CAR-ThyTreg cells, compositions, and their use in immunotherapy

Abstract

The present invention provides thymic T regulatory cells (ThyTreg cells) that encode or alternatively express 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 cytoplasmic costimulatory domain having a sequence having at least 85% identity with SEQ ID NO: 1, and a cytoplasmic stimulatory domain. The present invention also provides compositions and uses in immunotherapy.

Description

[Technical Field]

[0001] This invention belongs to clinical immunology and advanced cell immunotherapy. In particular, this invention provides CAR-ThyTreg cells, compositions containing them, and their use in inducing specific immune tolerance. [Background technology]

[0002] The primary function of the immune system is to defend organisms from pathogens, and it is also responsible for eliminating tumor cells and preventing cancer development. However, it can also lead to inadequate responses through autoimmune processes, allergies, or the emergence of transplant rejection. Proper function of the immune system is only possible when it is in balance or homeostasis. An excessive response can lead to pathological conditions such as allergies or rejection, while an incomplete response can allow infections and cancer to progress.

[0003] Autoimmune diseases, hyperinflammatory processes, and immune rejection are currently treated with immunosuppressants. Despite improvements, they still do not provide a definitive solution to these diseases and continue to cause side effects that affect the clinical progress of patients. Specifically, long-term immunosuppression causes chronic toxicity, which affects the achievement of treatment in addition to significantly impacting the patient's quality of life. Because most immunosuppressants act non-selectively, the entire system is suppressed and / or deregulated, which can interfere with normal immune system function, and this can have lifelong consequences.

[0004] To this end, achieving immune tolerance—which would allow for the indefinite avoidance of graft rejection or the symptoms of autoimmune disease affecting millions of people—has become a major challenge in modern medicine. The current scientific consensus is that a single immune tolerance induction, including retraining the recipient's immune response, could enable indefinite graft survival, limited autoimmune symptoms, or restoration of homeostasis in hyperinflammatory processes.

[0005] However, no therapies capable of inducing immune tolerance, preventing or curing autoimmune diseases, allograft rejection, or suppressing hyperinflammatory conditions have been developed or found at a commercial level. One of the most promising alternatives is inducing immune tolerance through cellular immunotherapy (Sicard et al., 2015).

[0006] The discovery of a subset of lymphocytes with inhibitory capabilities that can induce this tolerance has generated widespread enthusiasm in the clinical field. These cells, called regulatory T cells (Tregs), constitute an essential part of the immune system and can play a crucial role in maintaining beneficial immune homeostasis for patients. Tregs can suppress the effector functions of many cells, including T CD4+ and CD8+ lymphocytes, natural killer cells (NK cells), B cells, macrophages, and dendritic cells (DCs) (Sakaguchi et al., 2008).

[0007] The primary role of Tregs in transplantation has been confirmed by various studies in animal models of skin and cardiac transplants, demonstrating that Tregs present in the vessel at the time of transplantation are crucial for inducing and maintaining tolerance to the graft (Juneja et al., 2022). These Treg cells inhibit the activation and proliferation of effector T cells, which are responsible for cell rejection. Tregs can also induce B cell death and prevent humoral rejection, as already demonstrated in cardiac xenograft models (Ma et al., 2008). Therefore, cell therapy with Tregs is hypothesized to offer great hope in treating diseases mediated by excessive or insufficient immune responses, 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). The mechanism of this therapy is thought to be based on the idea that more circulating functional Tregs can prevent the activation and proliferation of effector cells that cause these diseases. Treg cell transplantation substantially increases their number in circulation and thus enhances the recipient's inherent tolerance mechanisms within receptors to the transplanted organ or its own tissues.

[0008] The safety and potential efficacy of Treg therapy in humans have been reflected in previously conducted Phase 1 / II trials. Most clinical trials using Tregs have been conducted within the context of bone marrow transplantation in patients with hematological neoplasms, demonstrating that Treg infusion in these patients reduces or prevents graft-versus-host disease (GvHD) (Trzonkowski et al., 2009). Treg-based therapies have also been tested, showing partial responses after 1–3 years of follow-up (Gliwinski et al., 2020), and favorable results have been obtained in children with type 1 diabetes (Marek-Trzonkowska et al., 2014) or multiple sclerosis (Chwojnicki et al., 2021).

[0009] Previous data have shown that antigen-specific Treg cells are more effective than nonspecific polyclonal Tregs obtained by ex vivo proliferation in mitigating graft rejection (Sagoo et al., 2011). To date, antigen-specific Tregs (Ag-specific) have been obtained by co-culturing Treg cells with allogeneic antigen-presenting cells (APCs) such as dendritic cells or B cells, which allows for the enrichment of alloreactive Treg cells in vitro. The main limitation of these strategies is the reduced number of Tregs and APCs that can be isolated. CAR (chimeric antigen receptor) technology may be suitable for generating Ag-specific ThyTregs to overcome these limitations (Beheshti et al., 2022). CARs are synthetic constructs that mimic T cell receptor activation and redirect specificity and effector function to specific antigens when expressed on T cells. CARs include an extracellular domain (scFv) responsible for binding to a specific antigen and an intracellular domain that promotes T cell activity and proliferation.

[0010] Efforts have also been made to design modified structures to enhance the innate properties of Treg cells. In this regard, Levings MK and colleagues (Dawson et al., 2020) showed that the incorporation of the CD28 domain into the cytoplasmic region of CAR significantly enhanced the properties of Treg cells, in contrast to other constructs incorporating other fragments such as CD137 (also known as 4-1BB).

[0011] However, despite these efforts, there is still a need for further Treg cell-based therapeutic approaches suitable for managing immune-related conditions. [Overview of the Initiative]

[0012] Prior to the present invention, the design of CAR-Treg cells based on the integration of the CD28 domain into the cytoplasmic region was well established, and their innate properties were enhanced.

[0013] However, the inventors have found that in the case of thymic Treg cells (hereinafter also called ThyTreg cells), incorporating the CD28 domain into the cytoplasmic domain of CAR provides CAR-ThyTreg cells (hereinafter also called "CARCD28") with a low ability to suppress the CD8+ cell population. Figure 7 shows that CARCD28 provided approximately 50% of the CD8+ cell suppression effect.

[0014] The inventors have surprisingly found that when CAR-ThyTreg cells incorporate the 4-1BB domain (SEQ ID NO: 1) into the cytoplasmic region of the CAR (hereinafter also referred to as CAR41BB-ThyTreg cells), their ability to reduce the CD8+ cell population is significantly improved, reaching over 90% of the population (Figure 7). Furthermore, it was found that not only is proliferation downregulated, but the viability of CD8+ cells is also significantly reduced (Figure 5). Therefore, administration of ThyTreg cells using this particular CAR construct, based on the incorporation of the 4-1BB sequence into the cytoplasmic domain, provides a dual negative effect on the target CD8 population, affecting not only the level of viability but also the level of proliferation.

[0015] The inventors also found that CAR41BB-ThyTreg significantly affects the suppression of the CD4 cell population (see Figure 7).

[0016] These experimental data support the immune tolerance-enhancing effect provided by the integration of the 4-1BB domain into the cytoplasmic domain of CARs in the ThyTreg population.

[0017] The above is surprising considering the previous technique (Dawson et al., 2020) which reported that the incorporation of the 4-1BB domain resulted in decreased activity, while significant activity was achieved when CD28 was included instead. Moreover, this previous technique did not provide any consideration for the incorporation of 4-1BB when designing CAR-Treg cells that enhance immune tolerance.

[0018] In summary, the data provided below indicate that the incorporation of the 4-1BB domain significantly enhances the innate properties of ThyTreg cells and is suitable for the management of diseases and conditions induced or associated with overactivity of the immune system.

[0019] Thus, in a first aspect, the present invention relates to a thymic T regulatory cell (hereinafter referred to as "ThyTreg cell") encoding a chimeric antigen receptor (CAR) comprising an extracellular domain, a hinge region, a transmembrane domain, and an intracellular domain, or alternatively expressing a chimeric antigen receptor (CAR) on its surface, wherein the intracellular domain comprises - a cytoplasmic co-stimulatory domain having a sequence with at least 85% identity to SEQ ID NO: 1 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL), and - a cytoplasmic stimulatory domain and a ThyTreg cell (also referred to herein as a CAR ThyTreg cell), or a ThyTreg cell population comprising or consisting only of the same. is provided.

[0020] Without being bound by theory, the inventors believe that the significantly different behavior of ThyTreg cells expressing a CAR containing 4-1BB is due to a specific marker profile, CD4+CD8+FOXP3, expressed on its surface.

[0021] In a second aspect, the present invention provides a method for preparing a population of CAR-ThyTreg cells of the present invention, the method comprising the step of transfecting or transducing ThyTreg cells with an expression construct encoding the CAR as defined in the first aspect of the present invention.

[0022] In a third aspect, the present invention provides a CAR-ThyTreg cell or cell population obtainable by the method of the second aspect of the present invention.

[0023] In a fourth aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the ThyTreg cell object of the present invention together with one or more pharmaceutically acceptable excipients or carriers.

[0024] In a fifth aspect, the present invention provides a combination comprising (a) a population of ThyTreg cells provided by the present invention, functionalized with a first member of a pair of specific binding moieties, particularly a member having the ability to bind to biotin, more specifically streptavidin, and (b) a binding moiety specific to activated immune cells, conjugated to a second member of a pair of specific binding moieties, particularly conjugated to biotin or a fragment thereof.

[0025] In a sixth aspect, the present invention provides CAR-ThyTreg cells or cell populations as defined in a first or third aspect of the present invention, or a combination of those in a fifth aspect, for use in therapeutic or diagnostic purposes.

[0026] In a seventh aspect, the present invention provides CAR-ThyTreg cells or cell populations or combinations of the present invention for use in inducing or restoring tolerance to the immune system. This aspect may be formulated as the use of CAR-ThyTreg cells or combinations of the present invention for the manufacture of pharmaceuticals in the induction or restoration of tolerance to the immune system. This aspect may also be formulated as an alternative method for inducing or restoring tolerance to the immune system in a subject, the method comprising administering a therapeutically effective amount of the CAR-ThyTreg cells, pharmaceutical composition or combination of the present invention to a subject in need thereof.

[0027] Antigen-specific Tregs have been shown to be more effective than polyclonal Tregs in suppressing immune responses. Our research indicates that CAR-ThyTregs have great potential due to their stable phenotype and function, as well as their ability to provide more potent and specific immunosuppression compared to polyclonal Tregs. Furthermore, we found that intracellular 4-1BB signaling is more effective in inducing suppressive function. This is important because 4-1BB reduces cellular exhaustion, ensures their durability in treated individuals, and allows them to effectively perform their immunosuppressive function. This is particularly important in autoimmune diseases, immune rejection, allergies, and chronic inflammatory processes where CAR-ThyTregs must counteract chronic inflammatory states.

[0028] Accordingly, in its final embodiment, the present invention provides CAR-ThyTreg cells, pharmaceutical compositions, or combinations of the present invention for use in the treatment of diseases selected from autoimmune diseases, inflammatory processes, allergies, graft-versus-host diseases, or immune rejection to transplantation. This embodiment may be formulated as the use of the CAR-ThyTreg cells, pharmaceutical compositions, or combinations of the present invention for the production of pharmaceuticals for treating diseases selected from autoimmune diseases, inflammatory processes, allergies, graft-versus-host diseases, or immune rejection to transplantation. This embodiment may also be formulated alternatively as a method for treating diseases selected from autoimmune diseases, inflammatory processes, allergies, graft-versus-host diseases, or immune rejection to transplantation. This method comprises the step of administering a therapeutically effective amount of CAR-ThyTreg cells, pharmaceutical compositions, and combinations of the present invention to a subject in need thereof. [Brief explanation of the drawing]

