression.

13. The ThyTreg cell population of claim 12, wherein the ThyTreg cell population derives from a male subject and has at least 70%, preferably at least 75%, at least 80%, at least85% or more preferably at least 90% of demethylation in the Treg-specific demethylated region (TSDR) of the FOXP3 gene.

14. The ThyTreg cell population of claim 12, wherein the ThyTreg cell population derives from a female subject and has at least 30%, preferably at least 35%, at least 40%, or more preferably at least 45% of demethylation in the Treg-specific demethylated region (TSDR) of the FOXP3 gene.

15. The ThyTreg cell population of any one of the preceding claims, wherein the cell population has a mean inhibitory capacity of CD4+ and / or CD8+ T cells proliferation of at least 60%, preferably at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or more preferably at least 90% at a thyTreg : responder cells ratio of 1 :1 ; and / or a mean inhibitory capacity of CD4+ and / or CD8+ T cells proliferation of at least 25%, preferably at least 30%, at least 35% or more preferably at least 40% at a thyTreg : responder cells ratio of 1 :4.

16. The ThyTreg cell population of any one of the preceding claims, wherein the cell population does not comprise effector T cells.

17. The ThyTreg cell or cell population of any of the preceding claims , wherein the extracellular domain comprises or consists of a first molecule from a pair of complementary affinity molecules, such as a biotin-binding moiety; preferably the biotinbinding moiety is selected from: neutravidin, bradavidin, tamavidin, shwanavidin, zebavidin, streptavidin, streptavidin derivatives, or any functional fragment thereof; particularly the biotin-binding moiety is streptavidin or a functional fragment thereof; particularly the biotin-binding moiety is monomeric streptavidin (mSA2).

18. The ThyTreg cell or cell population of any of the preceding claims, wherein the cytoplasmatic co-stimulation domain consists of SEQ ID NO: 1.

19. The ThyTreg cell or cell population of any one of the preceding claims, wherein the cytoplasmatic stimulation domain is CD3zeta.

20. The ThyTreg cell or cell population of any one of the preceding claims, wherein the transmembrane domain is selected from CD28, CD3, CD45, CD4, CD8, CD9, CD16,CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154; particularly is CD28 (SEQ ID NO: 4).

21. The ThyTreg cell or cell population of any one of the preceding claims, wherein the hinge region originates from CD8a (SEQ ID NO: 3).

22. The ThyTreg cell or cell population of any one of the preceding claims, wherein the CAR comprises (i) mSA2, (ii) CD8a hinge region, (iii) CD28 transmembrane region, (iv) SEQ ID NO: 1 , and (v) CD3zeta, wherein (i) to (v) are provided in N-terminal to C-terminal order; particularly it has at least 85% of identity with respect to sequences SEQ ID NO: 6.

23. A method for preparing a ThyTreg cell or cell population as defined in any one of the preceding claims which comprises the step of transfecting or transducing isolated ThyTreg cells with an expression vector codifying for the CAR as defined in any of the preceding claims.

24. The method of claim 23, wherein the expression vector: comprises or consists of a sequence with at least 85% of identity with SEQ ID NO: 7; particularly, the expression vector comprises a sequence 100% identical to SEQ ID NO: 7; or has sequence with at least 85% of identity with respect to sequence SEQ ID NO: 8; particularly, the expression vector is 100% identical to SEQ ID NO: 8.

25. The method of any one of the claims 23-24, wherein the isolated ThyTreg cells are obtained following the steps: a. mechanically disaggregating an isolated thymic tissue; b. filtering the product obtained after stage (a), and resuspending the precipitate comprising thymocytes in a culture medium; c. isolating CD25+ cells from the product obtained after stage (b); d. culturing the cell population obtained after stage (c) in a culture medium in the presence of a T cell activator and IL-2, wherein said T cell activator comprises at least CD3 and CD28 agonists; and e. removing the T cell activator from the culture medium of stage (d);the method optionally further comprising a step (f) culturing the regulatory T cells in a culture medium in the presence of IL-2; with the proviso that prior to step (d) the cell population has not been depleted from CD8+ cells.

26. The method of anyone of claims 23-25, wherein the thymic tissue is human thymic tissue.

27. A ThyTreg cell or cell population obtainable by the method of any one of claims 23- 26.

28. A pharmaceutical composition comprising a therapeutically effective amount of the ThyTreg cell population as defined in any one of claims 1 to 22 or 27, and one or more pharmaceutically acceptable excipients or carriers.

29. A combination comprising a ThyTreg cell population as defined in any one of claims 17-22 or 27, and a binding moiety specific of an activated immune cell, particularly an antibody or fragment thereof, this moiety being conjugated to the second molecule from a pair of complementary affinity molecules, such as to biotin, a biotin analog or a fragment of any thereof when the first molecule in the pair of complementary affinity molecules is a biotinbinding moiety.

30. The ThyTreg cell or cell population as defined in any one of claims 1-22 or 27, the pharmaceutical composition of claim 28 or the combination of claim 29, for use in therapy or diagnosis.

31. A ThyTreg cell or cell population as defined in any one of claims 1-22 or 27, the pharmaceutical composition of claim 28 or the combination of claim 29, for use in a method for the treatment of a subject in need thereof with immunotherapy, particularly in a method for inducing or restoring tolerance to the immune system in a subject in need thereof; preferably in a method for the treatment in a subject of a disease selected from: autoimmune disease, inflammatory processes, allergy, graft-versus host disease and immune rejection to transplant.

32. The combination of claim 29 for use according to any one of claims 30 or 31 , wherein the method comprises the separate administration of the ThyTreg cell population and binding moiety specific of an activated immune cell, to the subject.

33. The ThyTreg cell or cell population for use according to claims 30 or 31 , or the combination for use of claim 32, wherein said subject is a human

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