Method for culturing Treg cells

Abstract

Provided are in vitro methods for culturing regulatory T cells (Tregs), comprising contacting Tregs with an mTOR inhibitor, and products obtained and / or obtainable by the methods.

Description

[Technical Field]

[0001] The present invention generally relates to an in vitro method for culturing Tregs, comprising pretreating Tregs with an mTOR inhibitor prior to activation, and then culturing the activated Tregs in the presence of the mTOR inhibitor after activation. The in vitro method may be, in particular, an in vitro method for expanding Tregs, comprising pretreating Tregs with an mTOR inhibitor prior to activation, and then culturing the activated Tregs in the presence of the mTOR inhibitor after activation. In particular, the mTOR inhibitor may be added only once during the in vitro method, specifically to Tregs prior to activation, and may continue to be present for at least part of the culturing process after Treg activation. Thus, adding an additional mTOR inhibitor after pretreatment is not necessary. The present invention further relates to a Treg cell product obtainable by the in vitro method described herein. The present invention further relates to the use of the in vitro method described herein to increase Treg proliferation, reduce impurities in Treg cell products, promote desirable phenotypes, or increase the expression of heterologous nucleic acids that may have been introduced into Tregs. [Background technology]

[0002] Adoptive cell therapy (ACT), which involves the administration of functional immune cells to a target, has become an established and evolving immunotherapeutic approach for a variety of conditions, including malignancies and infectious diseases. Initially, tumor-infiltrating lymphocytes were shown to be effective in treating metastatic melanoma. Subsequently, redirected T cells or NK cells expressing chimeric antigen receptors (CARs) or heterologous T cell receptors (TCRs) to target distinct cellular targets were developed and applied clinically. Early approaches utilized immune cells with cytotoxic properties, such as cytotoxic T cells or NK cells, to target and destroy unwanted or harmful cells in the body. Recently, regulatory T cells (Tregs), which are CD4+CD25+FOXP3+, have been developed for ACT. Tregs have immunosuppressive functions. Tregs act to control cytotoxic immune responses and are essential for maintaining immune tolerance. The suppressive properties of Tregs can be therapeutically exploited to ameliorate and / or prevent immune-mediated organ damage, for example, in inflammatory disorders, autoimmune diseases, and transplantation.

[0003] The primary cell source for ACT is the patient's own blood (directly drawn from the vein or as a product of leukapheresis) or umbilical cord blood. A significant number of cells is required for therapeutic efficacy. To obtain sufficient numbers of cells for ACT, the desired cell population must be expanded ex vivo before administration to the subject. This is particularly important and challenging for Treg-based ACT, because Tregs account for a very small proportion of an individual's immune cells (approximately 3%–5% of peripheral blood), yet a significant number of cells are required to suppress the immune system. However, expansion of Tregs can lead to the expansion of other contaminating cells, such as CD4+CD25- T cells and CD8+ T cells. Depending on the level of contamination in the starting material, there is a risk that the contaminating cells may expand to levels inappropriate for ACT, potentially outgrowing the desired Tregs.

[0004] Currently, autologous rather than allogeneic cells are primarily used in ACT to reduce the risk of adverse immune reactions, such as transplant rejection and graft-versus-host disease (GvHD). However, patients with autoimmune diseases often have particularly low Treg numbers and / or impaired suppressive function (Brusko et al. 2005, Diabetes; 54: 1407-141; Lindley et al. 2005, Diabetes; 54: 92-99; Viglietta et al. 2004, J. Exp. Med; 199: 971-979; Kriegel et al. 2004, J. Exp. Med; 199; 1286-1291; Balandina et al. 2005, Blood; 105: 735-741). The present inventors have observed that immunosuppressed patients, such as liver transplant recipients, often have reduced numbers of circulating Tregs, making it relatively difficult to isolate and expand highly pure Tregs, especially under GMP conditions. Because of the low abundance of Tregs in these patients, the starting material selected for Tregs contains more non-Treg contaminants, further complicating the production of Tregs for ACT.

[0005] One approach to promote in vitro expansion of Tregs over other contaminating T cells is to culture the cells in the presence of the mTOR inhibitor rapamycin. In such protocols, rapamycin is typically added to the culture medium upon cell activation and maintained throughout the entire treatment period. Battaglia et al. (2006, J. Immunol; 177: 8338-8347) described a method for in vitro activation of bulk populations of human CD4+ T cells in the presence of rapamycin, promoting the expansion of CD4+CD25+FOXP3+ Tregs while suppressing the proliferation of CD4+CD25- and CD8+ T effector cells. However, rapamycin significantly reduced the rate of Treg expansion. Therefore, Treg expansion protocols in the presence of rapamycin require prolonged cell culture to obtain sufficient numbers of cells for ACT. This is particularly problematic in commercial processes, as the extended product manufacturing time increases the cost of ACT therapy and delays the start of production for the next patient. Furthermore, a longer protocol may negatively impact the quality and viability of Tregs.

[0006] It would therefore be desirable to provide new and improved methods for expanding Tregs in vitro, particularly methods for expanding Tregs in vitro that produce a product with a desired number of cells having a desired level of purity (e.g., low levels of T effector cells) and a desired phenotypic profile (e.g., FOXP3 expression level) in the shortest possible time. Summary of the Invention

[0007] The present inventors have developed an in vitro method for culturing or expanding Tregs. The method includes a first step of pretreating Treg starting material with an mTOR inhibitor prior to activation, and a further step of culturing the activated Tregs in the presence of an mTOR inhibitor. This method has the advantage of reducing the proportion of contaminating T effector cells and increasing the proportion of FOXP3+ cells in the product compared to methods that do not include these two steps. Furthermore, the Treg cell product has been confirmed to have an improved suppressive effect and cytokine expression profile compared to products prepared by methods that do not include these two steps.

[0008] Furthermore, the in vitro expansion method developed by the present inventors can increase cell proliferation compared to methods that do not include both of the above steps. The increased cell proliferation achieved by the in vitro method developed by the present inventors allows Treg cell products to be prepared at higher cell numbers and / or in shorter time periods while maintaining cell numbers compared to products prepared by methods that do not include both of the above steps. This is particularly surprising given that rapamycin has previously been shown to reduce the rate of Treg proliferation.

[0009] Compared to methods described in the art (adding rapamycin at the time of activation and maintaining rapamycin in the medium throughout the culture process), in certain embodiments, the inventors have developed a method by pre-treating cells with an mTOR inhibitor, which allows rapamycin to be diluted or removed from the medium after activation and before the end of the culture process, thereby increasing the growth rate and / or increasing total cell growth while maintaining a desirable level of purity.

[0010] The present inventors have further identified an in vitro method for genetically engineering and culturing Tregs that, in addition to the above advantages, enhances the expression of one or more transgenes introduced into Tregs. The present inventors have further determined that the method of the present invention results in higher transduction efficiency when one or more transgenes are introduced into a population of Tregs compared to methods that do not include both of the above steps. Surprisingly, however, despite this improved transduction efficiency, viral copy numbers (VCN) tend to be lower.

[0011] In a first aspect, (a) contacting a population of regulatory T cells (Tregs) with an mTOR inhibitor prior to activation; (b) activating the Tregs in the population; (c) culturing the population of Tregs obtained in step (b) in the presence of an mTOR inhibitor.

[0012] In a further aspect, (a) contacting a population of regulatory T cells (Tregs) with an mTOR inhibitor prior to activation; (b) activating the Tregs in the population; (c) culturing the population of Tregs obtained in step (b) in the presence of an mTOR inhibitor.

[0013] In certain embodiments, the method further comprises, prior to step (a), isolating CD4+CD25+ Tregs.

[0014] In certain embodiments, the mTOR inhibitor used in step (a) and / or step (c) may be rapamycin or a rapalog. The mTOR inhibitor used in step (a) may be the same as the mTOR inhibitor used in step (c). The initial concentration of the mTOR inhibitor used in step (a) and / or step (c) may be about 30 nM to about 500 nM.

[0015] In certain embodiments, the method does not include a step of removing the mTOR inhibitor used in step (a) before step (b) or step (c). In certain embodiments, the method does not include a step of washing the cells (especially Tregs) obtained in step (a) before step (b) or step (c). Thus, in certain embodiments, step (b) is carried out in the presence of an mTOR inhibitor. In certain embodiments, at least a portion of the mTOR inhibitor used in step (a) is also present in steps (b) and (c), and no additional mTOR inhibitor is added for step (c). Therefore, from an alternative perspective, a single addition of an mTOR inhibitor may be carried out at any time before the activation of Tregs (before step (b)) in the method of the present invention.

[0016] In certain embodiments, the population of Tregs is contacted with the mTOR inhibitor in step (a) for a period of time such that the product of the in vitro method after step (c) (e.g., after step (d) or step (e)) exhibits a decreased proportion of contaminating cells and / or an increased proportion of Tregs and / or an increased proliferation rate and / or an increased cell number compared to a corresponding method without step (a). In certain embodiments, the population of Tregs is contacted with the mTOR inhibitor in step (a) for at least about 15 minutes before step (b). For example, the population of Tregs may be contacted with the mTOR inhibitor in step (a) for about 30 to about 90 minutes before step (b). For example, the population of Tregs may be contacted with the mTOR inhibitor in step (a) for about 60 minutes before step (b).

[0017] In certain embodiments, step (b) occurs immediately after step (a).

[0018] In a specific embodiment, in step (b), the Tregs in the population are activated by contacting them with a TCR / CD3 activator and / or a TCR costimulatory activator. The TCR / CD3 activator may be, for example, an anti-CD3 antibody or a CD3-binding fragment thereof. The TCR costimulatory activator may be, for example, an anti-CD28 antibody or a CD28-binding fragment thereof.

[0019] In certain embodiments, the population of Tregs is cultured in step (c) for a period of time such that the product of the in vitro method exhibits a decreased proportion of contaminating cells and / or an increased proportion of Tregs compared to a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor.

[0020] In certain embodiments, in step (c), the Treg population is cultured in the presence of the mTOR inhibitor for at least about 6 hours. For example, in step (c), the Treg population may be cultured in the presence of the mTOR inhibitor for about 12 to about 72 hours, or about 12 to about 60 hours, or about 36 to about 60 hours. For example, in step (c), the Treg population may be cultured in the presence of the mTOR inhibitor for about 24 hours. For example, in step (c), the Treg population may be cultured in the presence of the mTOR inhibitor for about 48 hours.

[0021] In certain embodiments, step (c) is performed simultaneously with or immediately after step (b). For example, the TCR / CD3 activator and / or TCR costimulatory activator used to activate the Tregs may not be removed before step (c). For example, at least a portion of the TCR / CD3 activator and / or TCR costimulatory activator may be present throughout the entire period of step (c).

[0022] In certain embodiments, the method further comprises, at the end of step (c), reducing the concentration of the mTOR inhibitor used in step (c) or removing the mTOR inhibitor, and then culturing the Treg population after step (c), particularly expanding the Treg population after step (c).For example, the method may further comprise, after step (c), removing the mTOR inhibitor used in step (c), and then culturing (particularly expanding) the Treg population in the absence of an mTOR inhibitor.For example, the method may further comprise diluting the mTOR inhibitor used in step (c), and then culturing (particularly expanding) the Treg population in the presence of the mTOR inhibitor, the concentration of which is reduced in step (c).In particular, the step of reducing the concentration of the mTOR inhibitor or removing the mTOR inhibitor is an active step. For example, the reduction in concentration or removal of the mTOR inhibitor may be achieved by quantitatively adding the mTOR inhibitor to the Treg population in step (c), by replacing at least a portion of the culture medium (i.e., diluting the mTOR inhibitor in step (c)), or by washing the Treg population. In particular, the reduction in concentration or removal of the mTOR inhibitor is not a passive process of degradation of the mTOR inhibitor or consumption of the mTOR inhibitor by the cell population. Thus, even if degradation or consumption of the mTOR inhibitor occurs as described herein, the reduction in concentration or removal of the mTOR inhibitor is not specifically achieved by degradation or consumption.

[0023] In certain embodiments, the concentration of the mTOR inhibitor may be reduced to about 25 nM or less. In certain embodiments, the concentration of the mTOR inhibitor may be reduced or removed at least about 6 hours after the initiation of step (c). For example, the concentration of the mTOR inhibitor may be reduced or removed about 12 to about 72 hours, about 12 to about 60 hours, or about 36 to about 60 hours after the initiation of step (c). For example, the concentration of the mTOR inhibitor may be reduced or removed about 48 hours after the initiation of step (c).

[0024] In certain embodiments, the Treg population may be cultured (especially expanded) for at least about 6 days after the concentration of the mTOR inhibitor is reduced or removed. For example, the Treg population may be cultured (especially expanded) for about 8 to about 36 days, or about 8 to about 20 days, or about 10 to about 14 days after the concentration of the mTOR inhibitor is reduced or removed.

[0025] In certain embodiments, the method further comprises introducing a heterologous nucleic acid into the Tregs. In certain embodiments, the heterologous nucleic acid is introduced into the Tregs by transduction with a viral vector containing the heterologous nucleic acid. In certain embodiments, the heterologous nucleic acid encodes a chimeric antigen receptor (CAR) and / or a FOXP3 polypeptide and / or a safety switch and / or a polypeptide that increases the persistence of the cells.

[0026] In certain embodiments, the heterologous nucleic acid is introduced into the Tregs after the initiation of step (b) or after the initiation of step (c), e.g., at least about 6 hours after the initiation of step (b) or the initiation of step (c). For example, the heterologous nucleic acid may be introduced into the Tregs about 12 hours to about 72 hours, about 12 hours to about 60 hours, or about 36 hours to about 60 hours after the initiation of step (b) and / or step (c). For example, the heterologous nucleic acid may be introduced into the Tregs about 24 hours after the initiation of step (b) or the initiation of step (c). For example, the heterologous nucleic acid may be introduced into the Tregs about 48 hours after the initiation of step (b) or the initiation of step (c). Alternatively, the heterologous nucleic acid may be introduced into the Tregs (e.g., a population of Tregs) during step (b) or step (c).

[0027] In certain embodiments, before or at the same time that the heterologous nucleic acid is introduced into the Treg, the concentration of the mTOR inhibitor used in step (c) is reduced.For example, before or at the same time that the transduction of the Treg is carried out, the concentration of the mTOR inhibitor used in step (c) can be reduced.In certain embodiments, the concentration of the mTOR inhibitor can be reduced to about 25nM or less.

[0028] In certain embodiments, the mTOR inhibitor used in step (c) is removed before introducing the heterologous nucleic acid into the Treg, e.g., before transducing the Treg. In certain embodiments, the mTOR inhibitor used in step (c) is removed immediately before introducing the heterologous nucleic acid into the Treg, e.g., immediately before transducing the Treg.

[0029] In certain embodiments, the Treg population may be cultured (particularly expanded) for at least about 6 days after the heterologous nucleic acid is introduced into the Tregs. For example, the Treg population may be cultured (particularly expanded) for about 8 to about 36 days, or about 8 to about 20 days, or about 10 to about 14 days after the heterologous nucleic acid is introduced into the Tregs.

[0030] In a specific embodiment, the Treg population may be collected about 8 to about 36 days after the initiation of step (b). For example, the Treg population may be collected about 8 to about 22 days, about 10 to about 18 days, or about 12 to about 16 days after the initiation of step (b). The Treg population may be cryopreserved after collection, for example.

[0031] In a further aspect, there is provided a product obtained and / or obtainable by the method according to the first aspect.

[0032] In a further aspect, (a) contacting a population of Tregs with an mTOR inhibitor prior to activation; (b) activating the Tregs in the population; (c) culturing the population of Tregs obtained in step (b) in the presence of an mTOR inhibitor; Methods for reducing the proportion or amount of one or more contaminating cells (e.g., CD8+ T cells and / or CD4+CD25- T cells) in a population of Tregs and / or for inhibiting the proliferation of CD8+ T cells in a population of Tregs and / or for inhibiting the proliferation of CD4+CD25- T cells in a population of Tregs are provided.

[0033] The above method may relate to the first aspect of the present invention, including any embodiment thereof. In the method, the starting population of Tregs (i.e., the population of Tregs used in step (a)) may contain contaminating cells such as CD8+ T cells or CD4+CD25- T cells. As used herein, the term "contaminating cells" refers to cells that are not Tregs, including, for example, CD8+ T cells and CD4+CD25- T cells. The method reduces the proportion or amount of contaminating cells in the population of Tregs, where the reduction is relative to the starting population of Tregs (i.e., the population of Tregs used in step (a)). Alternatively, the reduction may be relative to the product of a corresponding method that does not perform step (a) and / or where step (c) is performed in the absence of an mTOR inhibitor.

[0034] In a further aspect, (a) contacting a population of Tregs with an mTOR inhibitor prior to activation; (b) activating the Tregs in the population; (c) culturing the population of Tregs obtained in step (b) in the presence of an mTOR inhibitor; Methods for increasing the proportion or amount of Tregs in a population of Tregs (e.g., methods for increasing the proportion or amount of FOXP3-expressing cells and / or methods for increasing the proportion or amount of cells having a demethylated Treg-specific demethylated region (TSDR)) are provided.

[0035] The above method may relate to the first aspect of the present invention, including any embodiment thereof. In the above method, the starting population of Tregs (i.e., the population of Tregs used in step (a)) may contain one or more contaminating cells, such as CD8+ T cells and / or CD4+CD25- T cells. The increase is in comparison with the starting population of Tregs (i.e., the population of Tregs used in step (a)). An increase in the proportion or amount of Tregs in a population of Tregs may refer to an increase in the purity of the population of Tregs. Alternatively, the increase may be in comparison with the product of a corresponding method that does not perform step (a) and / or a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor.

[0036] In a further aspect, (a) contacting a population of Tregs with an mTOR inhibitor prior to activation; (b) activating the Tregs in the population; (c) culturing the population of Tregs obtained in step (b) in the presence of an mTOR inhibitor; Methods for increasing the suppressive function of a population of Tregs are provided.

[0037] The method may be according to the first aspect of the invention, including any embodiment thereof. The increase in suppressive function is relative to the starting population of Tregs (i.e., the population of Tregs used in step (a)). Alternatively, the increase may be relative to the product of a corresponding method in which step (a) is not performed and / or in which step (c) is performed in the absence of an mTOR inhibitor.

[0038] In a further aspect, (a) contacting a population of Tregs with an mTOR inhibitor prior to activation; (b) activating the Tregs in the population; (c) culturing the population of Tregs obtained in step (b) in the presence of an mTOR inhibitor; A method for increasing expression of a heterologous nucleic acid is provided, further comprising the step of introducing a heterologous nucleic acid into the Tregs. The method, including any embodiment thereof, may relate to the first aspect of the present invention. The increased expression of the heterologous nucleic acid may be compared to a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor. In particular, the method may further comprise, after step (c) and before introducing the heterologous nucleic acid into the Tregs, reducing the concentration of the mTOR inhibitor in contact with the Treg population or removing the mTOR inhibitor from the Treg population. The Treg population may be further cultured (particularly expanded) in the presence of a reduced concentration of the mTOR inhibitor or in the absence of an mTOR inhibitor.

[0039] In a further aspect, (a) contacting a population of regulatory T cells (Tregs) with an mTOR inhibitor prior to activation; (b) activating the Tregs in the population; (c) culturing the population of Tregs obtained in step (b) in the presence of an mTOR inhibitor; (d) reducing the concentration of the mTOR inhibitor in contact with the population of Tregs or removing the mTOR inhibitor from contact with the population of Tregs; (e) further culturing (particularly expanding) the population of Tregs in the presence of reduced concentrations of the mTOR inhibitor or in the absence of an mTOR inhibitor, Methods for culturing (particularly expanding) Tregs are provided. The method may relate to the first aspect of the invention, including any embodiment thereof.

[0040] In a further aspect, (a) contacting a population of regulatory T cells (Tregs) with an mTOR inhibitor prior to activation; (b) activating the Tregs in the population; (c) culturing the population of Tregs obtained in step (b) in the presence of an mTOR inhibitor; (d) reducing the concentration of the mTOR inhibitor in contact with the population of Tregs or removing the mTOR inhibitor from contact with the population of Tregs; (e) introducing heterologous nucleic acid into said Treg; (f) culturing (particularly expanding) the population of Tregs in the presence of decreasing concentrations of the mTOR inhibitor or in the absence of an mTOR inhibitor, A method for culturing (particularly expanding) Tregs, comprising: step (e) is carried out before, simultaneously with or after step (d); A method for culturing (particularly expanding) Tregs is provided, wherein step (e) is performed before step (f). The method may relate to the first aspect of the invention, including any embodiment thereof.

[0041] In a further aspect, (a) contacting a population of Tregs with an mTOR inhibitor prior to activation; (b) activating the Tregs in the population; (c) culturing the population of Tregs obtained in step (b) in the presence of an mTOR inhibitor; (d) reducing the concentration of the mTOR inhibitor in contact with the population of Tregs or removing the mTOR inhibitor from contact with the population of Tregs; (e) further culturing (particularly expanding) the population of Tregs in the presence of reduced concentrations of the mTOR inhibitor or in the absence of an mTOR inhibitor, Methods for increasing the proliferation of the Tregs are provided. The method may be according to the first aspect of the invention, including any embodiment thereof. The increase in cell proliferation may be in comparison to a corresponding method in which step (a) is not performed and / or in which step (c) is performed in the absence of an mTOR inhibitor.

[0042] In a further aspect, (a) contacting a population of Tregs with an mTOR inhibitor prior to activation; (b) activating the Tregs in the population; (c) culturing the population of Tregs obtained in step (b) in the presence of an mTOR inhibitor; Methods for increasing the proportion or amount of CD45RA+ Tregs in a population of Tregs are provided. The method may be according to the first aspect of the present invention, including any embodiment thereof, wherein the starting population of Tregs (i.e., the population of Tregs used in step (a)) may comprise at least a portion of cells that are CD45RA-, and the increase is relative to the product of a corresponding method in which step (a) is not performed and / or in which step (c) is performed in the absence of an mTOR inhibitor.

[0043] In a further aspect, (a) contacting a population of Tregs with an mTOR inhibitor prior to activation; (b) activating the Tregs in the population; (c) culturing the population of Tregs obtained in step (b) in the presence of an mTOR inhibitor; Methods for increasing the proportion or amount of Helios+ Tregs in a population of Tregs are provided. The method may be related to the first aspect of the present invention, including any embodiment thereof. In the method, the starting population of Tregs (i.e., the population of Tregs used in step (a)) may contain at least a portion of cells that are Helios-. The increase may be in comparison with the starting population of Tregs (i.e., the population of Tregs used in step (a)). Alternatively, the increase may be in comparison with the product of a corresponding method that does not perform step (a) and / or a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor.

[0044] In a further aspect, (a) contacting a population of Tregs with an mTOR inhibitor prior to activation; (b) activating the Tregs in the population; (c) culturing the population of Tregs obtained in step (b) in the presence of an mTOR inhibitor; Methods for increasing the proportion or amount of CD27+ Tregs in a population of Tregs are provided. The method may be related to the first aspect of the present invention, including any embodiment thereof. In the method, the starting population of Tregs (i.e., the population of Tregs used in step (a)) may comprise at least a portion of cells that are CD27-. The increase may be in comparison with the starting population of Tregs (i.e., the population of Tregs used in step (a)). Alternatively, the increase may be in comparison with the product of a corresponding method in which step (a) is not performed and / or in which step (c) is performed in the absence of an mTOR inhibitor.

[0045] In a further aspect, (a) contacting a population of Tregs with an mTOR inhibitor prior to activation; (b) activating the Tregs in the population; (c) culturing the population of Tregs obtained in step (b) in the presence of an mTOR inhibitor; Further comprising the step of introducing a heterologous nucleic acid into the Treg. A method for increasing the transduction efficiency of a heterologous nucleic acid is provided. The method, including any embodiment thereof, may relate to the first aspect of the present invention. The increased transduction efficiency of a heterologous nucleic acid may be compared to a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor. In particular, the method may further comprise, after step (c) and before or simultaneously with the step of introducing the heterologous nucleic acid into the Tregs, reducing the concentration of the mTOR inhibitor in contact with the Treg population or removing the mTOR inhibitor from the Treg population. The Treg population may be further cultured (particularly expanded) in the presence of the reduced concentration of the mTOR inhibitor or in the absence of the mTOR inhibitor.

[0046] In a further aspect, (a) contacting a population of Tregs with an mTOR inhibitor prior to activation; (b) activating the Tregs in the population; (c) culturing the population of Tregs obtained in step (b) in the presence of an mTOR inhibitor; Further comprising the step of introducing a heterologous nucleic acid into the Treg. A method for reducing the vector copy number (VCN) of a heterologous nucleic acid is provided. The method, including any embodiment thereof, may relate to the first aspect of the present invention. The reduction in VCN of the heterologous nucleic acid may be in comparison with a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor. In particular, the method may further comprise, after step (c) and before or simultaneously with the step of introducing the heterologous nucleic acid into the Tregs, reducing the concentration of the mTOR inhibitor in contact with the Treg population or removing the mTOR inhibitor from the Treg population. The Treg population may be further cultured (particularly expanded) in the presence of the reduced concentration of the mTOR inhibitor or in the absence of the mTOR inhibitor. [Brief explanation of the drawings]

[0047] [Figure 1] Figure 1 shows the percentage of CD8+ cells at day 14 in bulk Tregs obtained from healthy donors, which were expanded under various conditions after adding 5% CD8+ cells. [Figure 2-1] FIG. 2 shows the percentage of CD8+ cells at day 14 in products of bulk Tregs obtained from patient-derived material expanded under various conditions. [Figure 2-2] Figure 2 (continued) [Figure 3] Figure 3 shows the percentage of FOXP3-expressing cells at day 14 in bulk Tregs obtained from healthy donors, which were expanded under various conditions with the addition of 5% CD8+ cells. [Figure 4] Figure 4 shows the Treg cell expansion rate at day 14 of bulk Tregs collected from healthy donors, which were expanded under various conditions with the addition of 5% CD8+ cells. [Figure 5] FIG. 5 shows the cell proliferation rate at day 14 of the bulk Treg expansion product obtained from patient-derived material. [Figure 6A] Figures 6A and 6B show IL-17 and IFNγ concentrations, respectively, in the products obtained by expanding bulk Tregs obtained from patient-derived material. [Figure 6B] Figure 6B [Figure 7] FIG. 7 shows the ability of Treg cells obtained by expanding patient-derived material to suppress T effector cell proliferation under a variety of conditions. [Figure 8] Figure 8 shows the mortality of products obtained by growing patient-derived material under various conditions. [Figure 9] FIG. 9 shows the total proliferation rate of RA+ and RA- Tregs grown under no rapa (control) or rapa conditions. [Figure 10] FIG. 10 shows the phenotype of RA+ Tregs grown under rapa-free (control) or rapa-free conditions, and the phenotype of RA- Tregs grown under rapa-free (control) conditions. [Figure 11]FIG. 11 shows the percentage of FOXP3+ cells at day 14 for products grown in a GMP-compliant process. [Figure 12] FIG. 12 shows the percentage of Helios+ cells at day 14 for products grown in a GMP-compliant process. [Figure 13] FIG. 13 shows the FOXP3-TSDR demethylation rate at day 14 for products grown in a GMP-compliant process. [Figure 14] FIG. 14 shows the percentage of CD8+ cells at day 14 for products grown in a GMP-compliant process. [Figure 15] FIG. 15 shows the transduction efficiency at 14 days for products grown in a GMP-compliant process. [Figure 16] FIG. 16 shows the VCN at day 14 of the product grown in a GMP-compliant process. DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention generally relates to an in vitro method for culturing (particularly expanding) regulatory T cells (Tregs), which can increase the proliferation of Tregs and / or reduce impurities in the final product. The in vitro method comprises pretreating a population of Tregs with an mTOR inhibitor prior to activation, and then culturing the activated population of Tregs in the presence of the mTOR inhibitor. In certain embodiments, the concentration of the mTOR inhibitor used in culturing the activated population of Tregs (step (c)) may be reduced (step (d)), and the Treg population may be further cultured (particularly expanded) in the presence of the reduced concentration of the mTOR inhibitor (step (e) or step (f)). In certain embodiments, the mTOR inhibitor used in culturing the activated population of Tregs (step (c)) may be removed (step (d)), and the Treg population may be further cultured (particularly expanded) in the absence of the mTOR inhibitor (step (e) or step (f)).

