Bead-free ex-vivo expansion of human regulatory t cells

Abstract

To provide methods for the production of regulatory T cells (Tregs) for use in adoptive cell therapy, in particular, simplified approaches for the expansion of Tregs ex vivo.SOLUTION: Provided is a method for the production of human regulatory T cells (Tregs), comprising: a) isolating CD4+, CD25+, CD127- / low T cells from a lymphocyte-containing biological sample obtained from a human subject; and b) culturing the T cells in medium comprising a CD28 superagonist (CD28SA) antibody, interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNF-alpha) under conditions effective in producing human Tregs that are CD4+, FOXP3+, HELIOS+, and have a demethylated Treg-specific demethylation region (TSDR).SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 841,215, filed April 30, 2019, the disclosure of which is hereby incorporated by reference in its entirety.

[0002] Federally Sponsored Research or Development Claims none.

[0003] The present disclosure relates generally to the production of regulatory T cells (Tregs) for use in adoptive cell therapy. In particular, the present disclosure relates to a simplified procedure for the ex vivo expansion of Tregs. Tregs generated by this method are suitable for use in a variety of immunotherapy regimens. [Background technology]

[0004] Regulatory T cells (Tregs) are a small subpopulation of peripheral blood lymphocytes that are critical for regulating immune system tolerance, inflammation, and homeostasis. Treg abnormalities have been observed in association with dysregulated inflammation and diverse autoimmune diseases. Thus, Tregs are being developed as adoptive cell therapy to treat autoimmune and inflammatory diseases, graft-versus-host disease after bone marrow transplantation, and solid organ transplant rejection (Bluestone and Tang, Science, 362:154-155, 2018).

[0005] Current methods for producing Tregs for preclinical and clinical trials are diverse (Ruchs et al., Frontiers in Immunol, 8:1844, 2018). Most methods use conventional CD4 + T cells and CD8 +It relies on strong antigenic or mitogenic stimulation of purified Tregs using methods developed for the expansion of T cells. In particular, such methods use antibodies against CD3 and CD28 immobilized on beads, artificial antigen-presenting cells, or polymer scaffolds that strongly activate Tregs to expand the cells with the help of IL-2. Under such non-native conditions in vitro, Tregs are at risk of losing their identity and function. Thus, there is a need in the art for a method of producing Tregs that results in consistent and stable expansion of Tregs without negatively affecting their identity and function. Moreover, it is desirable to develop a simplified and adaptable protocol for Treg expansion to reduce the complexity of the cell production method and make the method more automatable while maintaining the Treg phenotype of the starting cell population. Summary of the Invention

[0006] The present disclosure relates generally to the production of regulatory T cells (Tregs) for use in adoptive cell therapy. In particular, the present disclosure relates to a simplified procedure for the ex vivo expansion of Tregs. Tregs generated by this method are suitable for use in a variety of immunotherapy regimens. [Brief description of the drawings]

[0007] [Figure 1] 1 is a graph showing the extent of expansion of human Tregs generated using a standard protocol including anti-CD3 and anti-CD28 monoclonal antibodies conjugated to magnetic beads compared to the bead-free protocol of the present disclosure described in Example 1. Abbreviations are as follows: BF1 = protocol including anti-CD28SA Ab and IL-2; BF2 = protocol including anti-CD28SA Ab, IL-2 and IL-6; BF3 = protocol including anti-CD28SA Ab, IL-2 and TNF-alpha; and BF4 = protocol including anti-CD28SA Ab, IL-2, IL-6 and TNF-alpha. [Diagram 2]Graph showing expression levels of Treg lineage markers FOXP3, HELIOS, and CD27 on human Tregs generated using the bead-free protocol of the present disclosure as described in Example 1. Tregs were harvested on day 14. Abbreviations are as described in FIG. [Diagram 3] 1 is a flow cytometry histogram showing expression levels of Treg lineage markers FOXP3, HELIOS, CD62L, and CD27 on human Tregs generated using the bead-free protocol of the present disclosure described in Example 1. Tregs were harvested on day 14. Abbreviations are as described in FIG. [Figure 4] 1 is a flow cytometry histogram showing expression levels of Treg lineage markers HELIOS and CD27 on human Tregs generated using the bead-free protocol of the present disclosure described in Example 1. Tregs were harvested on day 14. Abbreviations are as described in FIG. [Diagram 5] Graph showing the extent of expansion of human Tregs generated using a standard protocol including magnetic beads and anti-CD3 and anti-CD28 monoclonal antibodies compared to the BF4 protocol of the present disclosure. Tregs were harvested on day 14. [Figure 6] 1 is a flow cytometry histogram showing the expression levels of Treg lineage markers FOXP3 and HELIOS on human Tregs generated using a standard protocol including magnetic beads and anti-CD3 and anti-CD28 monoclonal antibodies compared to the BF4 protocol of the present disclosure. Tregs were harvested on day 14. [Figure 7] 1 is a flow cytometry histogram showing the expression levels of Treg lineage markers HELIOS and CD27 on human Tregs generated using a standard protocol including magnetic beads and anti-CD3 and anti-CD28 monoclonal antibodies compared to the BF4 protocol of the present disclosure. Tregs were harvested on day 14. [Figure 8A]Graph showing the level of suppression of pre-activated effector T cell (Teff) proliferation by human Tregs generated using a standard protocol including magnetic beads and anti-CD3 and anti-CD28 monoclonal antibodies compared to the BF4 protocol of the present disclosure. [Figure 8B] Graph showing the level of suppression of autologous peripheral blood mononuclear cell (PBMC) proliferation by human Tregs generated using a standard protocol including magnetic beads and anti-CD3 and anti-CD28 monoclonal antibodies compared to the BF4 protocol of the present disclosure. [Figure 9] Graph showing the level of suppression of effector T cell (Teff) proliferation in the presence and absence of tumor necrosis factor alpha by human Tregs generated using a standard protocol including magnetic beads and anti-CD3 and anti-CD28 monoclonal antibodies, compared to the BF4 protocol of the present disclosure. [Figure 10] Graph showing the proliferation levels of human Tregs generated using magnetic beads and two rounds of stimulation with anti-CD3 and anti-CD28 monoclonal antibodies in the presence of IL-1 (beads) compared to the BF10 protocol of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] The present disclosure relates generally to the production of regulatory T cells (Tregs) for use in adoptive cell therapy. In particular, the present disclosure relates to an alternative approach for ex vivo expansion of Tregs to traditional magnetic bead or feeder cell-based protocols. Tregs generated in this manner are suitable for use in a variety of immunotherapy regimens.

