Cell therapy methods using regulatory cells stimulated with interleukin-33 and interleukin-2

Abstract

Disclosed herein are ex vivo activated and culture-expanded regulatory cells or effector cells that have been stimulated with IL- 2 and IL-33 or variants thereof, IL-2 and IL- 33 fusion proteins (IL-233) or variants thereof for the prevention or suppression of immune reactions in a host, including autoimmune responses. The resulting ex vivo culture-expanded regulatory cells can be used for therapeutic uses, including the preventing, suppressing, blocking or inhibiting rejection of transplanted tissue in a human or other animal host, or protecting against graft vs host disease (GVHD). The resulting ex vivo culture-expanded effector cells can be used for anti-cancer / anti-tumor therapeutic uses. Also provided are therapeutic and immunosuppressive methods utilizing the ex vivo culture-expanded regulatory cells or effector cells for human treatment.

Description

CELL THERAPY METHODS USING REGULATORY CELLS STIMULATEDWITH INTERLEUKIN-33 AND INTERLEUKIN-2CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application NO. 63 / 520,853 filed on August 21, 2023, entitled “ cell therapy methods using regulatory cells stimulated with Interleukin-33 and Interleukin-2”; the contents of which are incorporated herein by reference in their entirety.CROSS REFERENCE TO SEQUENCE LISTING

[0002] The present application is filed along with a Sequence Listing submitted electronically in XML format. The Sequence Listing file named SLB-005W01_SL created on August 20, 2024, is 17.1KB in size; the content of which is incorporated herein by reference for all purpose.BACKGROUND

[0003] Interleukin-2 (IL-2) is a cytokine which plays a role in maintenance of immune tolerance; regulation of T-helper (Th) cell function, especially Th2 function; the differentiation, survival, and function of Foxp3+CD4+regulatory T (Treg) cells; and interacts with innate lymphoid cells (ILCs).

[0004] Interleukin-33 (IL-33) is a cytokine, which in its wild type form contains a nuclear localization signal at its N-terminus. Protease cleavage yields an 18 kDa C-terminal fragment, which may be referred to herein as truncated IL-33. Truncated IL-33 can stimulate innate lymphoid cells (ILCs), Th2 cells, Treg cells, macrophages, dendritic cells and others, and thereby upregulate the expression of IL-2, IL-4, IL-5, and IL-13, and reduce pro- inflammatory Thl and Th 17 responses.

[0005] Fusion proteins comprising IL-2 domains and truncated IL-33 domains have been described in the art. See, for example, WO2018 / 140504, US2019 / 0365714, US 9,840,545; US 10,851,145; and US 20210261640; the disclosures of which are hereby incorporated by reference. Such fusion proteins can treat diseases such as autoimmune diseases and disorders, inflammation, etc. However, a number of such fusion proteins have been observed to haveone or more undesirable properties, such as aggregation, fast clearance in vivo, or loss of IL- 33 activity over time.

[0006] Accordingly, there is a need for fusion proteins comprising IL-2 domains and truncated IL-33 domains without one or more undesirable properties. IL-33 loses its activity due to spontaneous oxidation of certain residues. There is also a need for such fusion proteins to be easily expressed by recombinant organisms, easily purified, and have both IL-2 and IL-33 activity and stability comparable to wild type IL-2 and wild-type IL-33, respectively.SUMMARY

[0007] The present disclosure relates to regulatory and effector immune cell based cell therapy, for example, for immunomodulation, immunosuppression, treatment of autoimmune disorders, inflammation, cancer and other disorders. In particular, the present invention relates to activation and expansion of regulatory T cells (Treg) using IL-33, IL-2 and IL-33 fusion protein, or combination of IL-2 and IL-33. The present disclosure provides, among other things, compositions of Treg cells, methods of activating and expanding Treg cells and uses thereof.

[0008] In one aspect, the present disclosure provides methods of suppressing inflammation in a patient in need thereof comprising administering to the patient activated, expanded regulatory cells, wherein the culture-expanded regulatory cells are stimulated with IL-33, a combination of IL-2 and IL-33, or IL233 (SLT518) or variants thereof.

[0009] In another aspect, the disclosure provides methods of treating autoimmune disease in a patient in need thereof comprising administering to the patient activated, culture-expanded regulatory cells, wherein the culture-expanded regulatory cells are stimulated with IL-33, a combination of IL-2 and IL-33, or IL233 (SLT518) or variants thereof.

[0010] The expanded regulatory cells can be ex vivo culture-expanded regulatory cells. The expanded regulatory cells can be expanded in vitro.

[0011] The autoimmune disease can be type-1 diabetes, SLE (systemic lupus erythematosus), lupus nephritis, inflammatory bowel disease, Sjogren syndrome, Rheumatoid arthritis, or multiple sclerosis.

[0012] In another aspect, the present disclosure relates to methods of suppressing allograft rejection or graft survival in a patient in need thereof comprising administering to the patient activated, culture-expanded regulatory cells, wherein the culture-expanded regulatory cellsare stimulated with IL-33, a combination of IL-2 and IL-33, or IL233 (SLT518) or variants thereof. The allograft rejection can be acute allograft rejection. The allograft rejection can be chronic allograft rejection.

[0013] In another aspect, the present disclosure relates to a method of treating or preventing GvHD in a patient in need thereof comprising administering to the patient activated, ex vivo culture-expanded regulatory cells, wherein the culture-expanded regulatory cells are stimulated with IL-33, a combination of IL-2 and IL-33, or an IL233 fusion (e.g., SLT518) or variants thereof.

[0014] The treating, preventing, or suppression in the methods disclosed herein can include activating or inhibiting proliferation of auto-reactive T cells, auto-reactive B cells, alloreactive T cells, alloreactive B cells, syngeneic natural killer cells, allogeneic natural killer cells, innate lymphoid cells (ILC), macrophages, mast cells, ST2+ Tregs, and / or gamma-delta T cells.

[0015] In some embodiments, the culture-expanded regulatory cells are regulatory T cells (Tregs).

[0016] In some embodiments, the Treg cells are ST2+ Treg cells.

[0017] In some embodiments, he regulatory cells can be autologous CAR-Tregs, allogeneic CAR-Tregs, engineered CAR-Tregs, or CD4+ Tregs. The CD4+ Tregs can be ST2+ Tregs.

[0018] In some embodiments, the activated, ex vivo culture-expanded regulatory cells can be Foxp3+ Tregs, Foxp3 -negative IL-10+ (Tri Tregs), TGF-b-i- (Th3 Tregs), IL-35+ (Tr35 Tregs), CD8 Tregs, regulatory macrophages, regulatory dendritic cells, B-regulatory cells (Bregs), regulatory mast cells, regulatory basophils, regulatory eosinophils, regulatory NK cells (NK-regs), regulatory NK-T cells, or regulatory innate lymphoid cells (ILC).

[0019] In another aspect, the regulatory cells can be modified to express IL-33 receptor (ST2) by genetic manipulation including transfection, transduction, electroporation, CRISPR / Cas9 mediated gene-insertion, synthetic mRNA delivery.