[0029] [Figure 1](A) Scheme of the CAR-41BB cDNA sequence. (B) Scheme of the CAR-41BB protein structure. (C) Scheme of CAR-41BB-ThyTreg that recognizes a single biotinylated antibody specific to a cell surface marker. (D) Full-length nucleotide sequence encoding CAR4-1BB (h: hinge, tm: transmembrane, sd: signaling domain). [Figure 2] (For comparison purposes). (A) Scheme of the CARCD28 cDNA sequence. (B) Scheme of the CARCD28 protein structure. (C) Full-length nucleotide sequence encoding CARCD28 (h: hinge, tm: transmembrane, sd: signal transduction domain). [Figure 3] (A) A scheme for CAR-thyTregs that specifically recognize target cells via biotinylated antibodies. (B) A scheme for all co-culture conditions to test the phenotype and function of CAR-thyTregs. [Figure 4] (A) Dot plot of gated CD4 and CD8 on live CELLTRACE violet-positive target cells. (B) CD8:CD4 ratio of target PBMCs in co-culture experiments with thyTreg NT or engineered thyTreg combined with specific antibodies, nonspecific antibodies, biotinylated antibodies, and non-biotinylated antibodies. [Figure 5] This represents the frequency of target cell viability. (A) Frequency of gated CD8 viability on whole target cells. (B) Frequency of gated CD8 viability on whole target cells. [Figure 6] (A) Dot plots from flow cytometry analysis showing cell size (FSC) and 7AAD (viability) of untreated (NT) or virally modified cells. (B) Dot plots from flow cytometry analysis showing GFP and CAR expression (labeled with Atto665-biotin) of untreated (NT) or virally modified cells. [Figure 7] This graph shows the frequency of suppression of gated CD4+T (dark gray) and CD8+T (light gray) cell proliferation on whole-viable target cells. [Figure 8]Acquisition and characterization of thymocytes from human thymus. (A) Representative flow cytometry plots and summary data of thymocyte viability after mechanical dissection of thymic tissue. (B) Representative flow cytometry plots and summary of the CD4 / CD8 phenotype of thymocytes. (C) Representative flow cytometry plots showing the frequency of CD25+ thymocytes and FOXP3 expression in CD25+ thymocytes. Graphs show mean ± SEM. [Figure 9] Comparison of newly isolated thyTreg cells with and without prior CD8 depletion. The inventors compared thyTreg cells obtained using a standard protocol (no prior CD8 depletion, no depletion, n=17, orange) or using an additional step of CD8 depletion using complement-mediated lysis with anti-CD8a (OKT-8) and rabbit complement HLA-ABC before CD25+ selection (n=3, green). (A) Yield of thyTreg cells obtained on day 0. (B) Representative flow cytometry plots and (C) Summary of thyTreg CD4 / CD8 phenotypes with and without CD8 depletion. (D) Representative flow cytometry histograms and summary data of FOXP3 expression frequency in thyTreg cells isolated on day 0. Graphs show mean ± SEM. Comparisons between strategies were performed using the unpaired Mann-Whitney test. *, P<0.05, and **, P<0.01. The triangular symbol represents thyTreg cell products obtained from the same thymic tissue using both strategies in parallel. [Figure 10]Characteristics of the manufactured thyTreg. (A) Isolation and culture protocol for thyTreg acquisition. (B) Representative flow cytometry dot plots showing viability, purity, and CD4 / CD8 phenotype of thyTreg immediately after isolation (day 0) or after culture (day 7). (C) Summary of cell viability, purity, and CD4 / CD8 phenotype of n=16 thyTreg at day 0 (blue) and day 7 (orange). Graphs show minimum-intermediate-maximum values. **, P<0.01 and ***, P<0.001 (paired Wilcoxon test). (D) Representative flow cytometry histograms showing the expression of CD25 (left) and FOXP3 (right) in the thyTreg CD4 / CD8 subset. Fluorescence minus 1 (FMO) of FOXP3 is shown to determine background signal. (E) Correlation between the frequency of CD4+CD8+DP and the purity of the thyTreg product (Pearson correlation analysis). [Figure 11] Optimization of thyTreg culture conditions. Newly isolated thyTregs were cultured in parallel under standard conditions (orange) or test conditions (green), and their phenotypes were evaluated on day 7. (A) Purity, (B) phenotype, and (C) proliferation (n=4) of thyTregs cultured with or without rapamycin at a final concentration of 50 nM. (D) Purity, (E) phenotype, and (F) proliferation (n=3) of thyTregs cultured with or without human 5% AB serum. (G) Representative flow cytometry dot plots and (H) purity summary, (I) phenotype, and (J) proliferation of thyTregs stimulated with TransAct or Dynabeads in a 1:1 ratio (n=3). Graphs show mean ± SEM. The inventors did not determine significant differences between culture conditions by paired Wilcoxon tests. [Figure 12]Further characterization of thyTreg. (A) Doubling of thyTreg cells against the manufacturing protocol, n=16 (mean ± SEM). (B, C) Summary of changes in phenotypic and functional marker expression in thyTreg cells between day 0 (blue) and day 7 (orange). Graphs show minimum-intermediate-maximum. *, P<0.05, **, P<0.01, and ***, P<0.001 (unpaired Mann-Whitney test). (D) Total demethylation levels of 27 genomic regions located in 20 genes within n=4 thyTreg cell products and n=2 thyTconv cells cultured in parallel for 7 days (calculated as the mean of demethylation of CpG contained in that region). ID13 and ID14 are female donors. The left panel shows regions with different demethylation patterns within thyTreg and thyTconv. The right panel shows regions with similar demethylation patterns within thyTreg and thyTconv. [Figure 13] (A) Frequency of phenotypic and functional markers in thyTreg cells (day 7). (B) Frequency of homing markers in thyTreg cells (day 7). (C) Quantification of molecules secreted into thyTreg culture supernatant on day 7. Anti-inflammatory molecules are shown in blue, and pro-inflammatory molecules in red. (D) Representative flow cytometry histograms showing CD4 (green) and CD8T (purple) cell proliferation upon CellTrace Violet loss. C-, negative control of proliferation, PBMC cultured alone without stimulation; C+, positive control of proliferation, PBMC cultured alone with 1:1 to 1:8 anti-CD3 / anti-CD28 stimulation, stimulated PBMC cultured with thyTreg cells at different thyTreg:PBMC ratios. (E) Summary of thyTreg cell inhibitory ability, defined as the % inhibition of CD4 (green) and CD8T (purple) cell proliferation at the indicated ratios. Graphs show mean ± SEM. [Figure 14]Stability of thyTreg cell products. (A-D, G) ThyTreg cell products were restimulated under control conditions (CT, blue) or Th1 (orange) or Th17 (green) polarized conditions and evaluated after 3 days of culture. PBMCs were cultured in parallel under the same conditions. (A) Representative flow cytometry histogram showing FOXP3 expression. FOXP3 fluorescence minus 1 (FMO) is shown to determine the background signal. (B) Frequency of FOXP3, CTLA-4, CD39, and HLA-DR in thyTreg under different culture conditions. Paired Wilcoxon tests showed no significant differences between conditions. (C) Quantification of IFN-γ and IL-17A secreted by thyTreg or PBMC under different culture conditions. Paired Wilcoxon tests were used to compare culture conditions within the same cell type, and unpaired Mann-Whitney tests were used to compare thyTreg and PBMC under the same conditions (#, P<0.05). (D) Summary of the inhibitory ability of thyTreg cells cultured under different polarization conditions (n=4), defined as the percentage inhibition of CD4 (upper panel) and CD8T (lower panel) cell proliferation at the ratios shown. Graphs show mean ± SEM. Paired Wilcoxon tests showed no significant differences between conditions. (E) Demethylation levels of 11 conserved CpGs in the TSDR region of FOXP3 when thyTreg cell products (n=4) and thyTconv (n=2) were cultured in parallel for 7 days. ID13 and ID14 are female donors. (F) Total TSDR demethylation levels of thyTreg and ThyTconv (calculated as the mean of demethylation of 11 CpGs) immediately 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) Total TSDR demethylation levels of thyTreg cultured under different polarization conditions. [Figure 15]TSDR methylation patterns of all thyTreg, selected CD4+SP, and selected CD4+CD8+DP. (A) Flow cytometry plots show the CD4 / CD8 phenotype of a single thyTreg product before (input) and after selection of the CD4+SP subpopulation and CD4+CD8+DP subpopulation. The purity of the selected fraction exceeds 90%. (B) Percentage of methylated and demethylated CpG islands in the FOXP3 TSDR of the input and selected fractions. [Figure 16] Marker expression of ThyTreg cells on thyTreg cells, regardless of whether they are genetically modified or not. The histogram shows the frequency of living CD25+FOXP3+thyTreg cells expressing surface and intracellular markers in untransduced thyTreg (NT) and genetically modified thyTreg cells with a second-generation CAR having a 41-BB intracellular domain (CAR41BB-thyTreg). ThyTreg cells were labeled with surface (CD73, LAG-3, HLA-DR, CTLA-4, ICOS, GITR, Helios, LAP, CCR7, CCR4, CD62L, CXCR3, CD27, and CD103) and intracellular (IL-2, IFN-gamma, IL-10, and granzyme B) markers after activation 8 days after transduction and cell sorting. [Modes for carrying out the invention]

[0030] Detailed description of the invention Unless otherwise defined, all technical and scientific terms herein have the same meaning as they are commonly understood. The present invention can be tested using any methods and materials similar or equivalent to those described herein. However, preferred materials and methods are described herein. It should also be understood that the terms herein describe only specific embodiments and are not intended to limit them.

[0031] Throughout this specification and its appendices, the term “comprise” and variations such as “comprises” and “comprising” should be interpreted inclusively. That is, these words are intended to convey the possibility of including other elements or components not specifically listed, where the context allows. The word “comprise” also includes the term “consists of.”

[0032] In the context of this invention, "T regulatory cells," "T regulatory cells," and "Treg cells" are considered synonymous. "Thymic T regulatory cells," "thymic T regulatory cells," and "ThyTreg cells" are understood to be synonymous.

[0033] A "cell population" is understood as two or more cells, preferably a group of cells.

[0034] In a first aspect, the present invention provides a population of CAR-ThyTreg cells as defined herein, or a population of THyTreg cells comprising or consisting solely of such cells. Typically, the thyTreg cell population will contain at least 40%, preferably at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% CAR-ThyTreg cells. In some embodiments, the thyTreg cell population will contain 40% to 80% CAR-ThyTreg cells, for example, 50% to 70% CAR-ThyTreg cells.

[0035] The CAR of the present invention can redirect the specificity and reactivity of ThyTreg to cells associated with hyperactivation, autoimmune pathology, or transplant rejection. These cells express antigens recognized by tagged biomolecules (e.g., monoclonal or polyclonal antibodies, aptamers, nanoparticles, proteins, or peptides).

[0036] In the context of the present invention, “chimeric antigen receptor (CAR)” refers to a chimeric construct comprising a hinge-linked extracellular domain, a transmembrane domain, and one or more intracellular signaling domains.

[0037] In some embodiments, a cytoplasmic costimulatory domain having a sequence that is at least 85%, preferably at least 90%, at least 95%, 96%, 97%, 98%, 99%, and even 100% identical to SEQ ID NO: 1, mediates a signal that mediated a Treg cell-specific response, and Cytoplasmic stimulation domain, or a population of ThyTreg cells that includes or consist solely of them. It is further characterized by providing...

[0038] ThyTreg cell activation or ThyTreg cell-specific response can be assessed by measuring the expression of activation markers such as CD71, CD69, HLA-DR, PD-1, or ICOS by flow cytometry or other techniques, but are not limited to these. Furthermore, ThyTreg cell activation or ThyTreg cell-specific response can be determined by measuring the cellular ability to inhibit or block the activation or proliferation of effector T cells, as described in the examples herein.

[0039] In one embodiment of the first aspect, CAR-ThyTreg cells contain recombinant nucleic acid encoding CAR4-1BB of SEQ ID NO: 7 or 8, or having at least 85%, preferably at least 90%, at least 95%, 96%, 97%, 98%, 99%, or even 100% identity with SEQ ID NO: 7 or 8. In an alternative embodiment, ThyTreg cells express on their surface the CAR amino acid sequence of SEQ ID NO: 6 (CAR4-1BB), or having at least 85%, preferably at least 90%, at least 95%, 96%, 97%, 98%, 99%, or even 100% identity with SEQ ID NO: 6. In the present invention, the terms "4-1BB", "41-BB", and "41BB" are considered synonymous.

[0040] As used herein, the term “identity” refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence between two polynucleotide or polypeptide sequences. Two or more sequences (polynucleotides or amino acids) can be compared by determining their “identity percentage.” Whether nucleic acid sequences or amino acid sequences, the “identity percentage” of two sequences is calculated by dividing the number of exact matches between the two aligned sequences by the length of the shorter sequence and multiplying by 100. Appropriate programs for calculating the identity or similarity percentage between sequences are well known in the art, such as the NCBI BLAST program used with default parameters (http: / / www.ncbi.nlm.gov / cgi-bin / BLAST).

[0041] In some embodiments, the CAR is a universal CAR. Universal CAR-T cells may be allogeneic cells obtained from a healthy donor. T cells can be transduced using several gene editing methods, including, but not limited to, zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and CRISPR / Cas9. Universal CAR-T therapy can also be produced without gene editing.

[0042] The structure of a universal CAR can be modular. The CAR consists of two parts: (i) a signaling module on T cells, comprising only an extracellular domain that specifically binds to a switching module and an intracellular domain that transmits an activation signal; and (ii) a switching module, which is typically recognized by the signaling module on T cells and splits into bispecific antibodies or small molecules that bind to targets on target cells (e.g., activated immune cells). This divisible universal programmable (SUPRA) CAR system currently employs a variety of recognition modes, including but not limited to neoepitopes, SpyTag, biotin, and fluorescein isothiocyanate (FITC) and leucine zippers (Lin et al., 2021; Sutherland, Owens, and Geyer, 2020).

[0043] In one embodiment, the extracellular domain comprises molecules from a pair of complementary affinity molecules. In some embodiments, the pair of complementary affinity molecules is selected from the group consisting of biotin / biotin-binding moieties, antibody / antigen, enzyme / substrate, receptor / ligand, metal / metal-binding protein, carbohydrate / carbohydrate-binding protein, lipid / lipid-binding protein, and His-tag / His-tag-binding molecule. In some embodiments, the extracellular domain comprises a leucine zipper, an anti-fluorescein isothiocyanate (FITC) system, a bacterial toxin-antitoxin system, a FITC-folate adapter, a peptide neoepitope (PNE)-mAb system, or a photodegradable molecule adapter system. In a preferred embodiment, the pair of complementary affinity molecules is biotin / biotin-binding moieties. In this embodiment, the CAR-ThyTreg cells of the present invention would not directly bind to the target, but would require interaction with the other member of the specific binding moiety pair (biotinized intermediates in the following embodiments) to stimulate the specific function of the CAR-ThyTreg cells.

[0044] In another embodiment, the extracellular domain includes a biotin-binding moiety. Exemplary, non-limiting examples of suitable biotin-binding moieties in the context of the present invention include rizavidin, avidin, streptavidin, bladavidin, tamavidin, lentiavidin, zebavidin, neutraavidin, captavidin (trademark), combinations thereof, or functional fragments. A “functional fragment” is understood to be a fragment of the biotin-binding moiety that retains some or all of the binding capacity of the full-length portion. In this embodiment, the CAR-ThyTreg cells of the present invention do not bind to a target but rather to a biotinylated moiety (e.g., an antibody or fragment thereof, or an aptamer) to stimulate specific functions of the CAR-ThyTreg cells.

[0045] In one embodiment, the extracellular domain contains or consists solely of a fragment of streptavidin.

[0046] Streptavidin is a 52.8 kDa protein derived from the bacterium Streptomyces avidinii. Streptavidin exists naturally as a homotetramer. The secondary structure of the streptavidin monomer consists of eight antiparallel β-chains, which fold to give an antiparallel β-barrel tertiary structure. The biotin-binding site is located at one end of each β-barrel. Four identical streptavidin monomers (i.e., four identical β-barrels) associate to give the tetramer quaternary structure of streptavidin. The biotin-binding site within each barrel consists only of residues derived from within the barrel, along with a conserved Trp120 from the adjacent subunit. Thus, since each subunit contributes to the binding site on the adjacent subunit, the tetramer can also be considered a dimer of a functional dimer. The CAR-based streptavidin domain of the present invention can essentially consist of streptavidin monomer, dimer, or tetramer. In particular, it is a monomeric streptavidin having a sequence that has at least 85%, at least 90%, or at least 95% identity with respect to Sequence ID No. 2 (the high-affinity monomeric streptavidin domain known as mSA2, already disclosed in Lohmueller, JJ et al., 2017). Sequence ID 2 METDTLLLWVLLLWVPGSTGGAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFTKVKPSAASGS

[0047] In another embodiment of the first aspect of the present invention, the extracellular domain does not contain or is not fused to an antibody or any fragment thereof.

[0048] In one embodiment, the chimeric antigen receptor (CAR) of the present invention comprises a monomeric high-affinity streptavidin recognition domain, a hinge region, a transmembrane domain, and an extracellular domain having one or two cytoplasmic domains. In this embodiment, the extracellular domain does not contain or is not fused to an antibody or any fragment thereof.