[0009] The present disclosure provides a method for the generation of human regulatory T cells (Tregs), comprising: a) isolating CD4+, CD25+, CD127- / low T cells from a lymphocyte-containing biological sample obtained from a human subject; and b) culturing the T cells in a medium comprising a CD28 superagonist (CD28SA) antibody, interleukin 2 (IL-2) and tumor necrosis factor alpha (TNFalpha) under conditions effective for the generation of human Tregs that are CD4+, FOXP3+, HELIOS+ and have demethylated Treg-specific demethylated regions (TSDRs). The disclosure further provides a method for the generation of human regulatory T cells (Tregs), comprising: a) isolating CD4+, CD25+, CD127- / low T cells from a lymphocyte-containing biological sample obtained from a human subject; and b) culturing the T cells in a medium comprising CD28SA antibody, IL-2, IL-6 and TNFalpha under conditions effective for the generation of human Tregs that are CD4+, FOXP3+, HELIOS+ and have demethylated Treg-specific demethylated regions (TSDRs). The present disclosure also provides a method for the generation of human regulatory T cells (Tregs), comprising: a) isolating CD4+, CD25+, CD127- / low T cells from a lymphocyte-containing biological sample obtained from a human subject; and b) culturing T cells in a medium containing CD28SA antibody, IL-2, IL-1beta and TNFalpha under conditions effective for the generation of human Tregs that are CD4+, FOXP3+, HELIOS+ and have demethylated Treg-specific demethylation regions (TSDRs). In a preferred embodiment, the human Tregs are CD3+, CD27+, CD62L+, CD8- and CD19-. Preferred stimulation conditions, including cell culture in the presence of IL-6, are referred to as BF4 and BF4a in the examples and figures. Preferred stimulation conditions, including cell culture in the presence of IL-1beta, are referred to as BF10 in the examples and figures.

[0010] It is believed that BF4 and BF10 conditions and their variants, which include T cell culture in media consisting of the same but different concentrations of cytokines, result in the generation of Treg populations with advantageous properties compared to Tregs generated under conditions in which anti-CD3 and anti-CD28 antibodies are immobilized using beads or artificial antigen-presenting cells. Without being bound by theory, it is believed that the immobilization of anti-CD3 and anti-CD28 antibodies is an excessively strong and non-physiological stimulus that causes instability of the Treg lineage and the acquisition of pro-inflammatory functions.

[0011] As used herein, the terms "CD28 superagonist antibody", "CD28SA antibody" and "superagonist anti-CD28 antibody" refer to CD28-specific monoclonal antibodies that can activate T cells in the absence of T cell receptor activators. Thus, in a preferred embodiment, step b) does not include the use of anti-CD3 antibody and / or does not include the use of magnetic beads or Fc receptor-expressing feeder cells that crosslink the CD28 and CD3 expressed on the surface of isolated T cells. In some embodiments, the medium further includes one or both of tumor necrosis factor receptor 2 agonist (TNFR2a) and interferon-gamma (IFN-gamma). In some embodiments, TNFR2a is an anti-TNFR2 antibody.