[0020] In another aspect, the present disclosure relates to methods for generating tolerogenic dendritic cells or reparative macrophages comprising contacting one or more cells with activated, ex vivo culture-expanded regulatory cells, wherein the culture-expanded regulatory cells are stimulated with IL-33, a combination of IL-2 and IL-33, or an IL233 fusion protein (e.g., SLT518) or variants thereof.

[0021] The culture-expanded regulatory cells in the methods disclosed herein can be stimulated with an interleukin-2 (IL-2) having at least 90% identity to SEQ ID NO: 13; and atruncated interleukin-33 (IL-33), comprising a sequence with at least 90% identity to SEQ ID NO: 1.

[0022] The culture-expanded regulatory cells in the methods disclosed herein can be stimulated with an interleukin-2 (IL-2) having the amino acid sequence of SEQ ID NO: 14 and a truncated interleukin-33 (IL-33), having the amino acid sequence of SEQ ID NO: 3.

[0023] The culture-expanded regulatory cells in the methods disclosed herein can be stimulated with a fusion protein, comprising an interleukin-2 (IL-2) domain having at least 90% identity to SEQ ID NO: 13 and a truncated interleukin-33 (IL-33) domain, comprising a sequence with at least 90% identity to SEQ ID NO: 1

[0024] The culture-expanded regulatory cells in the methods disclosed herein can be stimulated with a fusion protein, comprising an interleukin-2 (IL-2) having the amino acid sequence of SEQ ID NO: 14 and a truncated interleukin-33 (IL-33), having the amino acid sequence of SEQ ID NO: 3.

[0025] In some embodiments, the truncated IL-33 or the truncated IL-33 domain can have at least one of N60, C97, Cl 16, Cl 21 , and Cl 48 of SEQ ID NO: 1 be substituted by a single amino acid residue. As non-limiting examples, the N60 of SEQ ID NO: 1 can be substituted by Ser or Asp; C97 of SEQ ID NO: 1 can be substituted by Gly; and / or Cl 16 of SEQ ID NO: 1 can be substituted by Phe.

[0026] In some embodiments, the truncated IL-33 or the truncated IL-33 domain can have a sequence of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 7.

[0027] The fusion protein can further comprise a linker. The linker can comprise GGGGS (SEQ ID NO: 9). The linker can have the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 10).

[0028] In some embodiments, the IL-2 or the IL-2 domain has a sequence consisting of SEQ ID NO: 13, SEQ ID NO: 14; SEQ ID NO: 15; and SEQ ID NO: 16.

[0029] In some embodiments, the fusion protein further comprises a signal peptide. The fusion protein can comprise a signal peptide that has the sequence of SEQ ID NO: 11.

[0030] In some embodiments, the domains are, in any order from N-terminus to C-terminus, an optional signal peptide; the interleukin-2 (IL-2) domain; an optional linker; and the truncated interleukin-33 (IL-33) domain.

[0031] In one aspect, the present disclosure relates to methods for achieving an immunosuppressive effect in a patient with an alloresponse or autoimmune response comprising administering to said patient, an effective amount of activated long-term, ex vivoculture-expanded regulatory cells to achieve therapeutic suppression of said response, wherein the culture-expanded regulatory cells are stimulated with an interleukin-2 (IL-2) having at least 90% identity to SEQ ID NO: 13; and a truncated interleukin- 33 (IL-33), comprising a sequence with at least 90% identity to SEQ ID NO: 1 and the regulatory cells are capable of inhibiting proliferation of the alloreactive T cells in a mixed lymphocyte reaction (MLR) assay by at least 90%.

[0032] In another aspect, the present disclosure relates to methods for achieving an immunosuppressive effect in a patient with an alloresponse or autoimmune response comprising administering to said patient, an effective amount of activated long-term, ex vivo culture-expanded regulatory cells to achieve therapeutic suppression of said response, wherein the culture-expanded regulatory cells are stimulated with an interleukin-2 (IL-2) having the amino acid sequence of SEQ ID NO: 14 and a truncated interleukin-33 (IL-33), having the amino acid sequence of SEQ ID NO: 3 and the regulatory cells are capable of inhibiting proliferation of the alloreactive T cells in a mixed lymphocyte reaction (MLR) assay by at least 90%.

[0033] In another aspect, the present disclosure relates to methods for achieving an immunosuppressive effect in a patient with an alloresponse or autoimmune response comprising administering to said patient, an effective amount of activated long-term, ex vivo culture-expanded regulatory cells to achieve therapeutic suppression of said response, wherein the culture-expanded regulatory cells are stimulated with a fusion protein, comprising an interleukin-2 (IL-2) domain having at least 90% identity to SEQ ID NO: 13 and a truncated interleukin-33 (IL-33) domain, comprising a sequence with at least 90% identity to SEQ ID NO: 1 and the regulatory cells are capable of inhibiting proliferation of the alloreactive T cells in a mixed lymphocyte reaction (MLR) assay by at least 90%.

[0034] In another aspect, the present disclosure relates to methods for achieving an immunosuppressive effect in a patient with an alloresponse or autoimmune response comprising administering to said patient, an effective amount of activated long-term, ex vivo culture-expanded regulatory cells to achieve therapeutic suppression of said response, wherein the culture-expanded regulatory cells are stimulated with a fusion protein, comprising an interleukin-2 (IL-2) having the amino acid sequence of SEQ ID NO: 14; and a truncated interleukin-33 (IL-33), having the amino acid sequence of SEQ ID NO: 3 and the regulatory cells are capable of inhibiting proliferation of the alloreactive T cells in a mixed lymphocyte reaction (MLR) assay by at least 90%.

[0035] The truncated IL-33 or the truncated IL-33 domain can have at least one of N60, C97, C116, C121, and Cl 48 of SEQ ID NO: 1 be substituted by a single amino acid residue. The N60 of SEQ ID NO: 1 can be substituted by Ser or Asp; C97 of SEQ ID NO: 1 can be substituted by Gly; and / or Cl 16 of SEQ ID NO: 1 can be substituted by Phe. The truncated IL-33 or the truncated IL-33 domain can have a sequence consisting of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 7.

[0036] In some embodiments, the fusion protein further comprises a linker. The linker can comprise GGGGS (SEQ ID NO: 9). The linker can have the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 10).

[0037] In some embodiments, the IL-2 or the IL-2 domain can have a sequence consisting of SEQ ID NO: 13, SEQ ID NO: 14; SEQ ID NO: 15; or SEQ ID NO: 16.

[0038] In some embodiments, the fusion protein further comprises a signal peptide. The fusion protein can comprise a signal peptide that has the sequence of SEQ ID NO: 11.

[0039] In some embodiments, the domains are, in any order from N-terminus to C-terminus, an optional signal peptide; the interleukin-2 (IL-2) domain; an optional linker; and the truncated interleukin-33 (IL-33) domain.