[0049] As used herein, the terms "culturing" and "cell culture" refer to in vitro methods for maintaining at least a portion of the cells in a starting material or population, including expanding cells (especially Tregs), particularly increasing the total number of cells (especially Tregs) compared to the starting material or population (cell expansion). It is understood that cells in a culturing process or method may undergo a resting or recovery phase from previously exposed conditions before entering an active expansion phase. The term "culturing" as used herein includes such a recovery or resting phase (e.g., before expansion begins) in which cells do not expand and cell numbers may temporarily decrease, as well as a phase in which cells are expanding but the total number of cells has not yet reached the number of cells in the starting material. At least a portion of the cells in the starting material or population refers to maintaining at least 50% of the Tregs, particularly in a Treg population. For example, at least about 55%, or at least about 60%, or at least about 65% of the Tregs, particularly in a Treg population, may be maintained. In particular, in embodiments in which cells are expanded, the terms "culturing" and "cell culture" may be used synonymously with "expanding" and "cell expansion" when the total number of cells (e.g., Treg total number) is increased compared to the total number of cells (e.g., Treg total number) in the starting material. Thus, the in vitro methods described herein may be referred to as in vitro methods for expanding Tregs.

[0050] The in vitro methods (e.g., culturing or expanding) described herein may be performed in closed and / or sterile cell culture systems. Appropriate equipment and conditions for cell culture of Tregs are known to those of skill in the art, particularly those used in Good Manufacturing Practice (GMP) protocols for culturing Tregs, and are applicable for use in combination with the in vitro methods disclosed herein. Suitable tissue culture flasks are also known to those of skill in the art, including gas-permeable static culture flasks such as G-rex™. Tregs may be cultured at temperatures of about 30°C to about 40°C, e.g., about 32°C to about 38°C, or about 33°C to about 37°C, or about 36°C to about 38°C, e.g., around about 37°C. Tregs may be cultured at a CO2 concentration of about 2% to about 8%, e.g., about 3% to about 7%, e.g., about 5% CO2. Tregs may be cultured at an O2 concentration of about 3% to about 30%, e.g., about 5% to about 25%, or about 15% to about 25%. Any one or more (e.g., all) of the steps of the methods described herein, particularly culture step (c) and subsequent culture steps, may be performed under any one or more of these conditions. It will be understood that the addition of materials to the Treg population necessary to perform various steps of the method (e.g., addition of an activating agent in step (b), addition of heterologous nucleic acid, addition of fresh medium, etc.) may be performed outside the desired conditions, but the Treg population can be restored to the desired conditions once the necessary materials have been added to the Tregs.

[0051] In vitro methods for culturing or expanding Tregs can achieve an expansion rate of at least about 2-fold over the starting population of Tregs. For example, in vitro methods for culturing or expanding Tregs can achieve an expansion rate of at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold over the starting population of Tregs. For example, in vitro methods for culturing or expanding Tregs can achieve an expansion rate of at least about 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold over the starting population of Tregs. For example, in vitro methods for culturing or expanding Tregs can achieve an expansion rate of up to about 3000-fold, up to about 2000-fold, or up to about 1000-fold over the starting population of Tregs. The expansion rate of the product (compared to the starting material) is determined by calculating the total number of cells or Tregs at the time of collection and dividing by the total number of cells or Tregs, respectively, seeded at the beginning of step (a).

[0052] especially, (a) contacting a population of regulatory T cells (Tregs) with an mTOR inhibitor prior to activation; (b) activating Tregs in the population; (c) culturing the population of Tregs obtained in step (b) in the presence of an mTOR inhibitor; In vitro methods for culturing or expanding Tregs are provided.

[0053] The present invention further relates to an in vitro method for culturing or expanding Tregs, comprising the step of introducing a heterologous nucleic acid into Tregs (e.g., by transduction with a viral vector), which can increase expression of the transgene. These methods are useful for preparing Tregs for adoptive cell transfer.

[0054] Treg starting material "Regulatory T cells or T regulatory (Treg) cells" are immune cells with immunosuppressive function that control cytotoxic immune responses and are essential for maintaining immune tolerance. As used herein, the term Treg refers to T cells with immunosuppressive function. The term regulatory expression cells refers to cells with immunosuppressive function. Preferably, the immunosuppressive function may refer to the ability of Tregs to reduce or inhibit one or more of the numerous physiological and cellular effects promoted by the immune system in response to stimuli such as pathogens, alloantigens, or autoantigens. Examples of such effects include increased proliferation of conventional T cells (Tconv) and secretion of inflammatory cytokines. Both of these effects can be used as indicators of the strength of an immune response. A relatively weaker immune response mediated by Tconvs in the presence of Tregs is considered to indicate the ability of Tregs to suppress an immune response. For example, a relative decrease in cytokine secretion is considered to indicate a weakened immune response, and thus, is considered to indicate the ability of Tregs to suppress an immune response. Tregs can also suppress an immune response by regulating the expression of costimulatory molecules on antigen-presenting cells (APCs), such as B cells, dendritic cells, and macrophages. The expression levels of CD80 and CD86 can be used to assess the in vitro suppressive efficacy of activated Tregs after co-culture.

[0055] Assays for measuring the strength of immune responses and thus the suppressive ability of Tregs are known in the art. Specifically, antigen-specific Tconv cells are co-cultured with Tregs, and a peptide of the corresponding antigen is added to the co-culture to stimulate a response from the Tconv cells. The degree of proliferation of Tconv cells and / or the amount of cytokines, such as IL-2, secreted by Tconv cells in response to the addition of peptides can be used as an indicator of the suppressive ability of the co-cultured Tregs. The amount of cytokines can be detected in the cell culture medium by ELISA or flow cytometry. Examples of suppressive assays and cytokine analysis are described in the Examples below. Several different Treg subpopulations have been identified, which may express different specific markers or different levels of specific markers. Tregs are generally T cells that express the markers CD4, CD25, and FOXP3 (CD4 + CD25 + FOXP3 + ).

[0056] Tregs may also express CTLA-4 (cytotoxic T lymphocyte-associated molecule-4) or GITR (glucocorticoid-inducible TNF receptor).

[0057] Treg cells are present in peripheral blood, lymph nodes, and tissues, and as used herein, Treg includes thymus-derived natural Treg (nTreg) cells, peripherally generated Treg, and inducible Treg (iTreg) cells.

[0058] Tregs express the cell surface markers CD4 and CD25 in the absence or in combination with low expression levels of the surface protein CD127 (CD4 + CD25 + CD127 - or CD4 + CD25 + CD127 low The use of such markers to identify Tregs is known in the art and is described, for example, in Liu et al. (JEM; 2006; 203; 7(10); 1701-1711).

[0059] Tregs are CD4 + CD25 + FOXP3 + T cells, CD4 + CD25 + CD127 - T cells, or CD4 + CD25 + FOXP3 + CD127 - / low It may be a T cell. Preferably, Tregs may be natural Tregs (nTregs). As used herein, the term "natural Tregs" refers to thymus-derived Tregs. Natural Tregs are CD4 + CD25 + FOXP3 + Helios + Neuropilin 1 + Compared with iTregs, nTregs have high expression of PD-1 (programmed cell death-1, pdcd1), neuropilin 1 (Nrp1), Helios (Ikzf2), and CD73. nTregs can be distinguished from iTregs based on the distinct expression of Helios protein or neuropilin 1 (Nrp1).

[0060] Tregs may have demethylated Treg-specific demethylation regions (TSDRs), which are important methylation-sensitive factors that regulate Foxp3 expression (Polansky, JK, et al., 2008. European Journal of Immunology, 38(6), pp.1654-1663).

[0061] Further preferred Tregs include Tr1 cells (which do not express Foxp3 and have high IL-10 production), CD8 + FOXP3 + T cells, and γδFOXP3 + These include, but are not limited to, T cells.

[0062] Tregs include naive Tregs (CD45RA + FoxP3 low ), effector / memory Treg (CD45RA - FoxP3 high ), and cytokine-producing Tregs (CD45RA - FoxP3 low It is known that there exist different subpopulations of Tregs, including those that express CD45RO. +These cells are believed to have increased levels of CD45RO compared to naive Tregs (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more CD45RO) and preferably express no or reduced levels of CD45RA (mRNA and / or protein) compared to naive Tregs (e.g., at least 80%, 90%, or 95% less CD45RA compared to naive Tregs). "Cytokine-producing Tregs" are Tregs that do not express CD45RA (mRNA and / or protein) or express a very low level of CD45RA compared to naive Tregs (e.g., at least 80%, 90%, or 95% less CD45RA compared to naive Tregs) and have a low level of FOXP3 compared to memory Tregs, e.g., FOXP3 is less than 50%, 60%, 70%, 80%, or 90% compared to memory Tregs. Cytokine-producing Tregs may also be Tregs that can produce interferon gamma and have a lower in vitro suppressive ability compared to naive Tregs (e.g., suppressive ability is less than 50%, 60%, 70%, 80%, or 90% compared to naive Tregs). The expression level herein may refer to mRNA or protein expression. In particular, with respect to cell surface markers such as CD45RA, CD25, CD4, and CD45RO, expression may refer to cell surface expression, i.e., the amount or relative amount of the marker protein expressed on the cell surface. Expression levels can be determined by methods known in the art. For example, mRNA expression levels can be determined by Northern blotting / array analysis, and protein expression can be determined by Western blotting or, preferably, FACS using antibody staining for cell surface expression.

[0063] In particular, Tregs may be naive Tregs. As used interchangeably herein, "naive regulatory T cells, naive T regulatory cells, or naive Tregs" refer to Treg cells that express CD45RA (particularly, that express CD45RA on the cell surface). Thus, naive Tregs are Treg cells that express CD45RA + Naive Tregs generally refer to Tregs that have not been activated via endogenous TCR by peptide / MHC, whereas effector / memory Tregs refer to Tregs that have been activated via endogenous TCR by stimulation. Typically, naive Tregs may express at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more CD45RA than non-naive Treg cells (e.g., memory Treg cells). Alternatively, naive Treg cells may express at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, or 100-fold more CD45RA than non-naive Treg cells (e.g., memory Treg cells). The level of CD45RA expression can be easily determined by methods in the art, for example, by flow cytometry using commercially available antibodies. Typically, non-naive Treg cells do not express CD45RA or have low levels of CD45RA.

[0064] In particular, naive Tregs may not express CD45RO, and CD45RO - Thus, naive Tregs may express at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less CD45RO than memory Tregs, or, alternatively, may express at least 2-, 3-, 4-, 5-, 10-, 50-, or 100-fold less CD45RO than memory Treg cells.

[0065] As described above, naive Tregs express CD25, but depending on the source of naive Tregs, the CD25 expression level may be lower than that of memory Tregs. For example, in the case of naive Tregs isolated from peripheral blood, the CD25 expression level may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower than that of memory Tregs. Such naive Tregs may be considered to express intermediate to low levels of CD25. However, those skilled in the art will understand that such differences may not be observed in naive Tregs isolated from umbilical cord blood.

[0066] Naive Tregs, as defined herein, are typically CD4 + , CD25 + , FOXP3 + , CD127 low , CD45RA + may be.

[0067] As used herein, low expression of CD127 refers to the expression of CD4 + Naive Tregs are characterized by a lower level of CD127 expression compared to non-regulatory or Tcon cells. Specifically, naive Tregs are characterized by a lower level of CD4 Tregs from the same subject or donor. + The expression of CD127 may be less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of that of non-regulatory or Tcon cells. CD127 levels can be assessed by methods standard in the art, including flow cytometry of cells stained with anti-CD127 antibodies.

[0068] Typically, naive Tregs do not express or express low levels of CCR4, HLA-DR, CXCR3, and / or CCR6. Specifically, naive Tregs may express lower levels of CCR4, HLA-DR, CXCR3, and CCR6, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower levels, compared to memory Tregs.

[0069] Naive Tregs also express CCR7 + and CD31 + Additional markers may be expressed, including:

[0070] The isolated naive Treg can be identified by methods known in the art, such as determining the presence or absence of any one or more of the above-mentioned markers on the cell surface of isolated cells.For example, CD45RA, CD4, CD25, and CD127low can be used to determine whether a cell is naive Treg.The methods for determining whether isolated cells are naive Treg and determining the presence and / or expression level of cell markers are well known in the art, such as flow cytometry using commercially available antibodies.

[0071] Tregs subjected to the in vitro method steps described herein are included in a cell population containing multiple Tregs ("population of Tregs" or "Treg population"). The term "population of Tregs" as used herein may be used synonymously with "population of cells containing Tregs." It is understood that not all cells in a Treg population express the above-mentioned Treg markers to the same extent. Thus, a population of Tregs may contain different identifiable subpopulations of Tregs as defined above. Furthermore, a population of Tregs need not be a pure Treg population and may contain some contaminating cells (non-Treg cells), such as, for example, CD8+ T cells, CD4+ T effector cells, NK cells, and APCs. For utility in the methods and applications of the present invention, it is sufficient that at least about 50% of the cells in the population are identifiable as Tregs, and preferably at least about 60%, 70%, 75%, 80%, 90%, or 95% of the population are identifiable as Tregs, preferably naive Tregs.

[0072] The starting population of Tregs used in the in vitro methods described herein (i.e., the population of Tregs used in step (a)) may be a Treg-enriched population of cells. In certain embodiments, the in vitro methods described herein further comprise, prior to step (a), a step of preparing a Treg-enriched population of cells. This enriched population of cells is used as the source of the population of Tregs for step (a). The population of cells may be enriched for any of the Treg phenotypes described above.

[0073] An "enriched" population of cells refers to a population of cells in which the proportion of a particular target cell (e.g., Treg) in the population is higher (e.g., at least 10%, 20%, 30%, 40%, or 50% higher) than the proportion in the material from which the cell population is derived. A Treg-enriched population of cells can be prepared by any method known to those skilled in the art and may include enrichment and / or depletion steps. For example, a Treg-enriched population of cells may be obtained by FACS and / or magnetic bead sorting. A Treg-enriched sample may be generated from a cell-containing sample by any method known to those skilled in the art, for example, from Tcon cells by introducing DNA or RNA encoding FOXP3 and / or from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells.

[0074] The starting population of Tregs (used in step (a)) can comprise, for example, at least about 40% CD4+CD25+ Tregs, e.g., at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80% CD4+CD25+ Tregs. For example, the starting population of Tregs can comprise about 95% or less, or about 90% or less, or about 85% or less, or about 80% or less CD4+CD25+ Tregs. For example, the starting population of Tregs can comprise about 50% to about 95%, or about 55% to about 90% CD4+CD25+ Tregs.

[0075] The starting population of Tregs can comprise, for example, at least about 25% FOXP3+ T cells, e.g., at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 75%, or at least about 80% FOXP3+ T cells. For example, the starting population of Tregs can comprise about 95% or less, or about 90% or less, or about 85% or less, or about 80% or less FOXP3+ T cells. For example, the starting population of Tregs can comprise about 50% to about 95%, or about 55% to about 90% FOXP3+ T cells.

[0076] Tregs in the starting population of Tregs may specifically express FOXP3. For example, the starting population of Tregs may contain at least about 25% FOXP3+ Tregs. For example, the starting population of Tregs may contain at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 70%, or at least about 75%, or at least about 80% FOXP3+ Tregs. For example, the starting population of Tregs may contain about 95% or less, or about 90% or less, or about 85% or less FOXP3+ Tregs. For example, the starting population of Tregs may contain about 50% to about 95%, or about 55% to about 90% FOXP3+ Tregs. Tregs may have any of the Treg phenotypes described herein, e.g., CD4+CD25+FOXP3+.

[0077] The starting population of Tregs can comprise, for example, at least about 15% CD45RA+ Tregs (e.g., CD4+CD25+CD127- / lowCD45RA+ Tregs), e.g., at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40% CD45RA+ Tregs (e.g., CD4+CD25+CD127- / lowCD45RA+ Tregs). For example, the starting population of Tregs can comprise no more than about 95%, or no more than about 90%, or no more than about 85%, or no more than about 80%, or no more than about 75%, or no more than about 70%, or no more than about 65%, or no more than about 60% CD45RA+ Tregs (e.g., CD4+CD25+CD127- / lowCD45RA+ Tregs). For example, the starting population of Tregs can contain about 50% to about 95%, or about 55% to about 90% CD45RA+ Tregs (e.g., CD4+CD25+CD127- / lowCD45RA+ Tregs).

[0078] The starting population of Tregs may contain, for example, about 1% or more CD8+ T cells, e.g., about 2% or more, or about 3% or more, or about 4% or more, or about 5% or more CD8+ T cells. For example, the starting population of Tregs may contain about 15% or less, about 10% or less, or about 5% or less CD8+ T cells. For example, the starting population of Tregs may contain about 1% to about 5%, 10%, or 15%, or about 2% to about 5%, 10%, or 15%, or about 3% to about 5%, 10%, or 15% CD8+ T cells.

[0079] The starting population of Tregs may contain, for example, about 1% or more CD4+CD25- T cells, e.g., about 2% or more, or about 3% or more, or about 4% or more, or about 5% or more CD4+CD25- T cells. For example, the starting population of Tregs may contain about 20% or less, about 10% or less, about 5% or less, about 3% or less, or about 2% or less CD4+CD25- T cells. For example, the starting population of Tregs may contain about 1% to about 20%, or about 2% to about 18%, or about 2% to about 15% CD4+CD25- T cells.

[0080] The in vitro methods described herein can be particularly beneficial when the starting material (population of Tregs) has particularly high levels of impurities (e.g., a high percentage of CD8+ T cells and / or CD4+CD25- T cells, and / or a low percentage of CD4+CD25+ T cells and / or FOXP3+ T cells). Such starting material tends to produce poor product quality (e.g., high impurity levels and relatively low Treg levels) when expanded under standard Treg culture conditions (e.g., in the absence of an mTOR inhibitor). In contrast, the methods of the present invention can generate expanded Treg populations with low impurity levels and high Treg numbers from starting material with high impurity levels and low Treg numbers. Thus, in certain embodiments, the starting population of Tregs (e.g., in step (a)) may comprise less than about 60% CD4+CD25+ Tregs, and / or less than about 60% CD4+CD25+CD127- / low Tregs, and / or less than about 75% FOXP3+ T cells, and / or more than about 5% CD8+ T cells, and / or more than about 10% CD4+CD25- T cells.

[0081] The in vitro methods described herein may be particularly useful for selectively expanding CD45RA+ Tregs (e.g., CD4+CD25+CD127- / lowCD45RA+ Tregs) relative to CD45RA- Tregs (e.g., CD4+CD25+CD127- / lowCD45RA- Tregs). Thus, the in vitro methods described herein may result in expanded Treg populations with higher levels of CD45RA+ Tregs (e.g., CD4+CD25+CD45RA+ or CD4+CD25+CD127- / lowCD45RA+) Tregs compared to expanded Treg populations obtained by corresponding methods without step (a) and / or corresponding methods in which step (c) is performed in the absence of an mTOR inhibitor. Selective expansion of CD45RA+ Tregs by the in vitro methods described herein may further result in expanded Treg populations with higher levels of markers indicative of a stable Treg phenotype. In particular, the selective expansion of CD45RA+ Tregs by the in vitro methods described herein may result in an expanded Treg population that has higher levels of Helios, and / or higher levels of CD27, and / or higher levels of TSDR demethylation compared to the starting population of Tregs used in step (a) and / or compared to an expanded Treg population obtained by a corresponding method that does not perform step (a) and / or a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor.

[0082] The population of Tregs cultured by the in vitro method described above may be isolated from peripheral blood mononuclear cells (PBMCs) obtained from a subject by leukapheresis. The subject from which the PBMCs are obtained may be a mammal, preferably a human. The Tregs may be matched (e.g., HLA-matched) or autologous to the subject to whom the cultured Tregs are administered. The subject to whom the cultured Tregs are administered may be a mammal, particularly a human. The Tregs may be obtained from either the patient's own peripheral blood (first party) or, in the case of hematopoietic stem cell transplantation, from the peripheral blood of a donor (second party) or from peripheral blood of an unrelated donor (third party). Preferably, the Tregs are autologous to the subject to whom the Tregs are administered. Alternatively, the Tregs cultured or expanded by the method of the present invention may be obtained by differentiation of autologous cells, such as pluripotent stem cells (particularly iPSCs).

[0083] mTOR inhibitors "mTOR" is also known as mammalian target of rapamycin, mechanistic target of rapamycin, FK506-binding protein 12-rapamycin complex-associated protein 1, FKBP12-rapamycin complex-associated protein, rapamycin and FKBP12 target 1, target of rapamycin protein 1, FRAP, FRAP1, FRAP2, RAFT1, and RAPT1. In some embodiments, the human mTOR protein corresponds to Uniprot No.: P42345. An mTOR inhibitor can inhibit, reduce, and / or decrease at least one activity of mTOR (e.g., serine / threonine protein kinase activity, etc.) against at least one substrate (e.g., p70S6 kinase 1, 4E-BP1, AT / PKB, and eEF2). In certain embodiments, mTOR inhibitors may inhibit, reduce, and / or decrease mTOR kinase activity, for example, by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99%, and / or inhibit, reduce, and / or decrease mTOR kinase activity. This can be determined by reducing the signaling of the mTOR pathway, as described in more detail below. In some embodiments, mTOR inhibitors directly bind to and inhibit mTORCl, mTORC2, or both mTORCl and mTORC2, and / or can directly bind to and inhibit mTORCl. The inhibition of mTOR activity by mTOR inhibitors may be reversible or irreversible.

[0084] mTOR is a conserved threonine and serine protein kinase and belongs to the phosphatidylinositol-3-kinase-related kinase (PIKK) family. mTOR is a protein kinase that phosphorylates threonine and serine residues in substrates. In certain embodiments, mTOR functions as a catalytic subunit of two multiprotein complexes called mTOR complex 1 (mTORC1) and complex 2 (mTORC2). In certain embodiments, despite the fact that both mTORC1 and mTORC2 are involved in the phosphoinositol-3 kinase (PI3K) and Akt signaling pathways, mTORC1 and mTORC2 function independently of each other. In some embodiments, an mTOR inhibitor can inhibit, reduce, and / or decrease mTORC1 activity (e.g., mTORC1 kinase activity) and / or mTORC2 activity, and / or inhibit, reduce, and / or decrease mTORC1 activity.

[0085] mTORC1 is a protein complex with five components: mTOR (the catalytic subunit of the complex), mTOR regulatory-associated protein (Raptor), mammalian lethal Secl3 protein 8 (mLST8, also known as GPL), proline-rich Akt substrate 40 kDa (PRAS40), and DEP domain-containing mTOR-interacting protein (Deptor). In some embodiments, the mTOR inhibitor suppresses the formation of the mTORC1 complex and / or reduces its stability.

[0086] mTORC2 comprises six distinct proteins, some of which are common to mTORC1 and mTORC2: mTOR, rapamycin-insensitive mTOR-associated protein (Rictor), mammalian stress-activated protein kinase-interacting protein (mSIN1), protein observed with Rictor-1 (Protor-1), mLST8, and Deptor. In certain embodiments, mTOR inhibitors suppress the formation of the mTORC2 complex and / or reduce its stability.

[0087] In some embodiments, an mTOR inhibitor is a compound, small molecule (e.g., a small organic molecule), polynucleotide, oligonucleotide, siRNA, polypeptide, or fragment, isoform, variant, analog, or derivative thereof that inhibits, reduces, suppresses, and / or is capable of inhibiting, reducing, or suppressing one or more activities of mTOR. In some embodiments, the inhibitor is a small molecule. In certain embodiments, the inhibitor is a small molecule having a molecular weight of less than 10 kD, less than 9 kD, less than 8 kD, less than 7 kD, less than 6 kD, less than 5 kD, less than 4 kD, less than 3 kD, less than 2 kD, less than 1 kD, less than 0.5 kD, or less than 0.1 kD. In some embodiments, the inhibitor is a small molecule that is or contains a nucleic acid, peptide, polypeptide, peptidomimetic, peptoid, carbohydrate, lipid, component thereof, or other organic or inorganic molecule. Libraries of chemical and / or biological mixtures (e.g., fungal, bacterial, or algal extracts) are known in the art and can be screened for mTOR inhibitor activity. Methods for synthesizing molecular libraries include, for example, (Carell et al., 1994a; Carell et al., 1994b; Cho et al., 1993; DeWitt et al., 1993; Gallop et al., 1994; Zuckermann et al., 1994).

[0088] An mTOR inhibitor may be a nucleic acid, peptide, chemical compound, or small organic molecule that inhibits at least one activity of the mTOR protein (e.g., serine / threonine protein kinase activity) toward at least one substrate (e.g., p70S6 kinase 1, 4E-BP1, AT / PKB, and eEF2). An mTOR inhibitor may be capable of directly binding to and inhibiting mTORC1, mTORC2, or both mTORC1 and mTORC2.

[0089] Inhibition of mTOR activity, for example, inhibition of mTORC1 and / or mTORC2 activity, can be determined by reducing the signal transduction of mTOR pathway.A variety of indicators can be used to reduce the output of such signal transduction pathway.Exemplary indicators include, but are not limited to: (1) reduction of Akt phosphorylation at residues, including but not limited to S473 and T308; (2) reduction of Akt activation, as indicated by reduction of the phosphorylation of Akt substrates, including but not limited to Fox01 / O3a T24 / 32, GSK3β; S21 / 9, and TSC2 T1462; (3) reduction of the phosphorylation of downstream signal transduction molecules of mTOR (for example, mTORC1), including but not limited to ribosomal S6 S240 / 244, 70S6K T389, and 4EBP1 T37 / 46. Measuring, detecting, and / or assessing proteins with site-specific phosphorylation is performed by any means known in the art, including, but not limited to, antibody staining techniques, immunoassays, enzyme-linked immunosorbent assays (ELISAs), enzyme-linked immunosorbent assays (EIAs), radioimmunoassays (RIAs), surface plasmon resonance (SPR), Western blotting, or protein arrays.

[0090] In certain embodiments, the mTOR inhibitor is 50 In certain embodiments, the mTOR inhibitor has an IC of less than 500 μM, less than 200 μM, less than 100 μM, less than 50 μM, less than 10 μM, less than 5 μM, less than 1 μM, less than 500 nM, less than 200 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 1 nM, or less than 500 pM. 50 In some embodiments, the IC 50 can be determined using any known standard and / or conventional technique. For example, in some embodiments, IC 50can be determined by measuring mTOR activity in the presence of various concentrations of the inhibitor under evaluation. The experimentally obtained values ​​of enzyme activity are then plotted against the concentration of inhibitor used. The inhibitor concentration that gives 50% enzyme activity (compared to activity in the absence of inhibitor) is known as the "IC 50 Similarly, other inhibitory concentrations can be defined by appropriate activity measurements. In some embodiments, the IC 50 is measured in a cell-free assay. In certain embodiments, IC 50 is measured in a cell culture assay. In certain embodiments, the cell culture is a T cell culture, e.g., a primary T cell culture.

[0091] Examples of mTOR inhibitors suitable for use in certain embodiments discussed herein include, but are not limited to, AZD8055, INK128, PF-04691502, and everolimus.

[0092] The mTOR inhibitor used in step (a) and / or step (c) of the in vitro methods described herein may in particular be rapamycin or a rapalog.

[0093] In certain embodiments, the mTOR inhibitor may be a selective mTOR inhibitor, selectively inhibiting at least one mTOR activity, but not other kinases (eg, PI3K), for example.

[0094] Thus, in some embodiments, the mTOR inhibitor does not inhibit PI3K activity. In certain embodiments, the mTOR inhibitor inhibits the IC 50 In certain embodiments, the mTOR inhibitor does not detectably reduce, inhibit, or decrease PI3K activity at an IC 50 IC of mTOR activity 50The mTOR activity is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 100%, at least 150%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold greater than mTORC1 and mTORC2 kinase activity. In some embodiments, the mTOR inhibitor inhibits, for example, selectively inhibits, mTORC1 and mTORC2 kinase activity relative to PI3K activity. In certain embodiments, the mTOR inhibitor is pyrazolopyrimidine, torin 1, torkinib (PP242), PP30, Ku-0063794, WAY-600 (Wyeth), WAY-687 (Wyeth), WAY-354 (Wyeth), or AZD8055.