[0012] While conventional anti-CD28 monoclonal antibodies bind to the exposed F″G loop of CD28, CD28SA monoclonal antibodies have been found to bind to the exposed C″D loop of the immunoglobulin-like domain of CD28, which is critical for B7 binding (Luhder et al., J ExpMed, 197:955-966, 2003). Exemplary CD28SA antibodies suitable for use in the methods of the present disclosure include, but are not limited to, ceralizumab (also known as TAB08, formerly known as TGN1412), developed by TheraMAB LLC (Moscow, Russia), and ANC28.1, sold by AncellCorp (Bayport, MN). The amino acid sequences of the variable regions of TGN1412 and its variants are described in U.S. Pat. No. 8,709,414.

[0013] The bead-free method of the present disclosure can be used in combination with antigen-specific expansion or selection of Treg to generate antigen-specific Treg.For example, the method for generating human regulatory T cells (Treg) can further include isolating antigen-specific T cells by staining with major histocompatibility complex (MHC) class II peptide multimers and / or culturing T cells in the presence of MHC class II peptide multimers in the presence of IL-2 before step b).Methods for antigen-specific expansion utilizing MHC class II peptide multimers and methods for adoptive transfer of Tregs are described in US Patent No. 7,722,862.

[0014] Alternatively, the method for generating human regulatory T cells (Tregs) may further comprise culturing the T cells in the presence of allogeneic stimulated B cells (sBc) in the presence of IL-2 prior to and / or during step b). In some embodiments, the T cells comprise a mismatch in HLA-DR associated with the allogeneic sBc. Methods for antigen-specific expansion and adoptive transfer utilizing allogeneic sBc are described in U.S. Pat. No. 9,801,911, the examples of which are incorporated herein by reference.

[0015] The disclosed method may further comprise a step c) of harvesting human Tregs, which in some embodiments begins 7-18 days after the initiation of step b). In some embodiments, step c) begins a minimum of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 days after the initiation of step b) and / or a maximum of 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, or 8 days after the initiation of step b). The disclosed method may further comprise a step c) of harvesting human Tregs, which in some embodiments begins 11-18 days after the initiation of step b). In some embodiments, step c) begins a minimum of 11, 12, 13, 14, 15, 16, or 17 days after the initiation of step b) and / or a maximum of 18, 17, 16, 15, 14, 13, or 12 days after the initiation of step b). The disclosed method is suitable for about 200 to about 2000-fold expansion of human Tregs. In preferred embodiments, the method results in the generation of at least 200, 600, 1000, 1400 or 1800 times more human Tregs than those present at the start of step a). In some embodiments, the expression levels of various markers by human Tregs are assessed by flow cytometry on the day of harvest. Markers assessed may include, but are not limited to, CD4, CD25, FOXP3, HELIOS, CD27, CD62L and CD8. Tregs are positive for CD4, CD25, FOXP3, HELIOS, CD27, CD62L and negative for CD8. TSDR demethylation is also quantified using methylation-specific PCR or pyrosequencing after bisulfide conversion. A high percentage of TSDR demethylation indicates that the generated cells are a stable Treg lineage.

[0016] References and claims to methods for treating or preventing a pathological immune response in a human subject in need thereof comprising administering to the subject human Tregs generated using the production methods of the present disclosure, in their general and specific forms, include: a) the use of human Tregs for the manufacture of a medicament for the treatment or prevention of a pathological immune response; and b) Pharmaceutical compositions comprising human Tregs for the treatment or prevention of pathological immune responses Similarly,

[0017] As used herein, the term "pathological immune response" includes autoimmune disease, autoinflammatory disease, allograft rejection and graft-versus-host disease. "Autoimmune disease" includes immune recognition that causes direct injury and dysfunction to self-tissue. Pathologically, autoimmune disease is typically caused by cells of the adaptive immune system. Autoimmune diseases include, but are not limited to, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, pemphigus, psoriasis, type I diabetes, celiac disease and Sjogren's syndrome. "Autoinflammatory disease" includes spontaneous activation or overreaction of the immune system to non-self antigens (e.g., environmental, dietary, commensal or other antigens) that cause indirect (bystander) injury and dysfunction to self-tissue. Pathologically, autoinflammatory disease is typically affected by cells of the innate immune system. Examples of autoinflammatory diseases include, but are not limited to, inflammatory bowel disease, amyotrophic lateral sclerosis and other neurodegenerative diseases, allergic airway disease, and chronic obstructive pulmonary disease.

[0018] The present disclosure further provides a pharmaceutical composition comprising human Tregs and a physiologically acceptable buffer, such as saline or phosphate buffered saline. An effective amount of the pharmaceutical composition for adoptive cell therapy is 10 7 ~10 11 (10 million to 100 billion) human Tregs (see, e.g., Tang and Lee, CurrOpinOrganTransplant, 17:349-354, 2012). In some examples, human Tregs are administered either locally to the affected tissue (e.g., by intra-articular injection into the affected joint when treating rheumatoid arthritis) or systemically (e.g., by intravenous injection when treating systemic lupus erythematosus). In some embodiments, Tregs are administered as either a single injection or multiple injections for better engraftment and sustained action. For local injection, 10 7 ~109 For systemic injection, this may involve administration of 10 9 ~10 11 In the treatment or prevention of solid organ transplantation, the Treg may comprise 10 9 ~10 11 in the treatment or prevention of graft-versus-host disease, may involve the administration of 10 10 ~10 11 This may include administration of Tregs.