[0049] The hinge region in CARs functions as a flexible spacer for the extracellular domain, improving the recognition / function of the CAR against antigens located closer to the surface of target cells. In many constructs, the hinge region may consist of the CH2CH3 domain of a human IgG antibody that can bind to Fc receptors on macrophages and other cells, thereby causing cross-activation and activation-induced cell death in vivo, independently of target antigen recognition. In one embodiment of the present invention, the hinge region is derived from CD8a (SEQ ID NO: 3). Sequence ID 3 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0050] The CAR must include a hinge and a transmembrane domain fused to an intracellular domain. The transmembrane domain may be of a natural or synthetic origin. If the transmembrane domain is of a natural origin, the domain may be derived from any membrane-binding or transmembrane protein. For example, transmembrane domains derived from CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, and CD154. Preferably, the transmembrane domain is a CD28 transmembrane domain having SEQ ID NO: 4: MFWVLVVVGGVLACYSLLVTVAFIIFWV.

[0051] In the context of this invention, the term "intracellular domain" refers to the signal transduction portion of a classical CAR. In the signal transduction system of this invention, the intracellular signal transduction domain (signal transduction domain) is located within the signal transduction component.

[0052] In the context of the present invention, the term “costimulatory domain” refers to a signaling portion that provides T cells with signals that mediate T cell responses, including but not limited to activation, proliferation, differentiation, and cytokine secretion, in addition to the primary signal provided by, for example, the CD3ζ chain of the TCR / CD3 complex. The costimulatory domain may include all or some ligands that specifically bind to CD27, CD28, OX40 (CD134), CD30, CD40, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83, in addition to 4-1BB (SEQ ID NO: 1). In some embodiments, the costimulatory signaling domain is an intracellular signaling domain that interacts with other intracellular mediators to mediate cellular responses, including activation, proliferation, differentiation, and cytokine secretion. In one embodiment of the present invention, the cytoplasmic costimulatory domain is CD3 zeta (SEQ ID NO: 5). RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0053] In one embodiment, the co-stimulatory domain is CD28 in addition to 4-1BB. In a preferred embodiment, the intracellular domain contains a fused CD28-4-1BB sequence called SEQ ID NO: 12(SEQ ID NO: 12):RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGGGKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0054] In an alternative embodiment, the intracellular domain consists solely of the 4-1BB sequence of Sequence ID No. 1.

[0055] In another embodiment of the first aspect of the present invention, the CAR comprises (i) mSA2, (ii) CD8a hinge region, (iii) CD28 transmembrane region, (iv) SEQ ID NO: 1 or 7, and (v) CD3 zeta, where (i) to (v) are provided in N-terminus to C-terminus order. In one embodiment, the CAR sequence is at least identical to SEQ ID NO: 6, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%. Sequence ID 6: METDTLLLWVLLLWVPGSTGGAEAGITGTWYNQHGSTFTTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFTKVKPSAASGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF ACDMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR The nucleic acid encoding the CAR of the present invention is a sequence having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with sequence number 7.

[0056] Prior to the expansion and genetic modification of regulatory T cells according to the present invention, a source of ThyTreg cells is obtained from the subject.

[0057] Preferably, suitable Treg cells in the context of the present invention are characterized by containing CD4+CD8+FOXP3. Populations having these suitable Treg cells can be obtained according to the method disclosed in European Patent No. 3759215A1, the contents of which are incorporated herein by reference. The resulting cells have a great ability to suppress effector cells of the immune system, an undifferentiated (naive) phenotype, high viability, and stable expression of FOXP3, as well as functional markers such as CTLA-4 and CD39, even under pro-inflammatory conditions.

[0058] In some embodiments, the ThyTreg cell population of the present invention comprises at least 60% 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% CD25+FOXP3+ cells.

[0059] In some embodiments, the ThyTreg cell population of the above embodiments is, as may be combined with any of the features or embodiments described herein, at least 10% CD4+CD8+ cells, preferably at least 15%, at least 20%, at least 25%, at least 30%, or at least 35%, and more preferably at least 40% CD4+CD8+ cells.

[0060] In some embodiments, 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 in a ThyTreg cell population express one or more 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.

[0061] In a preferred embodiment, at least 60%, preferably at least 65%, more preferably at least 70%, and even more preferably at least 75%, 80%, 85%, 90%, 95%, or even 100% of the cells in the ThyTreg cell population express one or more markers selected from the group consisting of cytotoxic T lymphocyte-associated protein (CTLA-4), inducible T cell costimulator (ICOS), HELIOS, latency-associated peptide (LAP), CCR4, and CD62L.

[0062] In another embodiment, 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 in the ThyTreg cell population express one or more markers selected from the group consisting of T cell immune receptor having Ig and ITIM domains (TIGIT), glucocorticoid-induced tumor necrosis factor receptor (GITR), latency-related 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 in the ThyTreg cell population express one or more markers selected from the group consisting of glucocorticoid-induced tumor necrosis factor receptor (GITR) and HLA-DR. In any of these preferred embodiments, the level of expression is 25% to 60%, preferably 30% to 50%.

[0063] In further embodiments, in combination with any of the features or embodiments described herein, 20% or less, preferably 15%, 10%, 5%, 2%, 1%, 0.5%, 0.25%, or even 0.1% or less of the cells in the ThyTreg cell population express one or more markers selected from the group consisting of CD39, CD73, lymphocyte activation gene 3 (LAG-3), and CXCR3. In preferred embodiments, 20% or less, preferably 15%, 10%, 5%, 2%, 1%, 0.5%, 0.25%, or even 0.1% or less of the cells in the ThyTreg cell population express one or more markers selected from the group consisting of CD73, lymphocyte activation gene 3 (LAG-3), and CXCR3.

[0064] Preferably, the ThyTreg cell population is characterized by the following: 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 in 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 in the ThyTreg cell population express HLA-DR and / or CD45RA, c) Less than 20%, preferably less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.25%, and even less than 0.1% of the ThyTreg cell population express CD73, LAG-3, and / or CXCR3.

[0065] In one embodiment, the ThyTreg cell population described herein is characterized by high levels of expression of anti-inflammatory cytokines such as IL-10 and / or TGF-β, and / or other inhibitory molecules associated with Treg function such as granzyme B, soluble LAG-3 and / or TIM-3, preferably with an expression level of at least 50 pg / ml, more preferably at least 100 pg / ml, and the expression level is determined as specified in the examples with respect to cytokine production analysis.

[0066] In one embodiment, the ThyTreg cell population described herein is characterized by an expression level of at least 100 pg / ml, preferably at least 150 pg / ml, of IL-10 and / or TGF-β, the expression level being determined as specified in the example with respect to cytokine production analysis.

[0067] In a preferred embodiment, the ThyTreg cell population described herein is a) Expression levels of granzyme B, soluble LAG-3 and / or TIM-3 of at least 100 pg / ml, and / or b) Expression levels of IL-17-A and / or PD-L1 less than 10 pg / ml The expression level is determined as specified in the examples with respect to cytokine production analysis.

[0068] In one embodiment, the ThyTreg cell population described herein is characterized by having stable FOXP3 expression. In some embodiments, the ThyTreg cell population is derived 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% demethylation in the Treg-specific demethylation region (TSDR) of the FOXP3 gene. In other embodiments, the ThyTreg cell population is derived from a female subject and has at least 30%, preferably at least 35%, at least 40%, or more preferably at least 45% demethylation in the Treg-specific demethylation region (TSDR) of the FOXP3 gene.

[0069] In another embodiment, the ThyTreg cell population described herein has an average inhibitory ability 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% of CD4+ and / or CD8+ T cell proliferation at a 1:1 thyTreg:responder cell ratio, and an average inhibitory ability of at least 25%, preferably at least 30%, at least 35%, or more preferably at least 40% of CD4+ and / or CD8+ T cell proliferation at a 1:4 thyTreg:responder cell ratio.

[0070] In one embodiment, the ThyTreg cell population described herein does not include effector T cells.

[0071] In one embodiment, thymic Treg cells are subjected to the following steps: a. A process of mechanically decomposing isolated thymic tissue, b. The product obtained after step (a) is filtered, and the precipitate containing thymocytes is resuspended in culture medium in the presence of IL-2, c. A step to isolate CD25+ cells from the product obtained after step (b), d. A step of culturing the cell population obtained after step (c) in a culture medium in the presence of a T cell activator and IL-2, wherein the T cell activator includes at least CD3 and CD28 agonists. e. A step to remove T cell activating factors from the culture medium in step (d), f. Depending on the case, a step of further culturing regulatory T cells in culture medium in the presence of IL-2. It is obtained by a method that includes or consists only of the following steps, provided that the cell population is not depleted of CD8+ cells before step (d).

[0072] ThyTreg cells obtained by the above isolation method are typically characterized by higher levels of CD27, CD45RA, and CCR4. Similar to lower levels of activated HLA-DR, the latter reduces their immunogenicity. In preferred embodiments, thyTreg cells obtained by the above isolation method are characterized as described herein.

[0073] In the context of the present invention, “thymic tissue” is any tissue sample from the thymus, which is a lymphoid gland where T cells or lymphocytes mature, and is located in front of the heart and behind the sternum. Thymic tissue can be removed by any method known in the art that serves such purposes, for example, by thymectomy, which may be transsternal, transcervical, or videoscope. Preferably, the thymic tissue is removed before performing the method, during a surgical intervention, more preferably with the intention of treating a heart disease, such as a congenital heart disease, or during a heart transplant. Even more preferably, the thymic tissue of the present invention is removed during a pediatric heart transplant.

[0074] Thymic tissue may be derived from humans or non-human mammals, such as, but not limited to, rodents, pigs, primates, ruminants, cats, or dogs. In preferred embodiments of the method of the present invention, the thymic tissue is derived from humans, more preferably from 0 to 16 years of age, even more preferably from 0 to 10 years of age, and particularly from 0 to 24 months of age.

[0075] In another preferred embodiment, the thymic tissue is derived from the same or a different individual from which the ThyTreg cells obtained at the end of the method of the present invention will subsequently be administered for cellular immunotherapy.

[0076] In this invention, "pediatric patient" or "child" is understood to be a human being aged 0 to 16 years, preferably 0 to 10 years, and more preferably 0 to 24 months.

[0077] Any commercially available tissue dissociation device using the latest technology can be used in step (a) of the method of the present invention. Examples of such dissociation devices include, but are not limited to, the gentleMACS Dissociator or gentleMACS Octo Dissociator from Miltenyi Biotec, the TissueLyser LT from Qiagen, or tissue dissociation devices from Worthington Biochemical, Sigma-Aldrich, or Roche Diagnostics. Preferably, the tissue dissociation device used in the present invention is, in one embodiment, the gentleMACS Octo Dissociator from Miltenyi Biotec, and step a) includes mechanically dissecting thymic tissue without the use of enzymes in the presence of culture medium.

[0078] T cells can be activated and proliferated before or after genetic modification of Treg cells to express a desired CAR. Generally, the T cells of the present invention are proliferated by contact with a surface to which a drug that stimulates CD3 / TCR complex-related signaling and a ligand that stimulates costimulatory molecules on the surface of the T cells are attached. In the present invention, once ThyTreg cells are newly isolated from the thymus and their purity is verified by flow cytometry (CD25+FOXP3+), the cells can be activated and proliferated using anti-CD3 / anti-CD28 magnetic beads (Dynabeads™) in a 1:1 (beads:cells) ratio. In one embodiment, the ratio of CD3:CD28 antibody bound to the beads is in the range of 100:1 to 1:100 and all integer values ​​in between. In one embodiment, the cells can be reactivated and restimulated for proliferation 7 days after the initial activation and proliferation. In this embodiment, reactivation and restimulation can be performed using anti-CD3 / anti-CD28 magnetic beads (Dynabeads®) or anti-CD3 / anti-CD28 matrix (Miltenyi Biotec®). The matrix-to-cell ratio is in the range of 1:50 to 1:1000, and all integer values ​​within that range. In one embodiment, cells (10 4 ~10 9The ThyTreg cells and matrix or beads are combined in a culture medium, preferably X-vivo15 or PBS (which does not contain divalent cations such as calcium and magnesium). Those skilled in the art will readily understand any cell concentration that can be used. For example, in one embodiment, a concentration of about 2 million cells / ml is used. In another embodiment, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, 50 million or 100 million cells / ml are used. The use of high concentrations may result in increased cell yield, cell activation, and cell proliferation.

[0079] In one embodiment, the mixture may be cultured for several hours (approximately 18 hours) to approximately 72 hours or any integer value in between. Several stimulation / growth cycles may also be desired so that the T cell culture time can be 28 days or more.

[0080] Suitable conditions for T cell culture include a suitable culture medium, preferably X-Vivo15(Lonza), but also minimal essential medium or RPMI Media1640(Lonza) that may contain factors necessary for proliferation and survival, such as serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), IL-7, or IL-15.

[0081] Other additives for cell growth include, but are not limited to, reducing agents such as 2-mercaptoethanol. The culture medium may contain RPMI1640, AIM-V, DMEM, MEM, C-MEM, F-12, X-Vivo15, and X-Vivo20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, and is either serum-free or suspended in an appropriate amount of serum (or plasma), and / or supplemented with sufficient amounts of cytokines for T cell growth and proliferation. Target cells are maintained under conditions necessary to support proliferation, e.g., appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2).

[0082] Twenty-four hours after activation, the viral vector encoding the CAR of the present invention is added to the cell mixture without removing the activation beads. Twenty-four hours after lentivector treatment (in one embodiment, forty-eight hours after lentivector treatment), the magnetic beads are removed with a magnet. The cells are then washed with warm culture medium, preferably X-vivo15 supplemented with 5% human serum, and tested for viability with 7-aminoactinomycin D (7-AAD, Figure 6A) and GFP expression by flow cytometry. The transduction rate is approximately 40% (20-70%, Figure 6B). If the cells show less than 30% transduction, GFP+ cells are sorted using a Tyto sorter (Miltenyi Biotec). The cells are then grown for 5 days in fresh medium RPMI1640 supplemented with 5% human serum (Lonza) and recombinant human IL-2 (600 U / ml, preferably Immunotools). Eight days later, cells were labeled with a biotinylation marker for CAR detection (preferably Atto665-biotin, Sigma-Aldrich) (Figure 6B). Since the CAR plasmid encodes mSA2 and GFP, the double-positive cells were sufficiently transduced and expressed GFP and CAR.

[0083] In one embodiment, the isolation of CD25+ cells in step c) includes the use of magnetic beads conjugated with an antibody against CD25.

[0084] In one embodiment, the T cell activator in step d) is a colloidal polymer nanomatrix (magnetic beads) conjugated with humanized CD3 and CD28 agonists. These magnetic beads promote efficient T cell activation while maintaining T cell viability.

[0085] In one embodiment, the T cell activator in step d) is used in a T cell activator:cell ratio ranging from 1:10 to 1:100.

[0086] In one embodiment, the cells are cultured for at least two or three days, preferably at least three days, in step d).

[0087] In one embodiment, cells are cultured in step d) in the absence of rapamycin.