[0095] In certain embodiments, the mTOR inhibitor is 50 In certain embodiments, the mTOR inhibitor selectively inhibits mTORC1 at an IC of less than 500 μM, less than 200 μM, less than 100 μM, less than 50 μM, less than 10 μM, less than 5 μM, less than 1 μM, less than 500 nM, less than 200 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 1 nM, or less than 500 pM. 50 inhibits mTORC1 activity at IC between 1 nM and 500 μM, between 1 nM and 500 nM, between 1 μM and 500 μM, between 10 μM and 100 μM, between 100 nM and 1 μM, between 250 nM and 750 nM, between 50 nM and 200 nM, or between 400 nM and 600 nM. 50 is measured in a cell-free assay. In certain embodiments, IC 50 is measured in a cell culture assay. In certain embodiments, the cell culture is a T cell culture, e.g., a primary T cell culture.

[0096] In some embodiments, the mTOR inhibitor selectively inhibits mTORC1 activity relative to mTORC2 and / or PI3K activity. In certain embodiments, the mTOR inhibitor selectively inhibits mTORC1 activity relative to mTORC2 and / or PI3K activity. 50IC of mTORC1 activity 50 at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 100%, at least 150%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold greater than mTOR. In some embodiments, the mTOR inhibitor is rapamycin (sirolimus). In certain embodiments, the mTOR inhibitor is a rapalog.

[0097] In one embodiment, mTOR inhibitor is active site inhibitor.These mTOR inhibitors bind to the ATP binding site (also called ATP binding pocket) of mTOR and inhibit the catalytic activity of both mTORC1 and mTORC2.One class of active site inhibitors is the dual specific inhibitor, which targets and directly inhibits both PI3K and mTOR.Dual specific inhibitors bind to both the ATP binding site of mTOR and PI3K.

[0098] Examples of such inhibitors include, but are not limited to, imidazoquinazoline, wortmannin, LY294002, PI-103 (Cayman Chemical), SF1126 (Semafore), BGT226 (Novartis), XL765 (Exelixis), and NVP-BEZ235 (Novartis).

[0099] Another class of mTOR active site inhibitors suitable for use in the methods discussed herein selectively inhibit mTORC1 and mTORC2 activity relative to one or more type I phosphatidylinositol 3-kinases, such as PI3 kinase α, β, γ, or δ. These active site inhibitors bind to the active site of mTOR but do not bind to PI3K. Examples of such inhibitors include, but are not limited to, pyrazolopyrimidine, Trinl (Guertin and Sabatini), PP242 (2-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-5-ol), PP30, Ku-0063794, WAY-600 (Wyeth), WAY-687 (Wyeth), WAY-354 (Wyeth), and AZD8055 (Liu et al., Nature Review, 8, 627-644, 2009).

[0100] In one embodiment, the selective mTOR inhibitor is a compound having a 50% inhibitory concentration (IC50) for mTORC1 and / or mTORC2. 50 ) is the IC of an inhibitor against one, two, or more than two type I PI3-kinases, or against all type I PI3-kinases. 50 It refers to an inhibitor that is at least 1 / 10, at least 1 / 20, at least 1 / 50, at least 1 / 100, at least 1 / 1000, or less than that.

[0101] mTOR has been shown to exhibit robust and specific catalytic activity towards its physiological substrate proteins, p70 S6 ribosomal protein kinase I (p70S6KI) and eIF4E-binding protein 1 (4EBP1), as measured by Western blotting with phospho-specific antibodies. In one embodiment, the mTOR pathway inhibitor is an S6 kinase inhibitor selected from the group consisting of BI-D1870, H89, PF-4708671, FMK, and AT7867.

[0102] Another class of mTOR inhibitors for use in the present invention are compounds that specifically bind to the mTOR FRB domain (FKBP rapamycin binding domain). Such compounds include rapamycin (also known as sirolimus, rapamune, Fyarro, and ABI-009) and rapalogs.

[0103] As used herein, the term "rapalog" refers to a compound that specifically binds to the mTOR FRB domain (FKBP rapamycin-binding domain), is structurally related to rapamycin, and retains mTOR inhibitory properties. The term "rapalog" does not include rapamycin. Rapalogs include esters, ethers, oximes, hydrazones, and hydroxylamines of rapamycin, as well as compounds in which functional groups in the basic rapamycin structure have been modified, for example, by reduction or oxidation. Pharmaceutically acceptable salts of such compounds are also considered rapamycin derivatives. Examples of rapalogs suitable for use in the methods discussed herein include, but are not limited to, temsirolimus (CC1779), everolimus (RAD001), deforolimus (AP23573), AZD8055 (AstraZeneca), and OSI-027 (OSI).

[0104] In some embodiments, the inhibitor is a molecule described in WO 2008 / 051493, WO 2008 / 051494, or WO 2010 / 062571, and / or U.S. Patent Nos. 7,981,893, 8,372,976, 7,968,556, 8,383,634, 8,110,578, or 8,492,381, all of which are incorporated herein by reference.

[0105] In certain embodiments, the mTOR inhibitor has formula (I): [ka] It has or includes the structural formula shown in In the formula, R 1 is a substituted or unsubstituted C alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl; R 2 is a substituted or unsubstituted C alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl; R 3 and R 4 are each independently H or C 1-8 It is alkyl.

[0106] In some embodiments, the mTOR inhibitor is or contains a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof. 1 In certain embodiments, the mTOR inhibitor is or contains a compound of Formula (I) where R is substituted aryl or substituted or unsubstituted heteroaryl (e.g., substituted phenyl). 2 is or contains a compound of formula (I), wherein the substituted group is a halogen group, a C alkyl group, a C 2-8 Alkenyl group, C 2-8 Alkynyl group, hydroxyl group, C 1-8or containing a compound of formula (I) substituted with one or more of the following groups, each of which is optionally substituted: alkoxyl, amino, nitro, thiol, thioether, imine, cyano, amido, phosphonato, phosphine, carboxyl, thiocarbonyl, sulfonyl, sulfonamide, ketone, aldehyde, ester, carbonyl, haloalkyl, B(OH), carbocyclic cycloalkyl, heterocycloalkyl, monocyclic or fused or non-fused polycyclic aryl or heteroaryl, amino, O-lower alkyl, O-aryl, aryl, aryl-lower alkyl, COCH, CONH, OCHCONH, NH, SONH, OCHF, CF, or OCF.

[0107] In some embodiments, the mTOR inhibitor having or comprising the structural formula shown in Formula (I) is Compound A. In certain embodiments, the mTOR inhibitor is Compound A. In some embodiments, Compound A is 2-(3-hydroxyphenyl)-9-(2-isopropylphenyl)-8-oxo-8,9-dihydro-7H-purine-6-carboxamide. In some embodiments, the mTOR inhibitor is 2-(3-hydroxyphenyl)-9-(2-isopropylphenyl)-8-oxo-8,9-dihydro-7H-purine-6-carboxamide, or a pharmaceutically acceptable salt or solvate thereof. In certain embodiments, Compound A has the structural formula: [ka] It has.

[0108] In certain embodiments, the mTOR inhibitor has the structural formula shown in Formula (II): [ka] having or including wherein L is a direct bond, NH, or O; Y is N or CR 3and R 1 is H, substituted or unsubstituted C1-8 alkyl, substituted or unsubstituted C2-8 alkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl; R 2 is H, substituted or unsubstituted C alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl; R 3 is H, substituted or unsubstituted C1-8 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, -NHR 4 , or -N(R 4 )2, R 4 is, at each occurrence, independently substituted or unsubstituted C alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl.

[0109] In some embodiments, the mTOR inhibitor is or contains a compound of formula (II), or a pharmaceutically acceptable salt or solvate thereof. In certain embodiments, the mTOR inhibitor is R 1 is substituted aryl (e.g., substituted phenyl). In certain embodiments, the mTOR inhibitor has or includes the structural formula shown in Formula (II) where Y is CH. In some embodiments, the mTOR inhibitor has or includes the structural formula shown in Formula (II) where L is a direct bond. In certain embodiments, the mTOR inhibitor has or includes the structural formula shown in Formula (II) where R 1 is a substituted or unsubstituted aryl, and R 2is substituted with one or more substituents selected from alkoxy, amino, hydroxy, cycloalkyl, or heterocycloalkyl; 1-8 It has or includes the structural formula shown in formula (II) where R is alkyl.

[0110] In certain embodiments, the mTOR inhibitor has or includes the structural formula shown in Formula (II), where the groups are "substituted or unsubstituted" and, if substituted, may be substituted with one or more optional substituents.Examples of substituents include those described in the exemplary compounds and embodiments disclosed herein, as well as halo groups (e.g., chloro, iodo, bromo, or fluoro), C alkyl groups, C alkenyl groups, C alkynyl groups, hydroxyl groups, C alkoxyl groups, amino groups, nitro groups, thiol groups, thioether groups, imine groups, cyano groups, amido groups, phosphonato groups, phosphine groups, carboxyl groups, carbamoyl groups, carbamate groups, acetal groups, urea groups, thiocarbonyl groups, sulfonyl groups, sulfonamide groups, sulfinyl groups, and the like. aryl groups, ketone groups, aldehyde groups, ester groups, acetyl groups, acetoxy groups, oxygen (=O) groups, haloalkyl groups (e.g., trifluoromethyl groups), substituted aminoacyl and aminoalkyl groups, carbocyclic cycloalkyl groups which may be monocyclic or fused or non-fused polycyclic (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl groups), or heterocycloalkyl groups which may be monocyclic or fused or non-fused polycyclic (e.g., pyrrolidinyl, piperidinyl, piperazinyl groups), , morpholinyl, furanyl, or thiazinyl), carbocyclic or heterocyclic, monocyclic or fused or non-fused polycyclic aryl groups (e.g., phenyl, naphthyl, pyrrolyl, indolyl, furanyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl, pyridinyl, quinolinyl, isoquinolinyl, acridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, benzimidazolyl, benzothienyl, or benzimidazolyl; zofuranyl group), amino group (primary, secondary, or tertiary), -O-lower alkyl group, -O-aryl group, aryl group, aryl-lower alkyl group, CO2CH3 group, CONH2 group, OCH2CONH2 group, NH2 group, N(C1-4 alkyl)2 group, NHC(O)C1-4 alkyl group, SO2NH2 group, SO2C1-4 alkyl group, OCHF2 group, CF3 group, and OCF3 group, which moieties may optionally be substituted with fused cyclic or bridging structures such as, for example, -OCHO- or -O-lower alkylene-O-.These substituents may be further substituted with a substituent selected from these groups as needed.

[0111] In certain embodiments, the mTOR inhibitor having or comprising the structural formula shown in Formula (II) is Compound B. In certain embodiments, the mTOR inhibitor is Compound B. In some embodiments, Compound B is 6-(4-(2H-1,2,4-triazol-3-yl)phenyl)-1-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one. In some embodiments, the mTOR inhibitor is 6-(4-(2H-1,2,4-triazol-3-yl)phenyl)-1-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one, or a pharmaceutically acceptable salt or solvate thereof. In certain embodiments, Compound B has the structural formula: [ka] It has.

[0112] In certain embodiments, the mTOR inhibitor has the structural formula shown in Formula (III): [ka] having or including In the formula, R 1 is substituted or unsubstituted C alkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, or substituted or unsubstituted heterocyclylalkyl; R 2 is H, substituted or unsubstituted C alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylalkyl, substituted or unsubstituted aralkyl, or substituted or unsubstituted cycloalkylalkyl; R 3 is H or substituted or unsubstituted C 1-8It is alkyl.

[0113] In some embodiments, the mTOR inhibitor is or contains a compound of formula (III), or a pharmaceutically acceptable salt or solvate thereof. 1 is a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl (e.g., R 1 In certain embodiments, the mTOR inhibitor has or includes the structural formula shown in formula (III): R is phenyl, pyridyl, pyrimidyl, benzimidazolyl, 1H-pyrrolo[2,3-b]pyridyl, indazolyl, indolyl, 1H-imidazo[4,5-b]pyridyl, 1H-imidazo[4,5-b]pyridin-2(3H)-onyl, 3H-imidazo[4,5-b]pyridyl, or pyrazolyl, each optionally substituted. 1 is phenyl substituted with one or more substituents each independently selected from the group consisting of substituted or unsubstituted C alkyl (e.g., methyl), substituted or unsubstituted heterocyclyl (e.g., substituted or unsubstituted triazolyl or pyrazolyl), aminocarbonyl, halogen (e.g., fluorine), cyano, hydroxyalkyl, and hydroxy. In another embodiment, R is pyridyl substituted with one or more substituents each independently selected from the group consisting of substituted or unsubstituted C alkyl (e.g., methyl), substituted or unsubstituted heterocyclyl (e.g., substituted or unsubstituted triazolyl), halogen, aminocarbonyl, cyano, hydroxyalkyl (e.g., hydroxypropyl), -OR, and -NR, where each R is independently H or substituted or unsubstituted C alkyl. In some embodiments, R 1is 1H-pyrrolo[2,3-b]pyridyl or benzimidazolyl optionally substituted with one or more substituents each independently selected from the group consisting of substituted or unsubstituted C alkyl and —NR, where each R is independently H or substituted or unsubstituted C alkyl.

[0114] In some embodiments, the mTOR inhibitor is R 1 but: [ka] The compound has or includes the structural formula shown in formula (III): wherein R, at each occurrence, is independently H or substituted or unsubstituted C 1-4 alkyl (e.g., methyl), and R 1 is each independently at each occurrence substituted or unsubstituted C alkyl (e.g., methyl), halogen (e.g., fluoro), cyano, —OR, or —NR, m is 0 to 3, and n is 0 to 3. It will be understood that any of the R' substituents is attached to a suitable atom of either ring in the fused ring system.

[0115] In some embodiments of the compound of Formula (III), R 2 is H, substituted or unsubstituted C alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted C 1-4 alkyl-heterocyclyl, substituted or unsubstituted C alkyl-aryl, or substituted or unsubstituted C 1-4 alkyl-cycloalkyl. For example, R 2 is H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, cyclopentyl, cyclohexyl, tetrahydrofuranyl, tetrahydropyranyl, (C 1-4 alkyl)-phenyl, (C 1-4 alkyl)-cyclopropyl, (C 1-4alkyl)-cyclobutyl, (C 1-4 alkyl)-cyclopentyl, (C 1-4 alkyl)-cyclohexyl, (C 1-4 alkyl)-pyrrolidyl, (C 1-4 alkyl)-piperidyl, (C 1-4 alkyl)-piperazinyl, (C 1-4 alkyl)-morpholinyl, (C 1-4 alkyl)-tetrahydrofuranyl, or (C 1-4 alkyl)-tetrahydropyranyl, each of which is optionally substituted.

[0116] In certain embodiments, R 2 is H, C1-4 alkyl, or (C1-4 alkyl)(OR): [ka] is. In the formula, R, at each occurrence, is independently H or substituted or unsubstituted C alkyl; R', at each occurrence, is independently H, -OR, cyano, or substituted or unsubstituted C alkyl; and p is 0 to 3.

[0117] In certain embodiments, the mTOR inhibitor having or comprising the structural formula shown in Formula (III) is Compound C. In certain embodiments, the mTOR inhibitor is Compound C. In some embodiments, Compound C is 7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-1-((1r,4r)-4-methoxycyclohexyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(1H)-one. In some embodiments, the mTOR inhibitor is 7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-1-((1r,4r)-4-methoxycyclohexyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(1H)-one, or a pharmaceutically acceptable salt or solvate thereof. In certain embodiments, Compound C has the structural formula: [ka] It has.

[0118] In certain embodiments, the mTOR inhibitor used in the in vitro methods described herein is rapamycin or a rapalog, particularly rapamycin. In certain embodiments, the mTOR inhibitor used in step (a) is the same as the mTOR inhibitor used in step (c).

[0119] In certain embodiments, the in vitro method does not include a step of removing the mTOR inhibitor used in step (a) before performing step (b) and / or step (c). For example, the in vitro method may not include a step of washing Tregs before performing step (b) and / or step (c). Accordingly, in certain embodiments, step (b) is performed in the presence of an mTOR inhibitor. Accordingly, in certain embodiments, the mTOR inhibitor used in step (a) may also be present in step (b) and / or step (c). Accordingly, in this embodiment, the mTOR inhibitor used in step (c) is the same as the mTOR inhibitor used in step (a). In certain embodiments, the mTOR inhibitor used in step (a) remains (is present) in the culture medium during steps (b) and (c), and an additional mTOR inhibitor for step (c) is not required. Alternatively, the above method may include only a single addition of an mTOR inhibitor (particularly during step (a)). The initial concentration of the mTOR inhibitor used in step (a) and / or step (c) may be the same or similar (e.g., the initial concentration of the mTOR inhibitor present in step (b) or step (c) may be at least 70%, 80%, 90%, or 95% of the initial concentration of the mTOR inhibitor present in step (a)). "Initial concentration" refers to the concentration at the start of the particular step being referred to. Note that the concentration of the mTOR inhibitor may naturally decrease over time, for example, due to degradation and / or cellular uptake, and therefore may differ from the concentration at the start and end of a particular step. The concentration of the mTOR inhibitor used in a particular step may only decrease naturally during the particular step (e.g., during one or more of steps (a), (b), and / or (c)). That is, the concentration of the mTOR inhibitor may not decrease as a result of non-natural or external factors, such as dilution (unless otherwise specified as a separate step). The concentration of the mTOR inhibitor may specifically refer to the concentration of the mTOR inhibitor in the culture medium. That is, the concentration of the mTOR inhibitor refers to the concentration of the mTOR inhibitor relative to the total volume that the cells are in. Generally, all concentrations mentioned herein refer to initial concentrations unless otherwise specified.The concentration of rapamycin in the medium can be determined by analytical chemistry techniques such as mass spectrometry (eg, liquid chromatography-mass spectrometry (LC-MS)).

[0120] The initial concentration of the mTOR inhibitor used in step (a) and / or step (b) and / or step (c) may be, for example, at least about 30 nM. For example, the initial concentration of the mTOR inhibitor used in step (a) and / or step (b) and / or step (c) may be at least about 40 nM, or at least about 50 nM, or at least about 60 nM, or at least about 70 nM, or at least about 80 nM, or at least about 90 nM, or at least about 100 nM.

[0121] The initial concentration of the mTOR inhibitor used in step (a) and / or step (b) and / or step (c) may be, for example, about 500 nM or less. For example, the initial concentration of the mTOR inhibitor used in step (a) and / or step (b) and / or step (c) may be about 450 nM or less, or about 400 nM or less, or about 350 nM or less, or about 300 nM or less, or about 250 nM or less, or about 200 nM or less, or about 150 nM or less, or about 120 nM or less.

[0122] The initial concentration of the mTOR inhibitor used in step (a) and / or step (b) and / or step (c) may be, for example, about 30 nM to about 500 nM, or about 50 nM to about 400 nM, or about 75 nM to about 300 nM, or about 75 nM to about 250 nM, or about 75 nM to about 200 nM, or about 75 nM to about 150 nM, or about 80 nM to about 300 nM, or about 80 nM to about 200 nM, or about 80 nM to about 150 nM, or about 80 nM to about 120 nM.

[0123] In certain embodiments, the medium is not replenished with an mTOR inhibitor after step (c), i.e., no further addition of an mTOR inhibitor is made in addition to the mTOR inhibitor present for step (c) (the mTOR inhibitor present in step (c) was added at the start of step (c) or was added as a single addition in step (a)).

[0124] Process (a) The in vitro methods described herein particularly include contacting a population of Tregs with an mTOR inhibitor prior to activation. Tregs may include, for example, those obtained from a subject as described above.

[0125] "Before activation" means that Tregs (in the Treg population) are contacted with an mTOR inhibitor before any in vitro activation step. That is, Tregs (in the Treg population) are contacted with an mTOR inhibitor before any ex vivo measures are taken to activate Tregs (for example, using anti-CD3 antibodies and / or anti-CD28 antibodies described below). This does not include natural in vivo activation of Tregs before the in vitro method described herein. Thus, some of the Tregs in the Treg population used in step (a) may be activated in vivo before being isolated from a subject.

[0126] The step of contacting the Treg population with an mTOR inhibitor before activation is carried out to pretreat the cells in the Treg population. " Pretreatment " means that the mTOR inhibitor is contacted with the Treg population to inhibit the mTOR in the cells in the Treg population.Therefore, step (a) can be carried out for any period and / or at any mTOR inhibitor concentration that results in inhibiting the mTOR in the cells in the Treg population.

[0127] Although it may be desirable to inhibit mTOR in as many Tregs as possible, mTOR may not be inhibited in all Tregs in the population, and it is not a requirement for this method or this step of this method that mTOR is inhibited in all Tregs.Therefore, in step (a), mTOR may be inhibited in some of the Tregs present.For example, mTOR may be inhibited in at least 50%, 60%, 70%, 80% or 90% of Tregs.

[0128] Inhibition of mTOR can be assessed by determining the phosphorylation level of mTOR itself or by determining the phosphorylation level of downstream targets of mTOR. For example, inhibition of mTOR can be assessed by determining the phosphorylation level of p70S6 kinase (p70 s6k Inhibition of mTOR can be assessed by determining the phosphorylation level of p70 ribosomal protein (S6) and / or the phosphorylation level of S6 ribosomal protein (S6). Inhibition of mTOR can be assessed by determining the reduced level of phosphorylated mTOR (P-mTOR) and / or phosphorylated p70 in the pretreated Treg population (i.e., Tregs after step (a) but before step (b)) compared to the starting population of Tregs (i.e., the starting population of Tregs used in step (a)). s6k (P-p70 s6k ) and / or reduced levels of phosphorylated S6 (P-S6).

[0129] The level of phosphorylated protein in the Treg population can be visually observed using Western blotting and / or quantified by densitometry quantification of Western blotting. This level can be normalized to a control protein such as tubulin and / or the corresponding non-phosphorylated protein (i.e., P-mTOR is normalized to non-phosphorylated mTOR). Alternatively or additionally, FACS can be used to determine the phosphorylated mTOR positive and / or phosphorylated p70 in the population. S6K The total number and / or percentage of cells that are positive and / or phosphorylated S6 positive may be determined. S6K, and suitable antibodies against S6 are commercially available.

[0130] For example, the percentage of phosphorylated mTOR-positive cells in the Treg population after step (a) may be reduced by at least about 5 percentage points, or at least about 10 percentage points, or at least about 15 percentage points, or at least about 20 percentage points, relative to the starting population of Tregs used in step (a). For example, the percentage of phosphorylated mTOR-positive cells in the Treg population after step (a) may be reduced by about 80 percentage points, or by about 75 percentage points, or by about 70 percentage points, or by about 65 percentage points, or by about 60 percentage points, or by about 55 percentage points, or by about 50 percentage points, relative to the starting population of Tregs used in step (a). For example, the percentage of phosphorylated mTOR-positive cells in the Treg population after step (a) may be reduced by 5 to 80 percentage points, or by 5 to 70 percentage points, or by 10 to 60 percentage points, or by 10 to 50 percentage points, relative to the starting population of Tregs used in step (a).

[0131] For example, the total number of phosphorylated mTOR-positive cells in the Treg population after step (a) can be reduced by at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60% compared to the total number of P-mTOR-positive cells in the starting Treg population used in step (a). For example, the total number of phosphorylated mTOR-positive cells in the Treg population can be reduced by up to about 100%, or up to about 95%, or up to about 90%, or up to about 85%, or up to about 80%, or up to about 75%, or up to about 70%, or up to about 65% compared to the total number of P-mTOR-positive cells in the starting Treg population used in step (a). For example, the total number of P-mTOR-positive cells in the Treg population after step (a) may be reduced by 5% to 100%, or 5% to 90%, or 10% to 80%, or 10% to 75%, or 15% to 70% relative to the total number of P-mTOR-positive cells in the starting population used in step (a).

[0132] For example, the proportion of phosphorylated mTOR positive cells and / or the total number of phosphorylated mTOR positive cells in the Treg population after step (a) can be reduced to the baseline level of phosphorylation.Baseline level refers to the lowest level of mTOR phosphorylation that can be achieved by mTOR inhibitor.That is, even if the concentration of mTOR inhibitor is increased and / or the contact time with cell is extended, the phosphorylation level of mTOR will not be further reduced.

[0133] For example, phosphorylated p70 in the Treg population after step (a) S6K The percentage of positive cells may be reduced by at least about 5 percentage points, or at least about 10 percentage points, or at least about 15 percentage points, or at least about 20 percentage points relative to the starting population of Tregs used in step (a). For example, the percentage of phosphorylated p70 in the population of Tregs after step (a) may be reduced by at least about 5 percentage points, or at least about 10 percentage points, or at least about 15 percentage points, or at least about 20 percentage points. S6KThe percentage of positive cells may be reduced by about 80%, or about 75%, or about 70%, or about 65%, or about 60%, or about 55%, or about 50% relative to the starting population of Tregs used in step (a). For example, the percentage of phosphorylated p70 in the population of Tregs after step (a) may be reduced by about 80%, or about 75%, or about 70%, or about 65%, or about 60%, or about 55%, or about 50%. S6K The percentage of positive cells may be reduced by 5 to 80 percentage points, or 5 to 70 percentage points, or 10 to 60 percentage points, or 10 to 50 percentage points relative to the starting population of Tregs used in step (a).

[0134] For example, phosphorylated p70 in the Treg population after step (a) S6K The total number of positive cells was determined based on the P-p70 expression in the starting population of Tregs used in step (a). S6K The total number of positive cells may be reduced by at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%. For example, phosphorylated p70 in a population of Tregs S6K The total number of positive cells is determined by the P-p70 count in the starting population used in step (a). S6K The total number of positive cells may be reduced by about 100%, or by about 95%, or by about 90%, or by about 85%, or by about 80%, or by about 75%, or by about 70%, or by about 65%. For example, the P-p70 in the population of Tregs after step (a) may be reduced by about 100%, or by about 95%, or by about 90%, or by about 85%, or by about 80%, or by about 75%, or by about 70%, or by about 65%. S6K The total number of positive cells is determined by the P-p70 count in the starting population used in step (a). S6K The total number of positive cells may be reduced by 5% to 100%, or 5% to 90%, or 10% to 80%, or 10% to 75%, or 15% to 70%.

[0135] For example, phosphorylated p70 in the Treg population after step (a) S6K Percentage of positive cells and / or phosphorylated p70 S6KThe total number of positive cells can be reduced to a baseline level of phosphorylation of p70 achievable with an mTOR inhibitor. S6K This means that increasing the concentration of mTOR inhibitor and / or the length of contact time with cells does not result in a significant increase in p70 phosphorylation. S6K The phosphorylation level of α-glucan no longer decreases.

[0136] For example, the percentage of phosphorylated S6-positive cells in the Treg population after step (a) may be reduced by at least about 5 percentage points, or at least about 10 percentage points, or at least about 15 percentage points, or at least about 20 percentage points relative to the starting Treg population used in step (a). For example, the percentage of phosphorylated S6-positive cells in the Treg population after step (a) may be reduced by about 80 percentage points, or by about 75 percentage points, or by about 70 percentage points, or by about 65 percentage points, or by about 60 percentage points, or by about 55 percentage points, or by about 50 percentage points relative to the starting Treg population used in step (a). For example, the percentage of phosphorylated S6-positive cells in the Treg population after step (a) may be reduced by 5 to 80 percentage points, or by 5 to 70 percentage points, or by 10 to 60 percentage points, or by 10 to 50 percentage points relative to the starting Treg population used in step (a).

[0137] For example, the total number of phosphorylated S6-positive cells in the Treg population after step (a) can be reduced by at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60% relative to the total number of P-S6-positive cells in the starting Treg population used in step (a). For example, the total number of phosphorylated S6-positive cells in the Treg population can be reduced by up to about 100%, or up to about 95%, or up to about 90%, or up to about 85%, or up to about 80%, or up to about 75%, or up to about 70%, or up to about 65% relative to the total number of P-S6-positive cells in the starting population used in step (a). For example, the total number of P-S6 positive cells in the Treg population after step (a) may be reduced by 5% to 100%, or 5% to 90%, or 10% to 80%, or 10% to 75%, or 15% to 70% relative to the total number of P-S6 positive cells in the starting population used in step (a).