[0040] In some embodiments, the method can include administering to a patient with an alloresponse or autoimmune response, which can suppress, block or inhibit in vivo alloresponses or autoimmune responses in the patient. The alloresponse or autoimmune response can follow tissue transplantation, and the method can suppress, block or inhibit graft- vs-host disease in the patient.

[0041] In another aspect, the present disclosure relates to a method for achieving a preventative therapeutic effect in a patient comprising administering to the patient, prior to onset of an alloresponse or autoimmune response, an effective amount of activated long-term, ex vivo culture-expanded regulatory cells to prevent the response, wherein the culture- expanded Treg cells are stimulated with an interleukin-2 (IL-2) having at least 90% identity to SEQ ID NO: 13; and a truncated interleukin- 33 (IL-33), comprising a sequence with at least 90% identity to SEQ ID NO: 1 and the regulatory cells are capable of inhibiting proliferation of the alloreactive T cells in a mixed lymphocyte reaction (MLR) assay by at least 90%.

[0042] In another aspect, the present disclosure relates to a method for achieving a preventative therapeutic effect in a patient comprising administering to the patient, prior to onset of an alloresponse or autoimmune response, an effective amount of activated long-term,ex vivo culture-expanded regulatory cells to prevent the response, wherein the culture- expanded regulatory cells are stimulated with an interleukin-2 (IL-2) having the amino acid sequence of SEQ ID NO: 14 and a truncated interleukin- 33 (IL-33) having the amino acid sequence of SEQ ID NO: 3; and the regulatory cells are capable of inhibiting proliferation of the alloreactive T cells in a mixed lymphocyte reaction (MLR) assay by at least 90%.

[0043] In another aspect, the present disclosure relates to a method for achieving a preventative therapeutic effect in a patient comprising administering to the patient, prior to onset of an alloresponse or autoimmune response, an effective amount of activated long-term, ex vivo culture-expanded regulatory cells to prevent the response, wherein the culture- expanded regulatory cells are stimulated with a fusion protein comprising: an interleukin-2 (IL-2) domain having at least 90% identity to SEQ ID NO: 13 and a truncated interleukin-33 (IL-33) domain having a sequence with at least 90% identity to SEQ ID NO: 1; and the regulatory cells are capable of inhibiting proliferation of the alloreactive T cells in a mixed lymphocyte reaction (MLR) assay by at least 90%.

[0044] In another aspect, the present disclosure relates to a method for achieving a preventative therapeutic effect in a patient comprising administering to the patient, prior to onset of an alloresponse or autoimmune response, an effective amount of activated long-term, ex vivo culture-expanded regulatory cells to prevent the response, wherein the culture- expanded regulatory cells are stimulated with a fusion protein comprising an interleukin-2 (IL-2) having the amino acid sequence of SEQ ID NO: 14 and a truncated interleukin-33 (IL- 33) having the amino acid sequence of SEQ ID NO: 3; and the regulatory cells are capable of inhibiting proliferation of the alloreactive T cells in a mixed lymphocyte reaction (MLR) assay by at least 90%.

[0045] The truncated IL-33 or the truncated IL-33 domain, can have at least one of N60, C97, Cl 16, C121, and C148 of SEQ ID NO: 1 is substituted by a single amino acid residue. As non-limiting examples, the N60 of SEQ ID NO: 1 can be substituted by Ser or Asp; C97 of SEQ ID NO: 1 can be substituted by Gly; and / or Cl 16 of SEQ ID NO: 1 can be substituted by Phe.

[0046] In some embodiments, the truncated IL-33 or the truncated IL-33 domain can have a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 7.

[0047] The fusion protein can further comprise a linker. The linker cn comprise GGGGS (SEQ ID NO: 9). The linker can have the sequence GGGGSGGGGSGGGGS (SEQ ID NO:10). The IL-2 or the IL-2 domain can have a sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14; SEQ ID NO: 15; and SEQ ID NO: 16.

[0048] The fusion protein can further comprise a signal peptide. The fusion protein can comprise a signal peptide that has the sequence of SEQ ID NO: 11. The domains can be, in order from N-terminus to C-terminus, an optional signal peptide; the interleukin-2 (IL-2) domain; an optional linker; and the truncated interleukin- 33 (IL-33) domain.

[0049] The patient can be treated prior to, at the time of, or immediately after tissue transplantation, and the method can further comprise preventing onset of graft- vs-host disease in the patient.

[0050] The patient can be treated prior to, at the time of, or immediately after tissue transplantation, and the method can further comprise blocking rejection of the transplanted tissue in the patient.

[0051] In any of the methods disclosed herein, the regulatory cells can comprise CD4+, CD25+ T cells. The regulatory cells can inhibit activation of cytotoxic T cells.

[0052] In one aspect, the present disclosure relates to a method of treating or preventing cancer in a patient in need thereof comprising administering to the patient activated, culture- expanded regulatory cells, wherein the culture-expanded regulatory cells are stimulated with IL-33, a combination of IL-2 and IL-33, or IL233 (SLT518) or variants thereof. The treating or preventing can include promoting the production of effector cells. The effector cells can include auto-reactive T cells, auto-reactive B cells, alloreactive T cells, alloreactive B cells, syngeneic natural killer cells, allogeneic natural killer cells, innate lymphoid cells (ILC), macrophages, mast cells, ST2+ Tregs, CD8 natural killer gamma cells, and / or gamma-delta T cells. The culture-expanded regulatory cells can be selected from the group consisting of autologous CAR-Tregs, allogeneic CAR-Tregs, engineered CAR-Tregs, and CD4+ Tregs. The CD4+ Tregs can be ST2+ Tregs. In other embodiments, the culture-expanded regulatory cells can be selected from the group consisting of Foxp3+ Tregs, Foxp3 -negative IL-10+ (Tri Tregs), TGF-b-i- (Th3 Tregs), IL-35+ (Tr35 Tregs), CD8 Tregs, regulatory macrophages, regulatory dendritic cells, B -regulatory cells (Bregs), regulatory NK cells (NK-regs), regulatory NK-T cells, and regulatory innate lymphoid cells (ILC). In one embodiment, the culture-expanded regulator cells are ST2+ Treg cells.

[0053] In another aspect, the present disclosure relates to a method of making activated, culture-expanded regulatory cells comprising the step of stimulating regulatory cells with aninterleukin-2 (IL-2) having at least 90% identity to SEQ ID NO: 13; and a truncated interleukin-33 (IL-33), comprising a sequence with at least 90% identity to SEQ ID NO: 1.

[0054] In another aspect, the present disclosure relates to a method of making activated, culture-expanded regulatory cells comprising the step of stimulating regulatory cells with an interleukin-2 (IL-2) having the amino acid sequence of SEQ ID NO: 14 and a truncated interleukin-33 (IL-33) having the amino acid sequence of SEQ ID NO: 3.

[0055] In another aspect, the present disclosure relates to a method of making activated, culture-expanded regulatory cells comprising the step of stimulating regulatory cells with an interleukin-2 (IL-2) domain having at least 90% identity to SEQ ID NO: 13 and a truncated interleukin-33 (IL-33) domain having a sequence with at least 90% identity to SEQ ID NO: 1.

