Method for obtaining regulatory t cells derived from thymus tissue and use of said cells as cellular immunotherapy in immune system disorders

Abstract

The present invention provides an in vitro method for obtaining and purifying regulatory T cells (or thyTreg cells) from thymus tissue, which makes it possible to obtain more than 10 billion cells from a single thymus. In addition to being safe from the clinical perspective, these thyTregs obtained in the present invention have a purity of more than 95% and very high inhibitory ability, survival rate and viability. The foregoing will not require the use of a large number of ex vivo cell amplification protocols. Transfer of these thyTreg cells to a patient can induce immune tolerance. Thus, the cells can be used as cell therapy to induce immune tolerance in the treatment and / or prevention of transplant rejection and autoimmune diseases.

Description

Technical Field

[0001] The present invention belongs to the field of clinical immunology and cellular immunotherapy, and in particular to protocols for obtaining and purifying regulatory T cells (Tregs) which can subsequently be transferred into patients in cell therapy for the purpose of inducing immune tolerance, e.g. in transplanted individuals or those undergoing autoimmune processes. Background Art

[0002] The main function long attributed to the immune system is that of protecting the organism from pathogens. However, we now know that the immune system is also responsible for eliminating tumor cells and preventing the development of cancer, and that it can also cause insufficient responses due to the occurrence of autoimmune processes, allergies or transplant rejection. The normal function of the immune system is only possible when there is a balance or homeostasis that is sufficient for normal function, so that excessive responses will cause pathological conditions such as allergies or rejection reactions, while insufficient responses will lead to the development of infections and cancer.

[0003] In recent years, we have witnessed the emergence of revolutionary cell therapies using immune cells in cancer treatment, which seek to induce or restore a specific immune response to the tumor and to repair the patient's immune defects that cause these pathologies. This is the case with the "CAR T cell" technology, in which the patient's immune cells are genetically modified to attack the tumor in a specific way. Companies like Novartis or Gilead have developed this cell therapy to treat B leukemia, achieving about 80% remission, and the therapy has recently been approved by the FDA and EMA, which represents a real revolution in cancer treatment (Maude SL, et al., 2014, N Engl J Med., 371 (16): 1507-17; Lee DW, et al., 2015, Lancet, 385 (9967): 517-528).

[0004] However, in the opposite sense, a cell therapy with similar effects, i.e., inducing immune tolerance to prevent or cure autoimmune diseases or allograft rejection, has not yet been developed. An immunotherapy that has been significantly successful in reducing excessive immune responses and restoring immune tolerance has not yet been developed. Currently, immunosuppressive drugs are used to treat autoimmune diseases and immune rejection. Since the emergence of these drugs in the 1960s, transplantation practice has become feasible, but, despite the improvement of these drugs, they still cannot provide a clear solution for rejection, and continue to cause side effects, which are the determining factors in the clinical evolution of patients. Specifically, long-term immunosuppression causes chronic toxicity, which, in addition to significantly affecting the quality of life of patients, also affects the completion of treatment, the overall success rate of patients and transplants, and survival. Since most immunosuppressants work in a non-selective manner, the entire system is suppressed and / or loses control, thereby losing its ability to defend the host against infection or tumor cell reproduction or producing vascular damage that causes transplant organ failure. In addition, in childhood, the continued use of immunosuppressants may interfere with normal development and interfere with the proper maturation of the immune system that occurs during this period, which may cause lifelong consequences in terms of changing the immune capacity of patients. These immunosuppressive drugs may continue to improve in the future, further reducing rejection rates; however, the downside of this strategy is often the degeneration and chronic damage to the immune system. Even if transplant rejection can be better prevented, conditions related to the degeneration of the immune system (such as infection, cancer, and autoimmune disease) will continue to limit the long-term survival of transplant patients receiving immunosuppressive drugs.

[0005] For this reason, achieving immune tolerance that would indefinitely avoid rejection or autoimmune diseases that affect millions of people has become a major challenge in modern medicine.

[0006] The current view of the scientific community is that only an induction of immune tolerance involving re-educating the immune response of the transplant recipient will allow the graft to survive indefinitely or prevent and cure the autoimmune process. Such tolerance induction would make it possible to tolerate the transplanted organ without the need for pharmacological immunosuppression, thereby eliminating the toxic effects of these therapies and their damaging effects on the immune system.

[0007] One of the most promising alternatives to increase the life expectancy of transplant patients is to induce immune tolerance with the help of cellular immunotherapy, which will allow the graft to survive indefinitely without the pathology associated with immunosuppressive therapy (Sicard A., et al., 2015, Front Immunol; 6: 149). This approach focuses on intentionally reducing the specific immune response to the transplant while eliminating or significantly reducing long-term pharmacological suppression, thereby maintaining an effective immune system. Immune tolerance is a fundamental feature of the immune system, and the discovery of lymphocyte subsets with suppressive ability that can induce this tolerance is causing widespread enthusiasm in the clinical field. These cells, known as regulatory T cells (Treg), constitute a fundamental part of the immune system and can play a vital role in preventing graft rejection or autoimmune diseases and maintaining immune homeostasis that is beneficial to patients. Treg can inhibit the effector function of a variety of cells, including T CD4+ and CD8+, NK cells, B cells, macrophages, and dendritic cells (Sakaguchi S., et al., 2008, Cell; 133: 775-87). Therefore, Treg cells have become an area of ​​interest in studying autoimmunity, allergy and transplantation.

[0008] Therefore, cell therapy using Tregs is assumed to be a great hope for the treatment of diseases mediated by excessive or insufficient immune system response, such as autoimmune processes (Bluestone JA, et al., 2015, Expert Opin. Ther. Targets; 19: 1091-103), graft-versus-host disease in bone marrow transplant patients (Brunstein CG, et al., 2016, Blood; 127: 1044-51), or transplant rejection (Safinia N., et al., 2015, Front Immunol; 6: 438).

[0009] The basic role of Tregs in transplants has been confirmed in various studies of skin and heart transplant animal models, proving that Tregs present in the receptacle during transplantation are essential for inducing and maintaining tolerance to the transplant (Wood KJ, Sakaguchi S., 2003, Nat. Rev. Immunol.; 3: 199-210). These Treg cells will hinder the activation and expansion of effector T cells, which are the cause of cellular rejection. In addition, Tregs can also induce B cell death, thereby preventing humoral rejection, as has been demonstrated in cardiac xenotransplantation models (Ma Y., et al., 2008, Xenotransplantation; 15: 56-63).

[0010] Current knowledge in this area points to the hypothesis that immune tolerance in transplant patients or those with autoimmune processes is determined by the balance of Treg cells and effector T cells. Therefore, it is expected that a greater number of circulating Tregs would be able to prevent the activation and proliferation of effector cells that trigger these diseases.

[0011] Therefore, a therapeutic strategy that can provide excellent results in preventing rejection or treating autoimmunity is to carry out cell therapy through Treg cell transfer to significantly increase the number of Treg cells in the circulation, and thereby strengthen the recipient's intrinsic tolerance mechanism in the recipient to the transplanted organ or to its own tissue. The transfer of autologous Treg in the patient is achieved by: extracting blood from the patient, purifying the Treg present in the blood, amplifying these Tregs in vitro to obtain a suitable number, and transferring the amplified autologous Tregs back to the patient. As described below, other groups have demonstrated their effectiveness in preventing rejection in animal models, and have even conducted clinical trials in other diseases, confirming their therapeutic use in humans.

[0012] The safety and potential effectiveness of Treg therapy in humans are reflected in the first batch of Phase I / II trials that have been conducted. Most clinical trials using Tregs are conducted in the context of bone marrow transplantation in patients with hematological tumors, showing that infusion of Tregs in these patients reduces or prevents graft-versus-host disease (GvHD) (Brunstein CG. et al., 2016, Blood. 127 (8): 1044-51; Di Ianni M. et al., 2011, Blood. 117 (14): 3921-8). The greatest risk of GvHD occurs in the first three months, and it has been demonstrated that immunosuppression by Treg therapy in this short period is sufficient to provide long-term tolerance. However, in the case of solid organ transplantation or autoimmune disease, this risk persists throughout the life cycle of the patient or transplant, which requires the protective effect of Tregs to persist in a timely manner to ensure the prevention of insufficient immune response.

[0013] An international consortium called The One Study is conducting a multicenter phase I / II study in which the safety and potential efficacy of infusing ex vivo expanded Tregs in the context of liver and kidney transplantation in adults is being studied. However, although the preliminary results of the consortium are promising, they demonstrate the difficulty of obtaining high-quality Tregs from the peripheral blood of adult patients and the limited survival and efficacy of Tregs present in adult patients due to their more differentiated phenotype (Safinia N., et al., 2015, Front Immunol., 6: 438). There are also Treg transfer trials in other diseases (such as type 1 diabetes), including trials in children (Marek-Trzonowska N., et al., 2012, Diabetes Care; 35: 1817-20). However, despite the great interest and expectations of this treatment alternative in recent years, treatment with Treg cells has not yet provided clear results in preventing human solid organ rejection.

[0014] Most strategies and trials conducted to date use Treg cells obtained from peripheral blood, which are then expanded ex vivo and transferred back to the patient as a cell therapy. The biggest limitation is obtaining a sufficient number of Tregs, as their frequency in peripheral blood is very low (4% to 10% of total T CD4+ lymphocytes), so it is essential to apply a large number of ex vivo Treg expansion protocols before being able to transfer the cells back to the patient. Wiesinger et al., 2017, Frontiers in Immunology, 8: 1371 describes the GMP-compliant production of ex vivo expanded Treg cells isolated from peripheral blood. The method involves depleting CD8+ cells before CD3 / CD28 stimulation.

[0015] Another important limitation is that the Treg cells present in adult peripheral blood are differentiated cells and may be highly aged. The survival period of these more differentiated cells is limited to a few months, the inhibitory ability is reduced, and the phenotype is unstable, which may cause them to lose the expression of Foxp3 molecules that determine their functionality and inhibitory ability, and even differentiate into pro-inflammatory effector T cells (Miyara M., et al., 2009, Immunity; 1-13). If these cells are also expanded in culture, their regulatory cell capacity will be further reduced (Hoffmann P., et al., 2009, Eur. J. Immunol.; 39: 1088-97).

[0016] Thus, ex vivo Treg cell expansion protocols have various issues that limit their usefulness in obtaining functional cells that can be successfully and safely used in human clinical trials:

[0017] a. Most expansion protocols do not meet the mandatory GMP (Good Manufacturing Practice) requirements for cells obtained for use in human immunotherapy.

[0018] b. It has been shown that, depending on the initial Treg phenotype, ex vivo expansion can lead to a loss of its suppressive capacity. Treg cells with an in vitro stimulated memory phenotype lose Foxp3 expression (responsible for their suppressive capacity) and their Treg phenotype, while Tregs with a naive phenotype are able to maintain Foxp3 expression and therefore their suppressive capacity after repeated stimulation and expansion. This fact has been confirmed by different authors, indicating that the naive Treg cell population, which is a minority in adults, will be most suitable for expansion, thereby retaining its suppressive properties.

[0019] c. In vitro expansion of differentiated Treg cells or Treg cells with a memory phenotype can lead to the loss of their suppressive phenotype and the acquisition of a pro-inflammatory phenotype that secretes cytokines such as IL-17 or IL-4, which may even worsen or promote the organ rejection process.

[0020] That said, the difficulty in obtaining sufficient numbers and quality of Tregs using current strategies limits the clinical utility of this therapy. As a result, ongoing human trials targeting tolerance induction via Treg transfer have encountered significant difficulties in generating sufficient cells with the necessary suppressive capacity to achieve clear short-term and long-term therapeutic effects.

[0021] To date, the use of Treg therapy to prevent solid organ rejection in humans has not produced clear clinical results. The limited number of Tregs that can be purified from peripheral blood, combined with the low survival rate and limited suppressive capacity of Tregs obtained from adults, may have made this therapeutic strategy unsuccessful.