[0138] For example, the proportion of phosphorylated S6 positive cells and / or the total number of phosphorylated S6 positive cells in the Treg population after step (a) can be reduced to the baseline level of phosphorylation.Baseline level refers to the minimum level of S6 phosphorylation that can be achieved by mTOR inhibitor.That is, even if the concentration of mTOR inhibitor is increased and / or the contact time with cells is extended, the phosphorylation level of S6 does not further decrease.

[0139] The ratio of phosphorylated to non-phosphorylated proteins can be determined by densitometric quantification of Western blotting.

[0140] The ratio of phosphorylated mTOR to unphosphorylated mTOR (P-mTOR / mTOR) after step (a) may be about 1.0 or less. For example, the ratio of P-mTOR / mTOR may be about 0.9 or less, or about 0.8 or less, or about 0.7 or less, or about 0.6 or less, or about 0.5 or less, or about 0.4 or less, or about 0.3 or less. The ratio of P-mTOR / mTOR after step (a) may be at least about 0.01, or at least about 0.05, or at least about 0.1. For example, the ratio of P-mTOR / mTOR after step (a) may be about 0.01 to about 1.0, or about 0.05 to about 0.8, or about 0.1 to about 0.5.

[0141] The ratio of P-mTOR / mTOR in the Treg population after step (a) can be reduced by at least about 0.1 relative to the ratio of P-mTOR / mTOR in the starting Treg population used in step (a). For example, the ratio of P-mTOR / mTOR in the Treg population after step (a) can be reduced by at least about 0.2, or at least about 0.3, or at least about 0.4 relative to the ratio of P-mTOR / mTOR in the starting Treg population used in step (a). For example, the ratio of mTOR / mTOR in the Treg population after step (a) can be reduced by about 0.9, or about 0.8, or about 0.7, or about 0.6, or about 0.5.

[0142] After step (a), the ratio of P-mTOR / mTOR in the Treg population can be reduced to the baseline level of phosphorylation.Baseline level refers to the minimum level of P-mTOR / mTOR that can be achieved by mTOR inhibitor.That is, even if the concentration of mTOR inhibitor is increased and / or the contact time with cells is extended, the ratio of P-mTOR / mTOR does not further decrease.

[0143] Phosphorylated p70 after step (a) S6K and non-phosphorylated p70 S6K Ratio to (P-p70 S6K / p70 S6K) may be about 1.0 or less. For example, P-p70 S6K / p70 S6K The ratio of P-p70 after step (a) may be about 0.9 or less, or about 0.8 or less, or about 0.7 or less, or about 0.6 or less, or about 0.5 or less, or about 0.4 or less, or about 0.3 or less. S6K / p70 S6K The ratio may be at least about 0.01, or at least about 0.05, or at least about 0.1. For example, after step (a), P-p70 S6K / p70 S6K The ratio may be from about 0.01 to about 1.0, or from about 0.05 to about 0.8, or from about 0.1 to about 0.5.

[0144] P-p70 in the Treg population after step (a) S6K / p70 S6K is the ratio of P-p70 in the starting population of Tregs used in step (a). S6K / p70 S6K For example, the ratio of P-p70 to P-p70 in the Treg population after step (a) may be reduced by at least about 0.1. S6K / p70 S6K is the ratio of P-p70 in the starting population of Tregs used in step (a). S6K / p70 S6K For example, the ratio of P-p70 in the Treg population after step (a) may be reduced by at least about 0.2, or at least about 0.3, or at least about 0.4. S6K / p70 S6K may be reduced by up to about 0.9, or up to about 0.8, or up to about 0.7, or up to about 0.6, or up to about 0.5.

[0145] P-p70 in the Treg population after step (a) S6K / p70 S6K The ratio of P-p70 to P-p70 can be reduced to a baseline level of phosphorylation, which is the level achievable with an mTOR inhibitor. S6K / p70 S6KThis means that increasing the concentration of mTOR inhibitor and / or the length of contact time with cells does not result in a significant increase in P-p70. S6K / p70 S6K The ratio of

[0146] The ratio of phosphorylated S6 to non-phosphorylated S6 (P-S6 / S6) after step (a) may be about 1.0 or less. For example, the P-S6 / S6 ratio may be about 0.9 or less, or about 0.8 or less, or about 0.7 or less, or about 0.6 or less, or about 0.5 or less, or about 0.4 or less, or about 0.3 or less. The P-S6 / S6 ratio after step (a) may be at least about 0.01, or at least about 0.05, or at least about 0.1. For example, the P-S6 / S6 ratio after step (a) may be about 0.01 to about 1.0, or about 0.05 to about 0.8, or about 0.1 to about 0.5.

[0147] The ratio of P-S6 / S6 in the population of Tregs after step (a) may be reduced by at least about 0.1 relative to the ratio of P-S6 / S6 in the starting population of Tregs used in step (a). For example, the ratio of P-S6 / S6 in the population of Tregs after step (a) may be reduced by at least about 0.2, or at least about 0.3, or at least about 0.4 relative to the ratio of P-S6 / S6 in the starting population of Tregs used in step (a). For example, the ratio of P-S6 / S6 in the population of Tregs after step (a) may be reduced by about 0.9, or by about 0.8, or by about 0.7, or by about 0.6, or by about 0.5.

[0148] After step (a), the ratio of P-S6 / S6 in the Treg population can be reduced to a baseline level of phosphorylation. Baseline level refers to the minimum level of P-S6 / S6 that can be achieved by the mTOR inhibitor. That is, increasing the concentration of the mTOR inhibitor and / or extending the contact time with the cells does not further reduce the ratio of P-S6 / S6.

[0149] Step (a) may be carried out in the presence of a medium suitable for Treg culture.Therefore, mTOR inhibitor may be present in the medium suitable for Treg culture.Examples of the medium suitable for Treg culture include XVIVO™ medium or TexMACS™ medium.The medium may be supplemented with, for example, serum, such as human serum, particularly human AB serum.

[0150] The step of contacting a population of Tregs with an mTOR inhibitor prior to activation (step (a)) may occur immediately before the step of activating the Tregs in the population (step (b)). That is, no additional steps may occur between steps (a) and (b). In particular, the method may not include a "resting" step between steps (a) and (b) in which the Tregs are left in the container without further action. The Tregs may be activated (step (b)) immediately after the time period in step (a) has elapsed.

[0151] In step (a), Tregs may be contacted with an mTOR inhibitor for any period of time that results in a product of the in vitro method of the invention (e.g., the product at the end of step (c), or the product at the end of any subsequent step after step (c), e.g., step (d), step (e), or step (f)) that has one or more of the following characteristics compared to the product of a corresponding method that does not involve step (a): -Reduction of the percentage of contaminating cells Decrease in the percentage of CD8+ T cells Decrease in the percentage of CD4+CD25- T cells Increased percentage of CD4+CD25+ T cells Increased proportion of FOXP3-expressing cells Increased proportion of cells with demethylated TSDR Increase in the proportion of Helios-expressing cells Increased percentage of CD27-expressing cells Increased proliferation rate Increased cell count Increased production of immunosuppressive cytokines (e.g., IL-17 and / or IFNγ) Enhanced inhibitory ability Increased expression of the transgene (if the method further comprises the step of introducing heterologous nucleic acid into Tregs) A "corresponding method omitting step (a)" is a method that is carried out using the same starting materials as the comparative method and is identical to the comparative method except that step (a) is not performed. Thus, the "corresponding method" may start from step (b). Therefore, if any period of step (a) brings about an improvement in the product (e.g., an improvement as shown in the above bullet points), that period is included in the present invention.

[0152] In particular, in step (a), Tregs may be contacted with an mTOR inhibitor for any period of time that results in a product of the method of the invention (e.g., the product at the end of step (c) or any subsequent step) that has one or more of the following characteristics compared to a product of a corresponding method that does not involve step (a): Decrease in the percentage of CD8+ T cells Increased proportion of FOXP3-expressing cells Increase in the proportion of Helios-expressing cells Increased percentage of CD27-expressing cells Increased proliferation rate Increased cell count Reduced production of inflammatory cytokines (e.g., IL-17) Enhanced inhibitory ability Increased expression of the transgene (if the method further comprises the step of introducing heterologous nucleic acid into Tregs)

[0153] In particular, in step (a), Tregs may be contacted with an mTOR inhibitor for any period of time that results in a product of the method of the invention (e.g., the product at the end of step (c) or any subsequent step) that has one or more of the following characteristics compared to a product of a corresponding method that does not involve step (a): Decrease in the percentage of CD8+ T cells Increased proportion of FOXP3-expressing cells Increase in the proportion of Helios-expressing cells Increased percentage of CD27-expressing cells Increased proliferation rate Increased cell count

[0154] As noted above, "product" refers to the Treg population obtained at the end of the method of the invention, in particular the cell population obtained at the end of any of steps (c), (d), (e), or (f).

[0155] The percentage of CD8+ T cells, CD4+CD25- T cells, CD4+CD25+ T cells, CD4+CD25+CD127- / low T cells, and FOXP3-expressing cells and / or Helios-expressing cells and / or CD27-expressing cells in the product (or any Treg population) can be determined using any known method, such as fluorescence-activated cell sorting (FACS).

[0156] The percentage of cells with demethylated TSDR can be determined using a methylation-specific qPCR assay.

[0157] The expansion rate (e.g., expansion rate of the product) (compared to the starting material) can be determined by calculating the total number of cells at the time of harvest and dividing it by the total number of cells seeded at the beginning of step (a). This calculation can be done for all cells or just Tregs.

[0158] The cell number in the product can be determined by measuring the number of cells in 1 mL of medium using an automated cell counter and multiplying the result by the total volume of the medium.

[0159] The amount of pro-inflammatory cytokine (e.g., IL-17 and / or IFNγ) produced (e.g., by the product) can be determined by collecting the cell culture medium and quantifying the cytokine in an equivalent assay, such as ELISA or flow cytometric quantification. Examples of suitable methods are described in the Examples below.

[0160] The suppressive ability (e.g., of a product) can be determined using any known suppressive assay. For example, as described above, the ability of Tregs to reduce or inhibit one or more of the numerous physiological and cellular effects promoted by the immune system in response to stimuli such as pathogens, alloantigens, or autoantigens can indicate immunosuppressive function. Examples of such effects include increased proliferation of conventional T cells (Tconv) and secretion of inflammatory cytokines. Both of these effects can be used as indicators of the strength of the immune response. A relatively weaker immune response mediated by Tconvs in the presence of Tregs is considered to indicate the ability of Tregs to suppress the immune response. Examples of suitable methods are described in the Examples below.

[0161] Transgene expression can be determined by measuring the effect of one or more transgenes, such as the effect of a safety switch as described in the Examples below. Cells with similar transduction efficiencies (e.g., within 5%, 4%, 3%, 2%, or 1% of each other) can be compared. Alternatively, transgene expression can be determined by qPCR.

[0162] The Tregs may be contacted with the mTOR inhibitor in step (a) for at least about 15 minutes. For example, the Tregs may be contacted with the mTOR inhibitor in step (a) for at least about 20 minutes, or at least about 25 minutes, or at least about 30 minutes, or at least about 35 minutes, or at least about 40 minutes, or at least about 45 minutes, or at least about 50 minutes, or at least about 55 minutes, or at least about 60 minutes.

[0163] The Tregs may be contacted with the mTOR inhibitor in step (a) for about 12 hours or less. For example, the Tregs may be contacted with the mTOR inhibitor in step (a) for about 11 hours or less, or about 10 hours or less, or about 9 hours or less, or about 8 hours or less, or about 7 hours or less, or about 6 hours or less, or about 5 hours or less, or about 4 hours or less, or about 3 hours or less.

[0164] For example, in step (a), Tregs may be contacted with the mTOR inhibitor for about 15 minutes to about 12 hours, or about 15 minutes to about 6 hours, or about 15 minutes to about 3 hours, or about 30 minutes to about 3 hours. For example, in step (a), Tregs may be contacted with the mTOR inhibitor for about 30 minutes to about 90 minutes, or about 45 minutes to about 75 minutes.

[0165] Step (a) may be performed entirely or partially at ambient temperature (e.g., about 18°C ​​to about 26°C). Step (a) may be performed entirely or partially in ambient atmosphere (e.g., about 78% nitrogen, about 21% oxygen, and about 1% other gases). Ambient temperature and ambient atmosphere refer to the temperature and atmosphere of the room in which step (a) is performed. In certain embodiments, step (a) is performed entirely at ambient temperature and ambient atmosphere. In other words, no specialized equipment is required to control the temperature and atmosphere to which the Treg population is exposed during all or part of step (a). In another embodiment, the initial addition of the mTOR inhibitor to contact the Treg population is performed at ambient temperature and ambient atmosphere, and the Treg population is then exposed to a specific temperature and atmosphere (e.g., the temperature, CO2, and / or O2 conditions described above for culturing Tregs), for example, by storing the Treg population in an incubator capable of maintaining the desired temperature and atmosphere.

[0166] Process (b) The in vitro method described herein further comprises a step of activating Tregs (in the Treg population) obtained in step (a). The step of activating Tregs may be performed immediately after contacting Tregs (in the Treg population) with an mTOR inhibitor (step (a)). That is, no additional step may be performed between steps (a) and (b). In particular, the method may not comprise a "resting" step between steps (a) and (b) in which Tregs are left in a container without further treatment. Tregs may be activated (step (b)) immediately after the time in step (a) has elapsed.

[0167] Although it may be desirable to activate as many Tregs as possible, it is not necessary to activate all Tregs in the population, and activating all Tregs is not a requirement of this method or this step of the method. Thus, in step (b), a portion of the Tregs present may be activated. For example, at least 50%, 60%, 70%, 80%, or 90% of the Tregs may be activated. As mentioned above, a portion of the Tregs may be activated in vivo before step (b). Other (non-Treg) cells in the Treg population may also be activated, but this is not a requirement of the method.

[0168] "Activating" Tregs refers to initiating a signaling pathway downstream of the TCR, or in other words, initiating a signaling pathway activated as a result of TCR binding to its ligand. This activation may be mediated by stimulating cells via the T cell receptor (TCR), which may be an endogenous TCR or a heterologous TCR, thereby mediating signaling through the TCR / CD3 complex. This activation may be achieved by contacting Tregs in a population with a TCR / CD3 activator and / or a TCR costimulatory activator. In particular, this activation may be achieved by contacting Tregs with a TCR / CD3 activator and a TCR costimulatory activator. Alternatively or additionally, Treg activation may be mediated by stimulating the cells via a heterologous receptor introduced into the cells and capable of initiating a signaling pathway downstream of the TCR or a signaling pathway activated as a result of TCR binding to its ligand. The heterologous receptor may, in particular, be a chimeric antigen receptor (CAR). CARs are described in detail below and typically contain an intracellular signaling domain that may be derived from the CD3 zeta chain. Therefore, Tregs can be activated by contacting the Tregs in the population with a CAR activator, which can be an antigen specifically recognized by the CAR. Other mechanisms that bypass TCR can also be used to activate Tregs. For example, ionophores such as ionomycin and / or phorbol myristate acetate (PMA) can be used to activate Tregs.

[0169] Step (b) may be performed in the presence of a medium suitable for Treg culture. Accordingly, the TCR / CD3 activator and / or TCR costimulatory activator may be present in the medium suitable for Treg culture. Examples of media suitable for Treg culture include XVIVO™ medium and TexMACS™ medium. The medium may be supplemented with, for example, serum, such as human serum, particularly human AB serum. The medium used in step (b) may be the same as the medium used in step (a). In certain embodiments, Tregs are not washed between steps (a) and (b), and / or the medium used in step (a) is not replaced and / or added before step (b). Accordingly, the medium used in step (a) may remain in contact with Tregs during step (b), and the TCR / CD3 activator and / or TCR costimulatory activator may be added to the medium.

[0170] A "TCR / CD3 activator" may be any activator that binds to one or more of the TCR alpha chain, TCR beta chain, CD3 gamma chain, CD3 delta chain, CD3 epsilon chain, and CD3 zeta chain of a Treg and stimulates signal transduction through the TCR / CD3 complex. Examples of TCR / CD3 activators include antigens specifically recognized by a TCR, anti-CD3 antibodies, and CD3-binding fragments of anti-CD3 antibodies. In particular, the TCR / CD3 activator may be an anti-CD3 antibody or a CD3-binding fragment of an anti-CD3 antibody. The TCR / CD3 activator (e.g., an anti-CD3 antibody or a CD3-binding fragment of an anti-CD3 antibody) may be immobilized on a surface, for example, the surface of beads. Such beads are commercially available, for example, ThermoFisher's Dynabeads™. Alternatively, the TCR / CD3 activator (e.g., an anti-CD3 antibody or a CD3-binding fragment of an anti-CD3 antibody) may be present in solution.

[0171] A "TCR costimulatory activator" may be any activator that binds to one or more TCR costimulatory receptors, such as CD28 and / or CD2, to stimulate signaling through the TCR / CD3 complex. Examples of TCR costimulatory activators include anti-CD28 antibodies, CD28-binding fragments of anti-CD28 antibodies, anti-CD2 antibodies, and CD2-binding fragments of anti-CD2 antibodies. In particular, the TCR costimulatory activator may be an anti-CD28 antibody or a CD28-binding fragment of an anti-CD28 antibody. The TCR costimulatory activator (e.g., an anti-CD28 antibody or a CD28-binding fragment of an anti-CD28 antibody) may be immobilized on a surface, e.g., the surface of beads. Such beads are commercially available, e.g., ThermoFisher's Dynabeads™. Alternatively, the TCR costimulatory activator, e.g., an anti-CD28 antibody or a CD28-binding fragment of an anti-CD28 antibody, may be present in solution.

[0172] In certain embodiments, the TCR / CD3 activator (e.g., an anti-CD3 antibody or a CD3-binding fragment of an anti-CD3 antibody) and the TCR costimulatory activator (e.g., an anti-CD28 antibody or a CD28-binding fragment of an anti-CD28 antibody) may be immobilized on the same surface, e.g., the same bead.

[0173] In certain embodiments, the TCR / CD3 activator and / or TCR costimulatory activator is not removed before step (c). In certain embodiments, after contacting the Tregs with the TCR / CD3 activator and / or TCR costimulatory activator, at least a portion of the TCR / CD3 activator and / or TCR costimulatory activator remains in contact with the Tregs throughout the remainder of the culture method (i.e., throughout step (c) and any additional steps) until the Tregs are harvested. In certain embodiments, the remaining portion of the TCR / CD3 activator and / or TCR costimulatory activator may be removed when the Tregs are harvested. If the TCR / CD3 activator and / or TCR costimulatory activator is present on the surface of beads, the beads may be separated from the remainder of the culture material. For example, if the beads are magnetic, they may be separated using magnetic force. If the TCR / CD3 activator and / or TCR costimulatory activator is an antibody (which may be present on the surface of the beads), the antibody may be internalized within the cells.

[0174] As used herein, the period defined by the reference to "activation" refers to the time point at which Tregs are first activated ex vivo, e.g., the time point at which Tregs are first contacted with a TCR / CD3 activator and / or a TCR costimulatory activator (the start of step (b)).

[0175] The initial addition of materials necessary to carry out step (b) (e.g., TCR / CD3 activators and / or TCR costimulatory activators) may be carried out at ambient temperature (e.g., about 18°C ​​to about 26°C) and in an ambient atmosphere (e.g., about 78% nitrogen, about 21% oxygen, and about 1% other gases). The Treg population may then be exposed to a particular temperature and atmosphere (e.g., the temperature, CO2, and / or O2 conditions described above for culturing Tregs), for example, by storing it in an incubator capable of maintaining the desired temperature and atmosphere. In particular, when step (b) is carried out simultaneously with step (c), step (b) may be carried out at the temperature and atmosphere desired for step (c) after the initial addition of materials necessary to carry out step (b) to the Treg population.

[0176] Process (c) The in vitro method described herein comprises culturing the Tregs obtained in step (b) (in vitro activated Tregs) in the presence of an mTOR inhibitor.

[0177] As used herein, the terms "culturing" and "cell culture" refer to in vitro methods for maintaining at least a portion of cells, including expanding cells (especially Tregs), particularly increasing the total number of cells (especially Tregs) compared to the starting material (cell expansion).

[0178] In certain embodiments, the terms "culturing" and "cell culture" may be used synonymously with "growing" and "cell growth."

[0179] Step (c) may be performed in the presence of a medium suitable for Treg culture. Thus, the mTOR inhibitor may be present in the medium suitable for Treg culture. Examples of media suitable for Treg culture include XVIVO™ medium or TexMACS™ medium. The medium may be supplemented with, for example, serum, such as human serum, particularly human AB serum. The medium used in step (c) may be the same as the medium used in step (a) and / or step (b). In certain embodiments, Tregs are not washed between step (b) and step (c), and / or the medium used in step (b) is not replaced and / or added before step (c). Thus, the medium used in step (b) may remain in contact with Tregs during step (c), and the mTOR inhibitor may be added to the medium or may remain in the medium from step (a). In certain embodiments, Tregs are not washed between step (a) and step (c), and / or the culture medium used in step (a) is not replaced and / or added before step (c). Thus, the culture medium used in step (a) can remain in contact with Tregs throughout step (b) and step (c) (and, if necessary, throughout any additional steps described herein). Thus, the mTOR inhibitor used in step (a) (e.g., present in the culture medium) can also be used in step (c) (e.g., present in the culture medium). In certain embodiments, no additional mTOR inhibitor is added to the culture medium used in step (c). Alternatively, in certain embodiments, a single addition of an mTOR inhibitor (e.g., added in step (a)) is sufficient in the in vitro methods described herein.

[0180] The step of culturing Tregs in the presence of an mTOR inhibitor (step (c)) may be performed simultaneously with or immediately after the step of activating Tregs (step (b)). In particular, if the TCR / CD3 activator and / or TCR costimulatory activator used in step (b) is not removed but is present throughout the entire period of step (c) (and, optionally, throughout the entire period of any additional steps described herein), the step of culturing Tregs in the presence of an mTOR inhibitor (step (c)) may be performed simultaneously with the step of activating Tregs (step (b)). That is, steps (b) and (c) may be initiated simultaneously. If the step of culturing Tregs in the presence of an mTOR inhibitor (step (c)) is performed immediately after the step of activating Tregs (step (b)), step (c) may be performed immediately after the removal of the TCR / CD3 activator and / or TCR costimulatory activator used in step (b), in which case there are no other steps of the method between steps (b) and (c).

[0181] In step (c), Tregs may be cultured in the presence of an mTOR inhibitor for any period of time that results in a product of the method of the invention that has one or more of the following characteristics compared to the product of a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor: a reduced proportion of contaminating cells; Decrease in the percentage of CD8+ T cells Decrease in the percentage of CD4+CD25- T cells Increased percentage of CD4+CD25+ T cells Increased proportion of FOXP3-expressing cells Increased proportion of cells with demethylated TSDR Increase in the proportion of Helios-expressing cells Increased percentage of CD27-expressing cells Increased proliferation rate Increased cell count Reduced production of pro-inflammatory cytokines (e.g., IL-17) Enhanced inhibitory ability Increased expression of the transgene (if the method further comprises the step of introducing heterologous nucleic acid into Tregs) A "corresponding method in which step (c) is performed in the absence of an mTOR inhibitor" is a method identical to the comparative method, except that it is performed using the same starting materials as the comparative method and step (c) is performed in the absence of an mTOR inhibitor. Thus, the "corresponding method" may include an alternative step (c) in which the activated Treg obtained in step (b) is cultured using the same materials (except for the mTOR inhibitor) and for the same period of time as those used in the comparative method. Therefore, if any period of step (c) brings about an improvement in the product, that period is included in the present invention.

[0182] In particular, in step (c), Tregs may be cultured in the presence of an mTOR inhibitor for any period of time that results in a product of the method of the invention having one or more of the following characteristics compared to the product of a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor: Decrease in the percentage of CD8+ T cells Increased proportion of FOXP3-expressing cells Increase in the proportion of Helios-expressing cells Increased percentage of CD27-expressing cells Increased proliferation rate Increased cell count Reduced production of pro-inflammatory cytokines (e.g., IL-17) Enhanced inhibitory ability Increased expression of the transgene (if the method further comprises the step of introducing heterologous nucleic acid into Tregs)

[0183] In particular, in step (c), Tregs may be cultured in the presence of an mTOR inhibitor for any period of time that results in a product of the method of the invention having one or more of the following characteristics compared to the product of a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor: Decrease in the percentage of CD8+ T cells Increased proportion of FOXP3-expressing cells Increase in the proportion of Helios-expressing cells Increased percentage of CD27-expressing cells Increased proliferation rate Increased cell count

[0184] A comparative method that does not include step (c) is a method that uses the same starting materials and includes all of the same steps as the present method except for step (c).

[0185] The percentage of CD8+ T cells, CD4+CD25- T cells, CD4+CD25+ T cells, FOXP3-expressing cells and / or Helios-expressing cells and / or CD27-expressing cells in the product, the proliferation rate, the number of cells in the product, the amount of immunosuppressive cytokines produced, the suppressive ability of the product, and the expression of the transgene can be determined as described above for step (a).

[0186] In step (c), Tregs may be cultured in the presence of an mTOR inhibitor for at least about 6 hours. For example, Tregs may be cultured in the presence of an mTOR inhibitor in step (c) for at least about 8 hours, or at least about 10 hours, or at least about 12 hours, or at least about 14 hours, or at least about 16 hours, or at least about 18 hours, or at least about 20 hours, or at least about 22 hours, or at least about 24 hours. In certain embodiments, Tregs may be cultured in the presence of an mTOR inhibitor in step (c) for at least about 26 hours, or at least about 28 hours, or at least about 30 hours, or at least about 32 hours, or at least about 34 hours, or at least about 36 hours, or at least about 38 hours, or at least about 40 hours, or at least about 42 hours, or at least about 44 hours, or at least about 46 hours, or at least about 48 hours.

[0187] The Tregs may be cultured in the presence of an mTOR inhibitor in step (c) for about 6 days or less. For example, the Tregs may be cultured in the presence of an mTOR inhibitor in step (c) for about 120 hours or less, or about 96 hours or less, or about 84 hours or less, or about 72 hours or less, or about 60 hours or less, or about 48 hours or less. For example, Tregs may be cultured in the presence of an mTOR inhibitor in step (c) for about 6 hours to about 6 days, or about 12 hours to about 72 hours, or about 12 hours to about 60 hours, or about 24 hours to about 72 hours, or about 24 hours to about 60 hours, or about 36 hours to about 60 hours. For example, Tregs may be cultured in the presence of an mTOR inhibitor in step (c) for about 24 hours or about 48 hours.

[0188] Further method steps After step (c), the in vitro methods described herein may further comprise the step of reducing the concentration of the mTOR inhibitor (e.g., by at least 50%, 60%, 70%, 80%, or 90% compared to the concentration of the mTOR inhibitor at the start of step (c), or by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the concentration of the mTOR inhibitor at the end of step (c)) or removing the mTOR inhibitor, and then further culturing (and in particular expanding) the Tregs. In particular, the in vitro methods described herein may further comprise the step of removing the mTOR inhibitor used in step (c) after step (c), and then further culturing (and in particular expanding) the Tregs in the absence of the mTOR inhibitor.

[0189] The further culturing step (in the presence of an mTOR inhibitor at a reduced concentration compared to the initial concentration applied in step (c) or in the absence of an mTOR inhibitor) may be carried out in the presence of a medium suitable for Treg culture. Examples of media suitable for Treg culture include XVIVO™ medium or TexMACS™ medium. The medium may be supplemented with, for example, serum, such as human serum, particularly human AB serum. The medium used in this further culturing step may be the same as the medium used in step (a) and / or step (b) and / or step (c). In certain embodiments, Tregs are not washed between step (c) and this further culturing step, and / or the medium used in step (c) is not replaced before this further culturing step (e.g., the concentration of the mTOR inhibitor may be reduced by dilution or by adding additional medium or other reagents to the medium). Thus, the medium used in step (c) may remain in contact with Tregs during this further culturing step. Alternatively, the medium used in step (c) can be completely or partially removed from Tregs, Tregs can be washed as needed, and new medium (which may not contain mTOR inhibitors) can be used for this further culture step (for example, the medium used in step (c) can be partially or completely replaced with new medium). In particular, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the medium present in step (c) can be replaced with new medium. If the medium used in step (c) is completely or partially removed before this further culture step, no mTOR inhibitor can be added to the new medium, so that this further culture step is carried out in the absence of mTOR inhibitor or in the presence of a reduced concentration of mTOR inhibitor. Alternatively, a reduced concentration of mTOR inhibitor (for example, the same or different mTOR inhibitor used in step (a) and / or step (c)) can be added to the new medium compared to the initial concentration of the mTOR inhibitor used in step (c).