[0056] The truncated IL-33 or the truncated IL-33 domain, can have at least one of N60, C97, Cl 16, C121, and C148 of SEQ ID NO: 1 be substituted by a single amino acid residue. As non-limiting examples, the N60 of SEQ ID NO: 1 can be substituted by Ser or Asp; C97 of SEQ ID NO: 1 can be substituted by Gly; and / or Cl 16 of SEQ ID NO: 1 can be substituted by Phe. In some embodiments, the truncated IL-33 or the truncated IL-33 domain can have a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 7.

[0057] The fusion protein can further comprise a linker. The linker can comprise GGGGS (SEQ ID NO: 9). The linker can have the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 10).

[0058] The IL-2 or the IL-2 domain can have a sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14; SEQ ID NO: 15; and SEQ ID NO: 16.

[0059] The fusion protein can further comprise a signal peptide. The fusion protein can comprise a signal peptide that has the sequence of SEQ ID NO: 11.

[0060] The domains can be, in order from N-terminus to C-terminus, an optional signal peptide; the interleukin-2 (IL- 2) domain; an optional linker; and the truncated interleukin- 33 (IL-33) domain.

[0061] In another aspect, the present disclosure relates to a pharmaceutical composition, comprising the culture-expanded regulatory cells disclosed herein. The pharmaceutical composition can further comprise a pharmaceutically-acceptable carrier, and optionally an additional therapeutic agent.

[0062] In another aspect, the disclosure provides methods of treating cancer in a patient in need thereof comprising administering to the patient activated, culture-expanded effectorcells, wherein the culture-expanded effector cells are stimulated with IL-33, a combination of IL-2 and IL-33, or IL233 (SLT518) or variants thereof.

[0063] The expanded effector cells can be ex vivo culture-expanded effector cells. The expanded effector cells can be expanded in vitro.

[0064] Treating or preventing cancer can include promoting the production of effector cells. The effector cells can include auto-reactive T cells, auto-reactive B cells, alloreactive T cells, alloreactive B cells, syngeneic natural killer cells, allogeneic natural killer cells, innate lymphoid cells (ILC), macrophages, dendritic cells, mast cells, ST2+ T cells, CD8 T cells, natural killer gamma cells, and / or gamma-delta T cells. The effector cells can be selected from the group consisting of autologous CAR-T cells, allogeneic CAR-T cells, engineered CAR-T cells, and CD4+ T cells. The CD4+ T cells can be ST2+ T cells. The effector cells can be selected from the group consisting of syngeneic or allogeneic Foxp3-negative T cells, IFNg+ T cells (Thl T cells), IL-4+ (Th2 T cells), IL-9+ T cells (Th9 T cells), IL-21+ T cells (Th21 T cells), TNFa-i- (Inflammatory T cells), CD8 Tregs. The effector cells may also include syngeneic or allogeneic antigen presenting cells including macrophages, dendritic cells or B cells loaded with tumor antigen or transfected with tumor-antigen expressing constructs.

[0065] In another embodiment, the effector cells described herein, can be modified to express IL-33 receptor (ST2) by genetic manipulation including transfection, transduction, electroporation, CRISPR / Cas9 mediated gene-insertion or synthetic mRNA delivery.

[0066] The cancer can be acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute myelogenous leukemia, adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, B-cell lymphoma, brain cancer, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer,macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, midline tract carcinoma involving NUT gene, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal cavity and par nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphocytic leukemia, T-cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms tumor.

[0067] The cancer can be melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, B-cell lymphoma, acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, bladder cancer, or lung cancer.

[0068] Depending upon the IL-2 domain, the method can stimulate proliferation and / or activation of T-regulatory (Treg) cells, T-helper2 (Th2) cells, innate lymphoid cells (ILC), cytotoxic T cells, natural killer (NK) cells, NK-T cells, ST2+ cells, dendritic cells, and / or macrophages.

[0069] Depending upon the IL-2 domain, the method can either promote anti-inflammatory M2 macrophages and inhibit pro-inflammatory Ml macrophages, or activate Ml macrophages to induce inflammation. The M2 macrophages can inhibit inflammation in a tissue or organ selected from the group consisting of pancreas, liver, kidneys, adipose tissue, salivary glands, central nervous system (CNS), and related organs. The Ml macrophages can promote anti-tumor immune response either systemically or locally.

[0070] Depending on the IL-2 domain, the method can either upregulate or down-regulate the expression of co-stimulatory molecules (including and not limited to CD80 (B7- 1), CD86 (B7-2), CD40, ICOS, MHC-I, MHC-II, PD1, PD-L1, GITR, BAFF-R, 0x40, 41BB, DR3, CR2) on the antigen-presenting cells including and not limited to dendritic cells,macrophages, B-cells, innate lymphoid cells, NK cells, epithelial cells, endothelial cells or stromal cells.

[0071] The method can further comprise administering an effective amount of a second therapy for the disease or disorder to the subject, wherein the second therapy is not a fusion protein described herein.

[0072] The second therapy can be another cell therapy or a gene therapy.BRIEF DESCRIPTION OF THE DRAWINGS

[0073] FIGs. 1A and IB show that SLT-518(h4) promotes dose dependent ST2+ Treg proliferation in fresh human PMBCs. Samples were provided by three healthy human PBMC donors. FIG. 1A illustrates that the percentage of ST2+ Tregs is higher for SLT518(h4)- treated cells as compared to those treated with wild- type IL2 alone. FIG. IB shows that the percentage of ST2+ Tregs in diseased patients is higher in cells treated with SLT518(h4) than those treated with an IL-2 mutein.

[0074] FIG. 2 shows sequences of wild type human IL-2 (SEQ ID NO: 13) and truncated wild type human IL-33 (SEQ ID NO: 1). In the IL-2 sequence, two residues each of interest in some embodiments, N88 and C125, are emphasized. In the truncated IL-33 sequence, three residues each of interest in some embodiments, N60, C97, and Cl 16, are emphasized.

[0075] FIG. 3 shows a sequence alignment between wild type human truncated IL-33 (SEQ ID NO: 1), mouse truncated IL-33 (SEQ ID NO: 17), and rat truncated IL-33 (SEQ ID NO: 18). In the human sequence, an N-glycosylation site comprising N60, and the four wild type Cys residues, C97, Cl 16, C121, and C148, are highlighted. In the mouse and rat sequences, Cys residues aligned with human Cys residues are emphasized.

[0076] FIG. 4 is a composite showing schematically that SLT-518(h4), an IL233 fusion protein, drives the local tissue environment to a more tolerogenic phenotype in mice. Supporting data shows that natural killer (NK) cells and co-stimulatory molecules on dendritic cells (DC) decrease after administration of the IL233 fusion protein. M2 macrophages, ST2+ Tregs, and ratios of ILlO / TNFa, Treg / Thl7, ILC2 all increase after administration of the 1L233 fusion protein.