[0022] A recently published "proof of concept" demonstrated that human thymus tissue is a potential source of Treg cells that can obtain cells with optimal phenotypes as an alternative to peripheral blood (Dijke IE, et al., 2016, American Journal of Transplantation, 16: 58-71; and MacDonald K. et al. 2017, Wolters Kluwer, Canadian national transplant research program, page S9; https: / / journals.lww.com / transplantjournal / Fulltext / 2017 / 05003). The study suggested the use of thymus tissue as a source of Tregs, confirming the quality of cells obtained from this tissue, its suppressive capacity, and a large number of cells that can be obtained relative to the use of peripheral blood as a cell source. However, since this was an in vitro study using an animal model, it applied a Treg purification protocol commonly used in research, which included the use of bovine serum, enzyme treatment, depletion of CD8 using complement-mediated lysis, rapamycin, particles coated with non-human anti-CD3 and anti-CD28 antibodies as Treg activators, etc., some of which are incompatible with the subsequent use of the obtained cells as immunotherapy in humans. In addition, the number of Treg cells obtained using this method is still not enough to implement adequate treatment in humans.

[0023] Based on the foregoing, it is necessary to develop an optimized method for obtaining and purifying Treg cells, which, in addition to making it possible to obtain cells in sufficient quantity and sufficient for subsequent use in human immunotherapy, will also make it possible to obtain cells with improved quality in terms of phenotype, purity, retained inhibitory ability, survival rate and vitality. Moreover, the method must provide cells that are safe for human cellular immunotherapy from a clinical perspective. The therapeutic use of Treg cells obtained by means of the enhancement method will make it possible to prevent chronic immune rejection, thereby achieving extended transplant survival. Summary of the invention

[0024] As mentioned above, one of the biggest limitations of Treg-based cell therapy is achieving sufficient numbers of Tregs. To date, protocols for purifying Tregs from peripheral blood have yielded yields of less than 20 million cells, requiring extensive expansion protocols that involve the use of different reagents and significantly reduce the quality of the cells.

[0025] The inventors provide for the first time a protocol for obtaining Treg cells without depleting CD8+ cells prior to CD3 / CD28 stimulation. The inventors demonstrate that this results in the presence of a CD4+CD8+ population of Foxp3+ Tregs in the final product (approximately 41%, Figure 4 B, 4C). Considering that about 80% of thymocytes in thymic tissue are cells expressing CD4 and CD8 markers ( Figure 4 A), CD8 depletion previously employed in other protocols would have eliminated the majority of these CD4+CD8+ Treg cells.

[0026] In a preferred embodiment, colloidal polymer nanomatrix conjugated to humanized CD3 and CD28 agonists is used for CD3 / CD28 activation, which is an innovative approach that provides levels of Treg cell activation and expansion comparable to those obtained using CD3 / CD28 beads ( Figure 6 ), while making it easy to remove the T cell activating agent from the culture medium, thereby minimizing the loss of Treg cells in this step.

[0027] As shown in the Examples, the protocol of the present invention for obtaining Treg cells from isolated thymic tissue enables the acquisition of more than 13 billion Treg cells with a purity greater than 95% (see Figure 1 C and 4B), vitality greater than 80% ( Figure 1 B), and the inhibitory ability is very high ( Figure 2 and 3 Such numbers of cells would make it possible to produce more than 1,000 doses of cell therapy for patients younger than 1 year old and hundreds of doses if used in older children or adults. These yields are unheard of in obtaining thyTreg cells.

[0028] Therefore, in a first aspect, the present invention relates to an in vitro method for obtaining a regulatory T (Treg) cell population from isolated thymic tissue, comprising the following steps:

[0029] a. Mechanical disintegration of thymic tissue;

[0030] b. filtering the product obtained after stage (a) and resuspending the pellet containing thymocytes in culture medium;

[0031] c. separating CD25+ cells from the product obtained after stage (b);

[0032] d. culturing the cell population obtained after stage (c) in a culture medium in the presence of a T cell activator and IL-2 or TGFβ (preferably IL-2), preferably wherein the T cell activator comprises at least a CD3 and CD28 agonist; and

[0033] e. removing the T cell activating agent from the culture medium of stage (d);

[0034] f. Optionally, further culturing regulatory T cells in the culture medium in the presence of IL-2 or TGFβ (preferably IL-2);

[0035] Provided that prior to step (d), the cell population is not depleted of CD8+ cells.

[0036] Another aspect of the invention relates to regulatory T cells or a population of regulatory T cells obtained or obtainable by a method of the invention.

[0037] Another aspect of the invention relates to the thyTreg cells and thyTreg cell populations of the invention as described herein for use as a medicament.

[0038] Another aspect of the present invention relates to a pharmaceutical composition comprising the thyTreg cells and thyTreg cell populations of the present invention as described herein, wherein the pharmaceutical composition further comprises an excipient and / or a pharmaceutically acceptable carrier.

[0039] Another aspect of the present invention relates to a kit (hereinafter referred to as "the first kit of the present invention"), which comprises all necessary reagents, media and devices for performing the method of the present invention as described herein.

[0040] Another aspect of the present invention relates to a kit (hereinafter referred to as the "second kit of the present invention"), which comprises the thyTreg cells or thyTreg cell populations of the present invention as described herein or the pharmaceutical composition of the present invention as described herein and a suitable medical device for administering the cells to an individual, preferably injecting the cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 .Show the results of purification and enrichment of thymus-derived thyTreg cells. Thymus tissue was obtained from a 5-month-old child. Shows the frequency of Treg cells in total thymocytes before the purification protocol (A), as well as the viability (B) and purity (C) of CD25+Foxp3+Treg cells in the final product obtained using the method of the present invention.

[0042] Figure 2.Show the suppressive capacity of thymic-derived thyTreg cells. PBMC cells were stained with CFSE, activated, and co-cultured alone or with thymic-derived thyTregs. The proliferation of reactive T cells was measured as a decrease in the fluorescence intensity of CFSE staining. After 3 days of culture, activated TCD4+ and TCD8+ alone proliferated by more than 87%. The presence of thyTregs reduced the proliferation of activated CD4+ and CD8+ T cells by more than 75%.

[0043] Figure 3 The efficacy of thyTreg in inhibiting the proliferation of allogeneic CD4+ and CD8+ T cells is shown. After 3 days of culture, non-activated (NA) T cells did not proliferate (white); more than 80% of activated CD4+ and CD8+ T cells proliferated, and several division cycles (peaks) were observed (gray area); however, the presence of thyTregs prevented these allogeneic T cells from proliferating by more than 80% (striped area) (A); the generated thyTreg populations showed suppressive capacity against allogeneic CD8+ and CD4+ T cells at various Treg to reactive cell ratios (B).

[0044] Figure 4 Phenotype of total thymocytes obtained from thymic tissue (A); purity of CD25+Foxp3+ cells in the final thyTreg product (B); distribution of thyTreg cells regarding CD4 and CD8 expression in the final thyTreg product (C).

[0045] Figure 5 Differences in CD25+Foxp3+ cell purity (A) and fold expansion (B) between the same regimen with rapamycin (+rapamycin) or without rapamycin (-rapamycin).

[0046] Figure 6 Differences in purity (AB), expression of functional Treg markers (C), and fold expansion (D) of CD25+Foxp3+ cells between the same protocol using Dynabeads or TransAct as cell activators.

[0047] Figure 7 thyTregs produced using our protocol (dark grey) express lower immunogenicity markers than conventional CD4+ T cells obtained from adult peripheral blood (grey). Isotype controls are represented by light grey areas.

[0048] Figure 8Compared with standard culture conditions, thyTregs produced using our protocol maintained stable Foxp3 expression (A) and IL-10 production capacity (B) in the presence of proinflammatory cytokines (IL-1β, IL-6, TNFα; inflammatory conditions). DETAILED DESCRIPTION

[0049] In a first aspect, the present invention provides an in vitro method for obtaining and purifying a novel subtype of Treg cells obtained from thymic tissue. In another aspect, the present invention relates to Treg cells or Treg cell populations obtained according to the methods described herein, which are referred to as "thyTreg" in the present invention.

[0050] In contrast to Tregs present in the blood or obtained by other methods, thyTregs obtained by the method of the present invention have been shown to exhibit differential characteristics that make them optimal for their clinical use in cell therapy. Thus, they have a greater ability to suppress effector cells of the immune system, an undifferentiated phenotype (since the cells contained in the thymus have not yet been exposed to foreign antigens), high motility and stable Foxp3 expression ( Figure 8 A) Stable IL-10 production ( Figure 8 B), and adequate expression of functional markers such as CTLA-4 and CD39 ( Figure 6 C). In addition, the protocol developed herein for obtaining, purifying, and processing thyTregs has enabled the production of more than 10 billion (1×10 10 ) thyTregs (see, for example, Example 1, in which 13 billion thyTregs were obtained). These numbers are much better than those that can be obtained from a single individual using other methods, whether peripheral blood (Safinia N., et al., 2015, Front Immunol., 6:438) or thymus tissue (Dijke IE, et al., 2016, American Journal of Transplantation, 16:58-71) as a cell source.

[0051] The commonly used Treg dose in therapeutic trials for adults is approximately 1×10 6 to 10×10 6Tregs. Thus, the yield of the protocol described herein will make it possible to produce more than 1,800 doses of thyTreg cells from a single thymus from, for example, a 6 month old pediatric patient (6-8 kg). This will not only make it possible to perform treatment without the need for a large number of cell expansions, but there will still be a sufficient number of cells to store several frozen doses that can be used for future reinfusion into the patient in the event of any signs of rejection or for use in other patients (allogeneic use).

[0052] As explained previously, a large number of thyTreg cells have been obtained using the protocol of the present invention, and the method is also compatible with the GMP conditions required for the subsequent use of the obtained cells as therapy in humans. In addition, the thyTreg obtained in the present invention can have a purity greater than 95% and very high suppressive capacity, survival rate and vitality. The Treg cell population and yield have been obtained without using products or methods that make them incompatible with their subsequent clinical use in humans. Therefore, this treatment method makes it possible to regulate immune homeostasis not only early in the pathological process, but also for a long time. Therefore, the transfer of thyTreg cells obtained using the method of the present invention to patients can achieve indefinite immune tolerance induction, that is, throughout the life of the graft.

[0053] Using thymic tissue, preferably thymic tissue removed from pediatric heart transplant patients as a cell source, a specific protocol has been developed by which a large number of thyTreg cells with optimal phenotype, high suppressive capacity and longer average lifespan are obtained without the need for a large amount of ex vivo expansion that would alter their quality. The cells thus obtained can be used for autologous or allogeneic transfer to patients, such as transplant patients, preferably solid transplant patients, and more preferably pediatric heart transplant patients. In this way, the intrinsic mechanisms of immune tolerance are activated. The results obtained show that this cellular immunotherapy can clearly prevent immune rejection of the transplant and ensure the reduction or complete elimination of the administration of immunosuppressive drugs and their associated toxicity. Moreover, the treatment strategy opens up new frontiers in the treatment of other types of disorders related to the immune system, such as autoimmune processes.

[0054] The present invention thus makes it possible to overcome the obstacles that have so far limited the success of Treg cell therapy in humans (preferably in the context of solid organ transplantation) and provides a solution to the need to provide optimized methods for obtaining and purifying Treg cells that are safe from a clinical point of view and in sufficient quantities and sufficient for their subsequent use in human cellular immunotherapy, preferably with an enhanced suppressive phenotype that is sustained in a timely manner, improved purity, retained suppressive capacity, high survival rate and viability.