[0190] The dilution or removal of the mTOR inhibitor may be performed immediately after step (c). That is, there are no other steps of the method between step (c) and this dilution or removal of the mTOR inhibitor, including a "rest" step in which the Tregs are left in the container without further action. The mTOR inhibitor may be diluted or removed immediately after the time of step (c) has elapsed.

[0191] The additional culturing (or expansion) step may be performed immediately after the mTOR inhibitor is diluted or removed. That is, there are no other steps of the method between the dilution or removal step and this additional culturing step, including a "resting" step in which Tregs are left in the container without further treatment. Immediately after the mTOR inhibitor is diluted or removed, Tregs may be further cultured or expanded in the presence of a reduced concentration of the mTOR inhibitor or in the absence of the mTOR inhibitor.

[0192] When the concentration of the mTOR inhibitor used in step (c) is reduced, the concentration of the mTOR inhibitor may be reduced compared to the initial concentration of the mTOR inhibitor used in step (c).In particular, the concentration of the mTOR inhibitor may be further reduced in addition to the natural reduction of the initial concentration of the mTOR inhibitor used in step (c) (for example, the reduction that may occur due to degradation as described above).That is, the concentration of the mTOR inhibitor used in step (c) is reduced as a result of intervention by non-natural or external factors.In other words, the step of reducing the concentration of the mTOR inhibitor or removing the mTOR inhibitor may be an active step.For example, the concentration may be reduced or the mTOR inhibitor may be removed by quantitatively adding (for example, quantitatively adding medium) to the Treg population in step (c), or by replacing at least a portion of the medium, or by washing the Treg population.In particular, the step of reducing the concentration of the mTOR inhibitor or removing the mTOR inhibitor is not a passive step of the degradation of the mTOR inhibitor or the consumption of the mTOR inhibitor by the cell population. Thus, as described herein, even if degradation or consumption of the mTOR inhibitor occurs, the process of reducing the concentration of or removing the mTOR inhibitor is not specifically achieved by degradation or consumption.

[0193] For example, the concentration of the mTOR inhibitor used in step (c) can be reduced to about 25nM or less.That is, the concentration of the mTOR inhibitor after or as a result of the active step of "reducing the concentration of mTOR inhibitor" as described above is about 25nM or less.For example, the concentration can be reduced to about 20nM or less, or about 15nM or less, or about 10nM or less, or about 5nM or less, or about 3nM or less, or about 2nM or less, or about 1nM or less (the concentration of the mTOR inhibitor after or as a result of the active step of "reducing the concentration of mTOR inhibitor" is about 20nM or less, or about 15nM or less, or about 10nM or less, or about 5nM or less, or about 3nM or less, or about 2nM or less, or about 1nM or less). In particular, when a single addition or treatment of mTOR inhibitor is carried out in the method of the present invention in step (a), the reduction in the concentration of the mTOR inhibitor after step (c) may be compared to the initial concentration applied in step (a), taking into account natural degradation.In particular, the concentration may be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.It should be understood that the concentration may be further reduced during additional culture or expansion steps after reducing the concentration of the mTOR inhibitor, for example, due to further dilution and / or degradation of mTOR.The active step of "reducing the concentration of the mTOR inhibitor" may be the result of adding materials required for introducing heterologous nucleic acid into Treg, as described below.

[0194] The concentration of the mTOR inhibitor used in step (c) may be reduced to about 0.01 nM or more (after or as a result of the active step of "reducing the concentration of the mTOR inhibitor" as described above). That is, the concentration of the mTOR inhibitor after the active step of "reducing the concentration of the mTOR inhibitor" is about 0.01 nM or more. For example, the concentration may be reduced to about 0.05 nM or more, or about 0.1 nM or more, or about 0.5 nM or more (after or as a result of the active step of "reducing the concentration of the mTOR inhibitor" the concentration of the mTOR inhibitor is about 0.05 nM or more, or about 0.1 nM or more, or about 0.5 nM or more). The step of "reducing the concentration of the mTOR inhibitor" may be the result of adding materials necessary for introducing heterologous nucleic acid into Treg, as described below.

[0195] For example, the concentration of the mTOR inhibitor may be reduced (after or as a result of the active step of "reducing the concentration of the mTOR inhibitor" as described above) to a concentration of about 0.01 nM to about 25 nM, or about 0.1 nM to about 20 nM, or about 0.5 nM to about 15 nM, or about 0.5 nM to about 10 nM. That is, the concentration of the mTOR inhibitor after or as a result of the active step of "reducing the concentration of the mTOR inhibitor" is about 0.01 nM to about 25 nM, or about 0.1 nM to about 20 nM, or about 0.5 nM to about 15 nM, or about 0.5 nM to about 10 nM. The step of "reducing the concentration of the mTOR inhibitor" may also be the result of the addition of materials necessary to introduce heterologous nucleic acid into Tregs, as described below. The resulting concentration of the mTOR inhibitor after the active reduction step can be determined relative to the concentration of the mTOR inhibitor used in step (c) (or step (a) if the mTOR inhibitor was added once), without considering natural degradation of the mTOR inhibitor or uptake of the mTOR inhibitor by cells.

[0196] The concentration of the mTOR inhibitor used in step (c) may be reduced or diluted by at least 1 / 5 (i.e., ×1 / 5) (after or as a result of the active step of "reducing the concentration of the mTOR inhibitor" as described above). For example, the concentration of the mTOR inhibitor used in step (c) may be reduced or diluted by at least 1 / 10, or at least 1 / 15, or at least 1 / 20. For example, the concentration of the mTOR inhibitor used in step (c) may be reduced or diluted by about 1 / 60, or about 1 / 50, or about 1 / 40. For example, the concentration of the mTOR inhibitor used in step (c) may be reduced or diluted by about 1 / 5 to about 1 / 60, or about 1 / 15 to about 1 / 50, or about 1 / 20 to about 1 / 40. The concentration reduction, i.e., the dilution factor, can be determined based on the concentration of the mTOR inhibitor used in step (c) (or step (a) if the mTOR inhibitor is added once), without taking into account natural degradation of the mTOR inhibitor or uptake of the mTOR inhibitor by cells.

[0197] As used herein, "removal" of mTOR inhibitor means removing mTOR inhibitor so that the concentration of remaining mTOR inhibitor is less than 0.01nM.When removing mTOR inhibitor, the method can include washing Treg population, and optionally centrifuging cells, for example, resuspending cells in new Treg medium.In particular, removal of mTOR can be complete removal.

[0198] At least about 6 hours after the start of step (c), the concentration of mTOR inhibitor may be reduced or the mTOR inhibitor may be removed.For example, at least about 8 hours, or at least about 10 hours, or at least about 12 hours, or at least about 14 hours, or at least about 16 hours, or at least about 18 hours, or at least about 20 hours, or at least about 22 hours, or at least about 24 hours after the start of step (c), the concentration of mTOR inhibitor may be reduced or the mTOR inhibitor may be removed.In certain embodiments, at least about 26 hours, or at least about 28 hours, or at least about 30 hours, or at least about 32 hours, or at least about 34 hours, or at least about 36 hours, or at least about 38 hours, or at least about 40 hours, or at least about 42 hours, or at least about 44 hours, or at least about 46 hours, or at least about 48 hours, or at least about 50 hours, 52 hours, or 54 hours after the start of step (c), the concentration of mTOR inhibitor may be reduced or the mTOR inhibitor may be removed.

[0199] The concentration of the mTOR inhibitor may be reduced or removed within about 6 days from the start of step (c). For example, the concentration of the mTOR inhibitor may be reduced or removed within about 120 hours, or about 96 hours, or about 84 hours, or about 72 hours, or about 60 hours, or about 48 hours from the start of step (c). For example, the concentration of the mTOR inhibitor may be reduced or removed about 6 hours to about 6 days, or about 12 hours to about 72 hours, or about 12 hours to about 60 hours, or about 24 hours to about 72 hours, or about 24 hours to about 60 hours, or about 36 hours to about 60 hours after the start of step (c).

[0200] This further culturing or expansion step (in the presence of reduced concentrations of an mTOR inhibitor or in the absence of an mTOR inhibitor) may be carried out for a period of time sufficient to achieve a desired cell number and / or a desired proliferation rate and / or a desired level of purity (e.g., percentage of Tregs and / or percentage of contaminating cells), which may be as described herein for the products of the above methods.

[0201] The duration of this further culturing or expansion step may depend, for example, on the length of time that step (c) was carried out. In particular, if the cells are cultured in the presence of an mTOR inhibitor for an extended period of time in step (c), it may take longer to recover the cells (and therefore this further culturing step may be longer) compared to if the cells are cultured in the presence of an mTOR inhibitor for a shorter period of time in step (c).

[0202] This further culturing or expanding step (in the presence of a reduced concentration of the mTOR inhibitor or in the absence of the mTOR inhibitor) may be carried out for at least about 6 days after the concentration of the mTOR inhibitor is reduced or the mTOR inhibitor is removed. For example, this further culturing or expanding step may be carried out for at least about 7 days, or at least about 8 days, or at least about 9 days, or at least about 10 days, or at least about 11 days, or at least about 12 days after the concentration of the mTOR inhibitor is reduced or the mTOR inhibitor is removed.

[0203] This further culturing or growing step (in the presence of a reduced concentration of an mTOR inhibitor or in the absence of an mTOR inhibitor) may be carried out for about 36 days or less, for example, about 30 days or less, or about 25 days or less, after the concentration of the mTOR inhibitor is reduced or the mTOR inhibitor is removed. This further culturing or growing step (in the presence of a reduced concentration of an mTOR inhibitor or in the absence of an mTOR inhibitor) may be carried out for about 20 days or less, after the concentration of the mTOR inhibitor is reduced or the mTOR inhibitor is removed. For example, this further culturing or growing step may be carried out for about 19 days or less, or about 18 days or less, or about 17 days or less, or about 16 days or less, or about 15 days or less, or about 14 days or less, after the concentration of the mTOR inhibitor is reduced or the mTOR inhibitor is removed.

[0204] This further culturing or expansion step (in the presence of a reduced concentration of an mTOR inhibitor or in the absence of an mTOR inhibitor) may be carried out for about 6 days to about 36 days, or about 6 days to about 20 days, or about 8 days to about 20 days, or about 8 days to about 16 days, or about 8 days to about 14 days, or about 10 days to about 14 days after reducing the concentration of the mTOR inhibitor or removing the mTOR inhibitor.

[0205] The in vitro method described herein may further comprise the step of introducing heterologous nucleic acid into one or more Tregs in the population.For example, the step of introducing heterologous nucleic acid into Tregs may be carried out after step (c).For example, the step of introducing heterologous nucleic acid into Tregs may be carried out simultaneously with the step of reducing the concentration of mTOR inhibitor.In particular, the step of introducing heterologous nucleic acid into Tregs may be carried out after the concentration of mTOR inhibitor is reduced or after the mTOR inhibitor is removed, and therefore may be carried out in the presence of reduced concentration of mTOR inhibitor or in the absence of mTOR inhibitor.

[0206] Although it may be desirable to introduce the heterologous nucleic acid into as many Tregs as possible, the heterologous nucleic acid need not be introduced into all Tregs in the population, and it is not a requirement of this method or this step of the method. The heterologous nucleic acid may be introduced into other (non-Treg) cells in the population, but this is not a requirement of the method. In certain embodiments, the heterologous nucleic acid is introduced into at least about 10% of the cells in the population (particularly Tregs) (e.g., resulting in a transduction efficiency of at least about 10%). For example, the heterologous nucleic acid may be introduced into at least about 15%, or at least about 20%, or at least about 25%, or at least about 30% of the cells in the population (particularly Tregs). For example, the heterologous nucleic acid may be introduced into up to about 100%, or up to about 95%, or up to about 90% of the cells in the population (particularly Tregs). In certain embodiments, the heterologous nucleic acid is introduced into at least about 10% of the Tregs (e.g., resulting in a transduction efficiency of at least about 10%). For example, the heterologous nucleic acid may be introduced into at least about 15%, or at least about 20%, or at least about 25%, or at least about 30% of Tregs. For example, the heterologous nucleic acid may be introduced into up to about 100%, or up to about 95%, or up to about 90% of Tregs. After introducing the heterologous nucleic acid into Tregs, a selectable marker encoded by the heterologous nucleic acid may be used to separate transduced Tregs from untransduced Tregs.

[0207] In certain embodiments, before or during the introduction of heterologous nucleic acid into Tregs, the concentration of the mTOR inhibitor used in step (c) is reduced or removed. For example, when the materials required for introducing heterologous nucleic acid into Tregs are added to the culture medium used in step (c), the mTOR inhibitor used in step (c) may be diluted as a result. The mTOR inhibitor used in step (c) may be diluted to a concentration of about 25 nM or less as described above. The mTOR inhibitor used in step (c) may be diluted to a concentration of at least about 1 / 2, 1 / 3, or 1 / 4. For example, the mTOR inhibitor used in step (c) may be diluted to a concentration of at least about 1 / 5, at least about 1 / 6, at least about 1 / 7, at least about 1 / 8, at least about 1 / 9, or at least about 1 / 10. Therefore, the step of introducing heterologous nucleic acid into Tregs may be performed under conditions in which the concentration of the mTOR inhibitor is reduced and / or may result in a reduction in its concentration.

[0208] Alternatively, the medium used in step (c) (and consequently the mTOR inhibitor used in step (c)) may be removed from Tregs, the Tregs may be washed as needed, and then the materials required for introducing heterologous nucleic acid into Tregs (which may be present in new medium such as XVIVO™ medium or TexMACS™ medium) may be contacted with Tregs. The materials used to introduce heterologous nucleic acid into Tregs may not contain mTOR inhibitors. Thus, the step of introducing heterologous nucleic acid into Tregs may be carried out in the absence of mTOR inhibitors.

[0209] The heterologous nucleic acid may be introduced into the Tregs at least about 6 hours after the initiation of step (b) and / or the initiation of step (c). For example, the heterologous nucleic acid may be introduced into the Tregs at least about 8 hours, or at least about 10 hours, or at least about 12 hours, or at least about 14 hours, or at least about 16 hours, or at least about 18 hours, or at least about 20 hours, or at least about 22 hours, or at least about 24 hours after the initiation of step (b) and / or the initiation of step (c). In certain embodiments, the heterologous nucleic acid may be introduced into the Tregs at least about 26 hours, or at least about 28 hours, or at least about 30 hours, or at least about 32 hours, or at least about 34 hours, or at least about 36 hours, or at least about 38 hours, or at least about 40 hours, or at least about 42 hours, or at least about 44 hours, or at least about 46 hours, or at least about 48 hours, or at least about 50 hours, or at least about 52 hours, or at least about 54 hours after the initiation of step (b) and / or the initiation of step (c).

[0210] The heterologous nucleic acid may be introduced into the Tregs within about 6 days of the initiation of step (b) and / or the initiation of step (c). For example, the heterologous nucleic acid may be introduced into the Tregs within about 120 hours, or within about 96 hours, or within about 84 hours, or within about 72 hours, or within about 60 hours, or within about 48 hours of the initiation of step (b) and / or the initiation of step (c).

[0211] For example, the heterologous nucleic acid may be introduced into Tregs about 6 hours to about 6 days, or about 12 hours to about 72 hours, or about 12 hours to about 60 hours, or about 24 hours to about 72 hours, or about 24 hours to about 60 hours, or about 36 hours to about 60 hours after the initiation of step (b) and / or step (c). For example, the heterologous nucleic acid may be introduced into Tregs about 24 hours or about 48 hours after the initiation of step (b) and / or step (c).

[0212] The in vitro method may further comprise the additional step of culturing or expanding Tregs after introducing the heterologous nucleic acid into Tregs, which may be an additional step of culturing or expanding Tregs in the presence of reduced concentrations of an mTOR inhibitor or in the absence of an mTOR inhibitor, as described above.

[0213] After introducing heterologous nucleic acid into Treg, the further step of culturing or expanding Treg can be carried out in the presence of a medium suitable for Treg culture.The example of the medium suitable for Treg culture is known as XVIVO™ medium or TexMACS™ medium.The medium can be supplemented with, for example, serum, for example, human serum, particularly human AB serum.

[0214] The additional step of culturing or expanding Tregs after introducing heterologous nucleic acid into Tregs may be carried out for at least about 6 days after introducing heterologous nucleic acid into Tregs. For example, this additional step of culturing or expanding may be carried out for at least about 7 days, or at least about 8 days, or at least about 9 days, or at least about 10 days, or at least about 11 days, or at least about 12 days after introducing heterologous nucleic acid into Tregs. "After introducing heterologous nucleic acid into Tregs" refers to the period after the start of the step of introducing heterologous nucleic acid into Tregs. That is, "at least 6 days after introducing heterologous nucleic acid into Tregs" refers to at least 6 days from the time when the material used to introduce heterologous nucleic acid into Tregs first comes into contact with Tregs.

[0215] The further step of culturing or expanding Tregs after introducing the heterologous nucleic acid into Tregs may be performed for about 36 days or less, for example, about 30 days or less, or about 25 days or less, after introducing the heterologous nucleic acid into Tregs. The further step of culturing or expanding Tregs after introducing the heterologous nucleic acid into Tregs may be performed for about 20 days or less, after introducing the heterologous nucleic acid into Tregs. For example, this further culturing or expanding step may be performed for about 19 days or less, or about 18 days or less, or about 17 days or less, or about 16 days or less, or about 15 days or less, or about 14 days or less, after introducing the heterologous nucleic acid into Tregs.

[0216] The further step of culturing or expanding Tregs after introducing the heterologous nucleic acid into Tregs may be carried out for about 6 days to about 36 days, or about 6 days to about 20 days, or about 8 days to about 20 days, or about 8 days to about 16 days, or about 8 days to about 14 days, or about 10 days to about 14 days after introducing the heterologous nucleic acid into Tregs.

[0217] As used herein, the term "introduced" refers to a method for inserting foreign nucleic acid, e.g., DNA or RNA, into a cell. As used herein, the term "introduced" includes both transduction and transfection methods. Transfection is the process of introducing nucleic acid into a cell by non-viral methods. Transduction is the process of introducing foreign DNA or RNA into a cell via a viral vector. Engineered cells can be generated by introducing the nucleic acids described herein using one of many means, including transduction with a viral vector or transfection with DNA or RNA. "Engineered cells" refer to cells that have been modified to contain or express a polynucleotide not naturally encoded by the cell. Methods for engineering cells are known in the art and include, for example, but are not limited to, genetic modification of cells using transduction methods such as retroviral or lentiviral transduction, transfection methods (e.g., DNA- or RNA-based transient transfection), including lipofection, polyethylene glycol, calcium phosphate, and electroporation, as described herein. Any suitable method can be used to introduce the nucleic acid sequence into the cell. Non-viral techniques, such as amphipathic cell membrane penetrating peptides, may also be used to introduce the nucleic acid.

[0218] In particular, the in vitro methods described herein may further comprise the step of transducing Tregs with a viral vector comprising a heterologous nucleic acid.

[0219] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. As used herein, and by way of example, some vectors used in recombinant nucleic acid technology allow an entity such as a nucleic acid segment (e.g., a heterologous DNA segment, such as a heterologous cDNA segment) to be transferred into a target cell. A vector may be a non-viral vector or a viral vector. Examples of vectors used in recombinant nucleic acid technology include, but are not limited to, plasmids, mRNA molecules (e.g., in vitro transcribed mRNA), chromosomes, artificial chromosomes, and viruses. A vector may be, for example, a naked nucleic acid (e.g., DNA). In its simplest form, the vector itself may be the nucleotide of interest.

[0220] A vector as used herein may be, for example, a plasmid, mRNA, or viral vector, and may include a promoter (as described herein) for expression of a nucleic acid molecule / polynucleotide, and optionally a regulator of said promoter.

[0221] In certain embodiments, the vector is a viral vector, such as a retroviral vector, and for example, a lentiviral or gammaretroviral vector.

[0222] The vector may further comprise an additional promoter; for example, in one embodiment, the promoter may be an LTR, such as a retroviral LTR or lentiviral LTR. Long terminal repeats (LTRs) are identical DNA sequences repeated hundreds or thousands of times at both ends of retrotransposons or proviral DNA formed by reverse transcription of retroviral RNA. LTRs are used by viruses to insert genetic material into the host genome. LTRs contain gene expression signals, such as enhancers, promoters (which may have both transcription enhancers and regulatory elements), transcription initiation (such as capping), transcription terminators, and polyadenylation signals. Preferably, the vector may comprise a 5'LTR and a 3'LTR.

[0223] A vector may contain one or more additional regulatory sequences that can act before or after transcription. A "regulatory sequence" is any sequence that promotes the expression of a polypeptide, for example, acts to increase the expression of a transcript or enhance mRNA stability. Suitable regulatory sequences include, for example, enhancer elements, post-transcriptional regulatory elements, and polyadenylation sites. Preferably, the additional regulatory sequence can be present in LTR(s). Suitably, the vector may include, for example, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) operably linked to the promoter.

[0224] A vector containing a heterologous nucleic acid can be introduced into a cell using various techniques known in the art, such as transformation and transduction. Some techniques are known in the art, including infection with a recombinant viral vector, such as a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a baculoviral vector, and a herpes simplex viral vector, direct injection of a nucleic acid, and transformation by biolistic methods.

[0225] Non-viral delivery system includes, but is not limited to, DNA transfection method.Here, transfection includes the process of using non-viral vector to deliver gene to target cell.Non-viral delivery system can include liposome-based cell membrane-permeable peptide or amphipathic cell membrane-permeable peptide, preferably complexed with nucleic acid molecule or construct.

[0226] Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic drug-mediated transfection, cationic facial amphiphiles (CFAs) (Nat. Biotechnol. (1996) 14: 556), and combinations thereof.

[0227] The heterologous nucleic acid molecule may be designed to be used as a single construct, and it is contemplated that it will be contained in a single vector, although it is not excluded that such a single construct may be introduced into a cell in combination with other vectors, for example, vectors encoding other polypeptides that are also desired to be introduced into the cell.

[0228] Heterologous nucleic acids that may be introduced into Tregs in the methods described herein may comprise sequences encoding a chimeric antigen receptor (CAR) or a TCR, and / or sequences encoding a FOXP3 polypeptide, and / or sequences encoding a safety switch, and / or sequences encoding a polypeptide that increases cell persistence.

[0229] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an engineered receptor that can confer antigen specificity to cells (e.g., Tregs). CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. CARs typically comprise an extracellular domain containing an antigen-specific targeting region, referred to herein as an antigen-binding domain, a transmembrane domain, an intracellular domain optionally containing one or more costimulatory domains, and an intracellular signaling domain. The antigen-binding domain is typically connected to the transmembrane domain by a hinge domain. The design of CARs and the various domains that CARs may contain are well known in the art.

[0230] When a CAR binds to its target antigen, it transmits an activation signal to the cell in which the CAR is expressed, thereby directing the specificity of the engineered cell to the target antigen, particularly the cell expressing the target antigen.

[0231] The antigen-binding domain of a CAR may be derived from or obtained from any protein or polypeptide that binds to (i.e., has affinity for) a desired target antigen, or more generally, a desired target molecule. It may be, for example, a ligand or receptor, or a physiological binding protein or portion thereof for the target molecule, or a synthetic or derivative protein. The target molecule may generally, but need not necessarily, be expressed on the surface of a cell, for example, the target cell or a cell in the vicinity of the target cell (due to bystander effect). Depending on the nature and specificity of the antigen-binding domain, the CAR may recognize a soluble molecule, for example, if the antigen-binding domain is based on or derived from a cellular receptor.

[0232] Antigen binding domains are most commonly derived from antibody variable chains (e.g., commonly in the form of an scFv), but may also be generated from other molecules such as T-cell receptor variable domains or receptors for ligands or other binding molecules, as described above.

[0233] CAR is typically expressed as a polypeptide that also includes a signal sequence (also known as a leader sequence), particularly a signal sequence that directs CAR to the plasma membrane of a cell. This is generally located next to or near the antigen-binding domain, generally upstream of the antigen-binding domain. Thus, the extracellular domain, or ectodomain, of CAR may include a signal sequence and an antigen-binding domain.

[0234] The antigen-binding domain confers on the CAR the ability to bind to a predetermined antigen of interest, preferably targeting an antigen of clinical interest or an antigen at the site of disease.

[0235] As described above, an antigen-binding domain may be any protein or peptide capable of specifically recognizing and binding to a biomolecule (e.g., a cell surface receptor or its component). Antigen-binding domains include any natural, synthetic, semi-synthetic, or recombinantly produced binding partner for the biomolecule of interest. Exemplary antigen-specific targeting domains include antibodies or antibody fragments or derivatives, extracellular domains of receptors, ligands for cell surface molecules / cell surface receptors or their receptor-binding domains, and tumor-binding proteins. As described below, the antigen-specific targeting domain may preferably be an antibody or antibody-derived, but other antigen-specific targeting domains are also encompassed, such as an antigen-specific targeting domain formed from a combination of an antigen-specific peptide and an MHC or HLA that can bind to the TCR of active Tcon cells at the site of transplantation, inflammation, or disease.

[0236] The CAR can be directed to any desired target antigen or target molecule. The antigen or molecule can be selected depending on the intended therapy and the condition to be treated. For example, it can be an antigen or molecule associated with a particular condition, or an antigen or molecule associated with a cell that is desired to be targeted to treat the condition. Typically, the antigen or molecule is a cell surface antigen or cell surface molecule.

[0237] The term "directed against" is synonymous with "specific for" or "anti." In other words, the CAR recognizes a target molecule. This means that the CAR can specifically bind to a designated or given antigen, i.e., target. In particular, the antigen-binding domain of the CAR can specifically bind to a target molecule or target antigen (more particularly, when the CAR is expressed on the surface of a cell, particularly an immune effector cell). Specific binding can be distinguished from nonspecific binding to a non-target molecule or non-target antigen. Thus, cells expressing the CAR are directed or redirected to specifically bind to target cells expressing the target molecule or target antigen, particularly target cells expressing the target antigen or target molecule on their cell surface.

[0238] Antigens that can be targeted by the present CAR include, but are not limited to, antigens expressed on cells associated with transplanted organs, autoimmune diseases, allergic diseases, and inflammatory diseases (e.g., neurodegenerative diseases). It will be understood by those skilled in the art that when the cells engineered to express a CAR are Treg cells or their precursors, the antigen may simply be present and / or expressed at the site of transplant, inflammation, or disease due to the bystander effect of Treg cells.

[0239] Antigens expressed on cells associated with neurodegenerative diseases include those presented on glial cells, such as MOG.

[0240] Antigens associated with organ transplantation and / or cells associated with the transplanted organ include, but are not limited to, HLA antigens present in the transplanted organ but absent from the patient, or antigens whose expression is increased during transplant rejection, such as CCL19, MMP9, SLC1A3, MMP7, HMMR, TOP2A, GPNMB, PLA2G7, CXCL9, FABP5, GBP2, CD74, CXCL10, UBD, CD27, CD48, and CXCL11. In one embodiment, the CAR is directed against an HLA antigen, particularly the HLA-A2 antigen.

[0241] Antibodies against such antigens are known in the art, and scFv can be conveniently obtained or generated based on known or available antibodies.In this regard, the VH sequence and VL sequence and CDR sequence that support the preparation of such antibody binding domains are published, for example, in WO2020 / 044055.The disclosure of this document is incorporated herein by reference.Can use any of the antigen binding domains or CDR sequences, VH sequences, and / or VL sequences disclosed in WO2020 / 044055 or WO2020 / 201230. For example, a CAR may be used to target HLA-A2 (herein, HLA-A2 is referred to as HLA-A * 02, HLA-A02, and HLA-A * The antigen-binding domain may contain an antigen-binding domain capable of binding to HLA-A (also referred to as HLA-A 2). * 02 is one particular group of class I major histocompatibility complex (MHC) alleles at the HLA-A locus.