[0077] FIG. 5 shows stimulated expansion of ST2+ Treg cells ex vivo using IL-33, IL-2 or IL-233 (SLT518(h4)) in each samples from Autoimmune Lupus Nephritis, Membranous Nephropathy, IgA Nephropathy and non- autoimmune CKD donors.

[0078] FIG. 6 shows stimulated expansion of ST2+ Treg cells ex vivo in all samples tested.As compared with IL-2 alone and IL-33 alone, Treg cells have a stronger response to IL-233, SLT-518(h4).DETAILED DESCRIPTION

[0079] This disclosure relates to regulatory immune cells based cell therapy and compositions and methods thereof. The methods described herein, in general, comprise administration of activated and expanded (e.g., ex vivo culture expanded, in vivo expanded) regulatory cells (e.g., ST2+ Treg cells). The activated and expanded regulatory cells (e.g., ST2+ Treg cells) can be produced by stimulating expanded Treg cells with (i) a composition comprising IL-2 and IL-33 or variants thereof, (ii) an IL-2 and IL-33 fusion protein or variants thereof, (iii) compositions or fusion proteins comprising an IL-2 polypeptide or domain, and a truncated IL-33 polypeptide or domain or variants thereof; or (iv) IL-33 or a truncated IL-33 polypeptide or domain or variant thereof. Methods for activating and expanding regulatory cells, the activated and enhanced expanded regulatory cells themselves, and methods for using the activated and expanded regulatory cells in immunotherapy and for suppression of autoimmune responses are described herein.

[0080] The fusion of IL-2 and IL-33 is key to enabling increased potency, tissue targeting and avidity. ST2 is the receptor for IL-33 and is expressed on the surface of a subset of regulatory T cells (Treg) among other cells such as macrophages, dendritic cells etc. This subset is more durable and active in modulating inflammatory suppression in tissues at the site of disease. Because IL-33 is naturally released in tissues, its function not only helps to expand regulatory cells (e.g., ST2+ Treg cells), but other ST2+ cells and promotes a local tolerogenic tissue micro-environment. Treatment with the activated and culture-expanded Treg cells and compositions described herein thus results in durable disease resolution.

[0081] All publications and patents cited in this disclosure are incorporated by reference in their entirety. To the extent, the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. The citation of any references herein is not an admission that such references are prior art to the present disclosure. When a range of values is expressed, it includes embodiments using any particular value within the range. Further, reference to values stated in ranges includes each and every value within that range. All ranges are inclusive of their endpoints and combinable.When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. The use of “or” will mean “and / or” unless the specific context of its use dictates otherwise.

[0082] Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4thed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer-defined protocols and conditions unless otherwise noted.

[0083] As used herein, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly indicates otherwise. The terms “include,” “such as,” and the like are intended to convey inclusion without limitation, unless otherwise specifically indicated.

[0084] Unless otherwise indicated, the terms “at least,” “less than,” and “about,” or similar terms preceding a series of elements or a range are to be understood to refer to every element in the series or range. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0085] An “individual,” “subject,” “patient” or “host” referred to herein is a vertebrate, preferably a mammal. More preferably, such individual is a human and the culture-expanded cells are human, although animals, including animal models for human disease states, are also included in this invention and therapeutic treatments of such animals are contemplated herein. Such animal model can be used to test and adjust the compositions and methods of this invention, if desired. Certain models involve injecting in-bred animals with established syngeneic cell lines. Also useful are chimeric animal models, described in U.S. Pat. Nos. 5,663,481, 5,602,305 and 5,476,993; EP application 379,554; and International Appl. WO91 / 01760. Non-human mammals include, but are not limited to, veterinary or farm animals, sport animals, and pets. Accordingly, as opposed to animal models, such animals may be undergoing selected therapeutic treatments.

[0086] As used herein, “allogeneic cells” (allogenicity) are those isolated from one individual (the donor) and infused into another (the recipient or host); whereas “autologous cells” (autology) refer to those cells that are isolated and infused back into the same individual (recipient or host). Accordingly, allogeneic T cell proliferation is stimulated by antigen presenting cells (“APC”) from another individual, while autologous T cell proliferation is stimulated by self-APCs. Unless specified, the APC in the present invention can, therefore, be of any type known in the art.

[0087] “Antigens” are entities that initiate immune responses. “Alloantigens” refer to agents that cause the immune system of one individual to recognize and destroy the cells of another individual when the two are mixed together. “Mitogens” are agents that induce all T cells to proliferate in an antigen nonspecific manner.

[0088] “Mixed lymphocyte reaction,” “mixed lymphocyte culture,” “MLR,” and “MLC” are used interchangeably to refer to a mixture comprising a minimum of two different cell populations that are allotypically different. At least one of the allotypically different cells is a lymphocyte. The cells are cultured together for a time and under suitable conditions to result in the stimulation of the lymphocytes, which in this particular invention are Treg cells. A frequent objective of an MLC is to provide allogeneic stimulation, such as may initiate proliferation of the Treg cells; but unless indicated, proliferation during the culture is not required. In the proper context, these terms may alternatively refer to a mixture of cells derived from such a culture. When cells from an MLC are administered as a bolus to a human, it is referred to as a “cellular implant.”

[0089] A “cell line” or “cell culture” denotes higher eukaryotic cells grown or maintained in vitro. It is understood that the descendants of a cell. May not be completely identical (either morphologically, genotypically, or phenotypically) to the parent cell.

[0090] By “long term” is meant a culture period lasting more than 1 week, preferably ^10 days, more preferably =2 weeks, more preferably =3-4 weeks, more preferably ^1 month, more preferably ^6-8 weeks, more preferablymonths, more preferablymonths, more preferably ^6 months, and most preferably 1 year or longer- so long as some level of cellular function, specifically suppressor activity is retained.

[0091] As used herein, “treatment” or “therapy” refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and may be performed either for prophylaxis or during the course of clinical pathology. Desirable effects include, but are not limited to, preventing occurrence or recurrence of disease, alleviating symptoms, suppressing, diminishing or inhibiting any direct or indirect pathological consequences of the disease, preventing metastasis, lowering the rate of disease progression, ameliorating or palliating the disease state, and causing remission or improved prognosis. The “pathology” associated with a disease condition is anything that compromises the well-being, normal physiology, or quality of life of the affected individual. This may involve (but is not limited to) destructive invasion of affected tissues into previously unaffected areas, growth at the expense of normal tissue function, irregular or suppressed biological activity, aggravation or suppression of an inflammatory or immunological response, increased susceptibility to other pathogenic organisms or agents, and undesirable clinical symptoms such as pain, fever, nausea, fatigue, mood alterations, and such other features as may be determined by an attending physician.