[0055] Therefore, some of the most prominent advantages of the method of the present invention and the thyTreg cells obtained by the method are as follows:

[0056] - In a preferred embodiment, the method does not perform any type of enzymatic digestion, or use reagents, animal serum or products that are incompatible with the subsequent therapeutic use of the obtained cells, which is contrary to other similar schemes described in the prior art, in which the culture medium is supplemented with bovine or human serum that makes the subsequent clinical use of the cells impossible. It is also common in the prior art to use particles coated with anti-CD3 and anti-CD28 antibodies to activate cells in Treg culture medium (see, for example, Dijke IE, et al., 2016, American Journal of Transplantation, 16: 58-71). However, the presence of this type of reagent requires complex additional methods to completely eliminate the above particles before transferring the cells to the patient. On the contrary, the preferred embodiment of the method of the present invention uses only reagents allowed in the production of cells for therapeutic use, because the reagent is 100% compatible and safe, thereby allowing the clinical and therapeutic use of the cells.

[0057] - Compared to the methods of the prior art (especially those methods in which peripheral blood is used as a cell source or the method described in Dijke I.E., et al., 2016, American Journal of Transplantation, 16: 58-71, in which only about 300 million cells are obtained (which is insufficient from a clinical point of view)), this method makes it possible to obtain a larger number of Treg cells (more than 10 billion thyTreg cells are obtained from a single thymus). This provides the possibility of preparing various doses that can be administered to the patient at different time intervals or can be stored for future exogenous or allogeneic administration.

[0058] - This approach does not require extensive ex vivo cell expansion, thereby maintaining the quality of the cells and the phenotype responsible for suppressive capacity (persistent Foxp3 expression).

[0059] - This method enables the differentiation of immature Treg cells (low Foxp3 expression) into stable and ideal Foxp3+ Tregs.

[0060] - ThyTreg cells with a purity of 95% can be produced, and there are no effector T cells, which not only have no inhibitory ability, but can even induce rejection or inflammatory reactions. CD25 antibodies (molecules present in Tregs) are usually used to separate Treg cells, but they also express T CD4+ effector cells. Therefore, in the current method, the purified population contains contaminating effector cells that have no inhibitory ability and have inflammatory effects. The advantage of the method of the present invention is that since the cells contained in the thymus are not exposed to exogenous antigens, there are no effector cells, so all purified cells will be thyTreg cells because they express CD25.

[0061] - The obtained thyTreg cells have high survival rate (higher than average lifespan) and viability greater than 80% or even 90% ( Figure 1 B).

[0062] - The obtained thyTreg cells have an excellent suppressive capacity, higher than 70%, due to their persistent Foxp3 expression. This is associated with the fact that the immune tolerance induced in patients after the transfer of these cells persists in due time and provides indefinite protection against immune rejection (long-term immune tolerance of the transplant).

[0063] - Compared with Treg cells obtained from peripheral blood (Dijke IE, et al., 2016, American Journal of Transplantation, 16: 58-71), the obtained thyTreg cells have the best suppressive phenotype.

[0064] In addition, this protocol of the present invention uses thymus tissue in which Treg cells are produced instead of peripheral blood as a cell source. The main advantage of thyTreg cells obtained from this tissue is that they have never migrated to the periphery, they all have a naive and undifferentiated phenotype, thereby having a greater survival rate, and are able to maintain Foxp3 expression and its inhibitory ability after repeated stimulation and amplification. Therefore, naive Treg cells, which are a minority in adults, are most suitable for cell therapy, thereby maintaining their inhibitory ability. In this regard, the analysis conducted by the inventors of the present invention demonstrated that thyTreg obtained using the protocol described herein:

[0065] a. The survival rate is 10 times higher than that of adult Treg cells obtained from peripheral blood.

[0066] b. having excellent inhibitory ability, thereby inhibiting the proliferation of TCD4 and TCD8 effector cells by more than 70%, or even 75% (see the examples of the present invention shown below),

[0067] c. Maintaining Foxp3 expression and a stable inhibitory phenotype after in vitro activation,

[0068] d. Even in the presence of cytokines that normally convert Treg phenotype to Th17 cells (IL-1β, IL-6, TNFα), Foxp3 expression and IL-10 production were maintained ( Figure 8 ).

[0069] Another advantage presented by the method of the present invention is that thymic tissue, which is usually removed and discarded during cardiac surgery (such as heart transplantation or different surgeries to alleviate congenital heart disease), can be easily obtained for use as a source of Tregs, preferably as a source of autologous Tregs, but also for its allogeneic use.

[0070] Furthermore, the administration of thyTreg cells obtained using the method of the invention makes it possible to reduce or completely eliminate the use of immunosuppressive drugs, which have associated chronic toxicity and side effects due to their nonspecific activity suppressing the entire immune system, such as the development of infections, tumor processes or autoimmune diseases.

[0071] Furthermore, the possibility of using the patient's own cells (autologous administration of thyTregs) significantly reduces potential side effects caused by the therapeutic use of allogeneic cells. In addition, this strategy is a low-cost therapy that can be made available to patients in a relatively short period of time.

[0072] In summary, the therapeutic strategy described herein makes it possible to prevent transplant rejection, to make the graft survive indefinitely, and to make transplant patients (especially children) have a longer life expectancy. In addition, it opens up the possibility of realizing a thyTreg cell biobank, which is obtained from thymus tissue preferably discarded in cardiac surgery using the method of the present invention, which will make it possible to develop cell therapies using allogeneic thyTregs for treating various immune-related diseases (such as autoimmune disorders or graft-versus-host disease) by inducing immune tolerance.

[0073] In a first aspect, the present invention relates to an in vitro method for obtaining a regulatory T (Treg) cell population from isolated thymic tissue, comprising the following steps:

[0074] a. Mechanical disintegration of thymic tissue;

[0075] b. filtering the product obtained after stage (a) and resuspending the pellet containing thymocytes in culture medium;

[0076] c. separating CD25+ cells from the product obtained after stage (b);

[0077] d. culturing the cell population obtained after stage (c) in a culture medium in the presence of a T cell activator and IL-2 or TGFβ (preferably IL-2), preferably wherein the T cell activator comprises at least a CD3 and CD28 agonist; and

[0078] e. removing the T cell activating agent from the culture medium of stage (d);

[0079] f. Optionally, further culturing regulatory T cells in the culture medium in the presence of IL-2 or TGFβ (preferably IL-2);

[0080] Provided that prior to step (d), the cell population is not depleted of CD8+ cells.

[0081] Depletion of CD8+ cells can be performed, for example, using complement mediated lysis.In a preferred embodiment, the thymocyte population is not depleted of CD8+ cells prior to the CD25+ cell isolation step.

[0082] The term regulatory T (Treg) cell used herein refers to a cell characterized by expressing CD4, CD25 and Foxp3 cell surface markers, also referred to as CD4+CD25+Foxp3+ cells. Preferably, the Treg cell population obtained by the method of the present invention is characterized in comprising at least 80%, preferably at least 85%, more preferably at least 90%, 95%, 97% or 99% CD4+CD25+Foxp3+ cells.

[0083] Preferably, step a) comprises mechanically dissociating the thymic tissue (eg by using a tissue dissociator as described below) in the presence of culture medium and without the use of enzymes.

[0084] This method will also be referred to as "the method of the present invention" in this specification.

[0085] Prior to performing the methods of the present invention, the isolated thymus tissue may be present, stored in a sterile container containing a saline solution, such as but not limited to sodium chloride, antibiotics, and antifungal agents.

[0086] In a preferred embodiment, optionally in combination with one or more of the features or embodiments described herein, the method of the present invention comprises a step prior to stage (a) comprising dividing the isolated thymic tissue into smaller portions or slices, more preferably into portions of 2 to 3 grams.

[0087] The method of the present invention is preferably carried out in a GMP-approved cell production unit, which ensures the biosafety of the finally obtained cells.

[0088] Preferably, the methods of the invention do not comprise the use of reagents such as bovine serum or proteins derived from bovine serum, human serum, enzymes, anti-CD28 or anti-CD3 coated beads, or rapamycin.

[0089] The "thymic tissue" referred to in the present invention is any tissue sample from the thymus, which is a gland of the lymphatic system located in front of the heart and behind the sternum, where T cells and lymphocytes mature. Thymic tissue can be removed by any method known in the art for the purpose of removal, such as by means of a thymectomy, which can be transsternal, transcervical or video-assisted (videoscopic). Preferably, the thymic tissue is removed prior to performing the method of the present invention, during surgical intervention, more preferably during surgical intervention intended to treat heart disease (e.g., congenital heart disease), or during heart transplantation. Even more preferably, the thymic tissue of the present invention is removed during pediatric heart transplantation.

[0090] Thymic tissue can be from human or non-human mammals, such as but not limited to rodents, pigs, primates, ruminants, felines or canines. In a preferred embodiment of the method of the present invention, thymic tissue is from human, more preferably people of age 0 (neonate) to 16 years old, even more preferably people of 0 to 10 years old, particularly people of 0 to 24 months old.

[0091] In another preferred embodiment, the individual from whom the thymus tissue comes is the same individual to whom the thyTreg cells obtained at the end of the method of the invention are subsequently administered for cellular immunotherapy. That is, the tissue is preferably autologous.

[0092] In the present invention, a "pediatric patient" or "child" is understood to be a person aged 0 to 16 years, preferably 0 to 10 years, even more preferably 0 to 24 months.

[0093] " Tissue dissociator " that can be used in the method for the present invention is a tissue dissociator that allows tissue to be mechanically decomposed without the use of enzyme. Any tissue dissociator commercially available in the prior art can be used for the stage (a) of the method for the present invention. The example of these dissociators is, but not limited to, the gentleMACS Dissociator or gentleMACS Octo Dissociator from Miltenyi Biotec, the TissueLyser LT from Quiagen or the tissue dissociator from Worthington Biochemical, Sigma-Aldrich or Roche Diagnostics. Preferably, the tissue dissociator used for the present invention is the gentleMACS Octo Dissociator from Miltenyi Biotec.

[0094] In step (a) of the method of the present invention, the previously removed thymic tissue can be mechanically decomposed using a tissue dissociator, thereby producing a cell suspension, the tissue dissociator being a unit that preferably allows semi-automatic and standardized tissue dissociation. The tissue sample to be dissociated can be added to a container (preferably a disposable container) that makes it possible to prepare and process the sample in a closed sterile system, thereby providing a high level of safety and minimizing the risk of contamination. The container may also include a GMP culture medium, and preferably includes an antibiotic, such as an antibiotic at a concentration of 2% to 20%, preferably 2% to 10%, more preferably 5%. Examples of antibiotics that may be present and used in the present invention include, but are not limited to, penicillin, streptomycin, amphotericin or any combination thereof. The GMP culture medium does not contain serum and components from animals, allowing the cultivation and amplification of human and mouse T cells and the amplification of thyTreg cells of the present invention.

[0095] "GMP (Good Manufacturing Practice) medium" is any cell culture medium that has been approved for maintaining cells, preferably T cells, more preferably human T cells, in a medium that will subsequently be used for human clinical studies, preferably for cell therapy. GMP medium does not contain components of animal origin, is serum-free and meets GMP requirements. Examples of such medium are, but are not limited to, X-Vivo TM 15 (Lonza, Ref.BE02-060), ImmunoCultTM-XF T cell expansion medium (StemCell Technologies, Ref.10981) or TexMACS GMP medium (Miltenyi Biotic, Ref.170-076-307). Preferably, the GMP medium for the method of the present invention also comprises an antibiotic. The antibiotics commonly present in the culture medium are, for example, those cited in the preceding paragraph. More preferably, the GMP medium for the method is the TexMACS medium of Miltenyi Biotic, which even more preferably comprises an antibiotic. Examples of suitable antibiotics and antibiotic concentrations are provided above.

[0096] "TexMACS medium" contains salts, amino acids, fatty acids, vitamins, pharmaceutical grade human albumin (ie, GMP or suitable for humans) and buffer. The pH of the medium is preferably maintained at 6.9 to 7.3, and may or may not contain phenol red.