[0088] In one embodiment, the removal in step e) is performed by centrifugal force or by a magnet.

[0089] In one embodiment, in step f), regulatory T cells are cultured for a further 1 to 7 days, preferably for a further 4 days, in the presence of IL-2.

[0090] In one embodiment, cells are cultured in step f) in the absence of rapamycin.

[0091] In one embodiment, the culture medium is a GMP culture medium.

[0092] In one embodiment, the culture medium further comprises an antibiotic, preferably 5% of an antibiotic.

[0093] CAR-ThyTreg cells of the present invention can be produced by introducing an expression construct containing a CAR as defined in any of the embodiments described above. In one embodiment, the expression construct contains a sequence having at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 7.

[0094] Nucleic acid CAR sequences can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Expression vectors may also be provided to cells as viral vectors. Viral vector technology is well known in the art. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, a suitable vector includes a functional origin of replication in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers.

[0095] The method of the present invention uses a DNA construct comprising a nucleic acid encoding a CAR as defined in any of the embodiments described above. In one embodiment, the expression vector is derived from a lentivirus. Lentiviruses are suitable tools for long-term gene transfer because they enable long-term and stable transgene integration and their proliferation in daughter cells. Lentiviral vectors have advantages over other vector sources: they can transduce non-proliferating cells and can have low immunogenicity.

[0096] The expression of nucleic acids encoding CAR targets in the present invention is typically achieved by operably ligating a nucleic acid encoding a CAR polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector. A typical cloning vector includes transcription and translation terminators, start sequences, and promoters useful for controlling the expression of a desired nucleic acid sequence.

[0097] Exemplary, non-limiting examples of promoter sequences include the cytomegalovirus (CMV), Simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long-terminal repeat (LTR) promoter, MoMul V promoter, avian leukemia virus promoter, Epstein-Barr virus pre-early promoter, Roussarcoma virus promoter, and human gene promoters, such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, human elongation factor 1 alpha (EF1a), and creatine kinase promoter. Furthermore, the present invention should not be limited to constitutive promoters. Inducible promoters are also considered as part of the present invention. The use of inducible promoters provides a molecular switch that can turn on the expression of an operablely linked polynucleotide sequence when such expression is desired and off when it is not desired.

[0098] In the context of this invention, the terms “operably linked” and “operably linked” refer to a functional linkage between a regulatory sequence and a heterogeneous nucleic acid sequence that results in the expression of the latter. For example, if a first nucleic acid sequence has a functional relationship with a second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. Generally, operably linked DNA sequences are contiguous and, if necessary, link two protein-coding regions within the same reading frame.

[0099] To evaluate the expression of a CAR polypeptide or a portion thereof, the expression vector introduced into cells may also contain either or both a selection marker gene and / or a reporter gene to facilitate the identification and selection of expressing cells from a population of cells to be transfected or transduced via a viral vector. Both the selection marker and reporter genes may be flanked by appropriate regulatory sequences to enable expression in the host cell.

[0100] The reporter gene identifies potentially transduced cells and evaluates the functionality of regulatory sequences. Reporter gene expression is assayed at an appropriate time after the DNA has been introduced into recipient cells. The reporter gene may be encoded in the same reading frame as the CAR sequence isolated by the T2A sequence, a 2A peptide derived from the Zosea asigna viral capsid protein. Alternatively, it may be under a promoter that may differ from the CAR promoter.

[0101] In another embodiment, the expression construct consists only of sequences that have at least 85%, at least 90%, or at least 95% identity with the sequence of Sequence ID No. 8.

[0102] Methods for introducing and expressing genes in cells are well known in the art. In relation to expression vectors, vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. Examples of biological methods for introducing a polynucleotide of interest into host cells include DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become one of the most widely used methods for inserting genes into mammalian cells, such as human cells. Other viral vectors may originate from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. Regardless of the method used to introduce exogenous nucleic acids into host cells, various assays can be performed to confirm the presence of recombinant DNA sequences in the host cells. Such assays include, for example, “molecular biological” assays well known to those skilled in the art, such as Southern blotting and Northern blotting, RT-PCR, and PCR. “Biochemical” assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA, flow cytometry, and Western blotting) or by the assays described herein for identifying agents that fall within the scope of the present invention, are also possible.

[0103] In a further embodiment, the present invention provides CAR-ThyTreg cells that can be obtained by the method of the present invention.

[0104] Cells resulting from the above process are characterized, among other things, by high expression of perforin, granzyme B, or both. Preferably, the cells exhibit overexpression of both perforin and granzyme B.

[0105] For the purposes of this invention, the expressions "can be obtained," "obtained," and equivalent expressions are used interchangeably, and in either case, the expression "can be obtained" encompasses the expression "obtained."

[0106] The present invention is based, in particular, on improving the immunosuppressive ThyTreg capacity in regulating the immune system state in hyperinflammatory processes, autoimmune diseases, or transplant rejection.

[0107] Accordingly, the present invention provides CAR-ThyTreg cells and methods for using them for adoptive immunotherapy. In one embodiment, the CAR-ThyTreg cells of the present invention can be produced by introducing a lentiviral vector into cells containing a desired CAR, for example, an anti-high affinity monomer streptavidin, CD8 alpha hinge, CD28 transmembrane domain, and human 4-1BB and CD3 zeta signaling domains. The CAR-ThyTreg cells of the present invention can replicate in vivo and exhibit long-lasting persistence that can result in sustained immunosuppressive control.

[0108] In one embodiment, the present invention relates to the administration of genetically modified ThyTreg cells expressing CAR for the treatment of patients with hyperinflammatory processes, autoimmune diseases, or transplant rejection. CAR-ThyTreg cells may be autologous or allogeneic. ThyTreg cells can be isolated from the human thymus of pediatric patients undergoing cardiac surgery.

[0109] In a further embodiment, the present invention provides a combination comprising a ThyTreg cell population as defined in any of the above embodiments, incorporating a first molecule from a pair of complementary affinity molecules such as a biotin-binding moiety (e.g., as the extracellular domain of a CAR), and an activated immune cell-specific binding moiety conjugated to a second molecule from a pair of complementary affinity molecules such as biotin, a biotin analog, or a fragment thereof. Biotin analogs may include, but are not limited to, α-dehydrobiotin, niositin, 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, and γ-imidazolidinone-4-hexanoic acid.

[0110] All embodiments provided under the first aspect of the present invention relating to the biotin-binding moiety and the CAR sequence contained therein are also embodiments of the combination of the present invention.

[0111] In the context of the present invention, the “activated immune cell-specific binding moiety” may be any molecule having the ability to bind to immune cells. As used herein, the term “immune cells” includes any cells 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, as well as neutrophils. Thus, the “activated immune cell-specific binding moiety” may be an antibody, aptamer, cytokine, any nucleic acid or peptide sequence, or a fragment thereof, having the ability to bind to a specific component (marker) associated with the activation of immune cells. Exemplary, non-limiting examples of surface markers already known in common knowledge in the art as being specific to activated immune cells include, among others, CD19, CD30, CD8, and interleukin-23 receptors.

[0112] In the context of this invention, the term “antibody” refers to an immunoglobulin molecule that specifically binds to an antigen. Exemplary, non-limiting examples of “antigen” in the context of this invention include, but are 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 desmosome core glycoprotein-3 (DSG3).

[0113] Antibodies can be intact immunoglobulins and immunoreactive moieties of intact immunoglobulins derived from natural or recombinant sources. Antibodies can exist in various forms, including polyclonal antibodies, monoclonal antibodies, multispecific antibodies such as bispecific antibodies, humanized antibodies, and fragment antibodies, as long as they exhibit the desired target-binding activity. Non-limiting examples of antibody fragments include Fv, Fab, Fab, Fab-SH, and F(ab)2, diabodies, linear antibodies, single-chain antibodies, and multispecific antibodies formed from antibody fragments.

[0114] In one embodiment, a biotin-binding moiety CAR protein (e.g., mSA2-CAR protein) can be designed to target a biotinylated aptamer.

[0115] In the context of this 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 that have been manipulated through repeated selection rounds to bind to a variety of molecular targets, such as small molecules, proteins, nucleic acids, cells, or tissues.

[0116] In one embodiment, a biotin-binding moiety CAR protein (e.g., mSA2-CAR) can be designed to target biotinylated cytokines.

[0117] In one embodiment, a biotin-binding moiety CAR protein (e.g., mSA2-CAR) can be designed to target a biotinylated receptor ligand.

[0118] In one embodiment, a biotin-binding moiety CAR protein (e.g., mSA2-CAR) can be designed to target a biotinylated nucleic acid sequence.

[0119] In biochemistry, "biotinylation" is the process of covalently attaching biotin to a protein, nucleic acid, or other biomolecule. There are known protocols in the art for biotinylating target molecules.

[0120] However, the present invention should not be construed as being limited only to the targets and diseases disclosed herein. Rather, the present invention should be construed as including any antigenic target related to a disease for which CAR may be suitable.

[0121] The CAR-modified T cells of the present invention may be administered alone or in combination with one or more pharmaceutically acceptable excipients or carriers as part of a pharmaceutical composition.

[0122] As used herein, “pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” means any solvent, dispersion medium, coating agent, antimicrobial and antifungal agent, isotonic agent and absorption retarder that is suitable for pharmaceutically active administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Acceptable carriers, excipients, or stabilizers that are nontoxic to the recipient at the dosage and concentration used and do not limit the scope of the present invention include, but are: additional buffers; preservatives; cosolvents; 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 and polyhydric sugar alcohols; amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, and threonine; lactitol, stachyol Organic sugars or sugar alcohols such as sucrose, mannose, sorbose, xylose, ribose, ribitol, myo-initose, myo-inititol, galactose, galactitol, glycerol, cyclitol (e.g., inositol), polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [alpha]-monothioglycerol, and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; and hydrophilic polymers such as polyvinylpyrrolidone.

[0123] In the context of the present invention, the term “therapeutic dose” refers to the amount of a target compound that elicits a biological or medical response in a tissue, system, or subject, as sought by researchers, veterinarians, physicians, or other clinicians. The term “therapeutic dose” includes an amount of a compound sufficient to prevent, or to some extent alleviate, the onset of one or more signs or symptoms of the disorder or disease being treated, when administered. The therapeutic dose varies depending on the compound, the disease and its severity, and the age, weight, etc., of the subject being treated. The dose required to obtain a therapeutic dose depends on various factors, such as the age, weight, sex, pathological condition, or tolerance of the individual to whom the composition of the present invention is to be administered. Preferably, the therapeutic dose of the present invention contained in the composition of the present invention is 10 4 ~10 9 Cells / kg body weight, preferably 10 5 ~10 7 The values ​​are between cells / kg body weight and include all integer values ​​within this range. T cell compositions can also be administered multiple times at these doses.

[0124] The compositions described herein can be administered to a patient subcutaneously, intradermally, intracranially, intranodulately, intramedullarily, intravenously (iv) or intraperitoneally. The CAR-ThyTreg cell composition of the present invention is preferably administered by iv injection.

[0125] In certain embodiments of the present invention, cells activated, proliferated, and manipulated using the methods described herein or other methods known in the art to proliferate T cells to therapeutic levels are administered to the patient together with any biotinylated biomolecule corresponding to the therapeutic need (e.g., before, simultaneously, or after). The pharmacopoeia of the present invention can be used alone and in combination with other drugs or compositions for inducing immune tolerance or for treating and / or preventing pathological conditions selected from the group consisting of autoimmune diseases, inflammatory processes, allergies, graft-versus-host diseases, and / or immune rejection in transplanted individuals. These other pharmacopoeia or compositions administered therapeutically in combination with the pharmacopoeia of the present invention may form part of the same composition or be administered using different compositions and may be administered simultaneously with or at different times to the pharmacopoeia of the present invention. The dosage of the above treatment administered to the patient will vary depending on the condition and the exact nature of the treatment recipient. Dosage scaling for human administration can be carried out according to established practices in the art.

[0126] In a more preferred embodiment, the pharmacopoeia of the present invention is administered after the completion of prior treatment with an immunosuppressant. At the time of administration of the pharmacopoeia of the present invention, the administration of the immunosuppressant can be excluded to make way for exclusive treatment with the pharmacopoeia of the present invention, or the dose of the immunosuppressant can be gradually reduced until it is completely excluded.

[0127] In the context of the present invention, as used herein, “combination therapy,” “in combination with,” or “together with” refers to any form of concurrent, parallel, simultaneous, sequential, or intermittent treatment by at least two distinct treatment modalities (i.e., compounds, components, targeting agents, or therapeutic agents). Therefore, the term refers to the administration of one treatment modality before, during, or after the administration of other treatment modalities to the subject. Combined modalities may be administered in any order. Therapeutically active modalities may be administered together (e.g., simultaneously in the same or separate compositions, formulations, or unit dosage forms) or separately (e.g., on the same or different days, in any order according to the appropriate administration protocol for separate compositions, formulations, or unit dosage forms) in the form and dosing regimen prescribed by the healthcare professional or in accordance with the regulatory authority. Generally, each treatment modality is administered in the dose and / or time schedule determined for that treatment modality. In some cases, three or more modalities may be used in combination therapy. Furthermore, the combination therapies provided herein may be used in conjunction with other types of treatment. For example, other anti-cancer treatments may be selected from among other treatments related to the subject's current standard of care, including chemotherapy, surgery, radiotherapy (radiotherapy), and / or hormone therapy.

[0128] Another aspect of the present invention relates to a method for inducing or restoring immune tolerance in an individual, more preferably in a transplanted or transplanted individual, or in an individual with an autoimmune disease, or in an inflammatory process, or in an allergy or graft-versus-host disease, comprising administering CAR-ThyTreg cells or cell populations, or a pharmaceutical composition or combination, to the individual.

[0129] As used herein, the terms “subject” or “individual” refer to a mammalian subject. Preferably, it is selected from humans, companion animals, non-domestic livestock, or zoo animals. For example, the subject may be selected from humans, mice, rats, dogs, cats, cattle, pigs, sheep, horses, bears, etc. In a preferred embodiment, the mammalian subject is a human subject.

[0130] Another aspect of the present invention relates to a method for treating and / or preventing a pathological condition selected from the group consisting of autoimmune diseases, inflammatory processes, allergies, graft-versus-host diseases, and / or immune rejection of a graft, comprising administering the CAR-ThyTreg cells or cell populations of the present invention or the pharmaceutical composition of the present invention to an individual suffering from the pathological condition.

