WO2024020531A1

WO2024020531A1 - Immune cell expansion and uses thereof

Info

Publication number: WO2024020531A1
Application number: PCT/US2023/070678
Authority: WO
Inventors: Joseph LEVENTHAL; James Mathew; Cheryl STRATTON
Original assignee: Tract Therapeutics, Inc.; Northwestern University
Priority date: 2022-07-21
Filing date: 2023-07-21
Publication date: 2024-01-25

Abstract

Methods, compositions, and kits for generating therapeutically relevant populations of immunosuppressive Treg cells and uses thereof are disclosed.

Description

IMMUNE CELL EXPANSION AND USES THEREOF [0001] FIELD [0002] The disclosure relates generally to the selection, expansion, and use of regulatory T cell (Treg cells) populations. [0003] BACKGROUND [0004] Significant limitations in the efficacy of organ and tissue transplant include the rejection of allografts by host immune systems and graft versus host disease. Pharmaceutical immunosuppressants are commonly used to treat these conditions; however, they are not always effective. Regulatory T cells (Treg cells) are potent suppressor regulatory T lymphocytes (CD4+/CD25+) that have been demonstrated to have importance in the active immune regulation/suppression in the processes of graft rejection and tolerance. Current methodologies for generating therapeutically relevant numbers of Treg cells rely on purification and ex vivo expansion of freshly isolated Treg cells. However, the logistics of these methodologies, including the requirement of freshly isolated Treg cells, the relatively low numbers of expanded Treg cells, as well as the time required for expansion and isolation of expanded populations of Treg cells present considerable disadvantages. Therefore, there is a need for new approaches for providing Treg cells for therapeutic purposes that overcome these disadvantages. SUMMARY [0005] Presented herein is a clinically applicable method for the expansion of regulatory T cells using a beadless matrix that eliminates the need for bead removal, and therefore is more efficient and is also associated with preservation of expanded cell numbers. [0006] In an aspect, this disclosure provides methods for expanding a population of T regulatory (Treg) cells. The method includes: (a) stimulating the population of Treg cells in a growth media supplemented with Interleukin-2 (IL-2), Transforming Growth Factor Beta (TGF-β), an inhibitor of mammalian target of rapamycin (mTOR), and one or more beadless reagents comprising an anti-CD3+ antibody and anti-CD28+ antibody, and culturing the population for about two days; (b) adding fresh growth media, IL-2, TGF-β, and the inhibitor of mTOR to the population about every two to three days; (c) after about 11 days, re-stimulating the population by adding additional growth media, IL-2, TGF-β, the inhibitor of mTOR to the population, and the beadless reagent, and culturing the population for about one day; (d) adding fresh growth media, IL-2 and TGF-β to the population about every two to three days; and (e) after about 21 days, counting the population of Treg cells and harvesting the population of Treg cells when the population of Treg cells comprises more than about 500 million cells, wherein the harvested population comprises the expanded population of Treg cells. [0007] In certain embodiments, if fewer than about 500 million cells are present at step (e) and the method further includes: (f) adding fresh growth media, IL-2 and TGF-β about every two to three days; and (g) on about Day 28 harvesting the expanded population of Treg cells. [0008] In certain embodiments, the one or more beadless reagents includes a biodegradable surface. In some embodiments, the one or more beadless reagents includes a colloidal matrix. [0009] In certain embodiments, the population of Treg cells is a CD4⁺/CD25⁺ population of Treg cells that is isolated from an apheresis sample. The apheresis sample may be previously- frozen. [0010] In certain embodiments of the methods disclosed herein: (a) the IL-2 concentration is maintained at about 500 to about 2,000 U/mL; (b) the TGF-β concentration is maintained at about 0.1 to about 10 ng/mL; and/or (c) the inhibitor of mTOR concentration is maintained at about 10 to about 200 ng/mL for at least about the first 8 days. [0011] In certain embodiments the inhibitor of mTOR is Everolimus, serolimus or rapamycin. [0012] In another aspect, this disclosure provides a composition comprising at least about 500,000,000 cells of the expanded population of Treg cells produced by the methods as disclosed herein. [0013] In yet another aspect, this disclosure provides a method for treating an organ transplant recipient, comprising administering to a patient the compositions of expanded Treg cells as disclosed herein. In certain embodiments, the organ transplant recipient is a kidney transplant recipient. [0014] In another aspect, this disclosure provides a method for expanding a population of T regulatory (Treg) cells. The method includes: (a) on Day 0 stimulate the population of Treg cells in a growth media, Interleukin-2 (IL- 2), Transforming Growth Factor Beta (TGF-β), Everolimus, and a beadless reagent comprising an anti-CD3+ antibody and anti-CD28+ antibody, and culture the population for about two days; (b) on Day 2 add fresh growth media, IL-2, TGF-β, and Everolimus to the population, and culture the population for about three days; (c) on Day 5 add fresh growth media, IL-2, TGF-β, and Everolimus to the population, and culture the population for about two days; (d) on Day 7 add fresh growth media, IL-2, TGF-β, and Everolimus to the population, and culture the population for about two days; (e) on Day 9 add fresh growth media, IL-2, TGF-β and, optionally, Everolimus to the population, and culture the population for about three days; (f) on Day 12 re-stimulate the population by adding additional growth media, IL-2, TGF-β, the beadless reagent and, optionally, Everolimus; (g) on Day 13 or day 14 add fresh growth media, IL-2, and TGF-β to the population, and culture the population for about two or three days; (h) on Day 16 add fresh growth media, IL-2, and TGF-β to the population, and culture the population for about three days; (i) on Day 19 add fresh growth media, IL-2, and TGF-β to the population, and culture the population for about two days; and (j) on Day 21, count the population of cells and harvest the population when the population comprises more than about 500,000,000 cells, wherein the harvested population comprises the expanded population of Treg cells; or when the population comprises fewer than about 4 billion cells continue to culture the population for about two days; (k) on Day 23 add fresh growth media, IL-2, and TGF-β, and culture the population for about three days; (l) on Day 26 add fresh growth media, IL-2, and TGF-β, and culture the population for about one day; (m) on Day 27 add fresh growth media, IL-2, and TGF-β, and culture the population for about one day; (n) on Day 28 harvest the expanded population of Treg cells. [0015] In certain embodiments: (a) the IL-2 concentration is added at about 500 to about 2,000 U/mL; (b) the TGF-β concentration is added at about 0.1 to about 10 ng/mL; and/or (c) the inhibitor of mTOR concentration is added at about 10 to about 200 ng/mL for at least about the first 7 days, or optionally 8, 9, 10, 11, 12, 13, or 14 days. [0016] In certain embodiments, the beadless reagent comprises a biodegradable surface. In certain embodiments, the beadless reagent comprises a colloidal matrix. [0017] In certain embodiments of the methods disclosed herein the population of Treg cells is expanded by at least 100 fold, or about 200 fold, or about 300 fold, or about 400 fold, or about 500 fold, or about 600 fold, or about 700 fold, or about 800 fold, or about 900 fold, or about 1,000 fold, or about 1,100 fold, or about 1,200 fold, or about 1,300 fold, or about 1,400 fold, or about 1,500 fold, or about 1,600 fold, or about 1,700 fold, or about 1,800 fold, or about 1,900 fold or about 2,000 fold, or more. [0018] In each of the foregoing aspects and embodiments, cells may be cultured in a serum- free media. In addition, the culture vessel may be about 800 to about 1200 mL. [0019] Other aspects, embodiments, and implementations will become apparent to those of ordinary skill in the art by reading the following detailed description, with reference where appropriate to the accompanying drawings. BRIEF DESCRIPTION OF THE FIGURES [0020] The following detailed description of the embodiments of the present disclosure can be best understood when read in conjunction with the following figures. [0021] Figure 1 illustrates phenotypic characterization of cell populations during the two- column cell selection process using the CliniMACS® Magnetic Column device, as described in Example No.1 and Table No.2. Data shown indicate percentages of CD20+, CD8+, CD4+, CD25+, and FoxP3+ cells before selection (apheresis), for the population of cells removed (Neg Sel), and for the population selected (Day 0/Pos Sel). [0022] Figure 2 shows the ‘growth kinetics’ of fresh versus frozen cells in a known process involving cell stimulation using a ExpAct beads (Miltenyi). See WO2017/132446. Apheresis products from a normal donor were split into two samples, where one sample was used fresh, and the other sample was cryopreserved before cellular subpopulation selection followed by Treg cell culture expansion. The cryopreserved sample was thawed using a thawing buffer including a DNAse containing PBS/EDTA buffer and then used for cellular subpopulation selection followed by Treg cell culture expansion. Cell population growth was measured at culture initiation (Day 0) during the growth process (Day 14), and at the end of the process upon reaching the final product (Day 21). [0023] Figure 3 shows enriched and expanded CD4+/CD25+ Treg cells that were generated from fresh or cryopreserved apheresis products in a known process involving cell stimulation using a ExpAct beads (Miltenyi). See WO2017/132446. Cells were evaluated for their functional ability to induce suppression in a mixed lymphocyte proliferation assay (MLR) assay. At Treg:T responder cell ratios of 1:2 through 1:32 there was no difference in the immunosuppressive function of Treg cells generated from fresh or frozen apheresis products. [0024] Figure 4 shows that Treg cells expanded for 21 days from peripheral blood lymphocytes of renal failure patients in a known process involving cell stimulation using a ExpAct beads (Miltenyi). See WO2017/132446. The cells had acceptable immune suppressive function at several Treg:T responder cell ratios. [0025] Figure 5 is a representation of a clinical protocol for the use of T-Reg therapy for kidney transplant recipients. [0026] Figure 6A shows growth curves of Treg cells (absolute number) in nine expansion cultures in the process involving cell stimulation using a ExpAct beads (Miltenyi). See WO2017/132446. [0027] Figure 6B shows the phenotype of Treg cells from an expansion culture shown in Figure 6A. [0028] Figures 7A and 7B show the results of an experiment to measure the immunoregulatory capabilities of expanded Tregs. Figure 7A shows the counts per minute (CPM) values with the various modulators at indicated modulator: T responder ratios. Figure 7B shows a mean ± SD percentage of suppression that were calculated for each individual experiment (n =9). [0029] Figures 8A, 8B, and 8C show the results of immune monitoring in blood of recipient of Treg obtained from a known process involving cell stimulation using a ExpAct™ beads (Miltenyi). See WO2017/132446. Figures 8A and 8B shows flow cytometric analyses performed using whole blood, and the absolute number of indicated subsets were serially monitored. Figure 8C shows the percent of Foxp3⁺ Tregs observed in the recipient PBMC at pre-transplant was considered as 1, and the fold change in relation to that was calculated during the post-transplant period for each patient. [0030] Figure 9 is a schematic representation of a protocol for large-scale, bead-free expansion of Treg cells in G-Rex 100M or G-Rex 10M using a bead-free method according to the disclosure. [0031] Figures 10A, 10B and 10C show the improved expansion (FIG.10A), MLR inhibition (FIG.10B), and flow cytometry profile (FIG.10C) of the a Treg expanded cell population with restimulation by CD3/CD28 at Day 11 versus Day 7 using a bead-free method according to the disclosure. [0032] Figure 11 shows testing of IL-2 cytokine combinations using a bead-free method according to the disclosure. [0033] Figure 12 shows the results of an experiment to determine the effect of Treg starting volume on cell expansion according to a method of the disclosure. [0034] Figure 13 shows the results of an experiment to determine the effect of vessel size on cell expansion according to a method of the disclosure. [0035] Figure 14A, 14B, and 14C show representative flow cytometry phenotyping of Treg products throughout culture using a bead-free method according to the disclosure. CD4⁺CD25⁺FOXP3⁺ expression and expression of high CD25 and FOXP3 on days 14 and 21 showed that the cells were true Tregs. CD25 expression was lower on day 0 possibly due to steric hindrance by antibodies used for purification and on day 28 due to rapid cell multiplication. [0036] Figure 15 shows potent inhibition of MLR by polyclonally expanded Tregs (n=4) produced according to a bead-free method of the disclosure.1x10⁵ Recipient PBMC; 1x10⁵ Irradiated Allo-PBMC; 50,000 – 1,562 Tregs (Additional R as Control) 6 Days; 3H-thymidine incorporation. Data Calculated as % of Inhibition = 1- (delta CPM in presence of Tregs/delta CPM in presence of control) x 100. [0037] Figure 16 shows the results of experiments to determine growth kinetics of human Treg expansion according to a method of the disclosure at day 21 and day 28. [0038] Figure 17 shows the results of an experiment to determine the effect of growth media on Treg growth kinetics using a bead-free method according to the disclosure. DESCRIPTION [0039] Ex vivo expansion of regulatory T cells to large numbers is necessary for the therapeutic use of these cells as an adoptive therapy. Previous methods to expand cells have used beads to display ligands capable of stimulating T cell proliferation. Removal of these beads is costly in terms of time and expense, and can lead to considerable loss of cell number. In order to address these and other issues, the disclosure is directed to is a clinically applicable method for the expansion of regulatory T cells using a beadless matrix that eliminates the need for bead removal, and therefore is more efficient and is also associated with preservation of expanded cell numbers. [0040] Before describing the present invention in detail, a number of terms will be defined. As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to a “nucleic acid” means one or more nucleic acids. [0041] As used herein, the term “about” refers to ± 10% of any particular value unless otherwise noted. [0042] As used herein, the terms “or” and “and/or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and/or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” [0043] As used herein, the term “viability” when used to describe a cell population, refers to the percentage of viable cells within the population. [0044] As used herein, the term “therapeutically relevant” in the context of administration of Treg cells to a recipient in need thereof, refers to the number of Treg cells that can be administered to the recipient to cause an ameliorating effect to the recipient. In one embodiment, a therapeutically relevant number of Treg cells is any number of Treg cells that at least reduces, stops, and/or prevents a cellular immune response that causes cellular, organ, or tissue rejection. In an embodiment, a therapeutically relevant number of Treg cells is about 0.5 x 109 to about 5 x 109 Treg cells administered at one time, post organ transplant to a lymphodepleted transplant recipient. The Treg cells can be administered at approximately 45-75 days post organ transplant, for instance, at 45, 50, 55, 60, 65, 70 or 75 days post transplant. [0045] The term “autoimmune disease” as used herein is defined as a disorder that results from an autoimmune response. An autoimmune disease is the result of an inappropriate and excessive response to a self-antigen. Examples of autoimmune diseases include, but are not limited to, Addison’s disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn’s disease, diabetes (Type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves’ disease, Guillain-Barr syndrome, Hashimoto’s disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren’s syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis, among others. [0046] For the purposes of describing and defining the present disclosure, it is noted that the term “substantially” is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term “substantially” is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue. [0047] All publications, patents and patent applications cited herein are hereby expressly incorporated by reference in their entirety for all purposes. [0048] Turning now to the various aspects of the disclosure, presented herein are methods for the selection and expansion of regulatory T cells using a beadless reagent for stimulating a starting selected population of cells. Previously known methods, for example U.S. Patent Application Publication No. 2009/0032013 (TRACT Therapeutics, Inc.) reflect the use of activated microbeads for the stimulation of selected cell populations. In contrast, the various aspects of the disclosure reflect a beadless protocol that eliminates the need for bead removal and also results in increased cell expansion. [0049] The compositions, methods, and kits disclosed herein are useful for preparing sterile, cellular therapy products for suppressing an individual’s immune system by administering the product to the individual. In one embodiment, a cellular therapy product intended for an individual is derived from an apheresis product taken from that individual. In another embodiment, a cellular therapy product intended for an individual is derived from an apheresis product taken from another individual or from another cell source. [0050] The apheresis products contemplated herein may be obtained from an individual, and are then frozen and stored until an enriched and expanded population of CD4+/CD25+ Treg cells may be needed, for example after an organ transplant. The apheresis product may be cryopreserved for days, months, weeks or years until approximately 21-28 days prior to the desired time of administration (e.g., post transplant) of the CD4+/CD25+ Treg cells to the individual. A frozen apheresis product can be thawed only when needed for use in the selection and expansion process as described herein. [0051] The methods presented herein are directed to expanding selected populations of Treg cells, for example purified CD4+CD25+ regulatory T cells (Tregs). To