[0019] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless otherwise indicated. For example, "an" excipient includes one or more excipients.

[0020] The phrase "comprising," as used herein, is open-ended and indicates that such embodiments may include additional elements. In contrast, the phrase "consisting of" is restrictive and indicates that such embodiments do not include additional elements (except for trace impurities). The phrase "consisting essentially of" is partially restrictive and indicates that such embodiments may include additional elements that do not substantially alter the basic properties of such embodiments. It is understood that aspects and embodiments described herein as "comprising" include "consisting of" and "consisting essentially of" embodiments.

[0021] The term "about" as used herein with respect to a value encompasses 90% to 110% of that value (eg, about 200-fold refers to 180-fold to 220-fold, inclusive).

[0022] An "effective amount" of an agent disclosed herein is an amount sufficient to perform a recited purpose. An "effective amount" can be empirically determined in relation to the claimed purpose. An "effective amount" or "sufficient amount" of an agent is an amount appropriate to affect a desired biological effect, e.g., a beneficial outcome, including a beneficial clinical outcome. The term "therapeutically effective amount" refers to an amount of an agent (e.g., human Treg) effective to "treat" a disease or disorder in a subject (e.g., a mammal, e.g., a human). An "effective amount" or "sufficient amount" of an agent can be administered in one or more doses.

[0023] The term "treating" or "treatment" of a disease refers to the implementation of a protocol that may include administering one or more drugs to an individual (human or otherwise) in an attempt to alleviate the signs or symptoms of the disease. Thus, "treating" or "treatment" includes protocols that do not require complete alleviation of signs or symptoms, do not require a cure, and in particular have only a palliative effect on an individual. As used herein, and as is well understood in the art, "treatment" is an approach to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms, reduction in the extent of the disease, stabilization (i.e., not worsening) of the disease state, prevention of the spread of the disease, delay or slowing of disease progression, improvement or palliative of the disease state, and remission. "Treatment" may also mean the prolongation of survival of an allogeneic transplant recipient as compared to the expected survival of an allogeneic transplant recipient not receiving treatment. "Palliation" of a disease or disorder means a lessening of the severity and / or undesirable clinical symptoms of the disease or disorder and / or a slowing of the time course of progression of the disease or disorder compared to the expected outcome without treatment.

[0024] Enumeration of embodiments In the embodiments below, any reference to Embodiment 1 includes one or both of Embodiment 1A and Embodiment 1B.

[0025] 1A. A method for the generation of human regulatory T cells (Tregs), comprising: a) isolating CD4+, CD25+, CD127- / low T cells from a lymphocyte-containing biological sample obtained from a human subject; and b) culturing T cells in medium containing CD28 superagonist (CD28SA) antibody, interleukin 2 (IL-2), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFalpha) under conditions effective to generate human Tregs that are CD4+, FOXP3+, HELIOS+ and have demethylated Treg-specific demethylation regions (TSDRs); and optionally wherein the human Tregs are CD62L+ and TNFR2+.

[0026] 1B. A method for the generation of human regulatory T cells (Tregs), comprising: a) isolating CD4+, CD25+, CD127- / low T cells from a lymphocyte-containing biological sample obtained from a human subject; and b) culturing T cells in medium containing CD28 superagonist (CD28SA) antibody, interleukin 2 (IL-2) and tumor necrosis factor alpha (TNFalpha) under conditions effective to generate human Tregs that are CD4+, FOXP3+, HELIOS+ and have demethylated Treg-specific demethylation regions (TSDRs); 1. A method for the generation of human Tregs, comprising:

[0027] 2. The method of embodiment 1, wherein step b) does not include the use of an anti-CD3 antibody.

[0028] 3. The method of embodiment 1 or embodiment 2, wherein step b) does not involve the use of magnetic beads or Fc receptor-expressing feeder cells that crosslink CD28 and CD3 of the isolated T cells.

[0029] 4. The method of any one of embodiments 1 to 3, wherein the culture medium further comprises one or both of a tumor necrosis factor receptor 2 agonist (TNFR2a) and interferon-gamma (IFN-gamma), optionally wherein TNFR2a is an anti-TNFR2 antibody.

[0030] 5. The method of any one of embodiments 1B to 4, wherein the medium further comprises one or both of IL-6 and IL-1 beta, optionally wherein the medium further comprises IL-1 beta but not IL-6, optionally wherein the medium further comprises IL-6, IL-1 beta but not IL-1 beta.

[0031] 6. The method of any one of embodiments 1 to 5, wherein the lymphocyte-containing biological sample is selected from the group consisting of whole blood, leukoreduced transfusion products and peripheral blood mononuclear cells (PBMCs), and optionally the biological sample is fresh or cryopreserved after being obtained from the human subject and then thawed prior to step a).