[0242] The antigen recognition domain may bind, preferably specifically bind, to one or more regions or epitopes within HLA-A2. An epitope, also known as an antigenic determinant, is a part of an antigen that is recognized by an antigen recognition domain (e.g., an antibody). In other words, an epitope is a specific part of an antigen to which an antibody binds. Preferably, the antigen recognition domain binds, preferably specifically binds, to one region or epitope within HLA-A2.

[0243] The heterologous nucleic acid can include, for example, a sequence encoding a safety switch polypeptide that provides a suicide moiety to cells in which the safety switch polypeptide is expressed. This is a useful safety mechanism that allows cells administered to a subject to be eliminated if the need arises, indeed more generally as desired or needed, e.g., once the cells have exerted or completed their therapeutic effect.

[0244] A suicide moiety has the ability to induce cell death, or more generally, cell loss or elimination. One example of a suicide moiety is a suicide protein encoded by a suicide gene, which can be expressed in or on a cell together with a desired transgene, and upon expression, turns off the expression of the transgene, allowing for cell elimination. A suicide moiety in this context refers to a suicide polypeptide, which is a polypeptide that can cause cells to be eliminated under permissive conditions, i.e., under induced or turned-on conditions.

[0245] The suicide moiety can be a polypeptide or amino acid sequence that can be activated to exert cell-eliminating activity by an activating agent administered to a subject, or that can be activated to exert cell-eliminating activity in the presence of a substrate that can be administered to a subject. In certain embodiments, the suicide moiety can be targeted by a separate cell-eliminating agent administered to a subject. The cell-eliminating agent can target the cells to be eliminated by binding to the suicide moiety. In particular, the suicide moiety can be recognized by an antibody, and when the safety switch polypeptide is expressed on the surface of a cell, binding of the antibody to the safety switch polypeptide will cause the cell to disappear or be eliminated.

[0246] The suicide moiety may be HSV-TK or iCasp9, as known in the art. However, in another example, the suicide moiety may be or contain an epitope recognized by a cell-eliminating antibody or other binding molecule capable of inducing cell elimination. The term "delete," as used herein in the context of cell elimination, is synonymous with "remove," "ablate," or "eliminate." This term is used to encompass cell death or inhibition of cell proliferation, which may reduce the number of cells in a subject. Complete 100% elimination may be desirable, but need not necessarily be achieved. Reducing the number of cells or inhibiting cell proliferation in a subject may be sufficient to achieve a beneficial effect.

[0247] In particular, the suicide moiety may be a CD20 epitope recognized by the antibody rituximab. Thus, in the safety switch polypeptide, the suicide moiety may comprise a minimal epitope based on the CD20-derived epitope recognized by the antibody rituximab. More particularly, the polypeptide may comprise two CD20 epitopes R1 and R2 separated by a linker L.

[0248] Safety switch polypeptides based on the rituximab epitope are described in International Publication Nos. WO 2013 / 15339 and WO 2021 / 239812, the contents of which are incorporated herein by reference. Peptides mimicking the epitope recognized by rituximab (so-called mimitopes) have been developed and used as suicide moieties in suicide-marker polypeptide constructs that further contain a CD34 minimal epitope as a marker site in WO 2013 / 15339. Specifically, WO 2013 / 15339 discloses a polypeptide called RQR8. This polypeptide contains two CD20 minimal epitopes separated from each other by a spacer sequence and a CD34 marker sequence interposed between them, and the two CD20 minimal epitopes are further linked to a stalk sequence that allows the polypeptide to protrude from the surface of the cell in which it is expressed. The safety switch polypeptide can be RQR8 or a variant thereof having at least about 80% sequence identity to the polypeptide, e.g., at least about 85%, 88%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. Other safety switch polypeptides that can be used as the basis for a safety switch domain include those described in commonly-owned, co-pending PCT Patent Application No. PCT / EP2021 / 064053 (WO 2021 / 239812), the contents of which are incorporated herein by reference.

[0249] FOXP3 is an abbreviation for forkhead box P3 protein. Expression of heterologous FOXP3 can increase FOXP3 expression in cells and help maintain the suppressive phenotype of Treg cells or cells with a regulatory phenotype. FOXP3 is a member of the FOX protein family of transcription factors and functions as a master regulator of the regulatory pathway in the development and function of regulatory T cells (Tregs). As used herein, "FOXP3" encompasses FOXP3 variants, isoforms, and functional fragments. A "FOXP3 polypeptide" is a polypeptide with FOXP3 activity, i.e., a polypeptide that can bind to FOXP3 target DNA and function as a transcription factor that regulates the development and function of Tregs.

[0250] By "increasing FOXP3 expression" is meant increasing the level of FOXP3 mRNA and / or FOXP3 protein in a cell (or population of cells) compared to a corresponding cell (or population of cells) that has not been modified by the introduction of a nucleic acid molecule or vector. For example, the level of FOXP3 mRNA and / or FOXP3 protein in a cell (or population of such cells) that has been modified according to the present invention may be at least 1.5-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, or at least 150-fold higher than the level in a corresponding cell (or population of such cells) that has not been modified according to the present invention.

[0251] Suitably, the level of FOXP3 mRNA and / or FOXP3 protein in the modified cells (or population of such cells) may be at least 1.5-fold, 2-fold, or 5-fold higher than in the corresponding cells (or population of such cells) that have not been so modified.

[0252] The technology for measuring the level of specific mRNA and protein is well known in the art.The level of mRNA in a population of cells such as Treg can be measured by techniques such as Affymetrix eBioscience PrimeFlow RNA assay, Northern blotting, serial analysis of gene expression (SAGE) or quantitative polymerase chain reaction (qPCR).The level of protein in a population of cells can be measured by techniques such as flow cytometry, high performance liquid chromatography (HPLC), liquid chromatography / mass spectrometry (LC / MS), Western blotting or enzyme-linked immunosorbent assay (ELISA).

[0253] "FOXP3 polypeptide" refers to a polypeptide with FOXP3 activity, i.e., a polypeptide that can bind to FOXP3 target DNA and function as a transcription factor that regulates the development and function of Treg.In particular, FOXP3 polypeptide may have the same or similar activity as wild-type FOXP3, for example, at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, or 150% of the activity of wild-type FOXP3 polypeptide.Therefore, the FOXP3 polypeptide encoded by the nucleotide sequence in the nucleic acid or vector described herein may have increased or decreased activity compared to wild-type FOXP3.Techniques for measuring transcription factor activity are well known in the art.For example, transcription factor DNA binding activity can be measured by ChIP.The transcriptional regulatory activity of transcription factor can be measured by quantifying the expression level of the gene it regulates. Gene expression may be quantified by measuring the level of mRNA and / or protein produced by the gene using techniques such as Northern blotting, SAGE, qPCR, HPLC, LC / MS, Western blotting, or ELISA. Genes regulated by FOXP3 include cytokines such as IL-2, IL-4, and IFN-γ (Siegler et al. Annu. Rev. Immunol. 2006, 24: 209-26, incorporated herein by reference). As described in detail below, FOXP3 or FOXP3 polypeptide includes its functional fragments, variants, and isoforms.

[0254] A "functional fragment of FOXP3" may refer to a portion or region of a FOXP3 polypeptide, or a portion or region of a polynucleotide (i.e., a nucleotide sequence) encoding a FOXP3 polypeptide, that has the same or similar activity as a full-length FOXP3 polypeptide or polynucleotide. A functional fragment may have at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the activity of a full-length FOXP3 polypeptide or polynucleotide. A person skilled in the art would be able to generate functional fragments based on the known structural and functional characteristics of FOXP3. These are described, for example, in Song, X., et al., 2012. Cell reports, 1(6), pp.665-675; Lopes, JE, et al., 2006. The Journal of Immunology, 177(5), pp.3133-3142; and Lozano, T., et al., 2013. Frontiers in oncology, 3, p.294. Additionally, N-terminally and C-terminally truncated FOXP3 fragments are described in WO 2019 / 241549 (incorporated herein by reference).

[0255] A "FOXP3 variant" may comprise an amino acid or nucleotide sequence that is at least 50%, at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical, and preferably at least 95%, at least 97%, or at least 99% identical, to a FOXP3 polypeptide or a polynucleotide encoding a FOXP3 polypeptide (e.g., wild-type FOXP3). A FOXP3 variant may have the same or similar activity as a wild-type FOXP3 polypeptide or polynucleotide, for example, at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, or 150% of the activity of a wild-type FOXP3 polypeptide or polynucleotide. Those skilled in the art can generate FOXP3 mutants based on the known structural and functional characteristics of FOXP3 and / or by using conservative substitutions.The FOXP3 mutant may have a similar or identical turnover time (or degradation rate) in Treg cells compared to wild-type FOXP3, for example, at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the turnover time (or degradation rate) of wild-type FOXP3 in Tregs.Some FOXP3 mutants may have a reduced turnover time (or degradation rate) compared to wild-type FOXP3, for example, FOXP3 mutants with amino acid substitutions at amino acid positions 418 and / or 422 of wild-type FOXP3, such as S418E and / or S422A, as described in WO2019 / 241549 (incorporated herein by reference). These represent the aa418 mutant, the aa422 mutant, and the aa418 and aa422 mutants, respectively.

[0256] Suitably, the FOXP3 polypeptide encoded by the nucleic acid molecules or vectors described herein may comprise or consist of the polypeptide sequence of human wild-type FOXP3, such as UniProtKB accession Q9BZS1, or a functional fragment or variant thereof.

[0257] In some embodiments of the present invention, the FOXP3 polypeptide comprises or consists of an amino acid sequence or a functional fragment thereof that is at least 70% identical to human wild-type FOXP3. Preferably, the FOXP3 polypeptide comprises or consists of an amino acid sequence or a functional fragment thereof that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to human wild-type FOXP3. In some embodiments, the FOXP3 polypeptide comprises or consists of human wild-type FOXP3 or a functional fragment thereof.

[0258] In some embodiments, as described above, the FOXP3 polypeptide may include a mutation at residue 418 and / or residue 422 of human wild-type FOXP3.

[0259] In some embodiments of the present invention, a FOXP3 polypeptide may be truncated at the N-terminus and / or C-terminus, thereby resulting in a functional fragment.

[0260] Preferably, the FOXP3 polypeptide may be a mutant of human wild-type FOXP3, such as a naturally occurring mutant. Preferably, the FOXP3 polypeptide is an isoform of human wild-type FOXP3. For example, the FOXP3 polypeptide may contain a deletion of amino acids 72 to 106 relative to human wild-type FOXP3. Alternatively, the FOXP3 polypeptide may contain a deletion of amino acids 246 to 272 relative to human wild-type FOXP3.

[0261] Those skilled in the art will understand that FOXP3 expression in Tregs can be indirectly increased by introducing into cells a polynucleotide encoding a protein that increases FOXP3 transcription and / or translation, or that increases the half-life of FOXP3 (e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%), or that increases FOXP3 function (e.g., as determined by the suppressive ability of transduced Tregs, measured as described above). For example, a polynucleotide that increases the transcription of endogenous FOXP3 by interacting with a non-coding sequence (CNS, e.g., CNS1, CNS2, or CNS3) present upstream of the endogenous FOXP3 promoter or coding region may be introduced into Tregs.

[0262] The heterologous nucleic acid may include a sequence encoding a polypeptide that increases the persistence of cells, for example, to provide a productive IL signal without the need for the addition of exogenous IL. Such IL signals may be constitutive or inducible. Exemplary techniques may include the use of engineered or chimeric receptors that can transmit IL signals without the need for the addition of exogenous IL. For example, inducible engineered receptors such as those described in WO 2018 / 111834, WO 2019 / 169290, and WO 2020 / 264039, or constitutively engineered receptors such as those described in WO 2018 / 038954, WO 2019 / 102207, WO 2019 / 053420, WO 2020 / 180694, and WO 2017 / 218850, or chimeric receptors such as those described in WO 2020 / 183131. No. WO 2017 / 029512, WO 2012 / 138858, WO 2014 / 172584, WO 2017 / 068360, WO 2021 / 023987, WO 2020 / 180664, and WO 2020 / 044239, or the use of engineered receptors with tethered activation molecules such as those described in WO 2017 / 201432 and WO 2019 / 183389.

[0263] The heterologous nucleic acid may further comprise one or more other coding sequences that may encode a protein of interest, e.g., a therapeutic protein. The one or more additional nucleotide sequences may encode, for example, a transcription factor, growth factor, or other factor that may help enhance cell function or survival. The one or more additional nucleotide sequences may encode, for example, an additional receptor, particularly an antigen receptor, such as a heterologous T cell receptor (TCR) or its derivative (e.g., a TCR-CAR construct, or a single-chain TCR construct).

[0264] The heterologous nucleic acid may further encode a selectable marker. Suitable selectable markers are well known in the art and include, but are not limited to, fluorescent proteins such as GFP. Preferably, the selectable marker is a fluorescent protein, such as GFP, YFP, RFP, tdTomato, dsRed, or a variant thereof. In some embodiments, the fluorescent protein is GFP or a GFP variant. Preferably, the selectable marker / reporter domain may be a luciferase-based reporter, a PET reporter (e.g., sodium-iodide symporter (NIS)), or a membrane protein (e.g., CD34, low-affinity nerve growth factor (LNGFR)).

[0265] The use of a selectable marker is advantageous because it allows cells into which the nucleic acid has been successfully introduced (e.g., Tregs) to be selected and isolated from the starting cell population using common methods, such as flow cytometry.

[0266] Preferably, the heterologous nucleic acid may be codon-optimized. Preferably, the heterologous nucleic acid may be codon-optimized for expression in human cells. Codon optimization has been previously described in International Publication Nos. 1999 / 41397 and 2001 / 79518. Different cells use particular codons at different frequencies. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in a cell type. Expression can be increased by altering the codons in the sequence to match the relative abundance of the corresponding tRNA. Similarly, expression can be decreased by deliberately selecting codons whose corresponding tRNAs are known to be rare in that particular cell type. This allows for even greater translational control.

[0267] When two or more coding sequences are expressed from a single nucleic acid molecule, the two or more coding sequences may be linked by a sequence that allows them to be coexpressed. In particular, the coexpression sequence, or coexpression site, may allow the encoded proteins or polypeptides to be expressed as separate entities. For example, the nucleic acid may include a sequence encoding an internal promoter, an internal ribosome entry sequence (IRES) sequence, or a cleavage site.

[0268] In particular, the co-expression sequence may encode a self-cleaving sequence between the encoded polypeptides. In particular, the self-cleaving sequence may be a self-cleaving peptide. Such a sequence automatically cleaves during protein production. Self-cleaving peptides that can be used are 2A peptides or 2A-like peptides, which are known in the art and are described, for example, in Donnelly et al., Journal of General Virology, 2001, 82, 1027-1041 (incorporated herein by reference). 2A peptides and 2A-like peptides are thought to cause ribosome skipping, resulting in a cleavage form that skips the formation of a peptide bond between the end of the 2A peptide and the downstream amino acid sequence. "Cleavage" occurs between the glycine and proline residues at the C-terminus of the 2A peptide. In other words, several residues are added to the end of the upstream cistron, and the downstream cistron starts with proline.

[0269] Suitable self-cleavage domains include P2A, T2A, E2A, and F2A sequences. These sequences may be modified to include the amino acid GSG at the N-terminus of the 2A peptide. Such modified alternative 2A sequences are known and reported in the art. Alternative 2A-like sequences that can be used are shown in Donnelly et al. (supra), such as TaV sequences.

[0270] Each self-cleaving sequence contained in the nucleic acid may be the same or different.

[0271] The self-cleaving sequence may contain additional cleavage sites that can be cleaved by common enzymes present in cells, which may help achieve complete removal of the 2A sequence after translation. Such additional cleavage sites may include, for example, furin cleavage sites.

[0272] Heterologous nucleic acid molecules and polynucleotides / nucleic acid sequences defined herein may comprise DNA or RNA. They may be single-stranded or double-stranded. Those skilled in the art will understand that, as a result of the degeneracy of the genetic code, many different nucleic acid molecules / polynucleotides can encode the same polypeptide. In addition, those skilled in the art will understand that, using routine methods, nucleotide substitutions that do not affect the polypeptide sequence encoded by the nucleic acid molecules / polynucleotides / nucleotide sequences defined herein can be made to reflect the codon usage of any particular host organism in which the polypeptide of the present invention is expressed.

[0273] Heterologous nucleic acids can be modified using any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or lifespan of the nucleic acid molecules defined herein.

[0274] Nucleic acid molecules, such as DNA nucleic acid molecules, can be produced recombinantly, synthetically, or by any means available to those of skill in the art. They can also be cloned by standard techniques.

[0275] Longer nucleic acid molecules / polynucleotides / nucleotide sequences are generally produced using recombinant means, for example, polymerase chain reaction (PCR) cloning techniques. This involves creating a pair of primers (e.g., about 15-30 nucleotides) that flank the target sequence to be cloned, contacting the primers with mRNA or cDNA obtained from animal or human cells, performing a polymerase chain reaction under conditions that allow amplification of the desired region, isolating the amplified fragment (e.g., by purifying the reaction mixture on an agarose gel), and recovering the amplified DNA. The primers may be designed to contain appropriate restriction enzyme recognition sites so that the amplified DNA can be cloned into an appropriate vector.

[0276] A heterologous nucleic acid may contain one or more regulatory sequences, such as a promoter. A "promoter" is a region of DNA that directs the initiation of transcription of a gene. The promoter is located upstream of the DNA (toward the 5' region of the sense strand) near the transcription start site of the gene. Any suitable promoter can be used, and its selection can be easily made by one of ordinary skill in the art. The promoter may be from any source and may be a viral promoter or a eukaryotic promoter, including a mammalian or human promoter (i.e., a physiological promoter). In one embodiment, the promoter is a viral promoter. Specific promoters include the LTR promoter, EFS (or functional truncations thereof), SFFV, PGK, and CMV. In one embodiment, the promoter is an SFFV or viral LTR promoter. In particular, an SFFV promoter may be used to enable transcription initiation of a nucleotide sequence within a nucleic acid molecule. When there is more than one nucleotide sequence, e.g., a nucleotide sequence encoding a CAR, a nucleotide sequence encoding FOXP3, a nucleotide sequence encoding a safety switch, and / or a nucleotide sequence encoding a polypeptide that increases cell persistence, the sequences may be operably linked to the same promoter.

[0277] "Operably linked to the same promoter" means that transcription of each polynucleotide sequence can be initiated from the same promoter (e.g., transcription of a first, second, and third polynucleotide sequence is initiated from the same promoter) and that each nucleotide sequence is positioned and oriented so that transcription is initiated from that promoter. A polynucleotide operably linked to a promoter is under the transcriptional control of that promoter.

[0278] The in vitro methods described herein may further comprise a step of harvesting the population of Tregs. By "harvesting" is meant any step of isolating the population of Tregs that is the product of the in vitro methods described herein in a form suitable for ACT. This may include, for example, removing the culture medium from the population of Tregs and / or removing any TCR / CD3 activators and TCR costimulatory activators, and / or further steps of removing impurities in the product. The harvesting step may be the final step of the in vitro methods described herein. Alternatively, the harvesting step may be followed by cryopreservation (as described below), and these may be the final two steps of the in vitro methods described herein.

[0279] The Treg population may be collected about 8 to about 36 days after the initiation of step (b). For example, the Treg population may be collected about 8 to about 30 days, about 8 to about 25 days, or about 8 to about 22 days after the initiation of step (b). For example, the Treg population may be collected about 10 to about 18 days, or about 12 to about 16 days after the initiation of step (b). The Treg population may be cryopreserved after collection, for example.

[0280] The population of Tregs produced by the in vitro method described herein may be cryopreserved, for example, so that it can be preserved until ACT. "Cryopreserving" or "cryopreservation" refers to freezing a population of Tregs under conditions that allow cells (e.g., Tregs) to remain viable (e.g., during freezing and after thawing). Cell viability can be measured by any method known in the art, for example, by flow cytometry using a viability stain (e.g., LIVE / DEAD™ Fixable Near-IR Dead Cell Stain (Thermofisher)). Typically, at least 50%, 60%, 70%, 80%, 90%, or 95% of the cells (e.g., at least 50%, 60%, 70%, 80%, 90%, or 95% of the Tregs) will survive during and after cryopreservation. Thus, viability refers to surviving cells. Those skilled in the art will understand that cryopreservation can maintain cells in a viable state by applying one or more conditions (for example, a specific temperature, a freezing rate and / or a thawing rate, and / or the use of a cryopreservative) that effectively stop cell death and maintain the structure of the cell. Such conditions are known in the art. The cryopreservation step can be the final step of the in vitro method described herein.

[0281] In vitro methods for culturing or expanding Tregs may require contacting the Tregs with interleukin-2 (IL-2) at some stage in the culture or expansion process, which may or may not be present at the same time as an mTOR inhibitor.

[0282] In certain embodiments, in the in vitro method described herein, step (a) and / or step (b) and / or step (c) may be performed in the absence of IL-2. In certain embodiments, IL-2 may be added to the culture medium after step (c). For example, IL-2 may be added to the culture medium simultaneously with the introduction of heterologous nucleic acid into Tregs (i.e., simultaneously with the first contact of the materials necessary for introducing heterologous nucleic acid into cells with Tregs). For example, IL-2 may be added to the culture medium simultaneously with or after the reduction of the concentration of the mTOR inhibitor used in step (c) (as described above), or simultaneously with or after the removal of the mTOR inhibitor used in step (c). The step of reducing the concentration of the mTOR inhibitor or removing the mTOR inhibitor and the step of introducing heterologous nucleic acid into Tregs are as described above.

[0283] IL-2 may be contacted with the Tregs in the population (e.g., by adding to the culture medium) at least about 6 hours after the initiation of step (b) and / or the initiation of step (c). For example, IL-2 may be contacted with the Tregs in the population at least about 8 hours, or at least about 10 hours, or at least about 12 hours, or at least about 14 hours, or at least about 16 hours, or at least about 18 hours, or at least about 20 hours, or at least about 22 hours, or at least about 24 hours after the initiation of step (b) and / or the initiation of step (c). In certain embodiments, IL-2 may be contacted with the Tregs in the population at least about 26 hours, or at least about 28 hours, or at least about 30 hours, or at least about 32 hours, or at least about 34 hours, or at least about 36 hours, or at least about 38 hours, or at least about 40 hours, or at least about 42 hours, or at least about 44 hours, or at least about 46 hours, or at least about 48 hours, or at least about 50 hours, or at least about 52 hours, or at least about 54 hours after the initiation of step (b) and / or the initiation of step (c).

[0284] Alternatively, IL-2 may be contacted with the Tregs in the population at the start of step (a) and / or at the start of step (b) and / or at the start of step (c).

[0285] The IL-2 may be contacted with the Tregs within about 6 days of the initiation of step (b) and / or the initiation of step (c). For example, the IL-2 may be contacted with the Tregs within about 120 hours, or within about 96 hours, or within about 84 hours, or within about 72 hours, or within about 60 hours, or within about 48 hours of the initiation of step (b) and / or the initiation of step (c). For example, IL-2 may be contacted with Tregs about 6 hours to about 6 days, or about 12 hours to about 72 hours, or about 12 hours to about 60 hours, or about 24 hours to about 72 hours, or about 24 hours to about 60 hours, or about 36 hours to about 60 hours after the start of step (b) and / or the start of step (c).

[0286] IL-2 is approximately 10 IU / mL to approximately 1 x 10 6 For example, IL-2 can be used at a concentration of about 50 IU / mL to about 1 × 10 5 IU / mL, or approximately 100 IU / mL to approximately 1 x 10 4 IL-2 may be used at a concentration of about 150 IU / mL to about 5000 IU / mL, or about 200 IU / mL to about 2500 IU / mL, or about 250 IU / mL to about 1500 IU / mL. A typical source of IL-2 is Proleukin (Clinigen Healthcare). International Units (IU) of IL-2 are calculated based on International Standard 86-500 available from the National Institute for Biological Standards and Control (NIBSC).

[0287] IL-2 may be further replenished as needed at various times during the culture (or expansion) process, for example, additional IL-2 may be added at intervals of about 12 hours to about 72 hours, e.g., about 24 hours to about 72 hours, e.g., about 48 hours to about 72 hours. For example, additional IL-2 may be added to the medium every 2 to 3 days.

[0288] The in vitro methods described herein may include one or more additional activation steps (in addition to step (b)). Tregs may be "reactivated" by any of the activation methods described above with respect to step (b). For example, Tregs may be reactivated by contacting them with a TCR / CD3 activator and / or a TCR costimulatory activator as described above with respect to step (b). For example, additional TCR / CD3 activators and / or TCR costimulatory activators (in addition to those originally used in step (b)) may be contacted with Tregs, for example, by adding them to the culture medium. Some of the original TCR / CD3 activators and / or TCR costimulatory activators may remain in contact with Tregs. That is, it is not necessary to remove the TCR / CD3 activators and / or TCR costimulatory activators used in step (b) before any additional activation steps. When the TCR / CD3 activators and / or TCR costimulatory activators are antibodies (which may be present on the surface of beads), the antibodies may be internalized within the cells. Additional TCR / CD3 activators and / or TCR co-stimulators may be added during the "reactivation" step to ensure an appropriate ratio of antibody (or beads) to cells.

[0289] For example, Tregs may be reactivated 4 to 12 days after the initiation of step (b). For example, Tregs may be reactivated 6 to 10 days after the initiation of step (b). The above method may further include one or more additional reactivation steps after this reactivation step. An additional reactivation step may be performed 4 to 12 days after the previous reactivation step, for example, 6 to 10 days after the previous reactivation step. The number of reactivation steps required depends on the length of the culture period before recovering the cells. In certain embodiments, the above method does not include more than one reactivation step (for a total of two activation steps).

[0290] final product Further provided herein is a product obtained and / or obtainable by the in vitro method described herein (including any embodiment thereof). The product is a population of cells that includes Tregs. Thus, the product comprises a population of Tregs.

[0291] The product may be, for example, at least about 50×10 6 For example, the product may contain at least about 100 x 10 cells. 6 pieces, or at least about 150 x 10 6 pieces, or at least about 200 x 10 6 For example, the product may contain approximately 80 x 10 cells. 8 Up to 75 x 10 8 Up to 70 x 10 8 The cell may contain up to 100 cells. The product may be, for example, at least about 10 x 10 6 For example, the product may contain at least about 15 x 10 Tregs. 6 pieces, or at least about 20 x 10 6 pieces, or at least about 25 x 10 6 pieces, or at least about 30 x 10 6 pieces, or at least about 35 x 10 6 pieces, or at least about 40 x 10 6 pieces, or at least about 45 x 10 6 pieces, or at least about 50 x 10 6 pieces, or at least about 55 x 10 6 pieces, or at least about 60 x 10 6 pieces, or at least about 75 x 10 6 pieces, or at least about 80 x 10 6 pieces, or at least about 85 x 10 6 pieces, or at least about 90 x 10 6 pieces, or at least about 95 x 10 6 pieces, or at least about 100 x 10 6 pieces, or at least about 150 x 10 6 pieces, or at least about 200 x 10 6 pieces, or at least about 250 x 106 pieces, or at least about 300 x 10 6 pieces, or at least about 350 x 10 6 pieces, or at least about 400 x 10 6 pieces, or at least about 450 x 10 6 pieces, or at least about 500 x 10 6 For example, the product may contain approximately 80 x 10 Tregs. 8 Up to 75 x 10 8 Up to 70 x 10 8 It may contain up to 10 Tregs.

[0292] When heterologous nucleic acid is introduced into Tregs, the product may be, for example, at least about 10 x 10 T cells containing the heterologous nucleic acid. 6 For example, the product may contain at least about 15 x 10 Tregs comprising heterologous nucleic acid. 6 pieces, or at least about 20 x 10 6 pieces, or at least about 25 x 10 6 pieces, or at least about 30 x 10 6 pieces, or at least about 35 x 10 6 pieces, or at least about 40 x 10 6 pieces, or at least about 45 x 10 6 pieces, or at least about 50 x 10 6 pieces, or at least about 55 x 10 6 pieces, or at least about 60 x 10 6 pieces, or at least about 75 x 10 6 pieces, or at least about 80 x 10 6 pieces, or at least about 85 x 10 6 pieces, or at least about 90 x 10 6 pieces, or at least about 95 x 10 6 pieces, or at least about 100 x 10 6 For example, the product may contain approximately 800 x 10 Tregs containing heterologous nucleic acid. 6 Up to 750 x 10 6 Up to 700 x 10 6 It may contain up to 10 Tregs.