obtain a CD4+/CD25+ enriched Treg cell population from an apheresis product, a two-step selection protocol may be used. For example, an initial negative selection step may be used to remove CD8+ and CD19+ cell populations from an apheresis product. Removal of CD8+/CD19+ cell populations is required since it eliminates the presence of these cell populations during the ex vivo expansion of the Treg cells. The CD8+/CD19+ populations can result in the outgrowth of “effector” cells that could result in organ rejection and negate the potentially beneficial outcomes of using Treg cells for the induction of immune tolerance. Subsequently, a positive selection step for CD4+/CD25+ cells is performed to capture only the Treg cells. The resultant CD4+/CD25+ enriched cells may then be expanded in culture by stimulating the cells with CD3/CD28 antibodies associated with a beadless reagent, such as a colloidal matrix. [0052] Expansion of the enriched CD4+/CD25+ Treg cell population increases the Treg cell population by about 100 fold, or about 200 fold, or about 300 fold, or about 400 fold, or about 500 fold, or about 600 fold, or about 700 fold, or about 800 fold, or about 900 fold, or about 1,000 fold, or about 1,100 fold, or about 1,200 fold, or about 1,300 fold, or about 1,400 fold, or about 1,500 fold, or about 1,600 fold, or about 1,700 fold, or about 1,800 fold, or about 1,900 fold or about 2,000 fold, or greater. Compared to the use of a process that uses antibodies attached to microbeads to stimulate the cells, the process using a beadless reagent can increase the resulting population of cells by at least 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%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500%, or more. [0053] Methods for selecting CD25+ T regulatory cells from an apheresis product are known. Fresh or frozen apheresis product may be used as a starting material. When a frozen product is used, the method may include thawing a cryopreserved apheresis product comprising T-cells. The thawed product is washed, for example, in a buffer comprising Human Serum Albumin (HSA), Magnesium Chloride (MgCl2), and Dornase alfa. Dornase alfa is a biosynthetic form of human deoxyribunuclease I (DNase I) enzyme and is commercially available under the tradename PULMOZYME®. The method also includes incubating the apheresis product (fresh or thawed) with one or more capture surfaces comprising a binding agent for CD8+ and CD19+ cells. Such capture surfaces are commercially available, for example, as CLINIMACS® system reagents (Miltenyi Biotec). After capturing the CD8+/CD19+ cells on the surfaces, a CD8/ CD19 depleted product can be collected by washing the one or more surfaces with the buffer. The CD8/CD19 depleted product can then be combined with a capture surface for CD25+ cells (e.g., CLINIMACS®). Cell captured on the capture surface for CD25+ cells can be eluted with the buffer to provide a CD25+ enriched product. During each of the foregoing steps, one or more buffers including HSA, MgCl2, and Dornase alfa can be used to wash or elute the surfaces and collect the cells. [0054] The method can provide a suitable population of cells between 0.5 x10⁹ to about 5 x10⁹ cells for use in treating an organ transplant patient, such as a solid organ transplant (SOT) patient (for example, kidney or liver). When the population is expanded according to the method of the disclosure, a percentage of CD4+ Treg cells in the expanded population of CD4+/CD25+ Treg cells may differ from a percentage of CD4+ cells in an expanded population of CD4+/CD25+ Treg cells selected from a fresh, non-frozen apheresis product by less than about 1% to about 10%, for example, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In addition, a percentage of CD25+ Treg cells in the expanded population of CD4+/CD25+ Treg cells may differ from a percentage of CD25+ cells in an expanded population of CD4+/CD25+ Treg cells selected from a fresh, non-frozen apheresis product by less than about 1% to about 10%, for example, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. [0055] In one aspect, the method of the disclosure is directed to expanding a CD25+ cell population using a beadless reagent to stimulate a starting population of CD4+/CD25+ cells. The method includes culturing CD25+ cells (which may be prepared according to the previous disclosure) in a growth media (e.g.,with a serum free supplement) supplemented with Interleukin-2 (IL-2), an mTOR inhibitor (e.g., serolimus, Everolimus, rapamycin), and Transforming Growth Factor Beta (TGF-β). An aspect of the disclosure includes a composition comprising the cells and the supplemented growth media. The cells may be suspended with the growth media in the presence of a beadless reagent having one or more antibodies comprising an anti-CD3+ antibody and anti-CD28+ antibody on day zero. In the various aspects of the disclosure, the antibodies of the beadless reagent are not attached to microbeads as known in the art. Instead, the antibodies herein are attached to a beadless matrix, for example, a colloidal polymeric nanomatrix. For example, the antibodies may be associated with the MACS® GMP T Cell TransAct™ matrix (Miltenyi Biotec) or other matrix that allows for sterile filtration and removal of excess reagent. Other bead free systems may also be used such as the ImmunoCult™ Human CD3/CD28 T Cell Activator (Stem Cell Technologies). [0056] Following about two days, additional IL-2 is added to the growth media, and the cells may be cultured for about another two to four days, for example, about three days. Next, additional growth media including IL-2, the mTOR inhibitor, and TGF-β is added to the culture for about one to three days, for example, about two days. Then, additional growth media and IL- 2, the mTOR inhibitor, TGF-β, and one or more antibodies comprising an anti-CD3+ antibody and anti-CD28+ antibody are added to the culture for about one to three days, for example, about two days. Afterwards, additional IL-2, the mTOR inhibitor, and TGF-β are added, and the cells are cultured for about two to four days, for example, about three days. Next, additional IL-2 is added, and the cells are cultured for about one to three days, for example, about two days. Next, additional growth media, IL-2, and TGF-β are added to the culture for about two to four days, for example, about three days. Subsequently, additional IL-2 is added to the culture for about two days. The total time for the culture according to the foregoing procedure may be about 20-22 days, for example about 21 days, or about 27-29 days, for example about 28 days. The amount of IL-2, mTOR inhibitor, TGF-β added depends on the size of the culture and one of skill in the art could readily extrapolate the amount of reagents for culturing the cells from the examples following below. [0057] In various aspects of the methods of the disclosure, the last day of addition of an mTOR inhibitor is Day 7 or Day 8. [0058] In another aspect of the disclosure, the cell culture is restimulated with a beadless reagent having an anti-CD3+ antibody and anti-CD28+ antibody on one of Day 9, 10, 11, or 12. [0059] In another embodiment, the method of the disclosure is directed to expanding a Treg cell population in a beadless medium. The method includes culturing CD25+ cells in a growth media supplemented with Interleukin-2 (IL-2), an mTOR inhibitor, and Transforming Growth Factor Beta (TGF-β). An aspect of the disclosure includes a composition comprising the cells and the supplemented growth media. The cells may be suspended with the growth media in the presence of one or more beadless reagents comprising an anti-CD3+ antibody and anti-CD28+ antibody. In certain embodiments, the method comprises expanding a population of T regulatory (Treg) cells, by: (a) stimulating the population of Treg cells in a growth media, Interleukin-2 (IL-2), Transforming Growth Factor Beta (TGF-β), an inhibitor of mammalian target of rapamycin (mTOR), and one or more beadless reagents comprising an anti-CD3+ antibody and anti-CD28+ antibody, and culturing the population for about two days; (b) adding fresh growth media, IL-2, TGF-β, and the inhibitor of mTOR to the population about every two to three days (without the mTOR inhibitor after about 7 days); (c) after about 10-12 days, for example 11 days, re-stimulating the population by adding additional growth media,IL-2, TGF-β , and the beadless reagent to the population, and culturing the population for about one day; (d) adding fresh growth media, IL-2 and TGF-β to the population about every two to three days; and (e) after about 21 days, counting the population of Treg cells and harvesting the population of Treg cells when the population of Treg cells comprises more than about 500 million cells, wherein the harvested population comprises the expanded population of Treg cells. In this embodiment, the growth media may be a serum-free growth media. [0060] In some embodiments, IL-2 concentration is maintained at about 500 to about 2,000 U/mL. In some embodiments, the TGF-β concentration is added at about 0.1 to about 10 ng/mL. In some embodiments, the inhibitor of mTOR (for example, Everolimus) concentration is added at about 10 to about 200 ng/mL. In some embodiments, IL-2 concentration is added at about 1,000 U/mL. In certain embodiments, TGF-β concentration added at about 1 ng/mL. In some embodiments, the inhibitor of mTOR (for example Everolimus) concentration added at about 100 ng/mL for at least about the first 7-8 days. In various embodiment, the growth media may be supplemented with a serum-free supplement. [0061] A schematic representation of an example of the bead-free method according to the disclosure is shown in Figure 10. In this embodiment: (a) on Day 0 stimulate the population of Treg cells in a growth media, Interleukin-2 (IL-2), Transforming Growth Factor Beta (TGF-β), Everolimus, and a beadless reagent (e.g., Miltenyi TransAct^®) comprising an anti-CD3+ antibody and anti-CD28+ antibody, and culturing the population for about two days; (b) on Day 2 add fresh growth media, IL-2, TGF-β, and Everolimus to the population, and culture the population for about three days; (c) on Day 5 add fresh growth media, IL-2, TGF-β, and Everolimus to the population, and culture the population for about two days; (d) on Day 7 add fresh growth media, IL-2, TGF-β, and Everolimus to the population, and culture the population for about two days; (e) on Day 9 add fresh growth media, IL-2, and TGF-β to the population, and culture the population for about three days; (f) on Day 12 re-stimulate the population by adding additional growth media, comprising IL-2, TGF-β, and the beadless reagent, and culturing the population for about one day; (g) on Day 13 add fresh growth media, IL-2, and TGF-β to the population, and culture the population for about three days; (h) on Day 16 add fresh growth media, IL-2, and TGF-β to the population, and culture the population for about three days; (i) on Day 19 add fresh growth media. IL-2, and TGF-β to the population, and culture the population for about two days; and (j) on Day 21, count the population of cells and harvest the population when the population comprises more than about 500,000,000 cells, wherein the harvested population comprises the expanded population of Treg cells. In the alternative, when the expanded population has fewer than about 4 billion cells, the method includes continuing to culture the population for about two days; (k) on Day 23 add fresh growth media, IL-2, and TGF-β, and culture the population for about three days; (l) on Day 26 add fresh growth media, IL-2, and TGF-β, and culture the population for about one day; (m) on Day 27 add fresh growth media, IL-2, and TGF-β, and culture the population for about one day; (n) on Day 28 harvest the expanded population of Treg cells. In this embodiment, the growth media may contain a serum- free supplement. Also, the Everolimus may be added at one of day 9 or day 12. [0062] In particular embodiments, no additional mTOR inhibitor is added to the cells beyond about 7-15 days of culture, for example, after about 7, 8, 9, 10, 11, 12, 13, 14, or 15 days of culture. [0063] Kits and compositions of the disclosure can include the cells, reagents, mixtures, and cultures described herein. For instance, in one embodiment, the disclosure is directed to a kit for providing an expanded and enriched CD4+/CD25+ Treg cell population, including the beadless reagent, mTOR inhibitor and remainder of the reagents as noted above. The kit may further comprise a buffer comprising HSA, MgCl2, and Dornase alfa and instructions for use. A composition can include ingredients of the kits and a thawed, previously frozen, apheresis product. The composition may include a selected cell population that is in the process of being expanded or is expanded as described herein. For instance, a composition may include a population of CD4+/CD25+ cells produced from a frozen apheresis product, wherein the population is depleted of CD8/CD9 cells and is cultured in a medium including Interleukin-2 (IL-2), an mTOR inhibitor, and Transforming Growth Factor Beta (TGF-β). In another embodiment of the disclosure, the cell expansion takes place in a bioreactor vessel of about 800- 1200 ml, for example 800, 1000 or 1200 mL. The bioreactor vessel may have a gas permeable membrane. [0064] In another embodiment, the disclosure is directed to a method for treating a patient that has had a solid organ transplant. For example, solid organ transplants can include, but are not limited to kidney, liver, heart, lungs, pancreas, and intestines. In some embodiments, a patient with one or more transplanted tissues can be treated with the expanded populations as described herein, for example, tissues such as bones, tendons, ligaments, skin, heart valves, blood vessels, and corneas. An example protocol of the method of the disclosure is shown in Fig. 6, which is applicable to both a population of T-regs produced using a bead-based or bead free stimulation reagent as described herein. The method includes administering to the patient the population of cells as described herein, in particular, a population of cells that has been selected and expanded from a frozen apheresis product and/or using a beadless reagent for stimulating the cell culture as described herein. Similarly, the disclosure is directed to a method of treating an autoimmune disease. The method includes administering to the patient the population of cells as described herein, in particular, a population of cells that has been selected and expanded from a frozen apheresis product. Administration may be through any means generally accepted for the administration of cells within an individual (e.g., intravenously). [0065] In one embodiment, a contemplated cellular therapy product includes an enriched and expanded CD4+/CD25+ T regulatory (Treg) cell population. For example, the population may include greater than about 80% CD4+ cells, or greater than about 85% CD4+ cells, or greater than about 90% CD4+ cells, or greater than about 95% CD4+ cells, or greater than about 98% CD4+ cells. Further, the population may include greater than about 80% CD25+ cells, or greater than about 85% CD25+ cells, or greater than about 90% CD25+ cells, or greater than about 95% CD25+ cells, or greater than about 98% CD25+ cells. Further, the population may include greater than about 20% FoxP3+ cells, or greater than about 25% FoxP3+ cells, or greater than about 30% FoxP3+ cells, or greater than about 35% FoxP3+ cells, or greater than about 40% FoxP3+ cells. [0066] Further, the enriched and expanded CD4+/CD25+ Treg cell population may have diminished amounts or may be devoid of other cells exhibiting specific antigens. For example, the population may include less than about 10% CD8+ cells, or less than about 5% CD8+ cells, or less than about 3% CD8+ cells, or less than about 2% CD8+ cells, or less than about 1% CD8+ cells. Similarly, the population may include less than about 10% CD20+ cells, or less than about 5% CD20+ cells, or less than about 3% CD20+ cells, or less than about 2% CD20+ cells, or less than about 1% CD20+ cells. Further, it should be noted that both CD19 and CD20 can be used as markers for B cells, and the use of either or both for phenotyping and/or targeting for depletion of B cells from Treg cell populations is contemplated herein. [0067] It is further contemplated that the enriched and expanded CD4+/CD25+ Treg cell population may have greater than about 80% viability, or greater than about 90% viability, or greater than about 95% viability, or greater than about 98% viability. [0068] In another embodiment, an enriched and expanded cell population may be frozen prior to administration. Accordingly, following enrichment and expansion, the cells may be frozen, thawed when needed, and then subsequently administered. It is also contemplated that the frozen expanded and enriched cell population may be frozen, and then re-enriched and re- expanded and then administered to the patient. [0069] In one embodiment, expanded and enriched CD4+/CD25+ Treg cell populations as disclosed herein are intended to be used as a therapeutic agent for the donor of the apheresis product from which the cells were derived. Alternatively, it is also contemplated that the therapeutic agent may be used for another individual in need thereof. It is also contemplated that such therapeutic agents may be used in multiple individuals in need thereof. It is further contemplated that further selection may be made of the Treg cell populations to reduce the risk of rejection or other complications in an individual caused by cells donated by another. [0070] The disclosure will be further characterized in the following examples, which do not limit the scope of the disclosure described in the claims. The Examples that follow are illustrative of specific embodiments of the disclosure, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting. The following examples establish protocols for successful enrichment, expansion, and use of therapeutically relevant populations of CD4+/CD25+ Treg cells from frozen apheresis samples. EXAMPLES [0071] Example 1: Selection of CD4+ and CD25+ T regulatory Cells [0072] Overview: This example establishes the selection protocols for successful enrichment of CD4+/CD25+ Treg cells. [0073] Declumping Buffers [0074] Three 1 L CLINIMACS^® PBS/EDTA buffer bags were labeled (Bags #1, #2, and #3) and prepared by adding 20 mL of 25% human serum albumin (HSA) and 1.8 mL of 200 mg/mL MgCl₂. To Bag #1, 54 mL of 1 mg/mL PULMOZYME^® (dornase alpha) was added. To each of Bags #2 and #3, 10.5 mL of 1 mg/mL PULMOZYME^® was added. From Bag #1, 200 mL of solution was transferred to a sterile culture bag, which was labeled as Bag #1A, and supplemented with 45 mL 25% HSA. The approximate volumes, percentages, and/or final concentrations of each component in the Bags are shown in Table 1. Table 1 Declumping Buffer Bag Components