[0097] Once step (a) is completed, the product obtained from the tissue decomposition is filtered, preferably using a filter with a pore size of 30 to 40 μm, more preferably wherein the filter is made of nylon mesh, to remove tissue residues and aggregates to obtain a uniform single cell suspension. Subsequently, the obtained product can be (optionally) centrifuged. Finally, the cell pellet containing thymocytes is resuspended in a GMP culture medium preferably containing antibiotics, for example, at a concentration of 2% to 20%, preferably 2% to 10%, more preferably 5% antibiotics. For the thymocyte suspension obtained, cell quality and viability control can be optionally performed using methods known in the art, usually if the viability is greater than 80% and there is no sign of contamination, then proceed to the next stage of the method of the present invention, which consists of purified Treg cells. Otherwise, the sample will be discarded.

[0098] In stage (c) of the method of the present invention, CD25+ cells (including Treg cells) are separated or purified from the product obtained in the previous stage. The purification of this stage (c) can be performed using, for example, but not limited to, any immunocytochemistry technique known in the art that can perform positive selection or separate cells expressing specific markers on the surface. For example, CD25+ cell populations can be separated by flow cytometry or using a magnetic cell separator. Although not yet approved by regulatory agencies, a single-use and close circuit cell sorter is still being developed, which may be suitable for producing cells for human therapy purposes (https: / / www.miltenyibiotec.com / ES-en / products / macs-flow-cytometry / cell-sorter.html). In a preferred embodiment, it is optionally combined with one or more of the features or embodiments described herein, and step (c) includes using magnetic beads conjugated to anti-CD25 antibodies.

[0099] This purification step is preferably performed in a clean room environment with the aid of a cell purification system authorized for use in human cell therapy, preferably using The purification is carried out in a purification system, preferably in the presence of a buffer solution (e.g. 0.9% sodium chloride supplemented with antibiotics or TexMACS medium or other) and a specific anti-human CD25 monoclonal antibody of the mouse IgG1 isotype conjugated to superparamagnetic particles (preferably CliniMACs CD25 reagent). That is, the product obtained in the previous stage is introduced into the purification system, preferably into system. The "CliniMACs plus" instrument is commercially available from Miltenyi Biotic and is an automated cell separation platform and a functionally closed sterile system." The "CD25 reagent" consists of a specific mouse IgG1 isotype anti-human CD25 monoclonal antibody conjugated to superparamagnetic iron dextran particles, preferably contained in a pyrogen-free sterile solution. The reagent is commercially available from Miltenyi Biotic and is used when combined with When the instrument or system is used in combination, it is possible to enrich CD25+ cells from a heterogeneous human cell population. Once CD25+ cells (including Treg cells) are purified in the method of the present invention, quality control can be optionally performed using, for example but not limited to, flow cytometry to verify the viability, quantity and purity of the obtained Treg cells.

[0100] "A cell purification system authorized for human cell therapy" is any instrument capable of separating and purifying a specific cell population from a heterogeneous cell suspension containing different cell types. These instruments must be based on the separation and purification of the cells of interest based on molecules or specific cell markers expressed on the cell surface, and it is essential for these instruments to retain the viability and functional capacity of the cells once purified. In addition, these instruments must be GMP authorized for human therapeutic use. Examples of these cell purification systems authorized for human cell therapy are, but are not limited to, CliniMACs plus (Miltenyi Biotec), CliniMACs Prodigy (Miltenyi Biotec), RoboSep (STEMCELL Technologies), MACSQuant Tyto (Miltenyi Biotec) or MoFlo Astrios Sorter (Beckman Coulter), preferably CliniMACs plus (Miltenyi Biotec).

[0101] At the end of step (c), the cell population obtained is usually analyzed for cell viability and purity of CD25+ cells. Preferably, the cell viability is at least 80%, preferably at least 85%, 90%, 95%, 97% or at least 99%. Moreover, the purity of CD25+ cells is preferably at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 97% or at least 99%. In a preferred embodiment, the cell viability is at least 80% and the purity of CD25+ cells is at least 70%.

[0102] In stage (d) of the method of the present invention, the Treg cells purified in the previous stage are cultured in the presence of a T cell activating agent. Step (d) may be performed for 1 to 5 days, preferably at least 2 or 3 days, more preferably at least 3 days.

[0103] "T cell activator" is any product that allows in vitro stimulation and proliferation of T cells (preferably humans) through its interaction with T cell activation receptors, for example, by engaging CD3 / CD28. The activation of T cells can be determined, for example, by determining the expression of CD25 and / or CD69, such as by using flow cytometry. Preferably, the activation of Treg cells can be assessed by determining the expression of CD25 and / or Foxp3. Preferably, the T cell activator comprises at least CD3 and CD28 agonists. The term "agonist" used herein refers to a ligand that binds to a receptor and activates the receptor. Examples of T cell activators are, but are not limited to, MACS GMP T Cell TransAct people (Miltenyi Biotec, Ref. 170-076-156), known as Dynabeads CD3 / CD28 CTS, a colloidal polymer nanomatrix as defined herein. TM Particles coated with anti-CD3 (ThermoFisher, Ref. 40203D) and antibody complexes (such as anti-CD3 / CD28 and anti-CD3 / CD28 / CD2 antibody tetramers) used by MacDonald et al. 2017 (Wolters Kluwer, Canadian national transplant research program, page S9; https: / / journals.lww.com / transplantjournal / Fulltext / 2017 / 05003).

[0104] As used herein, the term "antibody" may refer to an immunoglobulin or an antigen-binding fragment thereof. Unless otherwise indicated, the term includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, humanized antibodies, human antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutant antibodies, transplanted antibodies, and antibodies produced in vitro. In certain embodiments, the term "antibody" may also refer to antibody derivatives, such as antibody-based fusion proteins or antibodies further modified to include additional non-protein moieties such as water-soluble polymers, such as polyethylene glycol (PEG).

[0105] Preferably, the T cell activator used in the present invention is a T cell TransAct human reagent, which comprises a colloidal polymer nanomatrix. The T cell activator is a colloidal polymer nanomatrix conjugated with humanized CD3 and CD28 agonists, more preferably a T cell TransAct human reagent. For culture, Treg cells can be added to any culture container for culturing human cells. The "T cell TransAct human" reagent is commercially available from Miltenyi Biotec and can be used to activate and amplify human T cells. The reagent includes a nanomatrix, which is a colloidal polymer nanomatrix conjugated with agonists for CD3 and CD28 humanization supplemented with phosphate buffered saline (PBS) and containing poloxamer 188 (poloxamer 188) and recombinant human serum albumin. The pH value of the reagent is maintained, preferably maintained between 7.3 and 7.9. The nanomatrix promotes the effective activation of T cells while maintaining the viability of T cells. The use of polymer nanomatrix for CD3 / CD28 activation allows sterile filtration and removal of excess reagent by simply replacing the supernatant or by a washing step (e.g., by centrifugation). The T cell TransAct human reagent has been designed to activate T cells, however, in the present invention, the dosage has been adapted to produce activation of Treg cells. Therefore, preferably, the dosage of the T cell activator (e.g., T cell TransAct human) used in this stage of the method of the present invention is between 1.10 and 1:100. Also present in this culture step is a culture medium (preferably a GMP culture medium, more preferably a TexMACS culture medium), wherein the culture medium preferably comprises an antibiotic (e.g., 5% antibiotic) and also comprises interleukin 2 (IL-2) or TGFβ, preferably IL-2.

[0106] The cells may be cultured in step (d) in the presence or absence of rapamycin. For example, a concentration of rapamycin of 50 ng / mL to 200 ng / mL, preferably about 100 ng / mL, may be used. In a preferred embodiment, the cells are cultured in step (d) in the absence of rapamycin.

[0107] In step (e) of the method of the present invention, the T cell activating agent is preferably removed by centrifuging the culture of the previous stage. According to step (e) of the method of the present invention, the T cell activating agent can be removed by, for example, but not limited to, centrifugation, column separation, etc., preferably by centrifugation. The centrifugation is more preferably performed at room temperature at 1,500 rpm for 10 minutes.

[0108] After removal, the cells can be optionally maintained in culture for at least another 1 to 7 days, preferably for at least another 4 days, with regular changes of the culture medium containing antibiotics and IL-2. Expanding the cells on an additional day can provide the highest purity, but the number of Treg cells will be lower than expanding them for 2, 3 or preferably 4 days.

[0109] The cells may be cultured in step (f) in the presence or absence of rapamycin. For example, a concentration of rapamycin of 50 ng / mL to 200 ng / mL, preferably about 100 ng / mL, may be used. Preferably, the cells are cultured in step (f) in the absence of rapamycin.

[0110] In steps (d) and (f), IL-2 may be used at a concentration of 50 IU / ml to 2000 IU / ml, preferably 100 IU / ml to 1000 IU / ml, more preferably 500 IU / ml to 700 IU / ml, such as about 500 IU / ml or 600 IU / ml.

[0111] Preferably, in the culturing step of the method of the present invention, the culture medium is a GMP medium.

[0112] After the cultivation in stage (d) and / or (f) of the method of the invention, cell quality, sterility and safety control may be performed. Furthermore, the cultured cells may be distributed in different doses for storage and / or transportation.

[0113] As explained above, the large number of thyTreg cells that can be obtained with the scheme of the present invention makes it possible to store doses for future allogeneic or autologous administration. Therefore, in other preferred embodiments, the method of the present invention also includes an additional step (g), which includes storing, more preferably ultra-low temperature preservation, all or part of the thyTreg cells obtained after the cultivation of step (f) or after step (e). Even more preferably, this storage occurs in a sterile container, preferably a vial, with the same or different doses between the sterile containers. "Ultra-low temperature preservation" can preferably be carried out between -80°C and -196°C.

[0114] In a particular embodiment, the present invention relates to an in vitro procedure, protocol or method for obtaining regulatory T cells from isolated thymic tissue (or thyTreg cells), comprising the following stages:

[0115] a. mechanically dissociating thymic tissue using a tissue dissociator (preferably gentleMACS Octo Dissociator) in the presence of GMP medium (preferably TexMACS medium),

[0116] b. filtering (and preferably subsequently centrifuging) the product obtained after stage (a) and resuspending the pellet containing the thymocytes in a GMP medium (preferably a TexMACS medium),

[0117] c. purifying Treg cells from the product obtained after stage (b), said purification being carried out with the aid of a cell purification system authorized for use in cell therapy in humans, preferably using a CliniMACS plus instrument, more preferably in the presence of a specific anti-human CD25 monoclonal antibody of the murine IgG1 isotype conjugated to superparamagnetic particles, even more preferably in the presence of the CliniMACs CD25 reagent,

[0118] d. culturing the cell population obtained after stage (c) for at least 3 days in the presence of a T cell activator, a GMP medium (preferably a TexMACS medium), and IL-2, wherein the T cell activator is more preferably a colloidal polymer nanomatrix conjugated to humanized CD3 and CD28 agonists, and even more preferably a T cell TransAct human reagent,

[0119] e. removing the T cell activating agent from the culture medium of stage (d), preferably removing the nanomatrix used in stage (d), more preferably by centrifuging the culture medium, and

[0120] f. Optionally, maintaining the obtained thyTreg cells in culture medium for at least another four days.

[0121] Another aspect of the present invention relates to regulatory T cells or regulatory T cell populations obtained or obtainable by the method of the present invention (hereinafter, "regulatory T cells of the present invention", "thyTreg cells of the present invention" or "thyTreg cell populations of the present invention"). In a particular embodiment, the thyTreg cells of the present invention express at least the markers CD4, CD25 and Foxp3. That is, the thyTreg cells of the present invention are CD4+, CD25+ and Foxp3+. In another embodiment, the thyTreg cell population obtained by the method of the present invention is characterized in that it contains at least 80%, preferably at least 85%, more preferably at least 90%, 95%, 97% or 99% CD4+CD25+Foxp3+ cells.