[0032] 7. The method according to any one of the preceding claims, wherein the CD4+, CD25+, CD127- / low T cells of step a) are isolated from the biological sample by fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS).

[0033] 8. The method of any one of embodiments 1 to 7, further comprising a step c) of recovering human Tregs.

[0034] 9. The method of embodiment 8, wherein step c) begins 7 to 18 days after the initiation of step b), and optionally, step c) begins 11 to 18 days after the initiation of step b).

[0035] 10. The method of embodiment 9, wherein the human Tregs comprise about 200 to about 2000 times more CD4+, CD25+, CD127- / low T cells than at the start of step a).

[0036] 11. 10 produced using the method according to any one of embodiments 1 to 107 ~10 11 A pharmaceutical composition comprising human Tregs and a physiologically acceptable buffer.

[0037] 12. A method for treating or preventing a pathological immune response in a human subject in need thereof, comprising administering to the human subject an effective amount of the pharmaceutical composition of embodiment 11, optionally wherein the effective amount of the pharmaceutical composition is greater than or equal to 10 7 ~10 11 human Tregs, which are infused intravenously into a human subject over a period of 20 to 40 minutes.

[0038] 13. The method of embodiment 12, wherein the pathological immune response is an autoimmune or autoinflammatory disease.

[0039] 14. The method of embodiment 13, wherein the autoimmune or autoinflammatory disease is selected from the group consisting of rheumatoid arthritis, multiple sclerosis, amyotrophic lateral sclerosis, systemic lupus erythematosus, pemphigus, psoriasis, type I diabetes, celiac disease and inflammatory bowel disease, and optionally, the autoimmune or autoinflammatory disease is an inflammatory bowel disease selected from the group consisting of ulcerative colitis and Crohn's disease.

[0040] 15. The method of embodiment 13 or 14, which is effective in reducing symptoms or inhibiting the progression of an autoimmune or autoinflammatory disease, optionally wherein the inhibition of the progression of an autoimmune or autoinflammatory disease includes the inhibition of tissue destruction.

[0041] 16. The method of embodiment 12, wherein the pathological immune response is a rejection of a hematopoietic allograft or solid organ allograft.

[0042] 17. The method of embodiment 16, wherein the pathological immune response is a rejection reaction of a hematopoietic allograft, and the hematopoietic allograft is a bone marrow transplant or a peripheral blood stem cell transplant.

[0043] 18. The method of embodiment 16, wherein the pathological immune response is a rejection of a solid organ allograft, and the solid organ allograft is selected from the group consisting of heart, lung, heart / lung, kidney, pancreas, kidney / pancreas, liver, intestine, pancreatic islet and skin allografts.

[0044] 19. The method of embodiment 16, which is effective in reducing symptoms of acute and / or chronic rejection or in prolonging survival of an organ allograft.

[0045] 20. The method of embodiment 12, wherein the pathological immune response is graft-versus-host disease (GvHD).

[0046] 21. The method of embodiment 20, which is effective in reducing symptoms of acute and / or chronic GvHD or in inhibiting injury to the host's skin, liver, lungs and / or intestines.

[0047] 22. The method of embodiment 12, wherein the method is effective in increasing the percentage of Tregs above baseline in a human subject.

[0048] 23. A method for inhibiting the proliferation of human effector T cells (Teff), comprising contacting human Tregs generated using the method of any one of embodiments 1 to 10 with human CD4+, CD25-, CD127+ Teff under conditions effective to inhibit proliferation of Teff, optionally wherein the contacting is performed in the presence of TNF-alpha.

[0049] 24. The method or composition of any one of embodiments 1 to 23, wherein the method for the generation of human Tregs is compliant with Good Manufacturing Practice (GMP). EXAMPLES

[0050] The present disclosure will be described in further detail in the following examples, which are not intended to limit the scope of the present disclosure in any way.The attached figures are meant to be considered as an integral part of the specification and description of the present disclosure.The following examples are provided for illustration, but not for the limitation of the present disclosure.

[0051] In the experimental disclosure below, the following abbreviations are applied: Ab (antibody); allo (allogeneic); BF (bead-free); CD28 superagonist (CD28SA); FACS (fluorescence-activated cell sorting); IL-1β (interleukin 1 beta); IL-2 (interleukin 2); IL-6 (interleukin 6); IFNγ (interferon-gamma); PBMC (peripheral blood mononuclear cells); Teff (effector T cells); TNFα (tumor necrosis factor alpha); TNF receptor II agonist antibody (TNFR2a); Treg (regulatory T cells); TSDR (Treg-specific demethylated region); and UCSF (University of California, San Francisco).

[0052] Example 1 Development of a bead-free method for generating regulatory T cells (Treg) This example describes the development of a bead-free method for ex vivo expansion of human Tregs.