[0075] Leukapheresis Product Cryopreservation [0076] A collected leukapheresis sample was centrifuged at 2800 rpm for 11 minutes. Using a plasma extractor and an electronic scale, the supernatant was removed (i.e., plasma was expressed). Plasma was added back to the centrifuged sample to reach a volume of 60 mL. The leukapheresis sample was then transferred to a cryopreservation bag. A freezing solution of 50% DMSO (dimethyl sulfoxide) in Normal Saline (0.9% sodium chloride) was prepared, then placed in a 2-8°C refrigerator for at least 10 minutes. The 50% DMSO freezing solution was added to the cryopreservation bag to reach a final DMSO concentration of 10%. [0077] The cryopreservation bag was frozen in a controlled-rate freezer at an average cooling rate of 1°C/min, to an endpoint of -100°C. Once frozen, the bag was transferred to a monitored liquid nitrogen freezer and stored in the vapor phase. [0078] Leukapheresis Product Thawing [0079] Thawing of apheresis products generally results in some cellular clumping with associated decreased viability. Column separation of thawed apheresis products can be problematic due to the clumping of the product and the potential loss of cellular starting material. Therefore, a “thawing buffer” consisting of a PBS/EDTA buffer (phosphate–buffered saline, pH 7.2, supplemented with 1 mM EDTA), such as CliniMACS® PBS/EDTA buffer (Miltenyi Biotec), 5% human serum albumin, 3.5 mM MgCl₂ and Pulmozyme® 50 U/mL was used for the initial washing of the thawed apheresis product as well as for the selection process. [0080] The cassette holding the frozen leukapheresis bag was removed from the dry shipper and immersed completely in a 37 ± 2°C water bath. Once the cells thawed, but were still cold, the leukapheresis bag was removed from the cassette. [0081] CD8/CD19 Negative Selection [0082] Thawed leukapheresis cells were transferred to a 600 mL TRANSFER PACK^TM container (FENWAL^TM, Lake Zurich, IL). The entire contents of Bag #1A were added to the container. The container was incubated for 30 minutes, and then centrifuged at 1800 rpm for 15 minutes, all at room temperature. Using a plasma extractor and an electronic scale, the supernatant was removed and discarded. An aliquot of 200 mL of buffer from Bag #1B was added to the container. The container was centrifuged at 1800 rpm for 15 minutes at room temperature. Using a plasma extractor and an electronic scale, the supernatant was removed and discarded. The final product volume was adjusted to 85 ± 5 mL using buffer solution from Bag #1B. The cells were resuspended by gently agitating the container. [0083] The entire contents of a CLINIMACS^® CD8 microbead kit and the CD19 microbead kit were added to the resuspended cells. The microbead kits include a colloid of magnetic antibodies specific to the cells of interest (e.g., CD8+ CD19+ cells) and Iron-Dextran. The container was mixed gently before incubating for 30 minutes at room temperature on a rotator at 25 rpm. The incubated cells were diluted to 450 mL with buffer solution from Bag #1B. The container was centrifuged at 1800 rpm for 15 minutes. Using a plasma extractor and an electronic scale, the supernatant containing excess microbeads was removed and discarded. The product volume was adjusted to 100 ± 2 mL with buffer solution from Bag #1B. The cells were resuspended by gently agitating the container. [0084] The container and Buffer Bag #2 were connected to a CLINIMACS^® Plus instrument, and a CD8/CD19 depletion program was executed, which separates CD8+/CD19+ cells using a high-gradient magnetic separation column (Miltenyi). The magnetically-labeled CD8+CD19+ cells are retained in the magnetized column and separated from the unlabeled cells. The unlabeled cells are eluted out of the column and consist of a cell population depleted of CD8+/CD19+ cells. The CD8/CD19 depleted cells were collected in a “CD8/19 Depleted Product Preparation” Bag. [0085] CD4/CD25 Enrichment [0086] The CD8/CD19 depleted cells were diluted with cold (4-8°C) buffer (Buffer Bag #3) to a total volume of 380 mL. The cells were resuspended by gently agitating the CD8/19 Depleted Product Preparation Bag. The diluted product was cooled to 4-8^°C in a refrigerator for 20 minutes. [0087] The entire contents of a CLINIMACS^® CD25 microbead kit were combined with the diluted, cooled CD8/CD19 depleted cells in the CD8/19 Depleted Product Preparation Bag and the combination was mixed gently before incubating for 15 minutes at 4-8°C on a rotator at 25 rpm. The incubated cells were transferred to a 600 mL TRANSFER PACK^TM container, and then diluted with buffer solution from Bag #3. The container was centrifuged at 1800 rpm for 15 minutes at room temperature. Using a plasma extractor and an electronic scale, the supernatant containing excess Microbead reagent was removed and discarded. The product volume was adjusted to 100 ± 2 mL with buffer solution from Bag #3. The cells were resuspended by gently agitating the container. [0088] The container and Buffer Bag #3 were connected to a CLINIMACS^® Plus instrument, and a CD4/CD25 enrichment program was executed. This protocol is similar to that described above, with the exception that the desired CD4+/CD25+ cells are selected for and undesired cells are washed out. CLINIMACS® Magnetic Cell Separation Systems separate mixed cell populations in a magnetic field using an immunomagnetic label specific for the cells of interest (e.g., CD4+/CD25+ (bright), referred to as regulatory T-cells, or Treg). Thus, once the unlabeled cells were removed from the column, the retained cells (CD4+/CD25+ cells) were eluted by removing the magnetic field from the column, washing the cells out and collecting them. The resulting CD4+/CD25+ enriched cells were collected in a “CD25 Enriched Cell” Bag. Results [0089] Flow cytometry was used to determine the phenotypic characteristics of the selected population. The selection protocol yielded 98.7% CD4+ cells, 86.8% CD25+ cells, 0.0% CD8+ cells, and 0.1% CD20+ cells (see, Table 2 “Day 0,” which is a representative data set and Figure 1, which includes the data shown in Table No.2). FoxP3+ was used as a separate marker for Treg cells. Table 2 CD4+/CD25+ Enriched Cells