[0092] An “effective amount” is an amount sufficient to effect a beneficial or desired clinical result, particularly the generation of an immune response, or noticeable improvement in clinical condition. An “immunogenic amount” is an amount shown in the subject group being treated or tested (either diseased or not) that is sufficient to elicit an immunological response, which may comprise either a humoral response, a cellular response, or both. Preferably in accordance with the present invention, suppression is achieved at >30% over that which would occur absent practice of the culture-expansion methods disclosed herein. More preferably, the suppression achieved is =^40%, more preferably at 1150%, more preferably at ^70%, more preferably at >85%, more preferably at ^90%, more preferably at ^95%, more preferably at ^99, and most preferably at = 10017, so long as the ex vivo culture of the cells is sustainable long term.

[0093] The terms “suppression,” “inhibition” and “prevention” are used herein in accordance with accepted definitions, i.e., “suppression” results when an ongoing immune response is blocked or significantly reduced as compared with the level of immune response that results absent treatment by the present invention. Similarly, “inhibition” refers to blocking the occurrence of an immune response or significantly reduces such response as compared with the level of immune response that results absent treatment by the present invention. When administered prophylactically, such blockage may be complete so that no targeted immuneresponse occurs, typically referred to as a “prevention” with regard to completely blocking the immune response before onset; or in the present invention, the treatment may advantageously reduce the effect as compared to the normal untreated state, typically referred to as suppression or inhibition.

[0094] In terms of clinical response for subjects bearing a neoplastic disease, an effective amount is amount sufficient to palliate, ameliorate, stabilize, reverse or slow progression of the disease, or otherwise reduce pathological consequences of the disease. Similarly, in transplant patients experiencing the onset of GVHD, or those that are susceptible to GVHD, an effective amount is that amount which is sufficient to block or prevent its onset; or if GVHD pathology has begun, to palliate, ameliorate, stabilize, reverse or slow progression of the disease, or otherwise reduce pathological consequences of the disease. In any case, an effective amount may be given in single or divided doses. Preferred quantities and cell ratios for use in an effective amount are given elsewhere in this disclosure.

[0095] “Activation” refers to stimulating or enhancing cellular proliferation and the cell division necessary to produce progeny cells from the original cell population.

[0096] Additional description of the methods and guidance for the practice of the methods are provided herein.A. Embodiments i. Methods for stimulating proliferation and / or activation of regulatory or effector cells

[0097] In some embodiments, the present disclosure relates to methods for stimulating proliferation and / or activation of regulatory cells (e.g., T-regulatory (Treg) cells) of a subject. The regulatory cells can be autologous CAR-Tregs, allogeneic CAR-Tregs, engineered CAR- Tregs, or CD4+ Tregs. The CD4+ Tregs can be ST2+. The regulatory cells can be Foxp3+ Tregs, Foxp3-negative IL- 10+ (Tri Tregs), TGF-b+ (Th3 Tregs), IL-35+ (Tr35 Tregs), CD8 Tregs, regulatory macrophages, regulatory dendritic cells, B-regulatory cells (Bregs), regulatory NK cells (NK-regs), regulatory NK-T cells, or regulatory innate lymphoid cells (ILC). The stimulation can be performed in vivo, ex vivo, or in vitro.

[0098] In some embodiments, the present disclosure relates to methods for stimulating proliferation and / or activation of effector cells for treating or preventing cancer. The effector cells can include auto-reactive T cells, auto-reactive B cells, alloreactive T cells, alloreactive B cells, syngeneic natural killer cells, allogeneic natural killer cells, innate lymphoid cells (ILC), macrophages, dendritic cells, mast cells, ST2+ T cells, CD8 T cells, natural killergamma cells, and / or gamma-delta T cells. The effector cells can be selected from the group consisting of autologous CAR-T cells, allogeneic CAR-T cells, engineered CAR-T cells, and CD4+ T cells. The CD4+ T cells can be ST2+ T cells. The effector cells can be syngeneic or allogeneic Foxp3-negative T cells, IFNg+ T cells (Thl T cells), IL-4+ (Th2 T cells), IL-9+ T cells (Th9 T cells), IL-21+ T cells (Th21 T cells), TNFa+ (Inflammatory T cells), or CD8 Tregs. The effector cells may also include syngeneic or allogeneic antigen presenting cells including macrophages, dendritic cells or B cells loaded with tumor antigen or transfected with tumor-antigen expressing constructs.

[0099] In some embodiments, the regulatory cells or effector cells may be differentiated from stem cells (e.g., from iPSC).

[0100] In another embodiment, the regulatory or effector cells can be modified to express IL-33 receptor (ST2) by genetic manipulation including transfection, transduction, electroporation, CRISPR / Cas9 mediated gene-insertion or synthetic mRNA delivery.

[0101] For example, one in vitro method comprises isolating T cells from the subject; and contacting the isolated T cells with an effective amount of a composition or a fusion protein as described herein.

[0102] The present disclosure provides methods for manipulating and modulating a subpopulation of regulatory cells (e.g., Treg cells) or effector cells for suppression, inhibition and prevention of alloresponses and auto-immune responses, or as a component of cancer immunotherapy in solid cancer tumors. The activated and expanded regulatory cells can mediate potent inhibition of autologous T cell proliferation. Consequently, it is an object of the present disclosure to provide methods to modify and regulate the development of regulatory cells (e.g., Treg cells).

[0103] CD4+CD25+ cells play an important role in alloresponses in vivo, specifically graft- vs-host disease (GVHD) generation. Ex vivo depletion of CD4+CD25+ cells from donor T cell inoculum or in vivo CD25-depletion of recipient pre-transplant can result in increased GVHD responses.

[0104] It is an object of this disclosure to also provide methods to promote engraftment of human transplanted tissue, including whole or selected populations of blood or bone marrow transplants, particularly by suppressing, inhibiting, blocking or preventing GVHD by means of a population of activated and culture-enhanced regulatory cells administered in vivo. Advantageously such methods are achieved with reduced intensity regimens and little or no immune suppression. Such engraftment promotion effects can thus be applied to solid organtransplant patients as a means of achieving drug-free tolerance with reduced or no conditioning regimen requirements, or to allogeneic bone marrow or autoimmune patients with autologous or allogeneic bone marrow, as means of resetting the immune system.

[0105] It is also an object of the present invention to provide a method for treatment of effector cells, so that activated and culture-expanded cells may be infused into the host to produce an immunotherapeutic response. In its simplest form, the disclosure provides preferred, ex vivo, long-term, culture-expanding methods for human regulatory cells, while maintaining enhanced suppressive activity, comprising: a) obtaining regulatory cells from a patient or an allogeneic donor; b) isolating a population of regulatory cells from the obtained cells, followed by c) activating and enhancing the cultured regulatory cells, thereby increasing the number of modified suppressor cells in the culture.

[0106] In one embodiment the activated and expanded regulatory cells inhibit the autologous proliferation of peripheral blood cells. In another embodiment, the activated and expanded regulatory cells block or prevent GVHD, or inhibit or reverse the disease if already in progress. In yet another embodiment, the activated and enhanced cells are introduced into a different host; whereas in yet another embodiment, the cells are established as a cell line for continuous therapeutic use.