[0122] The Treg cells or Treg cell populations of the present invention may also express markers CTLA-4 and CD39. In some embodiments, at least 30%, 40%, 50%, 60%, 70% or at least 80% of the cells in the cell populations of the present invention express CD39 and / or CTLA-4.

[0123] The term "expression" in the present invention should be understood as protein or mRNA expression, preferably protein.

[0124] The thyTreg cells of the present invention express the above-mentioned cell markers (CD4+, CD25+ and Foxp3+) typical of the above-mentioned Treg cells; however, it also has such functional and structural characteristics: the characteristics distinguish the thyTreg cells from other Treg cells separated, amplified, purified and / or activated using other methods other than the method of the present invention. It is well known in the art that the gene expression profile of the cell and the results of its cell function vary depending on the protocol used to culture the cells in vitro and stimulate or activate the cells. Therefore, the method of the present invention uses specific culture conditions and reagents that affect the gene expression profile and the characteristics of the thyTreg cells finally obtained.

[0125] As explained previously in this specification, these cells of the present invention may have: i) greater suppressive capacity (they suppress the proliferation of activated T CD4+ and T CD8+ cells by more than 70% or even 75%); ii) stable Foxp3 expression over time; iii) higher survival rate; iv) greater frequency of IL-10 producing cells; v) undifferentiated optimal phenotype; vi) stable Foxp3 expression and retention of IL-10 production in the presence of proinflammatory cytokines (such as IL-1β, IL-6 or TNFα) that are able to convert the phenotype of other Treg cells obtained using other methods. Therefore, the cells of the present invention are significantly different from other Treg cells obtained by other methods of the prior art.

[0126] In a preferred embodiment, the cell population obtained by the method of the present invention has a viability of at least 80%, preferably at least 85%, more preferably at least 90%, 95%, 97% or 99%. Cell viability can be determined by any method known in the art, such as by excluding dead cells based on scatter signals and / or using fixable or non-fixable viability dyes for flow cytometry.

[0127] Moreover, in a preferred embodiment, the cell population of the present invention has an inhibitory capacity of at least 60%, preferably at least 65%, 70%, 75% or preferably 80% or more on the proliferation of activated T CD4+ and T CD8+ cells. The inhibitory capacity can be determined by any method known in the art, for example, by comparing T cell activation in the presence or absence of a Treg cell population, as shown in Example 2.

[0128] The thyTreg cells of the present invention can also be genetically modified by any method known in the art. For example, gene editing techniques (such as CRISP / CAS9) can be used to modify ThyTreg cells to eliminate immunogenic molecules and obtain "universality" ThyTreg cells. Such universal ThyTreg cells will be particularly useful for allogeneic purposes. The present invention also provides the possibility of developing the generation of CAR thyTreg cells (i.e., genetically modified to express chimeric antigen receptor thyTreg cells) so that they will have antigen specificity. Therefore, personalized therapy can be created with the thyTreg cells of the present invention, and the thyTreg cells specifically and exclusively inhibit the effector cells that react with the antigens of the transplanted organ or the autoantigens that mediate each patient's autoimmune process.

[0129] Therefore, in a preferred embodiment, the thyTreg cell or thyTreg cell population of the present invention comprises a recombinant nucleic acid sequence encoding and expressing a chimeric antigen receptor. Hereinafter, such a cell is referred to as "CAR thyTreg of the present invention". The nucleic acid sequence can be introduced into the cell in a recombinant manner by means of known molecular biology techniques.

[0130] "Chimeric antigen receptors" or "CARs" are T cell membrane receptors that are artificially introduced and expressed in the thyTreg cells of the present invention by means of genetic engineering. The chimeric receptor engages with a desired antigen present in another target cell, preferably in the effector T cell to be inhibited. These receptors are called chimeric because they are composed of parts of different molecules. The expression of chimeric antigen receptors is achieved by transferring the nucleic acid sequence encoding the receptor of interest to the thyTreg cells of the present invention using, for example, but not limited to, a viral vector.

[0131] The "antigen or epitope" recognized by the chimeric receptor can be, but is not limited to, a protein or protein fragment, a carbohydrate or a glycolipid. More preferably, the antigen or epitope is a molecule present in the effector cells of the immune system (i.e., expressed by the effector cells of the immune system), and even more preferably, it is a molecule expressed on the surface by the effector cells of the immune system, wherein even more preferably, the effector cells of the immune system are specific, i.e., the effector cells of the immune system attack epitopes present in organs transplanted to the host organism or autoantigens present in the organs or tissues of the host organism. Therefore, preferably, the CAR thyTreg cells of the present invention thus produced obtain the ability to recognize and inhibit the effector cells in the immune system that attack epitopes present in transplanted organs or autoantigens.

[0132] "Immune system effector cells" are understood to include, for example but not limited to, T CD4 or CD8 lymphocytes, NK cells, B cells, macrophages, phagocytes, neutrophils, basophils and eosinophils or dendritic cells. Preferably, the immune system effector cells involved in the present invention are T CD4 or T CD8 lymphocytes.

[0133] The thyTreg cells of the present invention (including genetically modified cells derived therefrom), the cell populations of the present invention or the CAR thyTreg cells of the present invention can be directly administered to an individual for use as a drug, such as for induction of immune tolerance, or can be added to a pharmaceutical composition for use as a cell therapy drug.

[0134] Therefore, another aspect of the present invention relates to a pharmaceutical composition (hereinafter, "the composition of the present invention" or "the pharmaceutical composition of the present invention"), which comprises the thyTreg cells, CARthyTreg cells or thyTreg cell populations of the present invention (preferably in a therapeutically effective amount).

[0135] The expression "therapeutically effective amount" refers to the amount of the cells of the present invention that produces the desired effect. The dosage used to obtain the therapeutically effective amount 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 therapeutically effective amount of the cells of the present invention contained in the composition of the present invention is 1 to 20 million cells per kg body weight of the individual (1-20×10 6 ).

[0136] In another preferred embodiment, the pharmaceutical composition of the present invention further comprises an excipient and / or a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise another active ingredient and / or an adjuvant.

[0137] The term "excipient" refers to a substance that contributes to the absorption of the elements of the composition of the invention, stabilizes said elements, activates or contributes to the preparation of the composition, for example in the sense of giving it consistency. Thus, the excipient may have the function of maintaining the viability of the cells contained in the composition and / or the inhibitory properties of said cells, etc., or a protective function for the composition, for example a function of isolating the composition from air and / or moisture, without excluding other types of excipients not mentioned in this paragraph.

[0138] Like excipients, a "pharmaceutically acceptable carrier" is a substance used in a composition to dilute the components of the invention contained therein to a certain volume or weight. A pharmacologically acceptable carrier is an inert substance or has a similar effect to any element of the invention. The function of the carrier is to facilitate the incorporation of other elements, achieve better dosage and administration, or give the composition consistency and form. When the form of expression is a liquid, the pharmacologically acceptable carrier is a diluent. The pharmacologically acceptable carrier that can be used in the present invention can be a liquid, such as water, a solvent, an oil or a surfactant, including those of petroleum, animal, plant or synthetic origin.

[0139] The term "adjuvant" refers to an agent that has no antigenic effect itself but can stimulate the effect of the composition of the present invention. There are many known adjuvants in the prior art, such as but not limited to aluminum phosphate, aluminum hydroxide, toll-like receptor agonists, cytokines, squalene, Freund's incomplete adjuvant or Freund's complete adjuvant.

[0140] As used herein, the terms "active essence", "active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient" mean any component that provides pharmacological activity or affects the structure, metabolism or function of the human or other animal body in the cure, reduction, treatment or prevention of a disease or pathological condition. The active ingredient involved in the present invention can be, for example, but not limited to, one or more immunosuppressive drugs or one or more anti-inflammatory agents. The active ingredient can be a therapeutic agent commonly used to treat autoimmune diseases, transplant rejection or graft-versus-host disease, i.e., "immunosuppressive drugs", such as, but not limited to: calcineurin inhibitors (cyclosporine, tacrolimus), mTOR inhibitors (sirolimus, everolimus), antiproliferative agents (azathioprine, methotrexate, mycophenolic acid), monoclonal antibodies (daclizumab, basiliximab), corticosteroids, anti-thymocyte and anti-lymphocyte globulins or any combination thereof, as long as these active ingredients are not incompatible with the survival and functionality of the cells of the present invention contained in the pharmaceutical composition.

[0141] The pharmaceutical composition of the present invention can also be present in the form of a sustained release formulation together with a carrier. The term "slow release" refers to a vehiculisation of the cells contained in the composition which is released gradually over a period of time, preferably but not necessarily with a relatively constant release level over the entire period of time. Illustrative examples of sustained release systems include, but are not limited to, liposomes, mixed liposomes, oleosomes, niosomes, ethosomes, millicapsule, microcapsule, nanocapsule, sponge, cyclodextrin, vesicle, micelle, mixed surfactant micelle, mixed phospholipid-surfactant micelle, millisphere, microsphere, nanosphere, lipid sphere, microemulsion, nanoemulsion, microparticle, milliparticle, microparticle, nanoparticle, lipid solid particle and nanostructured lipid medium.

[0142] The pharmaceutical composition of the present invention can be applied to animals, including mammals, and preferably to humans in various forms, including but not limited to parenteral, intraperitoneal, intravenous, intradermal, epidural, intraspinal, intrastromal, intraarticular, intrasynovial, intrathecal, intralesional, intraarterial, intracardiac, intramuscular, intranasal, intracranial, subcutaneous, intraorbital, intracapsular, by means of transdermal patches, percutaneous, nasal sprays, surgical implants, internal surgical coatings, infusion pumps, via catheters or by direct injection into the patient's lymph nodes. Preferably, the pharmaceutical composition of the present invention is formulated for its intravenous administration.

[0143] The pharmaceutical compositions of the present invention may be suitable for administration by means of medical devices that are capable of releasing the cells contained in the pharmaceutical compositions at concentrations sufficient to induce immune tolerance. These devices may be sufficient for local administration of the composition so that it acts in the affected area, thereby preventing the treatment from being dispersed. These devices may, for example, but not limited to, carry the pharmaceutical compositions of the present invention within the device or be coated with the pharmaceutical compositions of the present invention. These devices include, for example, circulatory assist, intravascular surgery, and cardiovascular surgical devices, including, but not limited to, stents, valves, rings, sutures, patches, or vascular grafts.

[0144] Another aspect of the present invention relates to a kit (hereinafter "the first kit of the present invention"), which includes all necessary reagents, media and tools for carrying out the method of the present invention as described above. Preferably, the kit comprises: (i) GMP culture medium, preferably TexMACS culture medium, more preferably also comprising antibiotics as described above; (ii) one or more specific antibodies to the response of surface markers expressed by the thyTreg cells of the present invention, such as the response to CD4, CD25, Foxp3, CTLA-4 and / or CD39 molecules, the kit preferably comprises anti-CD25 antibodies, more preferably specific mouse IgG1 isotype anti-human CD25 monoclonal antibodies conjugated to superparamagnetic particles, and the kit even more preferably comprises CliniMACS CD25 reagent; (iii) T cell activator, preferably a colloidal polymer nanomatrix with humanized CD3 and CD28 agonists, more preferably T cell TransAct human reagent; and optionally (iv) IL-2. In another preferred embodiment, the kit further comprises a filter, preferably the pore size of the filter is 30 μm to 40 μm, more preferably wherein the filter is made of nylon mesh. In another preferred embodiment, the kit further comprises saline or a buffer solution, preferably sodium chloride, antibiotics, antifungal agents or any other reagents to prevent contamination of cultured cells or tissues, one or more sterile containers for accommodating thymic tissue and / or cell suspensions, one or more vials for storing and / or cryopreserving the obtained thyTreg cells, and / or one or more containers for in vitro culture of Treg cells. The kit may also include instructions for implementing the method of the present invention.

[0145] Another aspect of the present invention relates to the use of the first kit of the present invention for carrying out the method of the present invention.