[0090] The data in Table No.2 demonstrate that the combined selection processes used for selecting Treg cells were successful. [0091] Example 2: T regulatory Cell Expansion [0092] Overview: Enriched populations of Treg cells obtained in Example No.1 are expanded in the present example to provide therapeutically relevant numbers of Treg cells using a bead-based stimulation reagent as previously described in WO2017. [0093] Preparation of Cell Media [0094] Growth medium was prepared by adding 100 mL 5% heat-inactivated AB serum (Valley Biomedical, Winchester, VA) to 1900 TexMACS medium (Miltenyi Biotec, San Diego, CA). A sample of 2.2 x 10⁷ IU of IL-2 (Prometheus Laboratories, San Diego, CA) was reconstituted in 1 mL of sterile water, then diluted with 9 mL growth medium to a final concentration of 2.2 x 10⁶ IU/mL (“diluted IL-2”). An aliquot of 1.5 mL of 2.5 mg/mL Rapamycin (Sigma-Aldrich, St. Louis, MO) was diluted with 1.35 mL growth medium to a final concentration of 0.25 mg/mL (“diluted rapamycin”). To 1 mL 5% acetic acid, 20 mL sterile water was added to reach a final concentration of 40 mM. A 100 µg sample of TGF-β (Invitrogen, Carlsbad, CA) was reconstituted in 1 mL 40 mM acetic acid, then diluted with 8.77 mL TexMACS buffer and 0.23 mL AB serum, to a final concentration of 10 µg/mL. Reconstituted and diluted TGF-β was stored in 0.5 mL aliquots frozen at -20°C. As needed, TGF-β aliquots were thawed and diluted with 4.5 mL growth medium to a final concentration of 1 µg/mL, and kept refrigerated at 2-8°C. Complete Growth Meeting (GM) was prepared by adding 900 µL diluted IL-2 solution, 800 µL diluted Rapamycin solution, and 2.0 mL thawed, diluted TGF-β solution to 2 L growth medium. [0095] Cell expansion protocol [0096] A population of CD4+/CD25+ enriched cells from Example No.1 was split into fractions containing 3 x 10⁷ total nucleated cells each in separate culture flasks (G-Rex100M, Wilson Wolf Manufacturing, New Brighton, MN) and diluted with 450 mL GM. CD3/CD28 ExpAct beads (Miltenyi) (0.6 mL, 2 x 10⁸ beads/mL) was added to each flask to reach a 4:1 bead:cell ratio. GMP ExpAct® beads are composed of MACS^® beads that have been coated with CD3 and CD28 antibodies. These beads provide non-specific stimulation signals required for the expansion of the Treg cell population. The culture flasks were then incubated at 37ºC, 5% CO2 throughout a 21-day expansion protocol as described below. [0097] On Day 2 (D2), 200 µL diluted IL-2 solution was added to each flask. [0098] On Day 5, 50 mL GM, 225 µL diluted IL-2 solution, 200 µL diluted Rapamycin, and 500 µL thawed, diluted TGF-β solution were added to each flask. [0099] On Day 7, a sample was taken from each culture flask and a manual cell count was performed. Following sample collection, 50 mL GM, 250 µL diluted IL-2 solution, 220 µL diluted Rapamycin, and 550 µL thawed, diluted TGF-β solution were added to each flask. CD3/CD28 ExpAct bead solution was added to the culture flasks at a 1:1 bead:cell ratio, based on the manual cell count. [00100] On Day 9, 50 mL GM, 272 µL diluted IL-2 solution, 240 µL diluted Rapamycin, and 600 µL thawed, diluted TGF-β solution were added to each flask. No further Rapamycin was added to the cultures after Day 9. [00101] On Day 12, 272 µL diluted IL-2 solution was added to each flask. [00102] On Day 14, aliquots were collected of the supernatant from each flask for sterility testing. Cells in each flask were then resuspended, and aliquots were collected for in-process testing. After aliquot collection, 100 mL GM, 318 µL diluted IL-2 solution, and 700 µL thawed, diluted TGF-β solution were added to each flask. [00103] On Day 16, 100 mL GM, 364 µL diluted IL-2 solution, and 800 µL diluted TGF-β solution were added to each flask. [00104] On Day 19, 364 µL diluted IL-2 solution was added to each flask. [00105] On Day 21, cells were resuspended, and aliquots were collected for a manual cell count. After aliquot collection, CD3/CD28 ExpAct beads were removed according to a manufacturer supplied Miltenyi Protocol. [00106] Results [00107] The cell expansion protocol yielded a 43-fold increase in cells, highly selected for CD4+ and CD25+ cells (see, Table 3, “Day 21.” “Day 0” cells are the same as from Table No.2 above). Table 3 CD4+/CD25+ Expanded Cells