[0053] Isolation of Tregs. Human peripheral blood mononuclear cells were isolated from peripheral blood samples using a Ficoll density gradient, washed twice, and stained with antibodies against CD4 (anti-CD4PerCP, clone SK3, BD Biosciences, Cat. No. 347324), CD25 (anti-CD25APC, clone 2A3, BD Biosciences, Cat. No. 340939), and CD127 (anti-CD127PE, clone HIL-7R-M21, BD Biosciences, Cat. No. 557938). CD4+CD25highCD127- / low Tregs were isolated by fluorescence-activated cell sorting (FACS).

[0054] Ex-vivo Treg expansion. 1×10 5 CD4+CD25+CD127- / low Tregs were seeded in 500 ml of T cell medium (RPMI with 5% FBS, penicillin / streptomycin, HEPES, sodium pyruvate, glutamax and non-essential amino acids) in a single well of a 48-well plate. Alternatively, use X-VIVO15 with human AB serum. T cells were stimulated with either 1-10 μg / mL of CD28SA Ab (ANC28.1, clone 5D10, Ancell Corp., Cat. No. 177-020) or magnetizable polymeric beads conjugated with anti-CD3 and anti-CD28 antibodies (anti-CD3 / CD28 beads) at a bead-to-cell ratio of 1:1. Anti-CD3 / CD28 beads are Dynabeads™ Human T-Activator CD3 / CD28 for T cell proliferation and activation (ThermoFisher Scientific, Cat. No. 111.31D). The test conditions without beads (BF) are shown in Table 1-1. Cells were supplemented with fresh medium on days 2, 5, 7, 9, 11 and 13. 300 IU / mL human recombinant IL-2 was supplemented on days 0, 2, 5, 7, 9, 11 and 13. 15, 50 and 150 ng / mL human recombinant IL-6 (Peprotech, Catalog No. 200-06) were supplemented on days 0, 2 and 5. 50 ng / mL human recombinant TNFα (Peprotech, Catalog No. 300-01A) was supplemented on days 0, 2 and 5. 2.5 μg / mL TNFR2a (clone MR2-1, HycultBiotech, Catalog No. HM2007-FS) was supplemented on days 0, 2 and 5. 40 ng / mL human recombinant IFNγ (Peprotech, Cat. No. 300-02) was supplemented on days 0, 2 and 5. 50 ng / mL human recombinant IL-1β (Peprotech, Cat. No. 200-01B) was supplemented on days 0, 2 and 5. Cells were counted on days 5, 7, 9, 11, 13 and 14 and harvested and analyzed on day 14. TIFF2025072363000002.tif89170

[0055] Flow cytometry. Ex-vivo expanded Tregs were cultured at 1 × 10 5 Cell-containing samples were harvested on day 14 of culture and immunophenotyped by staining with antibodies against CD4, CD27, FOXP3, and HELIOS.

[0056] Analysis of Treg-specific demethylated regions (TSDRs). Ex-vivo expanded Tregs were 5×10 5 Cell-containing samples were harvested on day 14 of culture, and methylation of the FOXP3 locus was assessed by pyrosequencing.

[0057] In-vitro suppression assays. Ex-vivo expanded Tregs cultured under various conditions (as above) were harvested, washed twice and then co-cultured with either pre-activated Teffs or autologous PBMCs. CD4+CD25lowCD127+ T cells isolated by FACS from PBMCs were stimulated with anti-CD3 / CD28 beads at a cell-to-bead ratio of 1:1. Fresh cell culture medium was added on days 2, 5, 7, 9, 11, 13 and 15 (or 2, 5 and 7) to obtain pre-activated Teff populations. PBMCs were cryopreserved and used after thawing. In vitro suppression assays were set up with 50,000 cells of pre-activated Teffs or PBMCs and various ratios of Tregs. In some assays, 50ng / ml TNFα was added to the co-culture wells. Tritiated thymidine was added on day 4 of co-culture for the last 16-18 hours and cell proliferation was determined by measuring tritiated thymidine incorporation.

[0058] result BF1 and BF1a conditions were compared with standard anti-CD3 / CD28 bead conditions in the presence or absence of IL-2. It was found that the proliferation of Tregs by stimulation with CD28 superagonist (CD28SA) Ab was dependent on the concentration of CD28SA Ab and the presence of IL-2. Briefly, better proliferation of Tregs was observed in the presence of 4 μg / ml of CD28SA Ab instead of 2 μg / ml. In addition, both BF1 and BF1a conditions resulted in better and longer-lasting Treg proliferation than applying standard anti-CD3 / CD28 bead conditions. Microscopic images taken on the fifth day of culture showed strong activation of Tregs by CD28SA Ab in the presence of IL-2 and a complete absence of activation-related cells clustering in the absence of IL-2. In contrast, anti-CD3 / CD28 beads activated Tregs in both the presence and absence of IL-2.