[0146] Another aspect of the present invention relates to a kit (hereinafter "the second kit of the present invention"), which comprises the thyTreg cells, CAR thyTreg cells or thyTreg cell populations of the present invention or the pharmaceutical composition of the present invention and a medical device sufficient for administering (preferably injecting) the cells in an individual.

[0147] A "medical device sufficient for injecting cells in an individual" is any device or instrument that can be used to inject cells into the bloodstream or tissue. Examples of this type of device or instrument are, but are not limited to, syringes, vials, catheters, needles, cannulas, or any instrument that can be used in cell therapy in general, including those known in the art.

[0148] In the second kit of the invention, the cells of the invention or the pharmaceutical composition of the invention may be packaged in vials, for example, at the same or different doses. Moreover, the elements may be labeled and / or fixed in any type of medium in the kit.

[0149] In addition, the second kit of the present invention may contain other elements that can be used to maintain the cells of the present invention or the composition of the present invention in vitro or in vitro, such as culture medium and / or reagents. The kit may also contain elements that help prevent the cells contained therein from being contaminated, such as antibiotics, bacteriostats, bacteroidetes, antifungal agents, etc.

[0150] Typically, the second kit of the present invention comprises all reagents necessary for inducing immune tolerance in an individual according to the present invention. In addition, the second kit may include all media and containers necessary for its initiation and optimization. Preferably, the kit also includes instructions for injecting the cell or pharmaceutical composition of the present invention into an individual.

[0151] Another aspect of the invention relates to the use of the thyTreg cells, CAR thyTreg cells or thyTreg cell populations of the invention or the pharmaceutical composition of the invention in the manufacture of a medicine. Alternatively, this aspect of the invention relates to the thyTreg cells, CAR thyTreg cells or thyTreg cell populations of the invention, or to the pharmaceutical composition of the invention for use as a medicine.

[0152] The term "drug" refers to any substance used to prevent, alleviate, treat or cure diseases or pathological conditions in humans and animals. The drugs involved in the present invention can be used for human or veterinary use. "Drugs for human use" refers to any substance or combination of substances that exhibits the properties of treating or preventing human diseases or pathological conditions, or is used in humans or administered to humans for the purpose of restoring, correcting or changing physiological functions (preferably the physiological functions of the immune system), thereby exerting pharmacological, immunological or metabolic effects. "Drugs for veterinary use" refers to any substance or combination of substances that exhibits the properties of curing or preventing animal diseases or pathological conditions, or can be administered to animals for the purpose of restoring, correcting or changing physiological functions (preferably the physiological functions of the immune system), thereby exerting pharmacological, immunological or metabolic effects.

[0153] The term "treat" refers to combating the effects caused by a disease or pathological condition of interest in a patient (preferably a mammal, more preferably a human), including:

[0154] (i) inhibiting a disease or pathological condition, i.e. stopping its development;

[0155] (ii) alleviate the disease or pathological condition, i.e., cause regression of the disease or pathological condition or its symptoms;

[0156] (iii) Stabilization of disease or pathological condition.

[0157] The term "preventing" includes preventing the disease or pathological condition from occurring, ie preventing the disease or pathological condition from occurring in a patient, preferably a mammal, more preferably a human, particularly when the patient is susceptible to the pathological condition but has not yet been diagnosed as such.

[0158] The drug of the present invention may be administered, but not limited thereto, by means of transplantation at a systemic level or by means of local injection in the affected tissue.

[0159] In a preferred embodiment of this aspect of the invention, the drug is a cell therapy drug, more preferably a cell immunotherapy drug.

[0160] "Cell therapy" or "cytotherapy" is to be understood as a therapy in which cellular material or cells (in the context of the present invention, ie the live thyTreg cells of the present invention) are administered to an individual.

[0161] Preferably, the medicament of the present invention is used for adoptively transferring the thyTreg cells, CAR thyTreg cells or thyTreg cell populations of the present invention to an individual, wherein the cells may be of autologous or allogeneic origin to the individual, although autologous is more preferred.

[0162] "Autologous" cells are understood to be those cells obtained from the same individual to whom the methods of the invention are subsequently administered after treatment or modification thereof. Thus, the term "autologous" means that the donor and recipient are the same individual.

[0163] Preferably, in the present invention, the thymus tissue used as starting material in the method of the present invention has been removed from the same individual who will subsequently benefit from cellular immunotherapy, ie administration of the medicament or pharmaceutical composition of the present invention.

[0164] In contrast, "allogeneic" is understood to refer to those cells, tissues, organs or biological samples that are typically obtained from an individual other than the recipient individual.

[0165] In another preferred embodiment, the medicament of the present invention is intended to induce or restore immune tolerance in an individual, more preferably an individual who has undergone a transplant or graft, or has an autoimmune condition, an inflammatory process or an allergy or a graft-versus-host disease. In general, the medicament of the present invention is intended to be used to treat and / or prevent any situation in which the activity of the immune system must be reduced or suppressed in an individual.

[0166] In another preferred embodiment, the drug of the present invention is intended to be used to treat and / or prevent pathological conditions associated with an overreaction, an undesirable or insufficient reaction of the immune system. More preferably, the pathological condition is selected from a list consisting of: autoimmune diseases, inflammatory processes, allergies, graft-versus-host disease and / or immune rejection in transplanted individuals. Alternatively, this aspect of the invention relates to thyTreg cells, CAR thyTreg cells, thyTreg cell populations, and pharmaceutical compositions of the present invention, which are used to treat and / or prevent pathological conditions selected from a list consisting of: autoimmune diseases, inflammatory processes, allergies, graft-versus-host disease and / or immune rejection in transplanted individuals. In a particular embodiment, the pathological condition is an autoimmune disease.

[0167] Examples of "autoimmune diseases" that can be treated and / or prevented with the medicaments of the present invention are, but are not limited to, type I diabetes, arthritis (e.g., rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis or juvenile idiopathic arthritis), multiple sclerosis, inflammatory bowel diseases of autoimmune origin (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), thyroiditis (e.g., Hyperadrenalism (Graves' disease), autoimmune adrenal insufficiency (Addison's disease), autoimmune oophoritis, autoimmune orchitis, autoimmune hepatitis, autoimmune hemolytic anemia, paroxysmal cold hemoglobinuria, autoimmune thrombocytopenia, autoimmune neutropenia, pernicious anemia, pure red cell aplasia, autoimmune coagulopathy, myasthenia gravis, autoimmune polyneuritis, pemphigus and other blistering disorders, rheumatic heart disease, Goodpasture syndrome, heart failure syndrome, lupus erythematosus, Sjögren's syndrome ( Syndrome, polymyositis, dermatomyositis, scleroderma, chronic obstructive pulmonary disease, chronic inflammatory disease, celiac disease, Churg-Strauss Syndrome, cardiovascular disease, polydermatomyositis, septic shock, rhinitis, psoriasis, cancer-related cachexia, eczema, vitiligo, Reiter Syndrome, Kawasaki disease, idiopathic thrombocytopenic purpura, Guillain-Barré Syndrome, antiphospholipid antibody syndrome (APS), or narcolepsy.

[0168] In the context of the present invention, the term "allergy" includes, but is not limited to, allergies to foods such as milk and egg proteins, pollens, mites, mold and fungal spores, animal fur, insect bites or medications.

[0169] In addition, the "transplanted (transplanted / grafted) individuals" involved in the present invention include both clinical transplant patients and animal models used in experiments of allogeneic or xenogeneic transplantation or allogeneic or xenogeneic transfer of organs or tissues.

[0170] The "transplantation" or "transfer" referred to in the present invention is preferably allogeneic or xenogeneic and relates to transplanted cells and to whole organs, parts of organs or tissues.

[0171] "Graft-versus-host disease" (GvHD) is a pathological immune condition that may occur after bone marrow or hematopoietic stem cell transplantation.

[0172] In another preferred embodiment of this aspect of the invention, the individual with a pathological condition, more preferably an individual undergoing a transplant or suffering from an autoimmune disease or condition is a human. Even more preferably, the human is a child or pediatric patient aged 0 to 16 years, preferably 0 to 10 years, more preferably 0 to 24 months. In the case where the autologous thyTreg cells of the present invention are used for treatment, the human is preferably a pediatric patient. In the case where allogeneic thyTreg cells are used for treatment, the human is preferably a patient of any age, including an adult.

[0173] In another preferred embodiment, the medicament of the invention is intended to treat and / or prevent immune rejection in transplanted individuals.More preferably, the transplant is a solid organ transplant.

[0174] "Solid organ transplantation" is understood to be the transplantation of organs rather than tissues (e.g., bone marrow or isolated cells). Examples of solid organs are, but are not limited to, the heart, kidney, liver, prostate, spleen, lung, gland (e.g., pancreas), skin, cornea, bone, digestive tract, etc. More preferably, the solid organ of the present invention relates to the heart.

[0175] In a particular embodiment, the medicament of the present invention is used to treat and / or prevent immune rejection in pediatric heart (preferably heart) transplant patients, wherein the medicament comprises the thyTreg cells of the present invention or the CARthyTreg cells of the present invention of autologous origin.

[0176] The medicament of the present invention can be used alone or in combination with other medicaments or compositions for inducing immune tolerance, or for treating and / or preventing pathological conditions selected from the list consisting of autoimmune diseases, inflammatory processes, allergies, graft-versus-host disease and / or immune rejection in transplanted individuals. These other medicaments or compositions to be administered as a therapy in combination with the medicament of the present invention may form part of the same composition, or may be administered by means of different compositions and may be administered simultaneously or at different times with the medicament of the present invention.

[0177] Therefore, in another preferred embodiment, the drug of the present invention is administered in combination with at least one immunosuppressant and / or with at least one anti-inflammatory drug, more preferably with at least one immunosuppressant. The administration may be simultaneous or sequential. Preferably, the administration is sequential, more preferably, the immunosuppressant is first administered in one or more doses distributed over time, and then, preferably, seven days after the start of treatment with the immunosuppressant, the drug of the present invention is administered, while gradually reducing or completely eliminating the administration of the immunosuppressant. Therefore, in a more preferred embodiment, once the previous treatment with the immunosuppressant is completed, the drug of the present invention is administered. When administering the drug of the present invention, the administration of the immunosuppressant may be completely eliminated to give way to exclusive treatment with the drug of the present invention, or the dosage of the immunosuppressant may be gradually reduced until completely eliminated. The immunosuppressant is preferably those previously described in this specification.

[0178] Another aspect of the invention relates to a method for inducing or restoring immune tolerance in an individual, more preferably in a transplanted or metastatic individual, or in an individual with an autoimmune disease, or in an inflammatory process or allergy or graft-versus-host disease, wherein the method comprises administering the thyTreg cells of the invention, the CAR thyTreg cells of the invention, the thyTreg cell population of the invention or the pharmaceutical composition of the invention to the individual, more preferably in a therapeutically effective amount.

[0179] Another aspect of the invention relates to a method for treating and / or preventing a pathological condition selected from the list consisting of: an autoimmune disease, an inflammatory process, an allergy, graft-versus-host disease and / or immune rejection in a transplanted individual, wherein the method comprises administering the thyTreg cells of the invention, the CAR thyTreg cells of the invention, the thyTreg cell population of the invention or the pharmaceutical composition of the invention to an individual suffering from said pathological condition.

[0180] Throughout the specification and claims, the word "comprises / comprising" and its variants are not intended to exclude other technical features, additives, components or steps. For those skilled in the art, other objects, advantages and features of the present invention will be inferred in part from the specification and in part from the practice of the present invention. The following examples and drawings are provided by way of example and are not intended to limit the scope of the present invention.

[0181] Example

[0182] The present invention is illustrated below by experiments performed by the inventors, which demonstrate the effectiveness of the method of the present invention in obtaining and purifying thyTreg cells for their subsequent therapeutic use in cellular immunotherapy by inducing immune tolerance.