[00108] The observed yield of enriched and expanded CD4+/CD25+ Treg cells is considerably greater than reported in the literature. Such an increase over the yields reported in the literature was unexpected. (Indeed, in subsequent clinical manufacturing of nine cellular products following the same protocols, the average yield was 91-fold (range 29-fold to 180 fold), which further demonstrates the repeatability of such unexpected yields (data not shown)). The results further show that there was not an outgrowth of undesirable CD8+ cells and that a high purity of CD4+/CD25+ Treg cells was retained. [00109] Example 3. Expansion of Fresh vs. Cryopreserved Leukapheresis Products [00110] Overview: this example sought to compare the efficacy of the present methodologies for CD4+/CD25+ cell selection and expansion in fresh versus frozen leukapheresis samples using a bead-based stimulation reagent as previously described in WO2017. [00111] A leukapheresis product from a healthy donor was split into two samples. The first sample [frozen sample] was treated according to the procedure in Example Nos.1 and 2, i.e., declumping buffer was prepared, and the cells were cryopreserved, thawed, Treg cells were selected, and Treg cells were expanded. For comparative purposes, the second sample [fresh sample] was not cryopreserved or exposed to buffer containing Pulmozyme^®, but was otherwise treated according to the procedure in Example Nos.1 and 2, i.e., the Treg cells were selected and expanded. [00112] Fresh vs. Frozen CD4+/CD25+ Treg cell Immunosuppressive Capacity [00113] The culture expanded CD4+/CD25+ Treg cells from fresh and frozen samples were evaluated for their functional activity to suppress T cell responsiveness in a standard mixed lymphocyte proliferation assay (MLR) (see Bresatz S, Sadlon T, Millard D, Zola H, Barry SC. Isolation, propagation and characterization of cord blood derived CD4+ CD25+ regulatory T cells. J Immunol Methods 2007; 327: 53–62). [00114] Results [00115] Cryopreservation and thawing with the declumping buffer before selection and expansion had a negligible effect on the final cellular product when compared to the selected and expanded fresh sample (see Table No.4 and Figure 2). Table No.4 indicates the detailed characterization of the initial cell product at culture initiation and the final product of fresh versus frozen expanded cell populations. As seen in Table No.4, characteristics of frozen cells and fresh cells are comparable both upon initiation of Treg cell expansion and after the Treg cells have undergone expansion. Table No.4 Fresh vs. Frozen Treg Cell Products