[0131] An "autoimmune disease" is a disorder resulting from an autoimmune response caused by an inappropriate and excessive response to an autoantigen. Examples of "autoimmune diseases" that can be treated and / or prevented by the pharmaceuticals of the present invention include type 1 diabetes, arthritis (e.g., rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, or juvenile idiopathic arthritis), multiple sclerosis, autoimmune inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), vasculitis (e.g., Wegener's disease or atherosclerosis), asthma, inflammatory autoimmune diseases of the bile duct (e.g., primary biliary cirrhosis or primary sclerosing cholangitis), autoimmune thyroiditis (Hashimoto's disease), hyperthyroidism (Graves' disease), autoimmune adrenal insufficiency (Addison's disease), autoimmune oophoritis, autoimmune orchitis, autoimmune hepatitis, autoimmune hemolytic anemia, paroxysmal cold hemoglobinuria, and auto Immunotoxic thrombocytopenia, autoimmune neutropenia, pernicious anemia, pure red cell dysplasia, autoimmune coagulation disorders, myasthenia gravis, autoimmune polyneuritis, pemphigus and other bullous disorders, rheumatic heart disease, Goodpasture syndrome, postcardiotomy syndrome, lupus erythematosus, Sjögren's syndrome, polymyositis, dermatomyositis, scleroderma, chronic obstructive pulmonary disease, chronic inflammatory diseases, celiac disease, Churg-Strauss syndrome, cardiovascular disease, polydermatomyositis, septic shock, rhinitis, psoriasis, cancer-associated cachexia, eczema, vitiligo, Reiter's syndrome, Kawasaki disease, idiopathic thrombocytopenic purpura, Guillain-Barré syndrome, antiphospholipid syndrome (APS), or narcolepsy.

[0132] In one embodiment, if the CAR-ThyTreg cells or cell population of the present invention include a pair of members (such as mSA2), any therapeutic use provided in the present invention would include administering an antibody, peptide, or aptamer tagged with a pair of specific binding sites (such as biotin) and administering the CAR-ThyTreg cells or cell population or pharmaceutical composition of the present invention. Both steps may be performed in any order (simultaneously or sequentially).

[0133] Any embodiment discussed herein can be carried out with respect to any ThyTreg cells or cell populations, methods for obtaining them, combinations, pharmaceutical compositions, medical or diagnostic uses, therapeutic methods, or methods for producing the pharmaceuticals described herein. It will be understood that the specific embodiments described herein are illustrative and not intended to be limitations of the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention. Those skilled in the art will be able to recognize or confirm many equivalents to the specific procedures described herein using only routine experiments. Such equivalents are considered to be within the scope of the invention and are encompassed by the claims.

[0134] All publications and patent applications are incorporated herein by reference to the same extent as each individual publication or patent application is specifically and individually incorporated by reference.

[0135] The use of the words "a" or "an" can mean "one," but also coincides with the meanings of "one or more," "at least one," and "one or more." The use of the term "another" can also refer to one or more. The use of the term "or" in a claim means "and / or" unless it explicitly refers only to substitutes or the substitutes are mutually exclusive.

[0136] As used herein and in the claims, the terms “comprising” (including any form of “comprising,” such as “comprise” and “comprises”), “having” (and any form of “having,” such as “have” and “has”), “including” (including any form of “including,” such as “includes” and “include”), or “containing” (including any form of “containing,” such as “contains” and “contain”) are inclusive or open-ended and do not exclude any additional unenumerated elements or method steps. The term “comprises” also includes and expressly discloses the terms “consists of” and “consists essentially of.” As used herein, the phrase "consisting essentially of" limits the claims to specific materials or steps and to elements that do not substantially affect the basic and novel features of the claimed invention. As used herein, the phrase "consisting only of" excludes any elements, steps, or components not expressed in the claims, except for, for example, impurities that are typically associated with that element or limitation.

[0137] Where used herein, approximate terms such as “about,” “around,” and “approximately” refer to conditions that, when modified in this way, are understood to be not necessarily absolute or complete, but are considered by those skilled in the art to be close enough to guarantee that a condition exists. The extent to which the description may change depends on the degree to which change can be initiated, and it is still recognized by those skilled in the art that the modified feature still possesses the necessary properties and capabilities of the unmodified feature. Generally, given the foregoing description, numerical values ​​in this specification modified by approximate terms such as “about” may vary by ±1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15% from the stated value. Preferably, the term “about” means the exact value (±0%).

[0138] The following embodiments are helpful in illustrating the present invention and should not be construed as limiting its scope. [Examples]

[0139] Example 1 - Determination of the generation, characterization, and suppression function of CAR-ThyTreg The present invention will be described in more detail with reference to the following experimental examples. This section is provided for illustrative purposes only and is not intended to limit unless otherwise specified. Accordingly, the present invention should not be construed as being limited in any way to the following examples, but rather as encompassing any variations that become apparent for the instructions provided herein. Without further explanation, it is assumed that the compounds of the present invention will be prepared and utilized and the claimed methods will be carried out using the foregoing description and the following exemplary examples. Accordingly, the following examples specifically point to preferred embodiments of the present invention and should not be construed as limiting the remainder of this disclosure.

[0140] We created a novel, potent CAR-ThyTreg cell consisting solely of highly functional and naive ThyTreg cells manipulated with high-affinity monomeric streptavidin (mSA2) protein for biotin. Higher affinity may lead to greater ThyTreg activity and response. We determined that the presence of biotinylated antibodies and target cells efficiently stimulates mSA2-ThyTreg cells. Furthermore, these enabled potent inhibitory functions related to direct target cell lysis.

[0141] material and method Lentiviral vector constructs and lentiviral vector production: Lentiviral vectors encoding the CAR(mSA2, SEQ ID NO: 2) coding region were listed in Figure 1 (CAR4-1BB-ThyTreg) and Figure 2 (CARCD28, for comparison), synthesized (Creative Biolabs), and cloned into Lenti-EF1a (Creative Biolabs). Viral vectors were constructed using psPAX2 and pMD2.G (both plasmids from Addgene, transfers from Didier Trono Addgene plasmids #12260 and #12259, respectively). All plasmids were transfected into 293T cells (reference number CRL-3216 from ATCC) using the lentiviral vector production protocol (Salmon and Trono, 2006). Lentiviral vectors were constructed by simultaneous transfection of 293T cells (ATCC, LGC Standards SLU, Barcelona, ​​Spain) with pCAR, psPAX2, and pMD2.G using a calcium phosphate transfection kit (Sigma-Aldrich, St. Louis, Missouri, USA). The physical titer of the vectors was assessed after 0.45 μm filtration (Corning, Corning, NY, USA) by quantifying HIV-1-p24gag using an ELISA kit (Abcam, Cambridge, UK). Briefly, on day 1, 1 million 293T cells were placed in a 10 cm dish. On day 2, the cells were transfected with three plasmids using the calcium phosphate-DNA precipitation method. The plasmids were mixed in sterile water to a final concentration of 1–10 μg / μl and added dropwise to a 2.5 M CaCl2 solution mixed with HBS. After incubation for 20-30 minutes, the DNA-CaCl2-HBS mixture was added to the cells and incubated at 37°C for 18 hours. The cells were then rinsed and cultured for 24-72 hours before proceeding to lentivector filtration and storage at -80°C.

[0142] Biotinylated antibodies—anti-CD8 (#REA734, Miltenyi Biotec), isotype control, rh-IgG1 (#REA293, Miltenyi Biotec), and anti-CD8 alpha (#OKT8, Creative Biolabs)—were all purchased already biotinylated. A non-biotinylated anti-CD8 antibody (#REA734, Miltenyi Biotec) was also purchased as an experimental control.

[0143] Cell line: Human cell line 293T (#CRL-3216) for lentivector production was cultured at 37°C. 293T cells were cultured in RPMI medium supplemented with a 1% mixed antibiotic (125 μg / mL ampicillin, 125 μg / mL cloxacillin, and 40 μg / mL gentamicin) and 5% fetal bovine serum (FBS).

[0144] Primary peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from unspecified human buffy coat samples obtained under a research contract from the Madrid Blood Center. PBMCs were cultured at 37°C in RPMI medium supplemented with 1% mixed antibiotics (125 μg / mL ampicillin, 125 μg / mL cloxacillin, and 40 μg / mL gentamicin) and 5% FBS.

[0145] Primary ThyTreg cells and lentiviral transduction: All experiments with primary cells were performed on ThyTreg isolated from unspecified thymuses (infants without thymic syndromes (such as Down syndrome or DiGeorge syndrome)) obtained from pediatric cardiac surgery, 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. The medium was also supplemented with 600 U / ml human recombinant IL-2 (Immunotools). Briefly, the thymus was then mechanically deaggregated (gentleMACS® Dissociators), and ThyTreg from thymocytes were selected using CD25 MicroBeads II (human, miltenyi Biotec). ThyTreg were stimulated and proliferated using anti-CD3 / CD28 beads (Dynabeads). For transduction, the lentivirus obtained above was added to the cells at a MOI of 1–50 24 hours after stimulation. After 48 hours, the cells were washed and resuspended in fresh ThyTreg medium containing 600 U / ml IL-2. After a further 2 days of stimulation and proliferation, the resulting cells were flow-selected by EGFP expression. The selected CAR-ThyTreg cells were then cultured for a further 2 days before assay.

[0146] Flow cytometry staining: ThyTreg and CAR-ThyTreg (CARCD28 or CAR4-1BB) were stained with the indicated antibody in staining buffer (PBS + 2% FBS) at 4°C for 30 minutes. Alternatively, CAR expression on ThyTreg cells was determined by using the biomolecule Atto665 biotin in PBS (Merck Sigma-Aldrich) at room temperature for 60 minutes. After washing twice with PBS, cells were stained with Fixable Viability Dye (Miltenyi Biotec) in PBS at 4°C for 30 minutes and washed twice with staining buffer. Live cells were gated based on signal negativity for forward-direction (FSC) and Fixable Viability Dye (Figure 6A). CAR-ThyTreg cells were gated with double-positive cells EGFP and Atto665 biotin (Figure 6B). A total of 50,000 events were recorded per sample. Flow cytometry parameters were adjusted using isotype controls and the fluorescence minus 1 (FMO) method.

[0147] Suppression function assay (Figure 4-5-7): Co-culture experiments were based on a series of controls, target cells cultured alone, and target cells cultured with or without biotinylated antibodies in ThyTreg or CAR-ThyTreg. In addition, specific biotinylated antibodies and several other antibodies: specific non-biotinylated antibodies and non-specific biotinylated antibodies were used as negative controls. The target cells were PBMCs. Since the biotinylated antibody was anti-CD8, CD8 T cells derived from PBMCs were considered the true target cells. Non-CD8 T cells were used as off-target controls. The day before co-culture, target PBMCs were stained with CELLTRACE® Violet (ThermoFisher) according to the manufacturer's instructions. Then, 1 million cells were activated in 2 ml of X-vivo15 supplemented with 5% human serum and 60 U / ml IL-2 for 18 hours in a 24-well plate using 1 million anti-CD3 / CD28 magnetic beads (Dynabeads®). Next, 1 million effector cells, ThyTreg or CAR-ThyTreg, were activated in 24-well plates for 18 hours in 2 ml of X-vivo15 supplemented with 5% human serum and 600 U / ml IL-2 using 1 million anti-CD3 / CD28 magnetic beads (Dynabeads®). After 18 hours of stimulation, the beads were removed, and the effector and target cells were washed with preheated X-vivo15 supplemented with 5% human serum and 600 U / ml IL-2. 50,000 CellTrace-violet-PBMCs were cultured alone or together with 100,000 ThyTreg or CAR-ThyTreg cells in 250 μl of X-vivo15 supplemented with 5% human serum and 600 U / ml IL-2 in a 96-well U-bottom plate (ThyTreg:target ratio 2:1), without antibody, or with 1.5 μg / ml of anti-CD8 biotin, anti-IgG-biotin, or anti-CD8 pure product, for 24 or 72 hours. Cells alone and co-cultured cells were incubated at 37°C for 24 or 72 hours in X-vivo15 supplemented with 5% human serum and 600 U / ml IL-2.After 72 hours of co-culture, cells were harvested and PBMCs were labeled with CELLTRACE violet by flow cytometry to identify effector cells (ThyTreg or CAR-ThyTreg) from target PBMCs. Harvested cells were labeled with different cytometry panels for surface markers: CD8-APC / Cy5 (Beckman Coulter) and CD4-ECD (Miltenyi Biotec). After target cell proliferation, the CELLTRACE violet signal was lost using flow cytometry analysis. Target cell viability was monitored using 7-aminoactinomycin D (7-AAD), which produces a positive signal in dead cells as measured by flow cytometry.

[0148] The inhibition rate was calculated by comparing the proliferation rate of target cells co-cultured with effector cells to the proliferation rate of target cells cultured alone.

[0149] result We constructed lenti vectors encoding two mSA2-CARs, each having two intracellular signaling domains (Figures 1 and 2): mSA2-CD28-CD3ζ (also known as "CARCD28," for comparison purposes) and mSA2-41BB-CD3ζ (also known as "CAR4-1BB," an embodiment of the present invention).

[0150] The CAR coding region, driven by the EF1 alpha promoter, is formed by a mouse igκ reader sequence, codon-optimized mSA2, CD8 alpha hinge domain, CD28 transmembrane domain, either 41BB or CD28 signaling domain, and CD3ζ cytoplasmic domain. Furthermore, the EGFP gene was added via a T2A cotranslating peptide. All vectors were packaged in lentiviruses and transduced into primary human ThyTreg cells.

[0151] After 3 days of stimulation and 2 days of transduction, transduced ThyTreg cells were evaluated for viability (analyzed in Figures 6A and 7AAD) and transduction frequency using ATTO665-biotin labeling, GFP expression, and subsequent flow cytometry (Figure 6B). Since ATTO665-biotin and EGFP expression are correlated, staining with ATTO665-biotin was found to be specific to CAR expression. Therefore, flow cytometry revealed that mSA2-CAR is expressed on the surface of ThyTreg cells. Cells expressing less than 30% CAR were then selected.

[0152] Next, we tested whether CAR-ThyTreg could mediate specific ThyTreg function in combination with specific antibodies. For these studies, ThyTreg cells (isolated from thymic tissue), CARCD28, and CAR4-1BB were co-cultured with primary allogeneic human PBMCs (w / o) (Figure 3), non-biotinylation-specific antibody (pure anti-CD8), biotinylation-nonspecific antibody (anti-IgG-biotin), or biotinylation-specific antibody (anti-CD8-biotin). After 3 days of co-culture of effector cells (ThyTreg) and target cells (PBMC-CELLTRACE violet), the cells were harvested and labeled to determine viability, CD4, and CD8 subsets in the target cells, which were determined to be CELLTRACE violet-positive cells.