[0293] When a heterologous nucleic acid encoding a CAR is introduced into Tregs, the product may be, for example, at least about 10 x 10 6 For example, the product may contain at least about 15 x 10 CAR-Tregs. 6 pieces, or at least about 20 x 10 6 pieces, or at least about 25 x 10 6 pieces, or at least about 30 x 10 6 pieces, or at least about 35 x 10 6 pieces, or at least about 40 x 10 6 pieces, or at least about 45 x 10 6 pieces, or at least about 50 x 10 6 pieces, or at least about 55 x 10 6 pieces, or at least about 60 x 10 6 pieces, or at least about 75 x 10 6 pieces, or at least about 80 x 10 6 pieces, or at least about 85 x 10 6 pieces, or at least about 90 x 10 6 pieces, or at least about 95 x 10 6 pieces, or at least about 100 x 10 6 For example, the product may contain approximately 800 x 10 CAR-Tregs. 6 Up to 750 x 10 6 Up to 700 x 10 6 It may contain up to CAR-Tregs.

[0294] The product may contain, for example, at least about 75% Tregs (in other words, at least about 75% of the cells in the product are Tregs). For example, the product may contain at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% Tregs. For example, the product may contain about 99% or less, or about 98% or less, or about 97% or less, or about 96% or less, or about 95% or less Tregs. For example, the product may contain about 75% to about 99%, or about 75% to about 95%, or about 80% to about 95% Tregs.

[0295] The product may contain, for example, at least about 75% FOXP3-expressing cells (in other words, at least about 75% of the cells in the product express FOXP3). For example, the product may contain at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% FOXP3-expressing cells. For example, the product may contain about 99% or less, or about 98% or less, or about 97% or less, or about 96% or less, or about 95% or less FOXP3-expressing cells. For example, the product may contain about 75% to about 99%, or about 75% to about 95%, or about 80% to about 95% FOXP3-expressing cells.

[0296] The product may contain, for example, at least about 75% FOXP3+ Tregs (in other words, about 75% or more of the cells in the product are FOXP3+ Tregs). For example, the product may contain at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% FOXP3+ Tregs. For example, the product may contain about 99% or less, or about 98% or less, or about 97% or less, or about 96% or less, or about 95% or less FOXP3+ Tregs. For example, the product may contain about 75% to about 99%, or about 75% to about 95%, or about 80% to about 95% FOXP3+ Tregs. Tregs may have any Treg phenotype as described herein, e.g., CD4+CD25+FOXP3+.

[0297] The product may contain, for example, at least about 2% CD45RA+ Tregs (e.g., CD4+CD25+CD45RA+ or CD4+CD25+CD127- / lowCD45RA+ Tregs) (in other words, about 2% or more of the cells in the product are CD45RA+ Tregs). For example, the product may contain at least about 3%, or at least about 4%, or at least about 5%, or at least about 6%, or at least about 7%, or at least about 8%, or at least about 9%, or at least about 10% CD45RA+ Tregs (e.g., CD4+CD25+CD45RA+ or CD4+CD25+CD127- / lowCD45RA+ Tregs). For example, the product may contain about 50% or less, or about 45% or less, or about 40% or less, or about 35% or less, or about 30% or less, or about 25% or less CD45RA+ Tregs (e.g., CD4+CD25+CD45RA+ or CD4+CD25+CD127- / lowCD45RA+ Tregs). For example, the product may contain about 2% to about 50%, or about 3% to about 40%, or about 4% to about 30%, or about 5% to about 20% CD45RA+ Tregs (e.g., CD4+CD25+CD45RA+ or CD4+CD25+CD127- / lowCD45RA+ Tregs). Tregs may have any Treg phenotype as described herein, e.g., CD4+CD25+FOXP3+.

[0298] The product may contain, for example, at least about 60% Helios+ Tregs (in other words, at least about 60% of the cells in the product are Helios+ Tregs). For example, the product may contain at least about 65%, or at least about 70%, or at least about 75%, or at least about 80% Helios+ Tregs. For example, the product may contain about 99% or less, or about 98% or less, or about 97% or less, or about 96% or less, or about 95% or less Helios+ Tregs. For example, the product may contain about 60% to about 99%, or about 65% to about 95%, or about 70% to about 95% Helios+ Tregs. Tregs may have any Treg phenotype as described herein, e.g., CD4+CD25+FOXP3+.

[0299] The product may contain, for example, at least about 50% CD27+ Tregs (in other words, at least about 50% of the cells in the product express CD27). For example, the product may contain at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80% CD27+ Tregs. For example, the product may contain about 99% or less, or about 98% or less, or about 97% or less, or about 96% or less, or about 95% or less CD27+ Tregs. For example, the product may contain about 50% to about 99%, or about 55% to about 95%, or about 60% to about 95% CD27+ Tregs. Tregs may have any Treg phenotype as described herein, e.g., CD4+CD25+FOXP3+.

[0300] The product may contain, for example, at least about 40% demethylated FOXP3-TSDR. For example, the product may contain at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70% demethylated FOXP3-TSDR. For example, the product may contain about 99% or less, or about 98% or less, or about 97% or less, or about 96% or less, or about 95% or less demethylated FOXP3-TSDR. For example, the product may contain about 40% to about 99%, or about 50% to about 95%, or about 60% to about 95% demethylated FOXP3-TSDR.

[0301] The product may contain, for example, about 5% or less CD8+ cells (in other words, about 5% or less of the cells in the product are CD8+ cells). For example, the product may contain about 4% or less, or about 3% or less, or about 2% or less, or about 1% or less CD8+ cells. For example, the product may contain about 0.1% to about 5%, or about 0.5% to about 4% CD8+ cells.

[0302] The product may contain, for example, about 5% or less CD4+CD25- cells (in other words, about 5% or less of the cells in the product are CD4+CD25- cells). For example, the product may contain about 4% or less, or about 3% or less, or about 2% or less, or about 1% or less CD4+CD25- cells. For example, the product may contain about 0.1% to about 5%, or about 0.5% to about 4% CD4+CD25- cells.

[0303] Further provided herein is a pharmaceutical composition comprising a product obtained and / or obtainable by the in vitro methods described herein (including any embodiment thereof).

[0304] A pharmaceutical composition is a composition comprising or consisting of a therapeutically effective amount of a pharmaceutically active agent, i.e., cells (e.g., Tregs). Pharmaceutical compositions preferably comprise a pharmaceutically acceptable carrier, diluent, or excipient (including combinations thereof). Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro, ed., 1985). The choice of pharmaceutical carrier, excipient, or diluent can be determined taking into account the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions may comprise any suitable binder, lubricant, suspending agent, coating agent, or solubilizing agent as, or in addition to, the carrier, excipient, or diluent.

[0305] "Pharmaceutically acceptable" means that the formulation is sterile and pyrogen-free. The carriers, diluents, and / or excipients must be "acceptable" in the sense of being compatible with the cells or vectors and not deleterious to the recipient thereof. Typically, the carriers, diluents, and excipients are physiological saline or infusion media that are sterile and pyrogen-free, although other acceptable carriers, diluents, and excipients may be used.

[0306] Examples of pharmaceutically acceptable carriers include, for example, water, saline, alcohol, silicone, wax, petrolatum, vegetable oils, polyethylene glycol, propylene glycol, liposomes, sugar, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, flavor oils, fatty acid monoglycerides, fatty acid diglycerides, petroethral fatty acid esters, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0307] use The in vitro methods described herein can be used to reduce the proportion or amount of one or more contaminating cells (e.g., CD8+ T cells and / or CD4+CD25- T cells) in a population of Tregs, and / or to inhibit the proliferation of CD8+ T cells, and / or to inhibit the proliferation of CD4+CD25- T cells.

[0308] Thus, further provided herein are methods for reducing the proportion or amount of one or more contaminating cells (e.g., CD8+ T cells and / or CD4+CD25- T cells) in a population of Tregs, and / or for inhibiting the proliferation of CD8+ T cells, and / or for inhibiting the proliferation of CD4+CD25- T cells. The methods include steps (a), (b), and (c) of the in vitro methods described herein. The methods may further include any of the other steps described herein and thus may be compatible with any embodiment of the in vitro methods described herein.

[0309] The percentage or amount of one or more contaminating cells is reduced in the final product of the above method compared with the starting material (the Treg population used in step (a)) and / or compared with the product of a corresponding method that does not perform step (a) and / or that performs step (c) in the absence of an mTOR inhibitor.The "corresponding method" is as described above.The percentage or amount of contaminating cells in the Treg population can be determined by FACS.

[0310] For example, the proportion or amount of one or more contaminating cells, particularly CD8+ T cells, may be reduced by at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, compared to the product of a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor, or compared to the starting material used in step (a). For example, the proportion or amount of one or more contaminating cells, particularly CD8+ T cells, may be reduced by up to about 99%, or up to about 95%, or up to about 90%, or up to about 85%, or up to about 80%, compared to the product of a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor, or compared to the starting material used in step (a). For example, the proportion or amount of one or more contaminating cells, particularly CD8+ T cells, may be reduced by about 50% to about 99%, or about 60% to about 95%, or about 65% to about 90%, compared to the product of a corresponding method that does not perform step (a) and / or a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor, or compared to the starting material used in step (a).

[0311] For example, the proportion of one or more contaminating cells, particularly CD8+ T cells, may be reduced by at least about 0.1 percentage points, or at least about 0.2 percentage points, or at least about 0.3 percentage points, or at least about 0.4 percentage points, or at least about 0.5 percentage points, or at least about 1 percentage point, or at least about 2 percentage points, or at least about 3 percentage points, or at least about 4 percentage points, or at least about 5 percentage points, or at least about 6 percentage points, or at least about 7 percentage points, or at least about 8 percentage points, or at least about 9 percentage points, or at least about 10 percentage points relative to the starting material used in step (a). For example, the proportion of one or more contaminating cells, particularly CD8+ T cells, may be reduced by up to about 20 percentage points, or up to about 15 percentage points relative to the starting material used in step (a).

[0312] Inhibition of the proliferation of CD8+ T cells and / or CD4+CD25- T cells means that CD8+ T cells and / or CD4+CD25- T cells may not proliferate, or proliferation may be reduced so that the total number or proportion of CD8+ T cells and / or CD4+CD25- T cells in the final product of the above method is not increased compared to the starting material (the Treg population used in step (a)) and / or compared to the product of a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor. The proportion and / or amount of CD8+ T cells and / or CD4+CD25- T cells in the final product may also be reduced as described above. The "corresponding method" is as described above. The total number and / or proportion of specific cells in the Treg population can be determined using an automated cell counter and FACS.

[0313] The in vitro methods described herein can be used to increase the percentage or amount of Tregs, and / or to increase the percentage or amount of FOXP3-expressing cells, and / or to increase the percentage or amount of cells with demethylated Treg-specific demethylated regions (TSDRs) in a population of Tregs, and / or to increase the percentage or amount of FOXP3+ Tregs, and / or to increase the percentage or amount of Helios+ Tregs, and / or to increase the percentage or amount of CD27+ Tregs.

[0314] Therefore, the present specification further provides a method for increasing the proportion or amount of Tregs, and / or increasing the proportion of FOXP3-expressing cells, and / or increasing the proportion or amount of cells with demethylated Treg-specific demethylation regions (TSDRs) in a Treg population, and / or increasing the proportion or amount of FOXP3+ Tregs, and / or increasing the proportion or amount of Helios+ Tregs, and / or increasing the proportion or amount of CD27+ Tregs.The method includes steps (a), (b), and (c) of the in vitro method described herein.The method may further include any of the other steps described herein, and thus may be compatible with any embodiment of the in vitro method described herein.

[0315] An increase in the proportion or amount of Tregs, and / or FOXP3-expressing cells, and / or cells with demethylated TSDRs, and / or FOXP3+ Tregs, and / or Helios+ Tregs, and / or CD27+ Tregs means an increase in the proportion or amount of Tregs, and / or FOXP3-expressing cells, and / or cells with demethylated TSDRs, and / or FOXP3+ Tregs, and / or Helios+ Tregs, and / or CD27+ Tregs, respectively, in the end product of the above method compared to the starting material (the population of Tregs used in step (a)), and / or compared to the product of a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor. The "corresponding method" is as defined above. The percentage or amount of Tregs, and / or FOXP3-expressing cells, and / or FOXP3+ Tregs, and / or Helios+ Tregs, and / or CD27+ Tregs, and the percentage or amount of cells with demethylated TSDRs in the Treg population can be determined by FACS or MS-qPCR as described above.

[0316] For example, the percentage or amount of CD4+CD25+FOXP3+ T cells may be increased by at least about 3%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, compared to the product of a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor, or normalized to the starting material used in step (a). For example, the percentage or amount of CD4+CD25+FOXP3+ T cells may be increased by up to about 150%, or up to about 100%, or up to about 90%, or up to about 80%, compared to the product of a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor, or normalized to the starting material used in step (a). For example, the percentage or amount of CD4+CD25+FOXP3+ T cells may be increased by about 3% to about 150%, or about 5% to about 100%, or about 10% to about 80%, compared to the product of a corresponding method without step (a) and / or a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor, or normalized to the starting material used in step (a).

[0317] For example, the percentage of Tregs can be increased by at least about 3 percentage points, or at least about 5 percentage points, or at least about 10 percentage points, or at least about 15 percentage points, or at least about 20 percentage points, or at least about 25 percentage points, or at least about 30 percentage points relative to the starting material used in step (a). For example, the percentage of Tregs can be increased by about 75 percentage points, or about 70 percentage points, or about 65 percentage points, or about 60 percentage points, or about 55 percentage points, or about 50 percentage points relative to the starting material used in step (a). Tregs can have any Treg phenotype as described herein, for example, CD4+CD25+FOXP3+.

[0318] For example, the percentage or amount of Helios+ Tregs can be increased by at least about 3%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20% compared to the product of a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor, or normalized to the starting material used in step (a).For example, the percentage or amount of Helios+ Tregs can be increased by up to about 150%, or up to about 100%, or up to about 90%, or up to about 80% compared to the product of a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor, or normalized to the starting material used in step (a). For example, the percentage or amount of Helios+ Tregs may be increased by about 3% to about 150%, or about 5% to about 100%, or about 10% to about 80%, compared to the product of a corresponding method that does not perform step (a) and / or a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor, or normalized to the starting material used in step (a). Helios+ Tregs may have any Treg phenotype as described herein, e.g., CD4+CD25+FOXP3+.

[0319] For example, the percentage of Helios+ Tregs can be increased by at least about 3 percentage points, or at least about 5 percentage points, or at least about 10 percentage points, or at least about 15 percentage points, or at least about 20 percentage points, or at least about 25 percentage points, or at least about 30 percentage points relative to the starting material used in step (a). For example, the percentage of Helios+ Tregs can be increased by about 75 percentage points, or about 70 percentage points, or about 65 percentage points, or about 60 percentage points, or about 55 percentage points, or about 50 percentage points relative to the starting material used in step (a). Helios+ Tregs can have any Treg phenotype as described herein, for example, CD4+CD25+FOXP3+.

[0320] For example, the percentage or amount of CD27+ Tregs may be increased by at least about 3%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, compared to the product of a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor, or normalized to the starting material used in step (a). For example, the percentage or amount of CD27+ Tregs may be increased by up to about 150%, or up to about 100%, or up to about 90%, or up to about 80%, compared to the product of a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor, or normalized to the starting material used in step (a). For example, the percentage or amount of CD27+ Tregs may be increased by about 3% to about 150%, or about 5% to about 100%, or about 10% to about 80%, compared to the product of a corresponding method without step (a) and / or a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor, or normalized to the starting material used in step (a).CD27+ Tregs may have any Treg phenotype as described herein, e.g., CD4+CD25+FOXP3+.

[0321] For example, the percentage of CD27+ Tregs can be increased by at least about 3 percentage points, or at least about 5 percentage points, or at least about 10 percentage points, or at least about 15 percentage points, or at least about 20 percentage points, or at least about 25 percentage points, or at least about 30 percentage points relative to the starting material used in step (a). For example, the percentage of CD27+ Tregs can be increased by about 75 percentage points, or about 70 percentage points, or about 65 percentage points, or about 60 percentage points, or about 55 percentage points, or about 50 percentage points relative to the starting material used in step (a). CD27+ Tregs can have any Treg phenotype as described herein, for example, CD4+CD25+FOXP3+.

[0322] For example, the rate or amount of TSDR demethylation in the product may be increased by at least about 3%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, compared to the product of a corresponding method that does not perform step (a) and / or a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor, or normalized to the starting material used in step (a). For example, the rate or amount of TSDR demethylation may be increased by up to about 150%, or up to about 100%, or up to about 90%, or up to about 80%, compared to the product of a corresponding method that does not perform step (a) and / or a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor, or normalized to the starting material used in step (a). For example, the rate or amount of TSDR demethylation may be increased by about 3% to about 150%, or about 5% to about 100%, or about 10% to about 80%, compared to the product of a corresponding method that does not perform step (a) and / or a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor, or normalized to the starting material used in step (a).

[0323] For example, TSDR demethylation can be increased by at least about 3 percentage points, or at least about 5 percentage points, or at least about 10 percentage points, or at least about 15 percentage points, or at least about 20 percentage points, or at least about 25 percentage points, or at least about 30 percentage points relative to the starting material used in step (a). For example, TSDR demethylation can be increased by up to about 75 percentage points, or up to about 70 percentage points, or up to about 65 percentage points, or up to about 60 percentage points, or up to about 55 percentage points, or up to about 50 percentage points relative to the starting material used in step (a).

[0324] The in vitro methods described herein can be used to increase the percentage of CD45RA+ Tregs in a Treg population. Accordingly, a method for increasing the percentage of CD45RA+ Tregs in a Treg population is further provided herein. The method comprises steps (a), (b), and (c) of the in vitro methods described herein. The method may further include any of the other steps described herein and thus may be compatible with any embodiment of the in vitro methods described herein. An increase in the percentage of CD45RA+ Tregs means that the percentage of CD45RA+ Tregs in the final product of the method is increased compared to the product of a corresponding method that does not perform step (a) and / or a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor. The "corresponding method" is as described above. The percentage of CD45RA+ Tregs in a Treg population can be determined by FACS.

[0325] For example, the percentage of CD45RA Tregs may be increased by at least about 3%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, compared to the product of a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor. For example, the percentage of CD45RA Tregs may be increased by about 150%, or about 100%, or about 90%, or about 80%, compared to the product of a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor. For example, the percentage of CD45RA Tregs may be increased by about 3% to about 150%, or about 5% to about 100%, or about 10% to about 80%, compared to the product of a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor.

[0326] For example, the percentage of CD45RA+ Tregs can be increased by at least about 2 percentage points, or at least about 3 percentage points, or at least about 4 percentage points, or at least about 5 percentage points, or at least about 10 percentage points, or at least about 15 percentage points, compared to the product of a corresponding method that does not perform step (a) and / or a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor. For example, the percentage of Tregs can be increased by about 75 percentage points, or about 70 percentage points, or about 65 percentage points, or about 60 percentage points, or about 55 percentage points, or about 50 percentage points, or about 45 percentage points, or about 40 percentage points, compared to the product of a corresponding method that does not perform step (a) and / or a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor. Tregs can have any Treg phenotype as described herein, for example, CD4+CD25+FOXP3+ or CD4+CD25-CD127- / low.

[0327] The in vitro methods described herein can be used to increase the suppressive function of a population of Tregs.

[0328] Therefore, a method for increasing the suppressive function of a Treg population is further provided herein. The method comprises steps (a), (b), and (c) of the in vitro method described herein. The method may further comprise any of the other steps described herein, and thus may be compatible with any embodiment of the in vitro method described herein.

[0329] An increase in the suppressive function of the Treg population means that the suppressive function of the Treg population is increased compared to the starting material (the Treg population used in step (a)) and / or compared to the product of a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor. The "corresponding method" is as defined above. An increase in the suppressive function can mean, for example, an increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the suppressive function of the starting material (the Treg population used in step (a)) and / or compared to the product of a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor.

[0330] The suppressive function of a population of Tregs can be determined by any method described herein, for example, by measuring the suppression of Teff proliferation by Tregs (e.g., at a particular Teff:Treg ratio) and / or by measuring product cytokine expression.

[0331] For example, cytokine expression (e.g., IL-17 or IFNγ) may be reduced by at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70% compared to the cytokine expression in the product of a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor, or compared to the starting material used in step (a). For example, cytokine expression may be reduced by up to 99%, or up to 95%, or up to about 90% compared to the cytokine expression in the product of a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor, or compared to the starting material used in step (a).

[0332] For example, the inhibition of Teff proliferation may be reduced by at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, compared to the inhibition of Teff proliferation by the product of a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor, or compared to the starting material used in step (a). For example, the inhibition of Teff proliferation may be reduced by up to 99%, or up to 95%, or up to about 90%, compared to the inhibition of Teff proliferation by the product of a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor, or compared to the starting material used in step (a).

[0333] The in vitro methods described herein can be used to increase the expansion of a population of Tregs and / or to increase the expansion of Tregs within a population of Tregs.

[0334] Therefore, the present specification further provides a method for increasing the proliferation of a Treg population and / or a method for increasing the proliferation of Tregs in a Treg population.The method comprises steps (a), (b), and (c) of the in vitro method described herein.The method may further comprise any of the other steps described herein, and thus may be compatible with any embodiment of the in vitro method described herein.

[0335] Increasing the population of Tregs or the proliferation of Tregs in a population of Tregs means increasing the proliferation rate of the population of Tregs or Tregs in a population of Tregs, respectively, obtained by the methods of the present invention compared to the proliferation rate obtained using a corresponding method without step (a) and / or a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor. The "corresponding methods" and methods for measuring the proliferation rate are as described above.

[0336] For example, the population of Tregs or the expansion rate of Tregs in the population of Tregs, normalized to the expansion rate of a corresponding method without step (a) and / or a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor, may be increased by at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%. For example, the expansion rate, normalized to the expansion rate of a corresponding method without step (a) and / or a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor, may be increased by up to about 100%, or up to about 90%, or up to about 80%, or up to about 70%, or up to about 60%, or up to about 50%.

[0337] When the in vitro method includes a step of introducing a heterologous nucleic acid into a cell, the method can increase the expression of the heterologous nucleic acid, and / or increase the transduction efficiency of the heterologous nucleic acid, and / or decrease the vector copy number (VCN) of the heterologous nucleic acid. Thus, a method for increasing the expression of a heterologous nucleic acid, and / or increasing the transduction efficiency of the heterologous nucleic acid, and / or decreasing the VCN of the heterologous nucleic acid is further provided herein. The method includes steps (a), (b), and (c) of the in vitro method described herein. The method may further include any of the other steps described herein, and thus may be compatible with any embodiment of the in vitro method described herein.

[0338] Increasing expression of a heterologous nucleic acid means increasing expression of the heterologous nucleic acid in a population of Tregs compared to the product of a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor. "Corresponding methods" and methods for measuring expression of the heterologous nucleic acid (particularly one or more transgenes encoded by the heterologous nucleic acid) are as described above.

[0339] For example, the expression of the heterologous nucleic acid (or one or more transgenes encoded by the heterologous nucleic acid) can be increased by at least about 5%, or at least about 10%, or at least about 15%, or at least about 20% compared to the product of a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor, or compared to the starting material used in step (a). For example, the expression of the heterologous nucleic acid (or one or more transgenes encoded by the heterologous nucleic acid) can be increased by up to about 100%, or up to about 90%, or up to about 80%, or up to about 70%, or up to about 60%, or up to about 50% compared to the product of a corresponding method in which step (a) is not performed and / or step (c) is performed in the absence of an mTOR inhibitor, or compared to the starting material used in step (a).

[0340] The increase in the transduction efficiency of heterologous nucleic acid refers to the increase in the transduction efficiency of heterologous nucleic acid in Treg population compared with the corresponding method that does not carry out step (a) and / or that carries out step (c) in the absence of mTOR inhibitor.The "corresponding method" is as described above.Transduction efficiency can be determined by detecting the presence of heterologous nucleic acid in cell or the presence of heterologous protein encoded by heterologous nucleic acid.

[0341] For example, the transduction efficiency of the heterologous nucleic acid (or one or more transgenes encoded by the heterologous nucleic acid) can be increased by at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, compared to the product of a corresponding method that does not perform step (a) and / or a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor. For example, the transduction efficiency of the heterologous nucleic acid (or one or more transgenes encoded by the heterologous nucleic acid) can be increased by up to about 100%, or up to about 90%, or up to about 80%, or up to about 70%, or up to about 60%, or up to about 50%, compared to the product of a corresponding method that does not perform step (a) and / or a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor.

[0342] For example, the transduction efficiency of the heterologous nucleic acid (or one or more transgenes encoded by the heterologous nucleic acid) can be increased by at least about 3 percentage points, or at least about 5 percentage points, or at least about 10 percentage points, or at least about 15 percentage points, compared to the product of a corresponding method that does not perform step (a) and / or a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor. For example, the transduction efficiency of the heterologous nucleic acid (or one or more transgenes encoded by the heterologous nucleic acid) can be increased by up to about 40 percentage points, or up to about 35 percentage points, or up to about 30 percentage points, or up to about 25 percentage points, or up to about 20 percentage points, compared to the product of a corresponding method that does not perform step (a) and / or a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor.

[0343] The VCN of heterologous nucleic acid is reduced, and this means that the VCN of heterologous nucleic acid in Treg population is reduced compared with the corresponding method that does not carry out step (a) and / or that does not carry out step (c) in the absence of mTOR inhibitor.The "corresponding method" is as described above.The VCN can be determined by digital / quantitative PCR.

[0344] For example, the VCN of the heterologous nucleic acid (or one or more transgenes encoded by the heterologous nucleic acid) may be reduced by at least about 0.5 copies per transduced cell, or by at least about 1 copy per transduced cell, compared to the product of a corresponding method that does not perform step (a) and / or a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor. For example, the VCN of the heterologous nucleic acid (or one or more transgenes encoded by the heterologous nucleic acid) may be reduced by up to about 5 copies, 4 copies, 3 copies, or 2 copies per transduced cell, compared to the product of a corresponding method that does not perform step (a) and / or a corresponding method in which step (c) is performed in the absence of an mTOR inhibitor.

[0345] This disclosure is not limited to the exemplary methods and materials disclosed herein; any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise specified, all nucleic acid sequences are written left to right in 5' to 3' orientation, and amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0346] When a range of values ​​is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly indicates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated or intervening value within a stated range and any other stated or intervening value within that stated range is encompassed within the present disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded from the range, and each range where either or both limits are included in the smaller range, where neither or both limits are included in the smaller range, is also encompassed within the present disclosure, excluding any specifically excluded upper or lower limit in the stated range. When a stated range includes one or both of the upper and lower limits, ranges excluding either or both of those included limits are also encompassed within the disclosure.

[0347] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0348] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," or "containing," and "contains," and are inclusive or open-ended and do not exclude additional, unrecited elements, elements, or method steps. The terms "comprising," "comprises," and "comprised of" also include the term "consisting of."

[0349] The publications discussed herein are solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that such publications constitute prior art to the appended claims.