[00116] Moreover, as shown in Figure 3, enriched and expanded CD4+/CD25+ Treg cells from fresh and frozen sources had nearly identical immunosuppressant effects. [00117] Therefore, these unexpected results indicate for the first time that cryopreserved leukapheresis products that are thawed using the declumping buffer described above may be effectively enriched and expanded for CD4+/CD25+ Treg cells and are equivalent to fresh cells. Indeed, using these techniques it has been demonstrated that Treg cells generated from fresh apheresis product are not significantly different (in terms of growth potential, immunosuppressive function, viability and phenotypic characterization) than those generated from cryopreserved apheresis product. [00118] Example 4. Immunosuppressive Capacity of Enriched and Expanded Treg Cells obtained from Renal Failure Patients [00119] Overview: to determine whether the enriched and expanded CD4+/CD25+ Treg cells obtainable by the present disclosure from renal failure patients would be effective for immunosuppressive therapy. In this example, using a bead-based stimulation reagent as previously described in WO2017, autologous apheresis products were taken from patients with renal failure who would be undergoing kidney transplants, CD4+/CD25+ Treg cells were enriched and expanded as described above, and the enriched and expanded CD4+/CD25+ Treg cells were tested for their immunosuppressive capacity. [00120] Treg cell enrichment and expansion [00121] Apheresis products from Renal Failure Patients (defined as end-stage kidney disease patients who are undergoing living donor kidney transplantation) that were used in this Treg cell feasibility study were obtained from consented donors under an IRB (Northwestern University) approved protocol (STU20666). The enrichment and expansion of CD4+/CD25+ Treg cells were carried out as described above. [00122] CD4+/CD25+ Treg cell Immunosuppressive Capacity [00123] The culture expanded CD4+/CD25+ Treg cells from renal failure patients were evaluated for their functional activity to suppress T cell responsiveness in an MLR inhibition assay, as described above. [00124] Results [00125] Expansion, viability, and phenotypic characterization were all comparable to that observed from previous results (see Table 5). Table 5 Renal Failure Patient CD4+/CD25+ Treg Cells

[00126] The culture expanded CD4+/CD25+ Treg cells from renal failure patients (see Figure 4) had suppressive activity comparable to that of Treg cells generated from normal donors (not shown). Therefore, these data indicate that enriched and expanded CD4+/CD25+ Treg cells from renal failure patients (i.e., individuals anticipating receiving a tissue transplant) may be useful as immunosuppressants and may be useful for treatment of rejection of allografts by host immune systems and graft versus host disease. [00127] Example 5: Ex Vivo Expanded Recipient Regulatory T cells in Living Donor Kidney Transplants [00128] Overview: [00129] A phase I dose-escalation clinical trial was initiated based on the above-described TReg Adoptive Cell Therapy (TRACT) for solid organ transplants (SOTs) as previously described in WO2017. [00130] Subjects: [00131] A Phase I trial of autologous, polyclonally expanded Treg Adoptive Cell Therapy (TRACT) was initiated in living donor kidney transplant recipients. This was a nonrandomized dose-ranging study with 3 tiers of cell dosing (0.5, 1, and 5 x 10E9 cells infused, n=3 subjects/tier). The clinical protocol for the trial is graphically shown in Figure 5. The inclusion and exclusion criteria were as follows: Inclusion Criteria • 18-65 years • No prior organ transplant • Single organ (kidney) recipients • Females: negative serum pregnancy test • Understand and give informed consent Exclusion Criteria • Sensitivity; Contraindication; or non-compliance to Sirolimus, Tacrolimus or MMF • Active Infection; Severely limiting secondary diseases; psychiatric illness; • Cardiovascular disease; or addiction • Positive flow cytometric crossmatch • PRA>20% • Current or historic donor-specific antibodies • 18 > BMI < 35 • Malignancy within 3 years of transplant (non-melanoma skin cancer excluded) • HIV or HBsAg positive • WBC<4,000/mm3; platelet <1000,000/mm3; Triglyceride <400mg/dl; total cholesterol >300 mg/dl • Anti-T cell therapy or other investigational drug within 30 days of transplant [00132] Enrolled subjects under-went a non-mobilized leukopheresis at least two weeks prior to kidney transplant. This leukopheresis product was cryopreserved for later isolation and manufacturing of Tregs. Kidney transplant recipients received alemtuzumab induction to achieve lymphodepletion and were started on tacrolimus and mycophenolate-based immunosuppression. Subjects were converted from tacrolimus to sirolimus at 30 days post- transplant to provide an immunosuppressive milieu conducive to the survival of infused Tregs. [00133] Ex Vivo GMP expansion of recipient Tregs: [00134] In a GMP compliant facility, CD4⁺CD25⁺ Tregs were isolated from the patient’s leukopheresis products using all CliniMACS^® reagents and systems, as using a bead-based stimulation reagent as previously described in WO2017. CD127 was not included in Treg isolation procedures due to a lack of a GMP compliant reagent. Treg expansion began with stimulation using MACS^® GMP ExpAct T Beads^® and IL-2, TGFβ, and Sirolimus on days 0 and 7, Sirolimus was not added to the culture after day 9 and expansion beads were removed before infusion into recipients. See Figures 6A and 6B. Robust expansion was observed, and cell threshold demands were met for the dose dependent study in all nine Treg samples, despite varying causes of end stage renal disease. Figure 6A shows growth curves of Treg cells (absolute number) in nine expansion cultures. Phenotypically, the expansion protocol generated a classic CD4⁺CD25⁺Foxp3⁺ with little contaminating CD8⁺ cell throughout culture (Figures 6B). Greater than 99% of cells were CD4+ most of which acquired CD25^High phenotype by day 14. However, a decrease in Foxp3 expression from day 14 to day 21 of culture was observed possibly due to the strain of rapid cellular expansion. Despite this reduction in the Foxp3 expression, DNA methylation analyses indicated that the Foxp3 promoter was still demethylated (data not shown), suggesting the expanded cell product retained the regulatory nature. Also, this loss of Foxp3 expression did not result in any adverse clinical events. [00135] Expanded Tregs retained clonal diversity: [00136] In conjunction with receptor expression, the effect of the expansion protocol on the clonal diversity of the Treg final product was analyzed. Using high-throughput sequencing, unique TCR rearrangements were analyzed from six patient apheresis and matched final Treg products. The clonal diversity was found to increase from initial apheresis product to the final Treg expanded product (Figure 7 and Table 6). This is likely due to uncovering of low frequency clones within the Treg product that were below the detection criteria in the apheresis product, not by the generation of new clones as expansion is an ex vivo procedure. Overall, the expansion protocol generated a Treg product that displayed key homing receptors and maintained a diverse T cell repertoire. Table 6 Number of Characteristic Clones