[0107] Preferably, the host is a human host and the culture-expanded cells are human, although animals, including animal models for human disease states, are also included in this disclosure and therapeutic treatments of such animals are contemplated herein.

[0108] In another embodiment, the activated and expanded regulatory cells suppress inflammation in a patient.

[0109] In another embodiment, the activated and expanded regulatory cells are used in a method of treating autoimmune disease (e.g., type-1 diabetes, SLE, lupus nephritis, inflammatory bowel disease, Sjogren syndrome, Rheumatoid arthritis, multiple sclerosis) in a patient.

[0110] In another embodiment, the activated and expanded effector cells are used in a method of treating cancer, tumors, or hematopoietic cancers in a patient.

[0111] The ex vivo stimulation methods of the present invention can have decided advantages. For example: 1) activation and enhancement can be CD4+CD25+ cell specific;2) stimulation in long-term culture can allow for the removal of the stimulating antigens prior to reintroduction of the cells in the host; 3) lack of systemic in vivo exposure to the activating and enhancing antigens can preclude significant interference with naturally occurring orinduced immunogenic responses or 4) activation and enhancement of anti-cancer effector cells for improved trafficking to the tumor microenvironment as well as activation of other anti-cancer cells. Moreover, success can be achieved with minimal host toxicity using the ex vivo activation and enhancement techniques, and the suppressor cell composition is prepared entirely in accordance with GMP conditions, meaning that the ex vivo, culture-enhanced CD4+CD25+ cells can be quickly approved for human infusion.

[0112] For clinical application, the cell product must achieve a certain effector to target ratio in vivo in order to obtain the desired beneficial effect. In many cases, the input cells at the start of cell culture will be limiting due to the rarity of suppressive precursor cells, or to limiting amounts of clinical material. The provided methods are designed to produce on the order of 109culture-enhanced cells for clinical applications. Furthermore, in order for tissue culture process to be clinically relevant, it must be scalable to large scale and be compliant with FDA approved procedures and reagents. Accordingly, the methods of the present invention, specifically designed to meet these requirements, advantageously produce an enhanced CD4+CD25+ suppressor cell infusion that is sufficiently specific to overcome volume- associated problems that would make using naturally occurring Treg cells impossible to achieve an immunosuppressive or preventative therapeutic effect in a patient, and the methods utilize conditions that have been previously-approved for human treatment.

[0113] The culture-expanded cells produced by the present long-term, culture-expansion methods can be viewed as a reagent provided by the present disclosure for treating GVHD, comprising activated and ex vivo culture-expanded regulatory or effector cells. Preferably, the T cells are suspended in media suitable for intravenous administration to a human transplant or cancer patient, such as a media comprising a physiological buffered solution. While not limited to any mechanism, it is believed that long-term culturing of the cells in the manner proposed results in potent subset enrichment and activation, thereby creating a sufficient population of the cells to effect therapeutic benefits to the patient.

[0114] In another embodiment, the disclosure relates to a method for inducing an anti- GVHD or anti-cancer response in vivo comprising contacting host regulatory cells or effector cells with activating and / or enhancing compositions ex vivo and infusing thus activated and / or enhanced regulatory cells or effector into the autologous host, who has or will undergo allogeneic transplant that can produce or has already initiated GVHD response in the transplant recipient. The cells are typically hematopoietic cells, such as peripheral blood lymphocytes (PBMCs), spleen cells, tumor-infiltrating lymphocytes or lymph node cells.

[0115] In another embodiment, the disclosure relates to a method for inducing an anticancer response in vivo comprising contacting host effector cells with activating and / or enhancing compositions ex vivo and infusing thus activated and / or enhanced effector cells into the autologous host, who has cancer. The cells are typically hematopoietic cells, such as peripheral blood lymphocytes (PBMCs), spleen cells, tumor-infiltrating lymphocytes or lymph node cells.

[0116] In one embodiment, the disclosure provides a method for human treatment that comprises: a) obtaining regulatory or effector cells from a patient or an allogeneic donor; b) isolating a population of cells from the obtained cells, followed by c) activating and longterm culture-enhancing the regulatory or effector cells, wherein the culture-enhancing and activating method for creating modified regulatory cells in a media comprise the presence of human IL-2 and IL-33 or other disclosed interleukin or compound to further enhance cell proliferation, thereby increasing the number of modified suppressor cells in the culture; and d) re-introducing at least a portion of the modified suppressor T cells into the host patient so as to induce an in vivo therapeutic response. Such response can prevent, block, suppress, inhibit or reverse GVHD or other autoimmune response, or peripheral blood cell proliferation, , cancer proliferation, or metastasis in a cancer patient.

[0117] In another embodiment, the method utilizes the culture-enhanced regulatory cells produced using the culture methods provided herein. While in still another embodiment, the method utilizes culture-enhanced regulatory (e.g., Treg cells) or effector cells derived from bone marrow, using the culture methods provided herein for peripheral blood, except in this case, bone marrow aspirates would be used to acquire the Treg cell or anti-cancer cell population.

[0118] In another embodiment, lymphocytes are obtained prior to the onset of cancer, from cancer-free hosts, and the cells are stored using conventional techniques until needed at the onset of disease, at which time the cells may be thawed, cultured and activated and enhanced as herein previously described for reinfusion into the host. Alternatively, an established cell line may be made from cancer-free hosts (allogeneic or autologous). The cell line can be stored as above, activated and culture-enhanced until needed. Similarly, it is an object to obtain lymphocytes prior to the allogeneic transplant from the transplant recipient, chemotherapy, or radiation therapy from the cancer patient, and store the cells using conventional techniques until the transplant is completed, at which time the cells may be thawed, CD25+ depleted and / or culture-enhanced and activated as herein described forreinfusion into the host to block, suppress, inhibit or prevent GVHD, or to reverse GVHD if already initiated or used for treatment of cancers. Alternately, an established cell line may be prepared from the host before transplant, and stored as above, or CD25+ depleted and / or culture-enhanced and activated as above, until needed.

[0119] In another embodiment, the method further comprises the step of administering human IL-2 and IL-33 to the host patient in vivo after re-introducing the modified regulatory or effector cells. The autologously re-introduced modified cells are thus considered to be a cellular implant. In an alternative embodiment, the host is similarly treated after allogeneic modified regulatory donor cells are introduced into the recipient. ii. Therapeutic Methods.

[0120] The methods of the present disclosure are particularly useful for humans, but may also be practiced on veterinary subjects.

[0121] In one embodiment, inflammation in a patient is suppressed by administering to the patient activated, ex vivo culture-expanded regulatory cells that are stimulated with IL-33, a combination of IL-2 and IL-33, or IL233 (SLT518) or variants thereof.

[0122] In another embodiment, autoimmune disease in a patient is treated by administering to the patient activated, ex vivo culture-expanded regulatory cells, wherein the culture- expanded regulatory cells are stimulated with IL-33, a combination of IL-2 and IL-33, or IL233 (SLT518) or variants thereof.