[0153] When co-cultured with modified CAR4-1BB ThyTreg cells, the CD8 population was found to disappear (Figure 4A). Interestingly, analysis of all culture conditions revealed that the CD8:CD4 ratio in target cells was modified only when co-cultured under PBMC + CAR4-1BB-ThyTreg + anti-CD8-biotin conditions, and the frequency of the CD8 subset was clearly reduced compared to the CD4 subset. Furthermore, this reduction in the ratio was not detected when co-cultured without ThyTreg treatment or when PBMCs were cultured alone with the antibody (Figure 4B). Therefore, importantly, the presence of specific biotinylated antibodies and target cells (in this case, CD8) was required to activate all genetically modified ThyTreg cells.

[0154] Cell death is one mechanism used to suppress effector cells. Therefore, in this co-culture experiment (Figure 4), we investigated whether the loss of CD8 was due to the induction of cell death. By analyzing the frequency of 7AAD+ cells, we showed that the suppressive function induced by genetically modified ThyTreg compared to unmodified ThyTreg (ThyTreg-NT) was due to specific cell death (Figure 5). Interestingly, analysis of all culture conditions revealed that when the co-culture was PBMC + CAR4-1BB-ThyTreg + anti-CD8-biotin conditions (Figure 5A), the lowest viability of the CD8 subset to all PBMC target cells was achieved, and a significantly lower viability of the CD8 subset was achieved compared to the maintenance of viability of the CD4 subset (Figure 5B). Furthermore, this decrease in frequency was not detected when the co-culture was performed with untreated ThyTreg or when PBMCs were cultured alone with the antibody. Specific biotinylated antibodies and target cells (in this case, CD8) were required for all genetically modified ThyTregs to be activated and function. It is important to note that cell death was not induced by polyclonal ThyTregs. This point introduces a novel difference in the function of CAR-ThyTregs compared to initial polyclonal and unmodified ThyTreg products.

[0155] On the other hand, the inhibitory function can be induced by the suppression of cell proliferation. The inventors observed that genetically modified thyTreg can induce proliferation suppression compared to non-modified ThyTreg (ThyTreg-NT). In fact, the disappearance of the CD8 target subset observed in Figure 4 compared to the CD4 subset was also due to the suppression of cell proliferation. Analysis of all culture conditions revealed that when the co-culture was PBMC + CAR4-1BB-ThyTreg + anti-CD8-biotin conditions (Figure 7, light gray), the proliferative capacity of the CD8 subset to all PBMC target cells was reduced compared to the suppression of cell proliferation of the CD4 subset (dark gray). Furthermore, the PBMC+CAR4-1BB-ThyTreg+anti-CD8-biotin condition exhibited higher inhibitory capacity (82% inhibition of CD8+ T cells (+ / -SEM: 7.1%) and 53.7% inhibition of CD4+ T cells (+ / -SEM: 18.8%)) and killing capacity (Figure 5). This inhibition was higher than that observed for ThyTreg NT, which showed 47.9% inhibition of CD8+ T cells (+ / -SEM: 6.2%) and 30.6% inhibition of CD4+ T cells (+ / -SEM: 22.1%).

[0156] mSA2-based cellular CAR-ThyTregs possess high potential for specific repression, improving upon what is found for polyclonal ThyTregs. Furthermore, the CAR-ThyTregs of this invention have been found to repress target cells and induce target cell death through a mechanism different from that of polyclonal ThyTregs. These cells can be used with antibodies already approved by the FDA or DEA, or currently in clinical development. Therefore, the cells can be used as ready-made reagents for in vitro preliminary selection of the best candidate antibodies against the antigen-binding domains of conventional CARs before proceeding with their construction.

[0157] Example 2 - Isolation and Characterization of ThyTreg Cell Population material and method Thymic tissue acquisition. The human thymus used in this study was excised and discarded during pediatric cardiac surgery at the Pediatric Cardiac Surgery Unit of Gregorio Maranon Hospital (HGUGM). Thymic tissue was collected in sterile containers containing TexMACS GMP medium (Miltenyi Biotec) supplemented with 1% antifungal antibiotic (penicillin-streptomycin-amphotericin B; Sigma-Aldrich) and kept at 4°C until processing. This study was conducted in accordance with the principles outlined in the Declaration of Helsinki, after approval by the HGUGM Ethics Committee. Written informed consent was obtained from legal guardians prior to patient enrollment.

[0158] Laboratory production of ThyTreg. Thymic tissue fragments were mechanically deaggregated in TexMACS GMP medium (Miltenyi Biotec) using a gentleMACS Dissociator (Miltenyi Biotec). The resulting whole thymocytes were filtered through a 40 μm pore, and CD25+ cells were immunomagnetically selected using human CD25 microbeads II and an LS column (Miltenyi Biotec). After isolation, CD25+ (thyTreg day 0) and CD25- (thyTconv day 0) were mixed in TexMACS GMP medium supplemented with 600 U / ml IL-2 (Miltenyi Biotec) for 10 minutes. 6 Cells were cultured at 37°C and 5% CO2 at a cell / ml rate. Cells were stimulated with the polymer nanomatrix T Cell TransAct (Miltenyi Biotec) to activate and proliferate human T cells via CD3 and CD28 according to 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 subculturing was performed as needed. Cells were harvested on day 7 and their phenotype, functionality, and stability were analyzed (Figure 10A). Furthermore, the dried cell pellet and culture supernatant were stored at -80°C for further analysis.

[0159] Flow cytometry and cell sorting. The inventors evaluated cell viability and phenotype at different stages of the procedure by flow cytometry. Briefly, after cell surface marker staining, cells were stained with Fixable Viability Dye-eFluor450 (eBioscience). Then, cells were fixed and permeabilized using the FOXP3 transcription factor staining kit (eBioscience) for intracellular staining. All antibodies are listed in Table 1. Flow cytometry analysis of labeled cells was performed using a MACSQuant16 cytometer (Miltenyi Biotec), acquiring at least 100,000 events, and the data was analyzed using Kaluza software (Beckman Coulter). TIFF2026518291000002.tif149170

[0160] To isolate CD4+ single positive (SP) and CD4+CD8+ double positive (DP) thyTreg cells, 50 × 10 6 total thyTreg cells were labeled with anti-CD4-VioBlue (Miltenyi Biotec) and anti-CD8-FITC (Beckman Coulter). Cells were washed and resuspended at 5 × 10 6 cells / ml in MACSQuant Tyto Running Buffer (Miltenyi Biotec) and sorted twice consecutively using High-Speed MACSQuant Tyto Cartridges (MACSQuant Tyto cell sorter, Miltenyi Biotec). After the first round, CD4+SP cells were recovered from the positive fraction. The negative fraction was loaded onto a second cartridge, and CD4+CD8+DP cells were recovered from the positive fraction.

[0161] [[ID=(13)]] In vitro suppression assay. Peripheral blood mononuclear cells (PBMCs) were obtained from the buffy coat of healthy donors from the Madrid Transfusion Center and frozen until use. Thawed PBMCs were cultured overnight in RPMI1640 (Biochrome) supplemented with 5% fetal bovine serum (FBS, Biochrome) and 60 U / ml IL-2 (ImmunoTools). The following day, PBMCs were stained with 1 μM CellTrace Violet (CTVio, Life Technologies). 1 × 10⁻⁶ 5 CTVio-labeled allogeneic PBMCs were co-cultured with thyTreg at different thyTreg:PBMC ratios (1:1, 1:2, 1:4, and 1:8) in X-VIVO15 (Lonza) round-bottom 96-well culture plates supplemented with 5% human serum AB (Sigma-Aldrich) and 600 U / ml IL-2 (ImmunoTools) in the presence of anti-CD3 / anti-CD28 coated beads (Dynabeads; Gibco) at a bead:PBMC ratio of 0.5:1. PBMCs cultured alone in or without Dynabeads were used as growth-positive (C+) and growth-negative (C-) controls, respectively. After 3 days of culture, live and dead cells were distinguished by labeling with anti-CD4-PC7 (Beckman Coulter), anti-CD8-FITC (Beckman Coulter), and 0.5 μg / mL 7AAD (Sigma-Aldrich). Cells were acquired using a MACSQuant16 cytometer (Miltenyi Biotec), and data analysis was performed using Kaluza software (Beckman Coulter). The rate of proliferation inhibition was calculated according to the "divide index method" (McMurchy and Levings, 2012) for CD4+ and CD8+ T cells.

[0162] Cytokine production analysis. Levels of different secreted cytokines or soluble proteins were measured in the culture supernatant (day 7) of thyTreg products using the ELLA Protein-Simple (Biotechne) immunoassay technique. The supernatant was thawed at room temperature and centrifuged to remove cellular debris. Samples were pre-treated (for TGF-β detection) and diluted according to the manufacturer's instructions (Simple Plex, Protein Simple). The samples were then loaded into SimplePlex cartridges with the necessary controls and quantified using the kit instructions for triplicate analysis. The graph shows the concentration of each molecule in pg / ml. Each point represents the mean of repeated measurements. The detection limits (LD) and quantitative ranges for each molecule evaluated were: IL-10, 0.14 (0.46-5530 pg / ml), TGF-β, 5.29 (20.8-12684 pg / ml), Granzyme-B, 0.385 (1.31-5000 pg / ml), LAG-3, 15 (39.6-151050 pg / ml), TI The values ​​were as follows: M-3, 0.33 (2.04-7780 pg / ml); IFN-γ, 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 ​​exceeding the limit of quantification are shown as the maximum limit of quantification. Values ​​below the limit LD are shown as 0.

[0163] Stability assay under pro-inflammatory conditions. thyTreg cell product (day 7) was subjected to 1 × 10⁶ ionization in TexMACS GMP medium supplemented with 600 U / ml IL-2. 6Cells were cultured at thyTreg / ml and restimulated with TransAct alone or with the following cytokines: 10 ng / ml IL-12 (for Th1 polarization), as well as 10 ng / ml IL-1β, 10 ng / ml IL-6, 10 ng / ml IL-23, and 20 ng / ml TNF-α (for Th17 polarization). All cytokines were purchased from ImmunoTools. PBMCs were cultured in parallel under the same conditions. Cells were cultured for 3 days, and excess TransAct matrix was removed on day 2. On day 3, thyTreg and PBMC culture supernatants were frozen at -80°C for cytokine analysis, and thyTreg was evaluated for cell viability, phenotype, and repressive ability as described above. The remaining cells were stored as a dry pellet at -80°C for TSDR methylation studies.

[0164] Methylation analysis. DNA was isolated from cell pellets using the DNeasy Blood & Tissue Kit (Qiagen). The methylation status of 141 CpG sites located in 29 different genomic regions, including 20 different genes containing Treg-specific demethylation regions (TSDRs), was analyzed by targeted next-generation bisulfite sequencing (NGS070V3 assay) performed by EpigenDx Inc. (Hopkinton, Massachusetts, USA).

[0165] Statistical analysis. Results are expressed as mean ± SEM (standard error of the mean) or minimum-median-maximum. 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 tests used to evaluate each experiment are specified in the legend of the respective figures. Statistical associations between variables were calculated using linear regression and Pearson correlation analysis. A p-value < 0.05 was considered statistically significant. The following criteria were used to distinguish significance levels: * =<0.05, ** =<0.01 and *** =<0.001.

[0166] result Isolation and phenotype of ThyTreg Thymocytes obtained by mechanical degradation from newly removed pediatric thymuses (n=20; age range 0-48 months, Table 2) showed a high viability rate (96.33%±0.99%) (Figure 8A). Most of them (76.68%±2.03%) showed the CD4+CD8+ double-positive (DP) phenotype, but 12.57%±1.23% were CD4+ single-positive (SP) cells and 6.98%±1.26% were CD8+ SP cells (Figure 8B). Since CD4+CD8+DP thymic Treg cells have been shown to significantly contribute to the human thymic Treg pool (Nunes-Cabacao et al., 2011; Martin-Gayo et al., 2010; Vanhanen et al., 2020), we decided to directly isolate CD25+ thymocytes (2.36%±0.34%) without prior depletion of CD8+ cells. The mean frequency of FOXP3+ cells on isolated CD25+ thymocytes was 67.08% ± 2.22% (representative plot in Figure 8C). The advantage of preserving DP thyTregs was supported by comparing thyTreg cells obtained with and without CD8+ depletion. While maintaining cell viability and the proportion of CD8+SP and FOXP3+ cells, the yield of thyTregs was significantly higher without CD8+ depletion (p=0.04; Figure 9A), and the proportion of DP cells (p=0.003; Figure 9B) was also significantly higher (Figures 9B-9D). Following this strategy, we found that after CD25+ immunomagnetic selection (Table 2), 10 9 6.54 × 10⁶ per thymocyte 6 The number of thyTreg(2.44 × 10) 6 From 11.65 × 10 6 (within this range) the cell viability exceeded 85%. Therefore, the estimated number of thyTregs per gram of thymus is approximately 9.96 × 10⁶. 6 (1.32 × 10 6 ~21.59×10 6 (This range is 200.3 × 10 for an average thymus weight of 20.10 grams) 6 This is equivalent to high-purity thyTreg. TIFF2026518291000003.tif146170

[0167] Characterization of ThyTreg culture and products After isolating thyTreg cells, they were activated for 3 days and cultured for an additional 4 days, as shown in Figure 10A. Note that the culture conditions were kept as simple as possible in order to maintain the immature nature of thyTreg cells and to avoid extraneous compounds commonly used during Treg proliferation, such as rapamycin and human AB serum, which did not show any advantage in terms of thyTreg purity, phenotype, or proliferation rate (Figures 11A-11F). These cellular characteristics were also maintained using TransAct instead of Dynabeads for cell activation to avoid cell loss associated with Dynabead removal (Figures 11G-11J). Cell phenotypes on day 0 and day 7 are shown in Figures 10B and C (n=16). ThyTreg cells collected on day 7 showed very high viability (92.41% ± 1.02%) and purity (95.2% ± 0.74%) with respect to CD25+FOXP3+. Both parameters were higher compared to day 0. During this short culture period, thyTreg proliferated 6.9 ± 1.42 times (Figure 12A). 200.3 × 10⁶ cells isolated on day 0 6 Considering the number of thyTregs and average doubling, the theoretical number of thyTregs that can be obtained from a single thymus is approximately 1,500 × 10⁶. 6 In the best case, 13,649 × 10⁻¹⁴ cells are produced from a single thymus. 6 The yield of thyTreg cells reaches [number].

[0168] The inventors observed that the proportion of CD4+SP thyTregs decreased during cell culture and was offset by an increase in the proportion of CD4+CD8+DP thyTregs (Figure 10B, Figure 10C, bottom panel). Notably, on day 7, these CD4+CD8+DP thyTregs exhibited a similar phenotype to CD4+SP thyTregs, characterized by high expression of CD25 and FOXP3 (Figure 10D). In fact, there was a positive correlation between the proportion of CD4+CD8+DPthyTregs and the frequency of CD25+FOXP3+thyTregs (Figure 10E).