[00137] Expanded Tregs were potently suppressive and induce infectious tolerance. [00138] The suppressive function of the Treg products was analyzed on days 0, 14 and 21 by using a classical mixed lymphocyte reaction (MLR) and measuring thymidine incorporation. Tregs were used as modulators in mixed lymphocyte reaction of autologous PBMC (R) stimulated with allogeneic irradiated PBMCs (Sx). Additional responder PBMC (Rx) was used as control modulators. After 7 days, a standard thymidine incorporation assay was performed. [00139] An increase in suppressive function in a dose dependent manner was observed as the Tregs expanded as shown by eight fold fewer Day 21 Tregs needed to achieve 50% suppression compared to Day 0 Tregs (See Figures 8A and 8B.) Figure 8B shows a mean ±SD percentage of suppression that were calculated for each individual experiment (n=9) using the formula described in Levitsky J, et al., Transplantation, 2009, 88(11):1303-11. [00140] Expanded Tregs met release criteria: [00141] The expanded Tregs underwent additional extensive testing to determine microbial sterility, endotoxins, cell viability, phenotypic characteristics, and number of residual beads used for stimulating the cells. It was found that the resultant cells met all the release criteria; i.e., negative aerobic, anaerobic and fungal contaminations, negative mycoplasma and negative gram stain; <5.0EU/kg endotoxin; >70% viable; >70% CD4⁺ CD25⁺; <10% CD8⁺ and CD19⁺; <3000 Exp-Act® beads/10E8 cells (Table 7). Table 7

[00142] In addition, potency was tested using MLR inhibition assays on days 14 and 21 of the culture and observed the expanded Tregs exceeded the expected regulatory capabilities. The day 14 assay was the in process testing and the data were available before the infusion of the expanded Tregs into the recipients. [00143] Infusion of expanded Tregs into kidney recipients resulted in amplification of Tregs in vivo: [00144] Kidney transplant recipients received alemtuzumab induction and this resulted in significant lymphodepletion (see Figure 9A). The lymphodepletion was important for the later effectiveness of Treg therapy. The subjects were begun on tacrolimus and mycophenolate-based immunosuppression and were converted from tacrolimus to sirolimus (rapamycin) at 30 days post-transplant (Figure 5) to provide an immunosuppressive milieu conducive to the survival of infused Tregs. By day +60 there was a recovery of the absolute numbers of NK cells, naïve B cells, Tregs (Figure 9A) and CD14⁺ monocytes (not shown) prior to Tregs infusion which was performed on day +60 post-transplant. Most importantly, Treg therapy resulted in 5-20 fold increase in the percentages of Tregs in all subjects and this increase remained stable in most patients until the end of the follow-up period of one year post-transplant (Figure 9B). [00145] Infusion of expanded Tregs into kidney transplant recipients was safe: [00146] There were no serious adverse events attributable to the Treg therapy in any subject (see Table 8). Protocol biopsies performed one month after Treg therapy have not shown rejection (NR) and no donor specific antibody (DSA) development was observed at the time point. At 1 year post-transplant, there was a subclinical rejection (subject #8) with C4d deposition and DSA in one patient due to non-compliance with his immunosuppression and this was successfully treated. Table 8

[00147] At one year post-op low titer DSA was also observed in the only African American subject (#1) of the study (African Americans have historically been shown to be highly reactive to the donor). There have been no infectious complications attributable to Treg infusion. Thus, the results from this phase I trial indicated that the foregoing Treg therapy is safe. [00148] Based upon these results, a dose of no less than 1 x10⁹ but no more than 5 x10⁹ cells for the Treg therapy is expected to be efficacious and safe. Example 6: Optimized Bead-free Expansion of Regulatory T cells [00149] Enriched populations of Treg cells obtained in Example No.1 are expanded in the present example to provide therapeutically relevant numbers of Treg cells in a bead-free expansion protocol according to an example aspect of the disclosure. [00150] Growth media (GM) was TexMACS medium (Miltenyi) supplemented with 10% heat-inactivated AB serum, 1,000 IU/ml IL-2 (Clinigen), and 1µg/ml TGF-β (Invitrogen). G- Rex^® bioreactor (Wilson Wolf) cultures were initiated with 5x10⁶ (G-Rex 10M) or 50x10⁶ (G- Rex 100M) cells and TransAct^® stimulation reagent (Miltenyi) at 60 µl/ml. GM, IL-2 and TGF- β are added every two to three days to maintain a cell density of 1x10⁶/ml. During days 0-9 the GM IL-2 and TGF-β are added with Everolimus (EVR, Novartis) at 100 ng/ml. On Day 11-12, the cells are restimulated with TransAct^® reagent at 60 µl/ml. On Day 14, in-process testing is performed for sterility, endotoxin detection, cell counts, viability, phenotyping, and the Treg suppression assay. EVR is not added after Day 9 and cells are harvested on Day 21. On Day 21, additional quality testing is performed, again for sterility, endotoxin detection, cell counts, viability, phenotyping, and the Treg suppression assay. The target Treg number at the end of the culture is 4-5x10⁹ and, if this number is not obtained, the culture is extended to another week to 28 days in total. This would ensure that sufficient cell numbers are obtained for Treg therapy. The expansion protocol is shown in Figure 10 and Tables 9 and 10 below. Table 9