[0350] The present invention will now be further described by way of examples, which are intended to aid those of ordinary skill in the art in practicing the present invention and are not intended to limit the scope of the present invention in any way. [Example]

[0351] Example 1 - Composition of Cellular Products material and method Starting materials Leukopak was supplied by BioIVT. PBMCs were isolated using a negative selection kit (StemCell Technologies). Briefly, unwanted fractions were targeted for removal by labeling with antibody conjugates and magnetic beads, followed by separation using a magnet. Treg populations were extracted using a Leukopak tube. Leukopak-derived PBMCs were subjected to CD25 positive selection using the Human CD25 Positive Selection Cocktail (Stemcell Technologies), followed by CD4 enrichment by negative selection using the Human CD4+ T Cell Enrichment Cocktail (Stemcell Technologies). The target cells were isolated using a magnet. The CD4+CD25+CD127- / low cell population was further separated using the CD127high Depletion Cocktail (Stemcell Technologies), and the target cells were isolated using a magnet. Cell fractions were stained with flow cytometry antibodies: anti-CD4 VioBlue (M-T466, Miltenyi), anti-CD25 PE (3G10, Miltenyi), anti-CD45RA FITC (T6D11, Miltenyi), and anti-CD127 APC (MB15-18C, Miltenyi), followed by FACS sorting. Where indicated, CD4+CD25+CD127- / low (bulk Treg) cells were used. To obtain CD8+ T cells for spiking, a small fraction of the leukopak-derived PBM was retained and subjected to negative selection using the human CD8+ T Cell Isolation Cocktail (StemCell Technologies). The target cells were isolated using a magnet. To confirm CD8 purity, the small cell fraction was stained with anti-CD3 APC (UCHT1, Biolegend) and anti-CD8a PE (RPA-T8, Biolegend) antibodies for flow cytometry. The isolated CD8+ cells were maintained in X-VIVO15 medium (Lonza) supplemented with 5% human AB serum (heat-treated, Merck) until the sorted Tregs were ready for spiking. Leukopaks were obtained from healthy volunteers or liver transplant patients. Where indicated, bulk Tregs obtained from healthy volunteers were supplemented with 5% or 10% CD8+ T cells obtained from the same subjects.

[0352] Lentivirus Cells were transduced using a lentiviral vector encoding the RQR8 safety switch, the FOXP3 polypeptide, and an HLA-A2-specific CAR.

[0353] Growth Protocol "1+24" or "1+48" condition Pre-activated Tregs were incubated for 1 hour in Treg medium (X-VIVO15 medium (Lonza) supplemented with 5% human AB serum (heat-treated, Merck)) containing 100 nM rapamycin. CD3 and CD28 beads were then added to activate the Tregs, and the cells were cultured for 24 or 48 hours (referred to as 1+24 and 1+48 conditions, respectively). After 24 or 48 hours, the cells were washed with fresh Treg medium and centrifuged. The supernatant was removed, and the cell pellet was resuspended in fresh Treg medium. Subsequently, the cells were expanded in fresh Treg medium (X-VIVO15 medium (Lonza) supplemented with 5% human AB serum (heat-treated, Merck)) containing IL-2 (Proleukin, Clinigen Healthcare) and lentiviral supernatant, but without rapamycin. The cells were replenished with Treg medium supplemented with IL-2 every 2–3 days. 14 days after activation, the CD3 and CD28 beads were removed and the cells were harvested.

[0354] "One hour pre-treatment" Pre-activated Tregs were incubated for 1 hour in Treg medium (X-VIVO15 medium (Lonza) supplemented with 5% human AB serum (heat-treated, Merck)) containing 100 nM rapamycin. After 1 hour, the cells were washed with fresh Treg medium and centrifuged. The supernatant was removed, and the cell pellet was resuspended in fresh Treg medium (without rapamycin). CD3 and CD28 beads were then added to the new Treg medium to activate the Tregs and culture them. After 48 hours, the medium was removed, and the cells continued to expand in fresh Treg medium (X-VIVO15 medium (Lonza) supplemented with 5% human AB serum (heat-treated, Merck)) containing IL-2 (Proleukin, Clinigen Healthcare) and lentiviral supernatant (but without rapamycin). The cells were replenished with Treg medium supplemented with IL-2 every 2–3 days. 14 days after activation, the CD3 and CD28 beads were removed and the cells were harvested.

[0355] "24-hour or 48-hour culture" Tregs were activated by adding CD3 and CD28 beads to Treg medium (X-VIVO15 medium (Lonza) supplemented with 5% human AB serum (heat-treated, Merck)) containing 100 nM rapamycin. After 24 or 48 hours (referred to as 24-hour and 48-hour conditions, respectively), cells were washed with fresh Treg medium (without rapamycin) and centrifuged. The supernatant was removed, and the cell pellet was resuspended in fresh Treg medium. Cells were then expanded in fresh Treg medium (X-VIVO15 medium (Lonza) supplemented with 5% human AB serum (heat-treated, Merck)) additionally containing IL-2 (Proleukin, Clinigen Healthcare) and lentiviral supernatant (but without rapamycin). Cells were replenished with Treg medium supplemented with IL-2 every 2–3 days. 14 days after activation, CD3 and CD28 beads were removed and cells were harvested.

[0356] "No Rapa" (control) Pre-activated Tregs were placed in Treg medium (X-VIVO15 medium (Lonza) supplemented with 5% human AB serum (heat-treated, Merck)) for 1 hour. CD3 and CD28 beads were then added to activate the Tregs and cultured for 24 or 48 hours. For the 24-hour condition or the control group (1 + 24-hour condition), Tregs were cultured for 24 hours. For the control groups (1-hour pretreatment condition, 48-hour condition, or 1 + 48-hour condition), Tregs were cultured for 48 hours. After 24 or 48 hours, the cells were washed with fresh Treg medium (without rapamycin) and centrifuged. The supernatant was removed, and the cell pellet was resuspended in fresh Treg medium. Cells were then expanded in fresh Treg medium (X-VIVO15 medium (Lonza) supplemented with 5% human AB serum (heat-treated, Merck)) containing IL-2 (Proleukin, Clinigen Healthcare) and lentiviral supernatant (but no rapamycin). Cells were replenished with Treg medium supplemented with IL-2 every 2–3 days. At 14 days post-activation, cells were harvested by removing CD3 and CD28 beads.

[0357] Analysis of cell populations The percentage of FOXP3-expressing cells and the percentage of CD8+ cells in the product harvested on day 14 were determined by FACS. First, Dextramer PE (CINGVCWTV-NS3 hepatitis-HLA-A) in FACS staining buffer. *Tregs were surface stained with 0202 (Immudex), LIVE / DEAD™ Fixable Near-IR Dead Cell Stain (Thermofisher) in PBS, and then anti-CD4 V450 (SK3, BD Bioscience), anti-CD8 BV605 (RPA-T8, Biolegend), anti-RQR8 FITC (QBEND, Invitrogen), anti-CD19 PE-Cy7 (HIB19, Biolegend), and anti-CD56 APC-R700 (NCAM16.2, BD Bioscience) in FACS staining buffer. For intracellular staining of FOXP3, cells were fixed, permeabilized, and stained with anti-FoxP3 AF647 (259D, Biolegend). Cells were washed before analysis on a BD FACsLyric™ flow cytometer. FOXP3 expression was analyzed using FlowJo software. The gating strategy was lymphocytes > single cells > viable cells > CD19-CD56- > CD4+ > CD4+CD25+ > FoxP3+.

[0358] result Figure 1 shows the percentage of CD8+ cells at day 14 in the product of bulk Tregs collected from healthy donors expanded with 5% CD8+ cells. Each bulk Treg starting material from a healthy donor was split in half and then expanded under either rapa-free (control) or rapa-free conditions (1 h, 24 h, 1 h + 24 h, 48 h, or 1 h + 48 h). Bars represent the average percentage of CD8+ cells across all donors measured. The 1 h and 24 h conditions did not significantly reduce the percentage of CD8+ cells in the expanded product compared to the rapa-free (control) condition [not significant (ns)]. The 1 + 24 h, 48 h, and 1 + 48 h conditions all significantly reduced the percentage of CD8+ cells in the expanded product compared to the rapa-free (control) condition (indicated by *, ***, and ****, respectively). The 1 + 24 h condition resulted in a greater improvement in the percentage of CD8+ cells compared to the 24 h condition. The 1+48h condition (****) resulted in a higher level of significance, indicating a greater improvement, compared to the 48h condition (***). Both the 1+24h and 1+48h conditions resulted in a greater improvement in the percentage of CD8+ cells compared to the 1h condition. Figure 2 shows the percentage of CD8+ cells at day 14 in bulk Treg-expanded products obtained from patient-derived material (four different patient samples: 0008, 0011, 0012, and 0013). The average percentage of CD8+ cells at day 14 across all patient donors, normalized to control, is also shown. The material was split and expanded under different conditions. Both the 1+24h and 1+48h conditions resulted in fewer CD8+ cells in the expanded product compared to the no-rapa (control) condition. Figure 3 shows the percentage of FOXP3-expressing cells at day 14 in the product of bulk Tregs collected from healthy donors expanded with 5% CD8+ cells. Each bulk Treg starting material from a healthy donor was split in half and then expanded either without rapa (control) or under rapa conditions (1 h, 24 h, 1 h + 24 h, 48 h, or 1 h + 48 h). The 1 h + 24 h, 48 h, and 1 h + 48 h conditions significantly increased the percentage of FOXP3+ cells in the expanded product, but the 1 h + 48 h condition (****) showed a greater improvement than the 48 h condition (**). The 1 h and 24 h conditions did not significantly improve the percentage of FOXP3+ cells in the expanded product. Table 1 shows the percentage of FOXP3+ cells in the growth product obtained from patient donor material using different conditions. It can be seen that both the 1+24h and 1+48h conditions yield improved products compared to the no Rapa (control) condition. [Table 1]

[0359] Example 2 - Total Cell Proliferation material and method The same starting material and the same growth protocol as in Example 1 were used. The expansion rate on day 14 was calculated by dividing the number of Tregs recovered on day 14 by the number seeded on day 0.

[0360] result Figure 4 shows the Treg cell proliferation rate at day 14 of bulk Tregs collected from healthy donors, expanded with 5% CD8+ cells. Each bulk Treg starting material from a healthy donor was split in half and then expanded in either control or no rapa conditions. The proliferation rate in the rapa condition was normalized to the no rapa condition by dividing the proliferation rate in the specific rapa condition by the proliferation rate in the no rapa condition. The 1+48h condition significantly improved proliferation rates compared to both the 1h and 48h conditions. Figure 5 shows the total cell proliferation rate of the bulk Treg-expanded product obtained from patient-derived material (LTX-0008) at day 14. Both the 1+24h and 1+48h conditions show improved proliferation rates compared to the no-rapa (control) condition.

[0361] Example 3 - IL-17 and IFNγ production material and method The same starting material and the same growth protocol as in Example 1 were used. IL-17 and IFNγ production were determined by cytometric bead array (CBA) assays on supernatants collected on day 14 from Tregs stimulated for 24 hours with CD3 / CD28 Dynabeads (Gibco, final dilution 1:40 in X-VIVO15 medium (Lonza) supplemented with 5% human AB serum (heat-treated, Merck)). Briefly, frozen supernatants were thawed and exposed to the LEGENDplex™ Hu Th cytokine panel (12-plex, with FP V02, BioLegend) using fluorescently coded beads according to the manufacturer's instructions and analyzed on an Attune™ NxT flow cytometer. Cytokine expression was analyzed using LEGENDplex™ data analysis software.

[0362] result Figure 6A shows the IL-17 concentration and Figure 6B shows the IFNγ concentration of the product obtained by expanding bulk Tregs obtained from patient-derived material. Both the 1 + 24h and 1 + 48h conditions show reduced concentrations of the pro-inflammatory cytokines IL-17 and IFNγ compared to the no-rapa (control) condition.

[0363] Example 4 - Inhibition Assay material and method The same starting material and the same growth protocol as in Example 1 were used. Tregs were stained with CellTrace Yellow (Invitrogen) and added at various ratios to a U-bottom 96-well plate along with T effector cells stained with CellTrace Violet (Invitrogen). Dynabeads were added at a 1:5 ratio of beads to T effector cells and incubated at 37°C in a 5% CO2 incubator for 5 days. Cells were harvested and incubated with Live / Dead NIR (Invitrogen) and Human Trustain FcX (Biolegend) in PBS. Cells were then stained with anti-CD4 BV510 (A161A1, Biolegend) and anti-CD34 FITC (QBEND10, Invitrogen) in FACS staining buffer. Cells were washed before analysis on an Attune™ NxT flow cytometer. T effector cell proliferation was detected by dilution of CellTrace Violet stain and analyzed using FlowJo software. The rate of inhibition of T effector proliferation was calculated using the following formula.

number

[0364] result Figure 7 shows the suppression of T effector cell (Teff) proliferation by Treg cells at various Treg:Teff ratios. At intermediate Treg:Teff ratios, Treg cells treated with rapamycin for 1+24h and 1+48h suppressed Teff proliferation more than untreated (control) cells.

[0365] Example 5 - Expression of transgenes material and method The same starting material and the same growth protocol as in Example 1 were used. Briefly, frozen Treg cells were thawed, counted, and then cultured at 2 × 10 cells in Treg medium (X-VIVO15 medium (Lonza) supplemented with 5% human AB serum (heat-treated, Merck)). 6 Resuspended at a concentration of 1 x 10 cells / mL 5 Treg cells were transferred into each well of a U-bottom 96-well plate. Rituximab (Midwinter) was serially diluted 2-fold with reconstituted rabbit complement (bRc) (Bio-Rad) to achieve rituximab concentrations of 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, and 12.5 μg / mL. Each concentration of rituximab was added in duplicate to the Treg cells in the plate and incubated at 37°C in a 5% CO2 incubator for 4 hours. After incubation, the plate was centrifuged and washed with FACS buffer. Cells were stained with Dextramer APC (CINGVCWTV, Immudex), Live / Dead Near IR (Invitrogen), CD4 BV510 (A161A1, Biolegend), Qbend PE (Qbend10, ThermoFisher), and Fc block (Biolegend), followed by FACS analysis. The depletion rate of cells expressing the transgene was determined by observing the death rate obtained at each concentration of rituximab. The death rate was calculated using the following formula:

number

[0366] result Figure 8 shows the kill rates at different concentrations of rituximab, normalized to the dextramer expression rate. The depletion rates are improved for growth products obtained from patient-derived material grown under the 1+24h and 1+48h conditions compared to growth products obtained from the same patient-derived material grown under the control condition without rapamycin. The transduction efficiencies of growth products obtained under all these conditions were very similar (see Table 2 below). Therefore, the improved kill rates are due to improved expression of the transgene. [Table 2]

[0367] Example 6 - Expansion of CD45RA+ and CD45RA- Tregs material and method Starting materials Leukopaks were collected from healthy volunteers and supplied by BioIVT. PBMCs were isolated using a negative selection kit (StemCell Technologies). Briefly, unwanted fractions were targeted for removal by labeling with antibody conjugates and magnetic beads, followed by separation using a magnet. Treg populations were extracted using a Leukopak tube. Leukopak-derived PBMCs were subjected to CD25 positive selection using the Human CD25 Positive Selection Cocktail (Stemcell Technologies), followed by CD4 enrichment by negative selection using the Human CD4+ T Cell Enrichment Cocktail (Stemcell Technologies). The target cells were isolated using a magnet. The CD4+CD25+CD127- / low cell population was further separated using the CD127high Depletion Cocktail (Stemcell Technologies), and the target cells were isolated using a magnet. Cell fractions were stained with flow cytometry antibodies: anti-CD4 VioBlue (M-T466Miltenyi), anti-CD25 PE (3G10, Miltenyi), anti-CD45RA FITC (T6D11, Miltenyi), and anti-CD127 APC (MB15-18C, Miltenyi), followed by FACS sorting. CD4+CD25+CD127- / low Treg populations were sorted into CD45RA+ and CD45RA- Treg populations. Each population (CD45RA+ and CD45RA-) from each healthy donor was split in half and then expanded under either rapa-free (control) or rapa-free (1h + 48h) conditions as described below.

[0368] Lentivirus Cells were transduced using a lentiviral vector encoding the RQR8 safety switch, the FOXP3 polypeptide, and an HLA-A2-specific CAR.

[0369] Growth Protocol "Lapa" condition Pre-activated Tregs were placed in Treg medium (X-VIVO15 medium (Lonza) supplemented with 5% human AB serum (heat-treated, Merck)) containing 100 nM rapamycin for 1 hour. CD3 and CD28 beads were then added for activation and cultured for 48 hours. After 48 hours, cells were washed with fresh Treg medium and centrifuged. The supernatant was removed, and the cell pellet was resuspended in fresh Treg medium. They were then expanded in fresh Treg medium (X-VIVO15 medium (Lonza) supplemented with 5% human AB serum (heat-treated, Merck)) containing IL-2 (Proleukin, Clinigen Healthcare) and lentiviral supernatant, but without rapamycin. The cells were replenished with Treg medium supplemented with IL-2 every 2–3 days. At 14 days after activation, the CD3 and CD28 beads were removed and the cells were harvested.

[0370] "Control" (no rapa) Pre-activated Tregs were placed in Treg medium (X-VIVO15 medium (Lonza) supplemented with 5% human AB serum (heat-treated, Merck)) for 1 hour. CD3 and CD28 beads were then added to activate the Tregs and cultured for 48 hours. After 48 hours, the cells were washed with fresh Treg medium (without rapamycin) and centrifuged. The supernatant was removed, and the cell pellet was resuspended in fresh Treg medium. They were then expanded in fresh Treg medium (X-VIVO15 medium (Lonza) supplemented with 5% human AB serum (heat-treated, Merck)) that further contained IL-2 (Proleukin, Clinigen Healthcare) and lentiviral supernatant (but did not contain rapamycin). The cells were replenished with Treg medium supplemented with IL-2 every 2–3 days. At 14 days after activation, the CD3 and CD28 beads were removed and the cells were harvested.

[0371] Analysis of cell populations The expansion rate on day 14 was calculated by dividing the number of Tregs recovered on day 14 by the number seeded on day 0. The percentages of FOXP3-, Helios-, and CD27-expressing cells in the product collected on day 14 were determined by FACS. Tregs were surface stained first with LIVE / DEAD™ Fixable Blue-Dead Cell Stain (Thermofisher) in PBS, followed by anti-CD4 BV510 (A161A1, Biolegend), anti-CCR7 APC / Fire810 (G043H7, Biolegend), anti-CD45RA PE (HI100, Biolegend), anti-CD45RO BV750 (UCHL1, Biolegend), anti-CD27 PerCP (0323, Biolegend), anti-CD25 PE-Cy7 (B696, Biolegend), anti-RQR8 FITC (QBEND, Invitrogen), and anti-CD62L BUV737 (DREG-56, BD Biosciences) in FACS staining buffer. For intracellular staining, cells were fixed, permeabilized, and stained with anti-FoxP3 BV421 (206D, Biolegend) and anti-HELIOS APC (22F6, Biolegend). Cells were washed before analysis on a Cytek Aurora Spectral Cytometer. Marker expression was analyzed using FlowJo software. The gating strategy was lymphocytes > single cells > viable cells > CD4+ > markers.

[0372] result The results are shown in Figures 9 and 10. Figure 9 shows the total proliferation rate of Tregs and RA-Tregs from donor 1 (Figure 9A) and donor 2 (Figure 9B) under control and Rapa conditions. Under Rapa conditions, RA-Tregs do not proliferate. Figure 10 shows the phenotypes of donors 1 and 2 on day 14. RA-Tregs cultured under Rapa conditions showed poor cell proliferation, resulting in insufficient cell numbers for phenotypic evaluation. However, RA-Tregs grown under control conditions exhibited a poorer phenotype than RA+Tregs, particularly lower expression of Helios and CD27. This is beneficial because RA+ Tregs have a greater ability to maintain FOXP3 expression than CD45RA- Tregs and are generally more stable than RA- Tregs, so that in a mixed population containing both RA+ and RA- Tregs, when expanded under Rapa conditions, RA+ Tregs can be selectively expanded over RA- Tregs, resulting in a product with a more desirable phenotype.

[0373] Example 7 - GMP Process method Leukopaks were collected from healthy volunteers and liver transplant recipients and supplied by BioIVT. Using a GMP-compliant process, Tregs were isolated and expanded under "rapa-free (control)" or "rapa" conditions in IL-2-containing medium suitable for Treg expansion. Tregs isolated from each donor were split, with half expanded under rapa-free conditions and the other half expanded under rapa-free conditions. Under "no rapamycin" (control) conditions, cells were activated with anti-CD3 / anti-CD28 beads on day 0 and expanded in a medium suitable for Treg proliferation. On day 2, cells were transduced with a lentiviral vector encoding the RQR8 safety switch, FOXP3, and an HLA-A2-specific CAR. Cells were replenished as needed. On day 14, cells were harvested and cryopreserved. Rapamycin was not present throughout the process. Under "rapa" conditions, cells were incubated for 1 hour in cell culture medium suitable for Treg expansion and containing 100 nM rapamycin. After 1 hour of pretreatment, cells were activated by adding anti-CD3 / anti-CD28 beads (day 0) and incubated in the same medium for an additional 2 days. On day 2, cells were transduced with a lentiviral vector encoding the RQR8 safety switch, FOXP3, and HLA-A2-specific CAR. Upon transduction, the volume in the flask was replenished to dilute the rapamycin so that the rapamycin concentration was 1 / 20 of the original 100 nM rapamycin concentration in the original medium. Cells were replenished as needed, but no additional rapamycin was added. On day 14, cells were harvested and cryopreserved.

[0374] Analysis of cell populations The phenotype of the product collected on day 14 was determined by FACS. The percentage of CD8+ cells in the product was determined before cryopreservation. For other markers, cells were thawed after cryopreservation. Tregs were surface stained first with LIVE / DEAD™ Fixable Blue-Dead Cell Stain (Thermofisher) in PBS, followed by anti-CD4 BV510 (A161A1, Biolegend), anti-CD8 BV605 (RPA-T8, Biolegend), anti-CD25 PE-Cy7 (B696, Biolegend), and anti-RQR8 FITC (QBEND, Invitrogen) in FACS staining buffer. For intracellular staining, cells were fixed, permeabilized, and stained with anti-FOXP3 BV421 (206D, Biolegend) and anti-HELIOS APC (22F6, Biolegend). Cells were washed before analysis on a Cytek Aurora Spectral Cytometer. Marker expression was analyzed using FlowJo software. The gating strategy was lymphocytes > single cells > viable cells > CD4+ > markers. Transduction efficiency was determined as described above by detecting the percentage of RQR8+ cells using FACS. Demethylation of the Treg-specific demethylated region (TSDR) of FOXP3 was determined using a methylation-specific qPCR assay. Vector copy number (VCN) was determined by detecting copies of WPRE (present in the lentiviral vector) per transduced cell using qPCR.

[0375] result The results are shown in Figures 11 to 16. Figures 11 and 12 show that the percentage of FOXP3+ cells and the percentage of Helios+ cells, respectively, are greater in products grown under rapa conditions compared to products grown under non-rapa conditions. FIG. 13 shows a greater rate of demethylation of FOXP3-TSDR in products grown under rapa conditions compared to products grown under no rapa conditions. FIG. 14 shows that there is a lower percentage of CD8+ cells in the products grown under rapa conditions compared to products grown under non-rapa conditions. FIG. 15 shows that transduction efficiency is greater in products grown under rapa conditions than in products grown under non-rapa conditions. FIG. 16 shows that VCN is lower in products grown under rapa conditions than in products grown under non-rapa conditions.

Claims

1. (a) contacting a population of regulatory T cells (Tregs) with an mTOR inhibitor prior to activation; (b) activating the Tregs in the population; (c) culturing the population of Tregs obtained in step (b) in the presence of an mTOR inhibitor.

2. The method of claim 1, further comprising the step of isolating a population of Tregs, particularly a population of CD4+CD25+ Tregs, prior to step (a).

3. 3. The method of claim 1 or claim 2, wherein the mTOR inhibitor used in step (a) and / or step (c) is rapamycin or a rapalog.

4. 10. The method of any one of the preceding claims, wherein the mTOR inhibitor used in step (a) is the same as the mTOR inhibitor used in step (c).

5. 10. The method of any one of the preceding claims, which does not include a step of removing the mTOR inhibitor used in step (a) prior to step (b) or step (c).

6. 10. The method of any one of the preceding claims, which does not include a step of washing the Tregs prior to step (b) or step (c).

7. 10. The method of any one of the preceding claims, wherein the mTOR inhibitor used in step (a) is also present in steps (b) and (c), and in particular no additional mTOR inhibitor is added for step (c).

8. 10. The method of any one of the preceding claims, wherein the initial concentration of the mTOR inhibitor used in step (a) and / or step (c) is from about 30 nM to about 500 nM.

9. 10. The method of any one of the preceding claims, wherein said Tregs in step (a) are contacted with said mTOR inhibitor for at least about 15 minutes, such as from about 30 minutes to about 90 minutes, prior to step (b).

10. 10. The method of any one of the preceding claims, wherein the Tregs are activated by contacting them with a TCR / CD3 activator and / or a TCR co-stimulatory activator.

11. The method of claim 10, wherein the TCR / CD3 activator is an anti-CD3 antibody or a CD3-binding fragment thereof, and / or the TCR costimulatory activator is an anti-CD28 antibody or a CD28-binding fragment thereof.

12. 10. The method of any one of the preceding claims, wherein in step (c) said Tregs are cultured in the presence of said mTOR inhibitor for at least about 6 hours, such as from about 12 hours to about 72 hours, such as from about 12 hours to about 60 hours, such as from about 36 hours to about 60 hours, such as about 48 hours.

13. 10. The method of any one of the preceding claims, wherein step (c) is carried out simultaneously with or immediately after step (b).

14. (d) reducing the concentration of the mTOR inhibitor in contact with the population of Tregs; (e) further culturing or expanding the population of Tregs in the presence of decreasing concentrations of the mTOR inhibitor; 10. The method of any one of the preceding claims, wherein steps (d) and (e) are performed after step (c).

15. (d) removing the mTOR inhibitor from contact with the population of Tregs; (e) further culturing or expanding the population of Tregs in the absence of an mTOR inhibitor; The method according to any one of claims 1 to 14, wherein steps (d) and (e) are carried out after step (c).

16. 15. The method of claim 14, wherein the concentration of the mTOR inhibitor is reduced to a concentration of about 25 nM or less.

17. 17. The method of any one of claims 14 to 16, wherein the concentration of the mTOR inhibitor is reduced or the mTOR inhibitor is removed at least about 6 hours, such as from about 12 hours to about 72 hours, such as from about 12 hours to about 60 hours, for example from about 36 hours to about 60 hours, after the start of step (c).

18. 18. The method of any one of claims 14 to 17, wherein the population of Tregs is cultured or expanded for at least about 6 days, such as from about 8 days to about 36 days, such as from about 10 days to about 14 days, after reducing the concentration of or removing the mTOR inhibitor.

19. 10. The method of any one of the preceding claims, further comprising the step of introducing heterologous nucleic acid into said Tregs, for example by transduction with a viral vector comprising said heterologous nucleic acid.

20. 20. The method of claim 19, wherein the heterologous nucleic acid encodes a chimeric antigen receptor (CAR), and / or a FOXP3 polypeptide, and / or a safety switch, and / or a polypeptide that increases persistence of the cells.

21. (d) reducing the concentration of the mTOR inhibitor in contact with the population of Tregs; (e) introducing a heterologous nucleic acid into said Treg; (f) further culturing or expanding the population of Tregs in the presence of decreasing concentrations of the mTOR inhibitor; 10. The method of any one of the preceding claims, wherein steps (d), (e), and (f) occur after step (c), step (e) occurs before step (f), and step (e) occurs before, simultaneously with, or after step (d).

22. (d) removing the mTOR inhibitor from contact with the population of Tregs; (e) introducing a heterologous nucleic acid into said Treg; (f) further culturing or expanding the population of Tregs in the absence of an mTOR inhibitor; 21. The method of any one of claims 1 to 20, wherein steps (d), (e), and (f) are performed after step (c), step (e) is performed before step (f), and step (e) is performed after step (d).

23. 22. The method of claim 21, wherein the concentration of the mTOR inhibitor is reduced to a concentration of about 25 nM or less concurrently with the step of introducing the heterologous nucleic acid into the Tregs in the population.

24. 24. The method of any one of claims 19 to 23, wherein the heterologous nucleic acid is introduced into the Tregs at least about 6 hours, such as from about 12 hours to about 72 hours, such as from about 12 hours to about 60 hours, such as from about 36 hours to about 60 hours, after the initiation of step (b) and / or the initiation of step (c).

25. 25. The method of any one of claims 19 to 24, wherein the population of Tregs is cultured or expanded for at least about 6 days, such as from about 8 days to about 36 days, such as from about 10 days to about 14 days after introducing the heterologous nucleic acid into the Tregs.

26. 10. The method of any one of the preceding claims, wherein the population of Tregs is harvested about 8 to about 36 days after the initiation of step (b), such as about 8 to about 22 days after the initiation of step (b), and optionally cryopreserved.

27. A product obtained and / or obtainable by the method of any one of claims 1 to 26.

Images