[0169] To further characterize thyTreg products, a series of cellular markers related to Treg phenotype and functionality were analyzed (Figure 13A). In summary, thyTreg products were characterized by high expression of cytotoxic T lymphocyte-associated protein (CTLA-4), inducible T cell costimulator (ICOS), thymic origin markers HELIOS and CD27, intermediate expression of T cell immune receptor (TIGIT) with Ig and ITIM domains, 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). Furthermore, to assess the homing ability of thyTreg cells, the expression of chemokine receptors CCR4, CXCR3 and CD62L selectin was determined (Figure 13B). ThyTreg cells exhibited high expression of CCR4, indicating their putative ability to migrate to organs with large epithelial surfaces (such as skin, intestines, or lungs) (Sather et al., 2007), while CD62L supported their location in lymph nodes (Lamarche and Levings, 2018). As previously shown (Dijke et al., 2016), the expression of several functional markers, including CTLA-4, CD73, ICOS, GITR, and LAP, significantly increased during cell culture. CD39 expression, on the other hand, decreased (Figure 12B). Furthermore, CD45RA expression increased, which may reflect the final switch from CD45RO to CD45RA that occurs as the final stage of maturation in the thymus (Fujii et al., 1992) (Figure 12B). Regarding chemokine receptor expression, CCR4 and CD62L significantly increased after 7 days of culture; on the other hand, CXCR3 expression decreased (Figure 12C), resulting in an undifferentiated phenotype (Groom and Luster, 2011).

[0170] Next, the inventors analyzed the profile of secreted molecules by thyTreg cells in the culture supernatant (Figure 13C). The inventors detected high levels of the anti-inflammatory cytokines IL-10 and transforming growth factor β (TGF-β) (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). In contrast, the inventors detected very low expression of pro-inflammatory cytokines, such as IFN-γ, IL-4, IL-17A, and PD-L1. Finally, the inventors evaluated the ability of thyTreg cells to suppress the proliferation of CD4+ and CD8+ stimulated T cells in vitro (Figures 13D and 13E). The inventors found that thyTreg exhibits an average inhibitory effect of over 80% at a thyTreg:responder cell ratio of 1:1 and a very high inhibitory effect of over 40% at a ratio of 1:4.

[0171] To determine the stability of thyTreg products, thyTreg cells were restimulated after being exposed to a cocktail of cytokines polarizing to Th1 (IL-2, IL-12) or Th17 (IL-2, IL-1β, IL-6, IL-23, TNF-α). We observed that the thyTreg cell phenotype remained stable with respect to FOXP3, CTLA-4, CD39, and HLA-DR expression (Figures 14A, 14B). Furthermore, thyTreg cells were not stimulated to produce IFN-γ or IL-17A under polarization conditions (Figure 14C), preserving their repressive functions (Figure 14D). To support these findings, we determined the stability of FOXP3 expression by analyzing the methylation profile of TSDR (Figures 14E-14F). The inventors observed intermediate levels of TSDR demethylation in thyTreg cells on day 0 (62.93% ± 4.3% for males and 22.74% ± 8.03% for females), which increased to 89.03% ± 2.57% for males and 51.26% ± 0.36% for females after culture. The difference in demethylation levels between sexes was attributed to methylation-mediated inactivation of one X chromosome in females. In contrast, TSDR demethylation of newly isolated (day 0) or cultured (day 7) thymic CD25-(thyTconv) was approximately 5%. Different methylation patterns between thyTreg and thyTconv were observed not only in the FOXP3 gene but also in eight other Treg-related genes, including CTLA-4, IKZF2, or ILR2A (Figure 12D). Notably, the TSDR demethylation status in thyTreg cells was maintained under pro-inflammatory conditions (Figure 14G).

[0172] One of the characteristics of our thyTreg products is the presence of the CD25+FOXP3+CD4+CD8+DP population. Therefore, we decided to evaluate their involvement in the Treg phenotype by analyzing the methylation status of TSDR. To this end, after 7 days of thyTreg culture, we selected the CD4+SP population and the CD4+CD8+DP population and analyzed their TSDR demethylation status compared to the overall thyTreg cell product (Figure 15). In fact, we confirmed that the percentage of CD4+CD8+DP cells with demethylated TSDR (94.1%) was similar to that observed in the CD4+SP or the total thyTreg population (92.8% and 91.6%, respectively), confirming the stability of FOXP3 expression in this cell subset.

[0173] Example 3: Immunophenotypic characterization of the CAR-ThyTreg population material and method After isolation, activation, transduction, sorting, and a total of 8 days of culture, thyTreg(NT) and CAR-thyTreg cells were stained with antibodies specified for surface markers in staining buffer (PBS + 2% FBS) and incubated at 4°C for 30 minutes. After washing twice with PBS, the cells were stained with Fixable Viability Dye (Miltenyi Biotec) in PBS at 4°C for 30 minutes. Excess dye was removed by washing the cells with staining buffer.

[0174] In some experiments, intracellular labeling was required. For this purpose, cells were treated with ionomycin (Sigma-Aldrich) and phorbol 12 myristate 13 acetate (Sigma-Aldrich) for 5 hours. During the last 2 hours of stimulation, cells were treated with GolgiStop (BD Biosciences), which stops Golgi apparatus-mediated cytokine secretion in stimulated cells. Cells were then harvested and stained at 4°C for 30 minutes using the specified antibody for surface markers in staining buffer (PBS + 2% FBS). After washing twice with PBS, cells were stained with Fixable Viability Dye (Miltenyi Biotec) in PBS at 4°C for 30 minutes and washed once with PBS. After viability labeling, cells were treated with Cytofix / Cytoperm® fixation / permebrania solution (BD Biosciences). This product is part of the BD Cytofix / Cytoperm® Plus kit (BD Biosciences) and includes cell fixation and permebrania at 4°C in the dark for 20 minutes. After cell fixation and permeabilization, the cells were washed twice with BD Perm / Wash® buffer. The intracellular staining antibody was diluted with this buffer and added, and the cells were stained in the dark at 4°C for 30 minutes. Finally, after two more washes, the cells were analyzed by flow cytometry. A total of 50,000 events were recorded per sample by flow cytometry.

[0175] Consideration In summary, our findings suggest that the phenotype and cytokine production of genetically modified thyTregs remain unchanged compared to unmodified thyTregs (NTs), regardless of the generation of CARs introduced into the thyTregs.

[0176] References Beheshti,S.A.,Shamsasenjan,K.,Ahmadi,M.,Abbasi,B.,2022.CAR Treg:A new approach in the treatment of autoimmune diseases.Int Immunopharmacol 102,108409. Bluestone,J.A.,Buckner,J.H.,Fitch,M.,Gitelman,S.E.,Gupta,S.,Hellerstein,M.K.,Herold,K.C.,Lares,A.,Lee,M.R.,Li,K.,Liu,W.,Long,S.A.,Masiello,L.M.,Nguyen,V., Putnam,A.L.,Rieck,M.,Sayre,P.H.,Tang,Q.,2015.Type 1 diabetes immunotherapy using polyclonal regulatory T cells.Sci Transl Med 7,315ra189. Brunstein,C.G.,Miller,J.S.,McKenna,D.H.,Hippen,K.L.,DeFor,T.E.,Sumstad,D.,Curtsinger,J.,Verneris,M.R.,MacMillan,M.L.,Levine,B.L.,Riley,J.L.,June,C.H.,Le,C.,Weisdorf,D.J.,McGlave,P.B.,Blazar,B.R.,Wagner,J.E.,2016.Umbilical cord blood-derived T regulatory cells to prevent GVHD:kinetics,toxicity profile,and clinical effect.Blood 127,1044-1051. Chwojnicki,K.,Iwaszkiewicz-Grzes,D.,Jankowska,A.,Zielinski,M.,Lowiec,P.,Gliwinski,M.,Grzywinska,M.,Kowalczyk,K.,Konarzewska,A.,Glasner,P.,Sakowska,J.,Kulczycka,J.,Jazwinska-Curyllo,A.,Kubach,M.,Karaszewski,B.,Nyka,W.,Szurowska,E.,Trzonkowski,P.,2021.Administration of CD4.BioDrugs 35,47-60. Dawson,N.A.J.,Rosado-Sanchez,I.,Novakovsky,G.E.,Fung,V.C.W.,Huang,Q.,McIver,E.,Sun,G.,Gillies,J.,Speck,M.,Orban,P.C.,Mojibian,M.,Levings,M.K.,2020.Functional effects of chimeric antigen receptor co-receptor signaling domains in human regulatory T cells.Sci Transl Med 12. Dijke IE,Hoeppli RE,Ellis T,Pearcey J,Huang Q,McMurchy AN,et al.Discarded Human Thymus Is a Novel Source of Stable and Long-Lived Therapeutic Regulatory T Cells.Am J Transplant.2016;16(1):58-71. Fujii Y,Okumura M,Inada K,Nakahara K,Matsuda H.CD45 isoform expression during T cell development in the thymus.Eur J Immunol.1992;22(7):1843-50. Gliwinski, M., Iwaszkiewicz-Grzes, D., Woloszyn-Durkiewicz, A., Tarnowska, M., Zalinska, M., Hennig, M., Zielinska, H., Dukat-Mazurek, A., Zielkowska-Debska, J., Zielinski, M., Jazwinska-Cury llo,A.,Owczuk,R.,Jarosz-Chobot,P.,Bossowski,A.,Szadkowska,A.,Mlynarski,W.,Marek-Trzonkowska,N.,Moszkowska,G.,Siebert,J.,Mysliwiec,M.,Trzonkowski,P.,2020.Proinsulin-specific T regulatory cells may control immune responses in type 1 diabetes:implications for adoptive therapy.BMJ Open Diabetes Res Care 8. Groom JR, Luster AD.T cell function of CXCR3.Exp Cell Res.2011;317(5):620–31. Juneja,T.,Kazmi,M.,Mellace,M.,Saidi,RF,2022.Utilization of Treg Cells in Solid Organ Transplantation.Front Immunol 13.746889. Lamarche C,Levings MK.Guiding regulatory T cells to the allograft.Curr Opin Organ Transplant.2018;23(1):106-13. Lin H,Cheng J,Mu W,Zhou J and Zhu L(2021)Advances in Universal CAR-T Cell Therapy Ma,Y.,He,K.M.,Garcia,B.,Min,W.,Jevnikar,A.,Zhang,Z.X.,2008.Adoptive transfer of double negative T regulatory cells induces B-cell death in vivo and alters rejection pattern of rat-to-mouse heart transplantation.Xenotransplantation 15,56-63. Marek-Trzonkowska,N.,Mysliwiec,M.,Dobyszuk,A.,Grabowska,M.,Derkowska,I.,Juscinska,J.,Owczuk,R.,Szadkowska,A.,Witkowski,P.,Mlynarski,W.,Jarosz-Chobot,P.,Bossowski,A.,Siebert,J.,Trzonkowski,P.,2014.Therapy of type 1 diabetes with CD4(+)CD25(high)CD127-regulatory T cells prolongs survival of pancreatic islets-results of one year follow-up.Clin Immunol 153,23-30. Martin-Gayo E,Sierra-Filardi E,Corbi AL,Toribio ML.Plasmacytoid dendritic cells resident in human thymus drive natural Treg cell development.Blood.2010;115(26):5366-75. McMurchy AN,Levings MK.Suppression assays with human T regulatory cells:a technical guide.Eur J Immunol.2012;42(1):27-34. Nunes-Cabaco H,Caramalho I,Sepulveda N,Sousa AE.Differentiation of human thymic regulatory T cells at the double positive stage.European Journal of Immunology.2011;41(12):3604-14. Safinia,N.,Vaikunthanathan,T.,Fraser,H.,Thirkell,S.,Lowe,K.,Blackmore,L.,Whitehouse,G.,Martinez-Llordella,M.,Jassem,W.,Sanchez-Fueyo,A.,Lechler,R.I.,Lombardi,G.,2016.Successful expansion of functional and stable regulatory T cells for immunotherapy in liver transplantation.Oncotarget 7,7563-7577. Lohmueller,J.J.,et al.,mSA2 affinity-enhanced biotin-binding CAR T cells for universal tumor targeting.Oncoimmunology,2017.7(1):p.e1368604. Sagoo,P.,Ali,N.,Garg,G.,Nestle,F.O.,Lechler,R.I.,Lombardi,G.,2011.Human regulatory T cells with alloantigen specificity are more potent inhibitors of alloimmune skin graft damage than polyclonal regulatory T cells.Sci Transl Med 3,83ra42. Sakaguchi,S.,Yamaguchi,T.,Nomura,T.,Ono,M.,2008.Regulatory T cells and immune tolerance.Cell 133,775-787. Salmon,P.,Trono,D.,2006.Production and titration of lentiviral vectors.Curr Protoc Neurosci Chapter 4,Unit 4.21. Sather BD,Treuting P,Perdue N,Miazgowicz M,Fontenot JD,Rudensky AY,et al.Altering the distribution of Foxp3(+)regulatory T cells results in tissue-specific inflammatory disease.J Exp Med.2007;204(6):1335-47. Sicard,A.,Koenig,A.,Morelon,E.,Defrance,T.,Thaunat,O.,2015.Cell therapy to induce allograft tolerance:time to switch to plan B? Front Immunol 6,149. Sutherland AR,Owens MN,Geyer CR.Modular Chimeric Antigen Receptor Systems for Universal CAR T Cell Retargeting.Int J Mol Sci(2020)21:7222.doi:10.3390 / ijms21197222 Clin Immunol 133,22-26. Vanhanen R,Leskinen K,Mattila IP,Saavalainen P,Arstila TP.Epigenetic and transcriptional analysis supports human regulatory T cell commitment at the CD4+CD8+thymocyte stage.Cellular Immunology.2020;347:104026.