Table 10

[00151] In the beadless protocol, Everolimus (EVR, Novartis) was found superior to Sirolimus (SRL, Pfizer) for its ability to inhibit the growth of effector T cells and to promote the expansion of Tregs and was chosen as the immunosuppressive agent to be added during the first nine days in this expansion protocol. Also, as described further herein, in some embodiments, Everolimus may be stopped after addition at one of Day 7-14. Example 7: Optimization of Cell Stimulation [00152] Aside from stimulating the Treg cultures on day 0, restimulation of the cultures for getting highest quantity and quality of the final product was optimized, and it was determined that restimulation at Day 11 resulted in improvement over day 7 restimulation. Figure 10A shows the difference in expansion in Tregs stimulation on Day 0 and restimulated on Day 7 or Day 11. Figure 10B shows the results of an MLR inhibition assay performed on day 14 or day 21 of the expansion protocol when the cultures were restimulated on either Day 7 or Day 14. Figure 10C shows the flow cytometric profile of Tregs expanded over 21 days when restimulated on Day 7 or on Day 11. Example 8: Selection of Concentration of Growth Media Supplements [00153] Differing concentrations and combinations of the growth factors IL-2 and TGF-β were evaluated. Figure 11 shows the fold expansion of culture when Tregs were supplemented with different combinations of TGF-β (1 ug/ml) and IL-2 at 1000U or 200U (vs.200U IL-2 alone). Example 9: Selection of Bioreactor Size and Starting Culture Volume for Treg Expansion [00154] In this experiment, the starting volume of the culture for maximal growth was evaluated. It was determined that 5x10⁶ for G-Rex^® 10M and 50x10⁶ for G-Rex^® 100M as the starting cell number was helpful. Figure 12 shows the fold expansion for the 5x10⁶ purified CD4+CD25+ cells were cultured in G-Rex 10M with starting volumes of either 5 mL or 50 mL using the bead-free expansion protocol as described herein. [00155] In addition, a frozen apheresis product was thawed and selected as described herein. Subsequently, the selected cells were separated into 1 G-Rex100M with 5x10⁷ cells and 3 G- Rex10M with 5x10⁶ cells per bioreactor. The same at-scale concentrations of growth media, mTor inhibitor, IL-2, and TGF-Beta were added as described herein. All flasks were restimulated on the same day (Day 11). Results are shown in Figure 13. Cell expansion in the G-Rex^®100 bioreactor provided more cells than the G-Rex^® 10 bioreactor. Example 10: Optimization of Expansion Duration [00156] In this experiment, the duration of the expansion culture was optimized. It was unexpectedly discovered that a dose of 5x10⁹ polyclonally expanded Tregs was safe in renal transplant recipients, so 5x10⁹ polyclonally expanded Tregs was chosen that as the target product number. In case the target of 5x10⁹ polyclonally expanded Tregs is not obtained by day 21, the culture is extended by another week to day 28. As shown in Figures 14A, B and C and Figure 15 the Tregs expanded to either 21 or 28 days had equivalent FOXP3 expression and in vitro suppression capabilities, both measures of their potency. [00157] In another experiment, MTR inhibit and 25+FoxP3+ expression was determined on human Treg expansion according to a method of the disclosure at day 21 and day 28. As shown in Figure 16 and Table 11, growth kinetics, including MLR inhibition and FoxP3+ expression were essentially the same in populations at day 21 in G-Rex 10 and G-Rex 100 and day 28 in G- Rex 100. Table 11

Example 11: Selection of Growth Media [00158] In this example, cell expansion in Ab serum growth media was compared to expansion with growth media supplemented with serum free supplement. The entire experiment was performed in G-Rex10 vessels according to the bead-free expansion protocol described herein. Three G-Rex10s (seeded at 5e6) contained Valley Biomedical AB Serum in the media and were compared to one Grex10 (seeded at 5e6) containing Cell-Vive™ T cell CD Serum Substitute. All other differences between the two conditions were minimized for a direct comparison. Figure 17 shows that cell expansion using serum-free supplement is both more consistent and better for Treg expansion. [00159] Serum-free supplement did not appear to have a growth delay within the first seven days of culture, leading to further expansion and better yield at harvest. Both conditions showed similar MLR inhibition (Figure 16) and similar phenotypes, including FoxP3 expressions (Table 12). Table 12

[00160] Having described the invention in detail and by reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. More specifically, although some aspects of the present invention are identified herein as particularly advantageous, it is contemplated that the present invention is not necessarily limited to these particular aspects of the invention. In some embodiment, percentages and amounts disclosed herein may vary in amount by ±10, 20, or 30%.

Claims

CLAIMS 1. A method for expanding a population of T regulatory (Treg) cells, comprising: (a) stimulating the population of Treg cells in a growth media, Interleukin-2 (IL-2), Transforming Growth Factor Beta (TGF-β), and an inhibitor of mammalian target of rapamycin (mTOR), and one or more beadless reagents comprising an anti-CD3+ antibody and anti-CD28+ antibody, and culturing the population for about two days; (b) adding fresh growth media, IL-2, TGF-β, and, optionally, the inhibitor of mTOR to the population about every two to three days; (c) after about 11 days, re-stimulating the population by adding additional growth media, IL-2, TGF-β, the beadless reagent, and optionally, the inhibitor of mTOR to the population; (d) adding fresh growth media, IL-2 and TGF-β to the population about every two to three days; and (e) after about 21 days, counting the population of Treg cells and harvesting the population of Treg cells when the population of Treg cells comprises more than about 500 million cells, wherein the harvested population comprises the expanded population of Treg cells.

2. The method of claim 1, wherein the about 11 days comprises 9 to 12 days.

3. The method of claim 1, wherein if fewer than about 500 million cells are present at step (e) and the method further comprises: (f) adding fresh growth media, IL-2 and TGF-β about every two to three days; and (g) on about Day 28 harvesting the expanded population of Treg cells.

4. The method of any of claims 1-3, wherein the one or more beadless reagents comprises a biodegradable surface.

5. The method of any of claims 1-3, wherein the one or more beadless reagents comprises a colloidal matrix.

6. The method of any of claims 1-5, wherein the population of Treg cells is a CD4⁺/CD25⁺ population of Treg cells that is isolated from an apheresis sample.

7. The method of claim 6, wherein the apheresis sample is a previously-frozen apheresis sample.

8. The method of any of claims 1-7, wherein, at least one of: (a) the IL-2 is added at about 500 to about 2,000 U/mL; (b) the TGF-β is added at about 0.1 to about 10 ng/mL; and (c) the inhibitor of mTOR concentration is added at about 10 to about 200 ng/mL.

9. The method of any of claims 1-8, wherein the inhibitor of mTOR is Everolimus.

10. A composition comprising at least about 500,000,000 cells of the expanded population of Treg cells produced by the method of any of claims 1-9.

11. A method for treating an organ transplant recipient, comprising administering to a patient the composition of claim 10.

12. The of method claim 11, wherein the organ transplant recipient is a kidney transplant recipient.

13. A method for expanding a population of T regulatory (Treg) cells, comprising: (a) on Day 0 stimulate the population of Treg cells in a growth media, Interleukin-2 (IL- 2), Transforming Growth Factor Beta (TGF-β), Everolimus, and comprising beadless reagent comprising an anti-CD3+ antibody and anti-CD28+ antibody, and culture the population for about two days; (b) on Day 2 add fresh growth media, IL-2, TGF-β, and Everolimus to the population, and culture the population for about three days; (c) on Day 5 add fresh growth media, IL-2, TGF-β, and Everolimus to the population, and culture the population for about two days; (d) on Day 7 add fresh growth media, IL-2, TGF-β, and Everolimus to the population, and culture the population for about two days; (e) on Day 9 add fresh growth media, IL-2, TGF-β and, optionally, Everolimus to the population, and culture the population for about three days; (f) on Day 12 re-stimulate the population by adding additional growth media, IL-2, TGF-β, the beadless reagent, and optionally, Everolimus to the population, and culture the population for about one day; (g) on Day 13 add fresh growth media, IL-2, and TGF-β to the population, and culture the population for about three days; (h) on Day 16 add fresh growth media, IL-2, and TGF-β to the population, and culture the population for about three days; (i) on Day 19 add fresh growth media, IL-2, and TGF-β to the population, and culture the population for about two days; and (j) on Day 21, count the population of cells and either (a) harvest the population when the population comprises more than about 500,000,000 cells, wherein the harvested population comprises the expanded population of Treg cells, or (b) continue to culture the population for about two days if the population has fewer than about 4 billion cells ; (k) on Day 23 add fresh growth media, IL-2, and TGF-β, and culture the population for about three days; (l) on Day 26 add fresh growth media, IL-2, and TGF-β, and culture the population for about one day; (m) on Day 27 add fresh growth media, IL-2, and TGF-β, and culture the population for about one day; (n) on Day 28 harvest the expanded population of Treg cells.

14. The method of claim 13, wherein the stimulating occurs in a 800-1200 ml vessel.

15. The method of claim 14, where the vessel comprises a gas permeable membrane.

16. The method of claim 13, wherein at least one of: (a) the IL-2 concentration is added at about 1,000 U/mL; (b) the TGF-β concentration is added at about 1 ng/mL; and (c) the inhibitor of mTOR concentration is added at about 100 ng/mL.

17. The method of any one of claims 13-16, wherein the beadless reagent comprises a biodegradable surface.

18. The method of any one of claims 13-16, wherein the beadless reagent comprises a colloidal matrix.

19. The method of any preceding claim wherein the growth media is supplemented with a serum free supplement.

20. The method of any of claims 1-9 or 13-19, wherein the population of Treg cells is expanded by at least 100 fold, or about 200 fold, or about 300 fold, or about 400 fold, or about 500 fold, or about 600 fold, or about 700 fold, or about 800 fold, or about 900 fold, or about 1,000 fold, or about 1,100 fold, or about 1,200 fold, or about 1,300 fold, or about 1,400 fold, or about 1,500 fold, or about 1,600 fold, or about 1,700 fold, or about 1,800 fold, or about 1,900 fold or about 2,000 fold, or more.