[0059] Three different T cell populations were isolated by FACS and stimulated for 7 days under BF1 or standard anti-CD3 / CD28 bead conditions. Microscopic images taken on day 7 of culture showed that CD28SA Ab selectively activated CD4+CD25+CD127- / low Treg over CD4+CD25-CD127high T effector cells (Teff) and CD8+ T cells. Selective activation of Treg was not observed when anti-CD3 / CD28 beads were utilized.

[0060] BF1 and BF2 conditions were compared to standard anti-CD3 / CD28 bead conditions. Ex vivo expansion rates of CD28SA Ab-stimulated Tregs were not found to be significantly affected by the addition of IL-6 to the cultures, and both BF1 and BF2 rates were higher than those observed with bead stimulation.

[0061] BF1 and BF3 conditions were compared to standard anti-CD3 / CD28 bead conditions. Ex vivo expansion rates of CD28SA Ab-stimulated Tregs were not found to be significantly affected by the addition of TNFα to the cultures, with both BF1 and BF3 rates being higher than those observed with bead stimulation.

[0062] BF1 and BF4 conditions were compared with standard anti-CD3 / CD28 bead conditions. The ex vivo expansion rate of CD28SA Ab-stimulated Tregs was improved by the addition of IL-6 and TNFα to the cultures. Microscopic images of bead-stimulated and BF4-stimulated Tregs on day 5 of culture showed large cell cluster formation in BF4 conditions, indicating strong activation and proliferation of Tregs. In addition, the ex-vivo expansion of CD28SA Ab-stimulated Tregs exposed to IL-6 and TNFα was found to be long-term and stable. This is advantageous for eliminating the need to restimulate Tregs and risk Treg destabilization.

[0063] BF4, BF4a and BF4b conditions were compared to standard anti-CD3 / CD28 bead conditions. IL-6 was found to enhance Treg proliferation over a wide range of concentrations (15, 50 or 150 ng / ml) by cells isolated from peripheral blood of three different human donors (a 50 year old female, a 21 year old male and a 33 year old male).

[0064] BF1 and BF6 conditions were compared to standard anti-CD3 / CD28 bead conditions. Ex vivo expansion rates of CD28SA Ab-stimulated Tregs were improved by the addition of IL-6 and TNFR2a to the cultures.

[0065] A comparison of ex vivo expansion of Tregs under BF1, BF2, BF3, BF4 and standard anti-CD3 / CD28 bead conditions is shown in Figure 1. A larger comparison of the total ex vivo expansion of Tregs after 14 days of culture is shown in Tables 1-2. TIFF2025072363000003.tif98170

[0066] BF8 and BF9 conditions were compared to standard anti-CD3 / CD28 bead conditions. Ex vivo expansion rates of CD28SA Ab-stimulated Tregs were improved by the addition of IL-6 and / or IFNγ to the cultures.

[0067] BF10 conditions were compared with standard anti-CD3 / CD28 bead conditions. The ex vivo expansion rate of CD28SA Ab stimulated Tregs was improved by the addition of both TNFα and IL-1β to the cultures. In addition, in the presence of CD28SA Ab and IL-2 and in the absence of TNFα and IL-1β, 62% of the Treg population generated under BF10 conditions was TNFR2+, CD25+, compared with 47% of the Treg population generated under BF1 conditions. Interestingly, Tregs generated under BF10 conditions expressed higher levels of CD71 than Tregs generated under BF1 conditions. CD71 is a transferrin receptor, which is upregulated in activated T cells, signifying cells that have entered a proteobolic state that contributes to proliferation.

[0068] As shown in Figure 2, ex vivo Treg expansion by stimulation with CD28SA Ab in the presence of proinflammatory cytokines results in a cell population with high expression of Treg lineage markers FOXP3, HELIOS and CD27. In addition, the expanded cell population is highly demethylated at the TSDR. A comparison of the phenotype of ex vivo expanded Tregs under BF1, BF2, BF3 and BF4 stimulation conditions is shown in Figures 3 and 4. Treg expansion under BF4 conditions resulted in the generation of over 1000-fold more cells than those present at the start of stimulation (day 0), whereas the extent of Treg expansion under standard anti-CD3 / CD28 bead conditions was much lower, as shown in Figure 5. Similarly, Treg expansion under BF10 conditions resulted in the generation of much more cells than expansion under standard anti-CD3 / CD28 bead conditions, as shown in Figure 10. A comparison of the phenotype of ex vivo expanded Tregs under BF4 and standard anti-CD3 / CD28 bead conditions is shown in FIG. 6 and FIG.

[0069] Ex vivo expansion of Tregs by stimulation with CD28SA Ab in the presence of proinflammatory cytokines under BF4 stimulation conditions results in a cell population with high suppressive capacity against preactivated Teffs and autologous PBMCs, as shown in Figures 8A and 8B. In addition, Tregs expanded ex vivo under BF4 stimulation conditions are more potent suppressors of Teff proliferation in the presence of the proinflammatory cytokine TNFalpha than Tregs expanded ex vivo under standard anti-CD3 / CD28 bead conditions, as shown in Figure 9.