[0177] Clause For completeness, various aspects of the present invention are described in the following numbered clauses. Clause 1 A thymic T regulatory cell (ThyTreg cell) that encodes a chimeric antigen receptor (CAR) comprising an extracellular domain, a hinge region, a transmembrane domain, and an intracellular domain, or alternatively expresses a chimeric antigen receptor (CAR) on its surface, wherein the intracellular domain is A cytoplasmic costimulatory domain having a sequence that is at least 85% identical to sequence number 1, Cytoplasmic stimulation domain and ThyTreg cells, including those mentioned above. Clause 2: The ThyTreg cells described in Clause 1, wherein the extracellular domain includes or comprises a biotin-binding moiety, the biotin-binding moiety being selected from neutraavidin, bladavidin, tamavidin, schwanavidin, zebaavidin, streptavidin, streptavidin derivatives, or any functional fragment thereof, the biotin-binding moiety being streptavidin or a functional fragment thereof, and in particular biotin-binding monomer streptavidin (mSA2). Clause 3: ThyTreg cells as described in Clause 1 or 2, wherein the cytoplasmic costimulatory domain consists solely of Sequence ID No. 1. Clause 4: ThyTreg cells in which the cytoplasmic stimulation domain is CD3 zeta, according to any one of the preceding clauses. Clause 5: ThyTreg cells 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, and in particular CD28 (SEQ ID NO: 4). Clause 6: A ThyTreg cell from any one of the preceding clauses, in which the hinge region is derived from CD8a (SEQ ID NO: 3). A ThyTreg cell from 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) CD3 zeta, and (i) to (v) are provided in N-terminus to C-terminus order, and has at least 85% identity with respect to the sequence of SEQ ID NO: 6 in particular. Clause 8: A ThyTreg cell of any one of the preceding clauses, in which the extracellular domain does not contain or is not fused to an antibody or any fragment thereof. Clause 9: ThyTreg cells as described in any one of Clauses 1 to 7, wherein the extracellular domain is fused to a binding site specific to activated immune cells, particularly an antibody or fragment thereof, more specifically to an anti-CD8+ antibody or fragment thereof. Clause 10 A method for preparing ThyTreg cells as defined in any one of Clauses 1 to 9, comprising the step of transfecting or transducing isolated ThyTreg cells with an expression vector encoding a CAR as defined in any one of Clauses 1 to 9. Clause 11 The method according to Clause 10, wherein the expression vector is a virus, particularly a lentivirus. Clause 12 The method according to Clause 10 or 11, wherein the expression vector contains a sequence having at least 85% identity with SEQ ID NO: 7, and in particular the expression vector contains a sequence having 100% identity with SEQ ID NO: 7. Clause 13 The method according to any one of Clauses 10 to 12, wherein the expression vector has a sequence that is at least 85% identical to the sequence of SEQ ID NO: 8, in particular the expression vector is 100% identical to SEQ ID NO: 8. Clause 14 Thymic Treg cells undergo the following steps: a. A process of mechanically decomposing isolated thymic tissue, b. The product obtained after step (a) is filtered, and the precipitate containing thymocytes is resuspended in the culture medium, c. A step to isolate CD25+ cells from the product obtained after step (b), d. A step of culturing the cell population obtained after step (c) in a culture medium in the presence of a T cell activator and IL-2, wherein the T cell activator includes at least CD3 and CD28 agonists. e. A step to remove T cell activating factors from the culture medium in step (d), The method according to any one of the claims 10 to 13, further comprising step (f) of culturing regulatory T cells in culture medium in the presence of IL-2, provided that the cell population is not depleted of CD8+ cells prior to step (d). The method according to Clause 14, wherein step (a) comprises mechanically degrading thymic tissue without the use of enzymes in the presence of a culture medium. The method according to any one of the claims 14 to 15, wherein the isolation of CD25+ cells in step (c) of claim 16 includes the use of magnetic beads conjugated with an antibody against CD25. The method according to any one of the claims 14 to 16, wherein the T cell activator in step (d) of claim 17 is a colloidal polymer nanomatrix conjugated with humanized CD3 and CD28 agonists. The method according to any one of the clauses 14 to 17, wherein the T cell activator of clause 18 (d) is used in a T cell activator:cell ratio in the range of 1:10 to 1:100. Clause 19 The method according to any one of Clauses 14 to 18, wherein the cells are cultured for at least two or three days, preferably at least three days, in step (d). Clause 20 The method according to any one of Clauses 14 to 19, wherein cells are cultured in step (d) in the absence of rapamycin. The method according to any one of the clauses 14 to 20, wherein the removal of step (e) of clause 21 is carried out by magnet or centrifugal force. The method according to any one of the claims 14 to 21, wherein in step (f) of claim 22, regulatory T cells are cultured for a further 1 to 7 days, preferably for a further 4 days, in the presence of IL-2. Clause 23 The method according to any one of Clauses 14 to 22, wherein cells are cultured in step (f) in the absence of rapamycin. Clause 24 The method according to any one of Clauses 14 to 23, wherein the culture medium is a GMP culture medium. Clause 25 The method according to any one of Clauses 14 to 24, wherein the culture medium further comprises an antibiotic, preferably 5% of an antibiotic. Clause 26 The method described in any one of Clauses 14 to 25, wherein the thymic tissue is of human origin. Clause 27 ThyTreg cells that can be obtained by the method described in any one of Clauses 10 to 26. A pharmaceutical composition comprising a therapeutically effective amount of CAR-ThyTreg cells as defined in any one of the clauses 1 to 8 or 27, and one or more pharmaceutically acceptable excipients or carriers. Article 29 A population of Treg cells defined in any of Articles 2-8, A binding site specific to activated immune cells, and a binding site conjugated with biotin. A combination that includes this. Clause 30: The combination described in Clause 29, wherein the cell-specific binding portion is an antibody or a fragment thereof. Clause 31: The combination described in Clause 30, wherein the antibody is anti-CD8+. Clause 32: The combination described in any one of Clauses 29-31, wherein the ThyTreg cells have an extracellular domain consisting solely of mSA2. Clause 33 A chimeric antigen receptor (CAR) comprising an extracellular domain, a hinge region, a transmembrane domain and an intracellular domain, The extracellular domain contains the antibody anti-CD8+ or a fragment thereof. The intracellular domain A cytoplasmic costimulatory domain having a sequence that is at least 85% identical to sequence number 1, and Cytoplasmic stimulation domain and Chimeric antigen receptors (CARs), including those mentioned above. Clause 34: A nucleic acid that codes for a CAR as defined in Clause 34. Clause 35 An expression vector containing the nucleic acid of Clause 35. Cells transduced with the vector of Clause 36. Clause 37: Treg cells, particularly thyTreg cells, as described in Clause 37. A pharmaceutical composition comprising a cell transduced with an effective amount of the vector described in Clause 36, or a therapeutically effective amount of the vector described in Clause 36, and one or more pharmaceutically acceptable vehicles or carriers. Clause 39 CAR-ThyTreg cells as defined in any one of Clauses 1-8, 27, or 36, pharmaceutical compositions as defined in Clause 28 or 38, or any combination of any one of Clauses 29-32, for use in treatment or diagnosis. Clause 40 CAR-ThyTreg cells as defined in any one of Clauses 1-8, 27, 36, or 37, pharmaceutical compositions of Clause 28 or 39, or any combination of Clauses 29-32, for use in methods of inducing or restoring tolerance to the immune system. Clause 41 CAR-ThyTreg cells as defined in any one of Clauses 1-8, 27 or 36 or 37, pharmaceutical compositions of Clause 28 or 39, or any combination of Clauses 29-32, for use in methods of treating diseases selected from autoimmune diseases, inflammatory processes, allergies, graft-versus-host diseases and immune rejection to transplants. Clause 42 A combination of any one of Clauses 29-31 for use as described in any one of Clauses 40-41, wherein the method involves separately administering a ThyTreg cell population and a binding site specific to activated immune cells.

Claims

1. A thymic T regulatory cell (ThyTreg cell) that encodes a chimeric antigen receptor (CAR) comprising an extracellular domain, a hinge region, a transmembrane domain, and an intracellular domain, or alternatively expresses a chimeric antigen receptor (CAR) on its surface, wherein the intracellular domain is A cytoplasmic costimulatory domain having a sequence that has at least 85% identity with SEQ ID NO: 1, which provides a signal that mediates a Treg cell-specific response, Cytoplasmic stimulation domain and ThyTreg cells, A population of ThyTreg cells that includes or consist solely of them.

2. The ThyTreg cell population according to claim 1, wherein the cell population comprises at least 60% 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% CD25+FOXP3+ cells.

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

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

5. The ThyTreg cell population according to any one of claims 1 to 4, wherein at least 25% of the cells in the ThyTreg cell population express one or more markers selected from the group consisting of glucocorticoid-induced tumor necrosis factor receptor (GITR) and HLA-DR.

6. The ThyTreg cell population according to any one of claims 1 to 5, wherein 20% or less of the cells in the ThyTreg cell population express one or more markers selected from the group consisting of CD73, lymphocyte activation gene 3 (LAG-3), and CXCR3.

7. d) At least 60% of the cells in the ThyTreg cell population express CCR4, e) At least 25% of the cells in the ThyTreg cell population express HLA-DR and / or CD45RA, f) The ThyTreg cell population according to any one of claims 1 to 6, wherein 20% or less of the cells in the ThyTreg cell population express CD73, LAG-3 and / or CXCR3.

8. The ThyTreg cell population according to any one of claims 1 to 7, characterized by high levels of expression of anti-inflammatory cytokines such as IL-10 and / or TGF-β, and / or other inhibitory molecules related to Treg function such as granzyme B, soluble LAG-3 and / or TIM-3, preferably with an expression level of at least 50 pg / ml, more preferably at least 100 pg / ml, the expression level being determined as specified herein.

9. The ThyTreg cell population according to any one of claims 1 to 8, characterized in that the ThyTreg cell population has an expression level of at least 100 pg / ml, preferably at least 150 pg / ml, of IL-10 and / or TGF-β, the expression level being determined as specified herein.

10. The ThyTreg cell population according to any one of claims 1 to 9, characterized by low expression levels of anti-inflammatory cytokines such as IFN-γ, IL-4, IL-17A and / or PD-L1, preferably 20 pg / ml or less, more preferably 10 pg / ml or less, and the expression level being determined as specified herein.

11. The aforementioned ThyTreg cell population, c) Expression levels of granzyme B, soluble LAG-3 and / or TIM-3 of at least 100 pg / ml, and / or d) Expression levels of IL-17-A and / or PD-L1 less than 10 pg / ml A ThyTreg cell population according to any one of claims 1 to 10, characterized in that the expression level is determined as specified herein.

12. The ThyTreg cell population according to any one of claims 1 to 11, characterized in that the ThyTreg cell population has stable FOXP3 expression.

13. The ThyTreg cell population according to claim 12, wherein the ThyTreg cell population is derived 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% demethylation in the Treg-specific demethylation region (TSDR) of the FOXP3 gene.

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

15. The ThyTreg cell population according to any one of claims 1 to 14, wherein the cell population has an average inhibitory ability on CD4+ and / or CD8+ T cell 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 1:1 thyTreg:responder cell ratio, and / or an average inhibitory ability on CD4+ and / or CD8+ T cell proliferation of at least 25%, preferably at least 30%, at least 35%, or more preferably at least 40% at a 1:4 thyTreg:responder cell ratio.

16. The ThyTreg cell population according to any one of claims 1 to 15, wherein the cell population does not contain effector T cells.

17. The ThyTreg cell or cell population according to any one of claims 1 to 16, wherein the extracellular domain comprises or consists of a first molecule from a pair of complementary affinity molecules such as a biotin-binding moiety, the biotin-binding moiety being selected from neutraavidin, bladavidin, tamavidin, schwanavidin, zebaavidin, streptavidin, streptavidin derivatives, or any functional fragment thereof, the biotin-binding moiety being streptavidin or a functional fragment thereof, and the biotin-binding moiety being monomeric streptavidin (mSA2).

18. The ThyTreg cell or cell population according to any one of claims 1 to 17, wherein the cytoplasmic costimulatory domain consists solely of SEQ ID NO:

1.

19. The ThyTreg cell or cell population according to any one of claims 1 to 18, wherein the cytoplasmic stimulating domain is CD3 zeta.

20. The ThyTreg cell or cell population according to any one of claims 1 to 19, wherein the transmembrane domain is selected from CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154, and is particularly CD28 (SEQ ID NO: 4).

21. The ThyTreg cell or cell population according to any one of claims 1 to 20, wherein the hinge region is derived from CD8a (SEQ ID NO: 3).

22. The ThyTreg cell or cell population according to any one of claims 1 to 21, wherein the CAR comprises (i) mSA2, (ii) CD8a hinge region, (iii) CD28 transmembrane region, (iv) SEQ ID NO: 1, and (v) CD3 zeta, and (i) to (v) are provided in the order from N-terminus to C-terminus, and have at least 85% identity with respect to the sequence of SEQ ID NO:

6.

23. A method for preparing a ThyTreg cell or cell population as defined in any one of claims 1 to 22, comprising the step of transfecting isolated ThyTreg cells with an expression vector encoding a CAR as defined in any one of claims 1 to 22.

24. The expression vector The expression vector contains or consists of a sequence having at least 85% identity with SEQ ID NO: 7, and in particular, the expression vector contains a sequence that is 100% identical to SEQ ID NO: 7, or The method according to claim 23, wherein the expression vector has a sequence that is at least 85% identical to the sequence of sequence number 8, and in particular the expression vector is 100% identical to sequence number 8.

25. The isolated ThyTreg cells undergo the following process: a. A process of mechanically decomposing isolated thymic tissue, b. A step of filtering the product obtained after step (a) and resuspending the precipitate containing the thymocytes in a culture medium, c. A step of isolating CD25+ cells from the product obtained after step (b), d. A step of culturing the cell population obtained after step (c) in a culture medium in the presence of a T cell activator and IL-2, wherein the T cell activator comprises at least CD3 and CD28 agonists. e. The step of removing T cell activating factors from the culture medium in step (d) The method according to claim 23 or 24, further comprising step (f) of culturing regulatory T cells in a culture medium in the presence of IL-2, provided that the cell population is not depleted of CD8+ cells prior to step (d).

26. The method according to any one of claims 23 to 25, wherein the thymic tissue is human thymic tissue.

27. ThyTreg cells or cell populations that can be obtained by the method described in any one of claims 23 to 26.

28. A pharmaceutical composition comprising a therapeutically effective amount of a 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 population of ThyTreg cells as defined in any one of claims 17 to 22 or 27, A binding site specific to activated immune cells, particularly an antibody or a fragment thereof, where the first molecule in a pair of complementary affinity molecules is a biotin-binding site, is conjugated to a second molecule from a pair of complementary affinity molecules such as biotin, a biotin analog, or a fragment thereof, and A combination that includes this.

30. A ThyTreg cell or cell population as defined in any one of claims 1 to 22 or 27, a pharmaceutical composition according to claim 28, or a combination according to claim 29, for use in treatment or diagnosis.

31. Preferably, for use in methods for treating subjects requiring immunotherapy, in a method for treating a disease selected from autoimmune diseases, inflammatory processes, allergies, graft-versus-host diseases and immune rejection to transplants, in a method for inducing or restoring tolerance to the immune system in subjects requiring induction or restoration of tolerance to the immune system, ThyTreg cells or cell populations as defined in any one of claims 1 to 22 or 27, the pharmaceutical composition according to claim 28, or the combination according to claim 29.

32. The combination according to claim 29 for use according to claim 30 or 31, wherein the method comprises separately administering a population of ThyTreg cells and a binding site specific to activated immune cells.

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

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