[0183] Example 1. Protocol for obtaining and purifying thyTreg cells from human thymus tissue

[0184] The scheme described herein is developed in the IISGM Immunomodulation Laboratory for purifying Treg cells from thymus tissue (thyTreg cells) removed from pediatric patients during cardiac surgery. The scheme makes it possible to obtain a large number of thyTreg cells with very high purity and optimal phenotype. The scheme is also GMP-compatible and meets the requirements for subsequent use of cells as human immunotherapy.

[0185] The phases of the programme are as follows:

[0186] 1. Removal and transportation of thymic tissue.

[0187] 2. Dissociate tissue and obtain thymocytes.

[0188] 3. Purify Treg cells.

[0189] 4. Cultivate and activate thyTreg cells.

[0190] 1.1. Removal and transportation of thymic tissue

[0191] During pediatric heart transplants and other congenital heart disease surgeries, thymus tissue is often removed to gain access to the heart. Due to the large size of the thymus in young children, which completely covers the heart, surgeons have to remove it to free up the surgical field, so this tissue, which is usually discarded, can be used as a source of thyTregs for pediatric heart transplant patients or for allogeneic use of thyTreg cells for immunotherapy in other patients and pathologies.

[0192] The thymus removed in the operating room was collected in a sterile container containing 50 ml of 0.9% sodium chloride saline solution supplemented with antibiotics and antifungals (Sigma, Cat. No. A5955). The container was sealed and protected in a biosafety plastic container, which was then transported to a GMP-certified cell production unit for processing.

[0193] 1.2. Tissue dissociation and thymocyte harvesting

[0194] Following the required biosafety protocols and meeting the specific requirements for GMP production of cells, the thymic tissue is removed from its container and transferred to sterile culture plates where the tissue is divided into 2-3 gram portions. All methods are performed by professionals in the "clean room" of the cell production unit and meet all requirements for using cells as a therapy for humans.

[0195] To mechanically dissociate the tissue, fragments of thymic tissue were introduced into a specific disposable container containing a blade for dissociating tissue (gentleMACS C tube, Miltenyi Biotic Ref.130-096-334) and also containing GMP TexMACS medium and 5% antibiotics. The thymic tissue was dissociated with a gentleMACS Octo Dissociator (Miltenyi Biotec). After the dissociation was complete, the tube was centrifuged, the supernatant was discarded, and the cell pellet containing the thymocytes was resuspended in 50 ml of Tex MACS medium containing 5% antibiotics and filtered using a filter with a pore size of 40 μm to remove aggregates and tissue residues. The obtained thymocyte suspension was subjected to quality and viability control. If the viability was greater than 80% and there was no sign of contamination, the thyTreg cells were purified. Otherwise, the sample was discarded.

[0196] The innovative element of the developed protocol is that it does not involve enzymatic digestion, nor any type of reagents, animal sera or methods that are incompatible with the subsequent use of the cells as a therapy in humans.

[0197] 1.3 Purification of thyTreg cells

[0198] Treg cells are purified in a clean room environment using processing equipment and magnetic cell separators (MACS) approved for human cell therapy by the corresponding regulations. In this case, it was decided to use the CliniMACS plus instrument (MiltenyiBiotec), for which purpose a closed circuit (CliniMACS tube kit, Miltenyi Biotec) and CliniMACS CD25 reagent (Miltenyi Biotec) were used. According to the manufacturer's instructions, all thymocytes separated in the previous step were processed and introduced into the MACS system to purify Treg. Once the cells are purified, quality control is performed using a flow cytometer to verify the viability, quantity and purity of thyTreg cells. If the viability is at least 80% and the purity of CD25+ cells is at least 70%, the purified cells are activated and cultured. Otherwise, the sample is discarded.

[0199] 1.4 Cultivation and activation of thyTreg cells

[0200] The purified CD25+ cells were distributed in large culture containers (flasks) containing TexMACS medium and 5% antibiotics, 600U / ml of GMP interleukin 2 (IL-2) and a colloidal polymer nanomatrix (T cell TransAct human, Miltenyi Biotec, Cat. No. 130-111-160) that makes it possible to contain and activate thyTregs. The dose of T cell TransAct used in humans was optimized in the present invention, and the range of the dose was 1:10 to 1:100.

[0201] After three days of culture, the nanomatrix was removed by centrifugation and the thyTreg cells were cultured for another four days, with the medium containing antibiotics and IL-2 being replaced regularly. The results showed that in this example, after tissue dissociation, the proportion of CD25+Foxp3+Treg cells in the total thymocytes was 9.10% ( Figure 1 A), after applying the scheme of the present invention, a final product was obtained, the cell viability of which was 96% ( Figure 1 B), the purity of thyTregs of CD25+Foxp3+ cells is greater than 95% ( Figure 1 C)( Figure 1). The frequency of CD25+Foxp3+ cells was determined by flow cytometry (Gallios, Beckman coulter). We permeabilized the cell membrane for intracellular staining of Foxp3 using an anti-human Foxp3 kit (eBioscience). We then stained the cells with CD25 (Beckton Dickinson) and Foxp3 (eBiosciences) antibodies conjugated to fluorescent dyes. Viability was calculated by including a fixable viability dye in the staining procedure. In this example, with 26.17 gr of processed whole thymus, we produced a final product that contained 13.7×10 9 (>13 billion) thyTregs of the above purity and viability.

[0202] Virtually all protocols currently available in the literature or for cell culture / expression supplement the culture medium with bovine serum which is incompatible with therapeutic use of cells in humans. TM ) to activate cells or use rapamycin to increase the Treg purity of the product. The use of these agents hinders the therapeutic use of the obtained cells, or requires additional methods to completely remove the above-mentioned particles before transferring the cells to patients. In contrast, the protocol described herein does not use any serum, drugs or reagents that make it incompatible with the therapeutic use of the cells in humans. Both the culture medium and IL-2 are GMP-compliant and can be used to produce cells for clinical treatment. In addition, through this protocol, the colloidal polymer nanomatrix used for its activation in thyTreg cells in this embodiment is completely removed by simple centrifugation, therefore, the quality and yield of the cells are not affected, and its subsequent use in cell therapy is allowed.

[0203] 1.5. Obtaining the final thyTreg product

[0204] After seven days of cultivation, the relevant quality, sterility and safety controls (presence of microplasma and genetic stability) were carried out, with successful results, thus confirming that the product obtained meets all the requirements for its therapeutic use in humans. Next, the necessary dose of thyTreg cells for cell therapy in patients was determined, taking into account the individual's body weight and the dose per kg of cells determined in each case. The remaining cells were cryopreserved for future repeated administration in patients or for use in other patients or diseases.

[0205] Schematically, the scheme of the present invention can obtain more than 13 billion thyTregs with a purity greater than 95%, a viability greater than 90%, and a very high inhibitory capacity. Such a number of cells will make it possible to prepare more than 1,000 doses of cell therapy for patients less than 1 year old and hundreds of doses if used for older children or adults. These yields of thyTreg cells are unheard of. To date, the yield obtained by the scheme for purifying Tregs from peripheral blood is less than 20 million cells, so a large number of expansion schemes are required, which require the use of different reagents and significantly reduce the quality of the cells. The purity obtained with other schemes is obviously poorer because there are activated cells in the peripheral blood, which may leave contaminants that become Tregs in the purification process. In addition, the higher differentiation or activation state of the cells present in the peripheral blood makes the obtained Treg cells have lower survival rate and lower inhibitory capacity than the Treg cells obtained by this thymus-based scheme of the present invention, which significantly drags down their therapeutic practicality.

[0206] Example 2. Analysis of the suppressive capacity of thyTreg cells obtained using the protocol described in Example 1

[0207] The suppressive capacity of the thyTreg cells generated in the previous example was analyzed by measuring their ability to inhibit the cell proliferation of TCD4 and TCD8 lymphocytes in vitro, which are the main mediators of cellular rejection of transplants and autoimmune diseases. For this purpose, thyTreg cells were incubated with CFSE (CellTrace TM Allogeneic peripheral blood mononuclear cells (PBMCs) stained with thyTregs (CFSE Proliferation Kit, Invitrogen) were co-cultured at a ratio of thyTreg:PBMC 1:2. PBMCs were first activated with PMA and ionomycin (Sigma) to trigger the proliferation of T CD4+ and T CD8+ lymphocytes, and the proliferation of these cells was compared with cultures that did not contain thyTregs. The proliferation of reactive T cells was measured by flow cytometry according to the decrease in fluorescence intensity of CFSE staining. Antibodies (Beckman Coulter) targeting specific surface markers CD3, CD4 and CD8 markers were used to distinguish CD4+ and CD8+ T cells. The results confirmed that the presence of thyTregs reduced the proliferation of activated TCD4 and TCD8 cells by more than 70% ( Figure 2 ).

[0208] The efficacy of the obtained thyTreg population in suppressing the proliferation of allogeneic CD4+ and CD8+ T cells was also investigated using different thyTreg: reactive cell ratios. ThyTregs showed very high suppressive capacity, suppressing the proliferation of allogeneic effector CD4+ and CD8+ T cells by more than 80% ( Figure 3 A, 3B). The data also showed that a reduced proportion of thyTregs in the co-culture resulted in a reduced inhibitory effect on proliferation, confirming that inhibition of proliferation is a specific effect of thyTregs ( Figure 3 B).

[0209] Example 3. Non-depletion of CD8+ positive cell population provides higher Treg cell yield

[0210] The vast majority (80%) of all thymocytes isolated from the thymus are double positive (CD4+CD8+) cells ( Figure 4 A). The inventors have surprisingly shown that a very high proportion of Treg cells are within this DP fraction. In fact, the inventors have shown that when following the protocol described in Example 1 (on day 7 of culture), about 41% of the thyTreg cell population obtained as a final product are DP cells ( Figure 4 B, 4C). Therefore, the previous depletion of CD8+ cells (selection of CD4+CD8-CD25+ followed by stimulation and expansion of Treg cells) as described in Dijke IE, et al., 2016 or MacDonald et al. 2017 will completely eliminate the DP fraction, so the Foxp3+DP population will not be present in the final product, resulting in a sharp drop in the recovery rate of CD25+Foxp3+ cells. Again, without being bound by theory, due to the less differentiated phenotype of Treg cells contained in the DP fraction, the cells may have a higher survival rate, plasticity and stability of their suppressive capacity.

[0211] Example 4. No need to use rapamycin to obtain high purity thyTreg products

[0212] Rapamycin is an immunosuppressant that inhibits T cell activation and is commonly used in Treg cell culture. In the presence of high doses of IL-2 in vitro, rapamycin can induce the proliferation of Tregs and attenuate the proliferation of effector T cells. Regarding CD25+Foxp3+ purity, we compared the presence or absence of 100ng / mL rapamycin in our cultures and obtained similar results ( Figure 5 A). Interestingly, in the absence of rapamycin, Treg proliferation was slightly higher after 7 days of culture ( Figure 5B). Avoiding the use of rapamycin in the process of producing thyTregs will likely facilitate obtaining authorization from government drug agencies to use these cells as therapy in humans. Therefore, avoiding the use of rapamycin provides a further advantage in our procedure.

[0213] Example 5. Comparison of colloidal nanomatrix (TransAct) and CD3 / CD28 beads for thyTreg stimulation

[0214] In most protocols, the Dynabeads CD3 / CD28 CTS from Invitrogen were used. TM ” to culture or expand Treg cells. It contains magnetic beads conjugated to agonistic monoclonal antibodies against human CD3 and CD28. These beads need to be removed by magnetic separation after Treg activation and before the cells are administered to patients, which results in a significant loss of cells (>25%) after removal of the Dynabeads by the magnetic column.

[0215] The protocol described in Example 1 replaces the use of dynabeads by MACS GMP T Cell TransAct (Miltenyi Biotec), which comprises a colloidal polymer nanomatrix for activating T cells. The inventors have become pioneers in the use of this product for the activation of Treg cells.