[0070] Moreover, ex vivo expansion of Tregs by stimulation with CD28SA Ab in the presence of proinflammatory cytokines does not increase the frequency of Tregs producing the proinflammatory cytokines IL-2, IL-17, IFN-gamma, and IL-4.

Claims

1. 1. A method for the generation of human regulatory T cells (Tregs), comprising: a) isolating CD4+, CD25+, CD127- / low T cells from a lymphocyte-containing biological sample obtained from a human subject; b) culturing T cells in medium containing a CD28 superagonist (CD28SA) antibody, interleukin 2 (IL-2) and tumor necrosis factor alpha (TNFalpha) under conditions effective to generate human Tregs that are CD4+, FOXP3+, HELIOS+ and have demethylated Treg-specific demethylation regions (TSDRs); A method comprising:

2. 2. The method of claim 1, wherein step b) does not include the use of an anti-CD3 antibody.

3. 3. The method of claim 2, wherein step b) does not involve the use of magnetic beads or Fc receptor-expressing feeder cells to crosslink CD28 and CD3 of the isolated T cells.

4. The method of claim 3, wherein the medium further comprises one or both of a tumor necrosis factor receptor 2 agonist (TNFR2a) and interferon-gamma (IFN-gamma).

5. The method of claim 3, wherein the medium further comprises one or both of IL-6 and IL-1 beta.

6. 2. The method of claim 1, wherein the lymphocyte-containing biological sample is selected from the group consisting of whole blood, leukoreduced transfusion products and peripheral blood mononuclear cells (PBMCs).

7. 6. The method of claim 5, wherein the biological sample is fresh or has been cryopreserved after being obtained from the human subject and then thawed prior to step a).

8. The method of claim 1, wherein the CD4+, CD25+, CD127- / low T cells of step a) are isolated from the biological sample by fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS).

9. The method of any one of claims 1 to 7, further comprising a step c) of harvesting human Tregs 7 to 18 days after the initiation of step b).

10. 10. The method of claim 9, wherein the human Tregs comprise about 200 to about 2000 times more CD4+, CD25+, CD127- / low T cells than at the start of step a).

11. 10. A method according to claim 1, ７ ~10 １１ 23. A pharmaceutical composition comprising human Tregs and a physiologically acceptable buffer.

12. 12. The pharmaceutical composition of claim 11 for use in the treatment or prevention of a pathological immune response in a human subject in need thereof.

13. The pharmaceutical composition for use according to claim 12, wherein the pathological immune response is an autoimmune or autoinflammatory disease.

14. 14. The pharmaceutical composition for use according to claim 13, wherein the autoimmune or autoinflammatory disease is selected from the group consisting of rheumatoid arthritis, multiple sclerosis, amyotrophic lateral sclerosis, systemic lupus erythematosus, pemphigus, psoriasis, type I diabetes, celiac disease and inflammatory bowel disease.

15. 14. The pharmaceutical composition for use according to claim 13, which is effective in reducing symptoms or inhibiting the progression of an autoimmune or autoinflammatory disease, optionally wherein the inhibition of the progression of an autoimmune or autoinflammatory disease includes the inhibition of tissue destruction.

16. 13. The pharmaceutical composition for use according to claim 12, wherein the pathological immune response is a rejection reaction of a hematopoietic allograft or a solid organ allograft.

17. 17. The pharmaceutical composition for use according to claim 16, wherein the pathological immune response is a rejection reaction of a hematopoietic allograft, and the hematopoietic allograft is a bone marrow transplant or a peripheral blood stem cell transplant.

18. 17. The pharmaceutical composition for use according to claim 16, wherein the pathological immune response is a rejection reaction of a solid organ allograft, and the solid organ allograft is selected from the group consisting of heart, lung, heart / lung, kidney, pancreas, kidney / pancreas, liver, intestine, pancreatic islet and skin allografts.

19. 17. A pharmaceutical composition for use according to claim 16, which is effective in alleviating the symptoms of acute and / or chronic rejection or in prolonging the survival of an organ allograft.

20. 13. The pharmaceutical composition for use according to claim 12, wherein the pathological immune response is graft-versus-host disease (GvHD).

21. 21. A pharmaceutical composition for use according to claim 20, which is effective in alleviating symptoms of acute and / or chronic GvHD or is effective in inhibiting damage to the skin, liver, lungs and / or intestines of the host.

22. 13. The pharmaceutical composition for use according to claim 12, which is effective in increasing the proportion of Tregs above baseline in a human subject.

23. 11. A method for inhibiting the proliferation of human effector T cells (Teffs), comprising contacting human CD4+, CD25-, CD127+ Teffs with human Tregs generated using the method of any one of claims 1 to 10 under conditions effective in inhibiting the proliferation of Teffs, optionally wherein the contacting is performed in the presence of TNF alpha.

24. The method of any one of claims 1 to 10, wherein the method for the generation of human Tregs is Good Manufacturing Practice (GMP) compliant.

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