[0216] The advantage of using nanomatrices for activation purposes is that the nanomatrices can be easily removed by simple centrifugation and medium replacement without causing a large loss of cells.

[0217] The inventors compared how the two activators affected the purity, expression of functional markers, and expansion of Treg cells after culture. As shown in the figure, the purity of CD25+Foxp3+ was slightly higher when using TransAct (89%) rather than Dynabeads (82%) ( Figure 6 A, 6B). When using TransAct, the activation marker HLA-DR as well as the fold expansion (number of CD25+Foxp3+ cells before and after activation and expansion) appeared comparable compared to Dynabeads, whereas markers associated with Treg function (e.g., CTLA-4 and CD39) showed higher expression ( Figure 6 C, 6D).

[0218] Example 6. Treg cells generated using the protocol of the present invention have stable Foxp3 expression and maintain IL-10 production Vitality

[0219] Numerous studies have shown that in the presence of proinflammatory cytokines, Treg cells obtained from peripheral blood may lose their Foxp3 expression and suppressive phenotype, and even Tregs can switch their phenotype under these conditions, acquiring the phenotype of Th1, Th17, or Th2 cytokine-secreting cells. Therefore, a potential limitation of Treg-based therapy is the instability and plasticity of therapeutic Tregs, especially under inflammatory conditions. We analyzed the stability of Foxp3 expression and the ability to produce IL-10 in the thyTregs obtained in the final product in the presence of an inflammatory environment. To this end, we cultured thyTregs in the presence of IL-1β, IL-6, and TNFα. The results showed that Foxp3 expression ( Figure 8 A) and the ability to produce IL-10 ( Figure 8 A) Comparable between standard culture conditions and in the presence of an inflammatory environment.

[0220] Example 7. Treg cells generated using the protocol of the present invention express low levels of immunogenic markers

[0221] The inventors have also determined that the Treg cells or cell populations of the present invention will be suitable for their use in treating autoimmune diseases, GVHD and other immune diseases in patients different from the subjects from which the cells are obtained (allogeneic use). In order to be suitable for use as allogeneic cell therapy, Treg cells must have low immunogenicity to avoid being recognized and destroyed by the recipient's immune system. Furthermore, allogeneic Tregs must have the ability to suppress immune cells from the recipient to exert their suppressive function (as shown in Example 2).

[0222] Typically, if the cells expressing MHC molecules (HLA-DR) have suitable co-stimulatory molecules (CD40, CD40L, CD80, CD86 ...), the cells can stimulate T cells and be recognized by T cells. The inventors determined the expression of CD40L, CD80, CD86 and HLA-DR (Beckman Coulter) in ThyTreg cells obtained as described in Example 1 and conventional CD4+ cells obtained from adult peripheral blood by flow cytometry. They demonstrated that the thyTregs produced by the scheme of the present invention expressed these immunogenic markers ( Figure 7 ). Since the obtained ThyTreg cell population has lower levels of co-stimulatory molecules, these may not be able to induce a strong immune response in allogeneic recipients by direct antigen presentation, or at least immune activation will occur to a lower extent than conventional CD4+ T cells.

[0223] Terms

[0224] 1. An in vitro method for obtaining regulatory T cells from isolated thymic tissue, comprising the following stages:

[0225] a. Mechanically dissociate thymic tissue using a tissue dissociator in the presence of GMP medium,

[0226] b. filtering the product obtained after stage (a) and resuspending the pellet containing the thymocytes in GMP medium,

[0227] c. purifying regulatory T cells from the product obtained after stage (b),

[0228] d. culturing the regulatory T cells obtained after stage (c) for at least 3 days in the presence of a T cell activator, a GMP medium, and IL-2,

[0229] e. removing the T cell activating agent from the culture medium of stage (d), and

[0230] f. Optionally, maintaining the regulatory T cells in culture for at least an additional four days.

[0231] 2. The method according to clause 1, wherein the purification in stage (c) is performed using a CliniMACS plus instrument in the presence of specific anti-human CD25 monoclonal antibodies of the murine IgG1 isotype conjugated to superparamagnetic particles.

[0232] 3. A method according to any one of clauses 1 or 2, wherein the T cell activator of stage (d) is a colloidal polymer nanomatrix conjugated to humanized CD3 and CD28 agonists.

[0233] 4. The process according to any one of clauses 1 to 3, wherein the removal of stage (e) is carried out by means of centrifugation.

[0234] 5. The method according to any one of clauses 1 to 4, wherein the thymus tissue is from a human.

[0235] 6. The method according to any one of clauses 1 to 5, wherein the thymus tissue is from a human aged 0 to 16 years.

[0236] 7. The method according to clause 6, wherein the thymus tissue is from a human aged 0 to 24 months.

[0237] 8. The method according to any one of clauses 1 to 7, further comprising a further step (g), said step (g) comprising cryopreserving the obtained regulatory T cells.

[0238] 9. A regulatory T cell or a population of regulatory T cells obtained by the method according to any one of clauses 1 to 8, wherein the regulatory T cells express at least CD3, CD25 and Foxp3 markers.

[0239] 10. The regulatory T cell or regulatory T cell population according to clause 9, wherein the cell comprises a nucleic acid sequence encoding a chimeric antigen receptor.

[0240] 11. A regulatory T cell or a population of regulatory T cells according to clause 10, wherein the antigen is an antigen present in an effector cell of the immune system.

[0241] 12. A pharmaceutical composition comprising a regulatory T cell or a regulatory T cell population according to any one of clauses 9 to 11.

[0242] 13. The pharmaceutical composition according to clause 12, further comprising an excipient and / or a pharmaceutically acceptable carrier.

[0243] 14. A kit for performing the method according to any one of clauses 1 to 8, comprising GMP medium, anti-CD25 antibody, a T cell activator and IL-2.

[0244] 15. The kit comprising a regulatory T cell or a regulatory T cell population according to any one of clauses 9 to 11, or a pharmaceutical composition according to any one of clauses 12 or 13, and a suitable medical device for injecting the cells into a subject.

[0245] 16. Use of a regulatory T cell or a regulatory T cell population according to any one of clauses 9 to 11 or a pharmaceutical composition according to any one of clauses 12 or 13 in the preparation of a medicament.

[0246] 17. The use according to clause 16, wherein the drug is a cell therapy drug.

[0247] 18. Use according to any one of clauses 16 or 17, wherein the medicament is for the treatment and / or prevention of a pathological condition selected from the list consisting of: an autoimmune disease, an inflammatory process, an allergy, a graft-versus-host disease and / or immune rejection in a transplanted individual.

[0248] 19. The use according to clause 18, wherein the individual suffering from the pathological condition is a human.

[0249] 20. The use according to any one of clauses 18 or 19, wherein the medicament is for treating and / or preventing immune rejection in a transplant recipient.

[0250] 21. The use according to clause 20, wherein the transplant is a solid organ transplant.

[0251] 22. The use according to clause 21, wherein the solid organ is the heart.

[0252] 23. The use according to any one of clauses 16 to 22, wherein the medicament is administered in combination with at least one immunosuppressive drug.

[0253] 24. The use according to any one of clauses 16 to 23, wherein the medicament is administered after completion of previous treatment with an immunosuppressive drug.

Claims

1. A population of regulatory T (Treg) cells obtained from isolated thymic tissue, wherein: The thyTreg cell population is characterized by comprising CD4+CD8+Foxp3+ cells; wherein the Treg cell population does not contain effector cells, and Wherein, the Treg cell population contains at least 90% CD4+CD25+Foxp3+ cells.

2. The thyTreg cell population according to claim 1, characterized in that The thyTreg cell population contained 95% CD4+CD25+Foxp3+ cells.

3. The thyTreg cell population according to claim 1 or 2, characterized in that The cells have been genetically modified.

4. The thyTreg cell population according to claim 3, characterized in that The cell comprises a nucleic acid sequence encoding a chimeric antigen receptor.

5. A pharmaceutical composition comprising the thyTreg cell population according to any one of claims 1 to 4, wherein The pharmaceutical composition further comprises an excipient and / or a pharmaceutically acceptable carrier.

6. Use of the thyTreg cell population according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 as a medicine.

7. Autologous or allogeneic use of the thyTreg cell population according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in a method for the treatment and / or prevention of a pathological condition selected from the list consisting of: autoimmune diseases, inflammatory processes, allergies, graft-versus-host disease and / or immune rejection in a transplanted individual.

8. Use of the thyTreg cell population according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 for use in the method according to claim 7, wherein: The individual having the pathological condition is a human. 9 . The use of the Treg cell population according to claim 7 , wherein the Treg cell population is used for treating and / or preventing immune rejection in a transplant subject.

10. The use of the Treg cell population according to any one of claims 7 to 9, characterized in that: The Treg cell population is administered in combination with at least one immunosuppressive drug.

11. The use of the Treg cell population according to any one of claims 7 to 10, characterized in that: The Treg cell population is administered after completion of previous treatment with an immunosuppressive drug.

12. The use of the Treg cell population according to any one of claims 7 to 11, characterized in that: The use of the Treg cell population is allogeneic use.

13. A kit comprising the thyTreg cell population according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5; and a suitable medical device for administering the cells to an individual.

14. An in vitro method for obtaining the regulatory T (Treg) cells of claim 1 from isolated thymic tissue.

15. The method according to claim 14, characterized in that The method comprises the following steps: a. mechanically disintegrate the thymus tissue; b. filtering the product obtained after stage (a) and resuspending the pellet containing thymocytes in culture medium; c. separating CD25+ cells from the product obtained after stage (b); d. culturing the cell population obtained after stage (c) in a culture medium in the presence of a T cell activator and IL-2, wherein the T cell activator comprises at least a CD3 and CD28 agonist; and e. removing the T cell activating agent from the culture medium of stage (d); f. Optionally, further culturing the regulatory T cells in the culture medium in the presence of IL-2; Provided that prior to step (d), the cell population is not depleted of CD8+ cells.

16. The method according to claim 15, characterized in that Step a) comprises mechanically disintegrating the thymus tissue in the presence of culture medium and without the use of enzymes.

17. The method according to any one of claims 15 or 16, characterized in that The separation of CD25+ cells in step c) comprises the use of magnetic beads conjugated with anti-CD25 antibodies.

18. The method according to any one of claims 15 to 17, characterized in that The T cell activator described in stage (d) is a colloidal polymer nanomatrix conjugated with humanized CD3 and CD28 agonists.

19. The method according to any one of claims 15 to 18, characterized in that The T cell activating agent described in stage (d) is used at a T cell activating agent:cell ratio of 1:10 to 1:

100.

20. The method according to any one of claims 15 to 19, characterized in that The cells are cultured in step (d) for at least 2 or 3 days, preferably at least 3 days.

21. The method according to any one of claims 15 to 20, characterized in that In step (d) the cells are cultured in the absence of rapamycin.

22. The method according to any one of claims 15 to 21, characterized in that The removal of stage (e) is carried out by means of centrifugation.

23. The method according to any one of claims 15 to 22, characterized in that In step f), the regulatory T cells are cultured in the presence of IL-2 for a further period of 1 to 7 days, preferably for a further period of 4 days.

24. The method according to any one of claims 15 to 23, characterized in that In step f) the cells are cultured in the absence of rapamycin.

25. The method according to any one of claims 15 to 24, characterized in that The culture medium is GMP medium.

26. The method according to any one of claims 15 to 25, characterized in that The culture medium also contains antibiotics, preferably 5% antibiotics.

27. The method according to any one of claims 15 to 26, characterized in that The thymus tissue is from human.

28. The method according to any one of claims 15 to 27, characterized in that The thymus tissue is from a human aged 0 to 16 years, preferably from a human aged 0 to 24 months.

29. The method according to any one of claims 15 to 27, characterized in that The method further comprises a further step (g), wherein the step (g) comprises cryopreserving the obtained regulatory T cells.

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