[0123] In yet another embodiment, allograft rejection is suppressed by administering to the patient activated, ex vivo culture-expanded regulatory cells, wherein the culture-expanded regulatory cells are stimulated with IL-33, a combination of IL-2 and IL-33, or an IL233 fusion protein (e.g., SLT518) or variants thereof.

[0124] The disclosure also includes a method of treating or preventing GvHD in a patient in need thereof comprising administering to the patient activated, ex vivo culture-expanded regulatory cells, wherein the culture-expanded regulatory cells are stimulated with IL-33, a combination of IL-2 and IL-33, or an IL233 fusion (e.g., SLT518) or variants thereof.

[0125] In another embodiment, a patient having cancer is treated by administering to the patient activated, ex vivo culture-expanded effector cells, wherein the culture-expanded effector cells are stimulated with IL-33, a combination of IL-2 and IL-33, or an IL233 fusion protein (e.g., SLT518) or variants thereof. For example, the cancer can be any one of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute myelogenous leukemia, adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cellcarcinoma, B-cell lymphoma, brain cancer, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, midline tract carcinoma involving NUT gene, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal cavity and par nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphocytic leukemia, T-cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms tumor.

[0126] The cancer can be selected from the group consisting of melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, B-cell lymphoma, acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, bladder cancer, and lung cancer.

[0127] Any of the methods disclosed herein can include inhibiting proliferation of auto- reactive T cells, auto-reactive B cells, alloreactive T cells, alloreactive B cells, syngeneic natural killer cells, allogeneic natural killer cells, innate lymphoid cells (ILC), ST2+ Tregs,and / or gamma-delta T cells. For example, the regulatory cells can be autologous CAR-Tregs, allogeneic CAR-Tregs, engineered CAR-Tregs, or CD4+ Tregs. In one embodiment, the ST2+ Tregs are CD4+ Tregs. The regulatory cells can be Foxp3+ Tregs, Foxp3-negative IL- 10+ (Tri Tregs), TGF-b+ (Th3 Tregs), IL-35+ (Tr35 Tregs), CD8 Tregs, regulatory macrophages, regulatory dendritic cells, B-regulatory cells (Bregs), regulatory NK cells (NK- regs), regulatory NK-T cells, or regulatory innate lymphoid cells (ILC).

[0128] In another aspect, the disclosure provides for a method for generating tolerogenic dendritic cells or reparative macrophages comprising contacting one or more cells with activated, ex vivo culture-expanded regulatory cells, for example cells stimulated with IL-33, a combination of IL-2 and IL-33, or an IL233 fusion protein (e.g., SLT518) or variants thereof.

[0129] The immune status of the recipient or host may be any of the following: (i) the individual may be immunologically naive with respect to certain antigen presenting cells (APC) or tumor-associated antigens (TAA) present in the composition; or (ii) the individual may not currently be expressing anti-tumor immunity, but may have immunological memory, particularly T cell memory relating to a particular antigen.

[0130] The compositions containing the present modified and / or activated and enhanced cell population, or a cocktail thereof, can be administered for the prophylactic and / or therapeutic treatments. In therapeutic application, compositions are administered to a patient in an amount sufficient to prevent, suppress, block or inhibit, or at least partially arrest the immunogenic response, such as that which takes place prior to the onset of GVHD or during the GVHD response and its complications. An amount adequate to accomplish this is defined as a “therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's own immune system, but generally range from about 0.05 mg / kg body weight to about 5 mg / kg body weight, preferably between about 0.2 mg / kg body weight to about 1.5 mg / kg body weight.

[0131] In prophylactic applications, compositions containing the modified, and / or activated and enhanced cell population, or a cocktail thereof, are administered to a patient not already in a disease state to enhance the patient's resistance. Such an amount is defined to be a “prophylactically effective dose.” In this use, the precise amounts depend upon the host's state of health and general level of immunity, but is generally in the ranges described above.

[0132] Single or multiple administrations of the compositions can be carried out with dose levels and frequency selected by the treating physician. In any event, the pharmaceuticalformulations should provide a quantity of the modified CD25 cells of this invention sufficient to treat the patient effectively.

[0133] The terms “immunogen,” or “immunogenic composition” or “vaccine” are used herein to refer to a compound or composition, as appropriate, that is capable of either: a) generating an immune response against an antigen in a naive individual; or b) reconstituting, boosting, or maintaining an immune response in an individual. The immunological response may comprise antibodies, immunoreactive cells (such as helper / inducer or cytotoxic cells), or any combination thereof.

[0134] “Inactivation” of a cell is used herein to indicate that the cell has been rendered incapable of cell division to form progeny. The cell may nonetheless be capable of response to stimulus, or biosynthesis and / or secretion of cell products such as cytokines. Methods of inactivation are known in the art. Preferred methods of inactivation are treatment with toxins, such as mitomycin C, or irradiation. Cells that have been fixed or permeabilized and are incapable of division are also examples of inactivated cells.

[0135] In genera], the practice of embodied methods of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques and necessary definitions are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, 2nd edition (Sambrook et al., 1989); Oligonucleotide Synthesis (Gait, ed., 1984); Animal Cell Culture (Preshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (Weir & Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (Miller & Calos, eds., 1987); Current Protocols in Molecular Biology (Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (Coligan et al., eds., 1991). General procedures for the preparation and administration of pharmaceutical compositions are outlined in Remington’s Pharmaceutical Sciences, 18th Edition (1990), Martin ed., Mack Publishing Co., Pa.

[0136] The treated individual may be exhibiting an active autoimmune response (either humoral or cellular immunity, or both) or GVHD. However, the subject should be at least partly immunocompetent, so as to minimize a GVHD reaction of pathological scope. However, it is recognized that cancer patients or those affected by autoimmune or other immunogenic diseases often show a degree of immunosuppression, and this does notnecessarily prevent the use of the compositions of the invention, as long as the compositions may be given safely and effectively.

[0137] Examples of diseases and disorders that can be treated according to this disclosure include, but are not limited to an autoimmune disorder, acute kidney injury, ankylosing spondylitis, autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune lymphoproliferative syndrome, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune urticaria, autoimmune uveitis, Crohn’s disease, dermatomyositis, diabetic nephropathy, diabetic retinopathy, graft versus host (GVH) disease, Hashimoto’s thyroiditis, idiopathic inflammatory bowel disease (IBD), and inflammatory demyelinating disease, inflammatory neuropathy, insulitis, interstitial cystitis, juvenile idiopathic arthritis, lupus, lupus erythematosus, lupus glomerulonephritis, lupus nephritis, IgA nephropathy, membranous nephropathy (MPGN), microscopic colitis, multiple sclerosis, myasthenia gravis, obesity, pancreatitis, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, renal ischemia reperfusion injury, rheumatoid arthritis, Sjogren's syndrome, systemic lupus erythematosus, transplant rejection, type 1 diabetes, type 2 diabetes, ulcerative colitis, vasculitis, and Wegener's granulomatosis.

[0138] In some embodiments, the disease or disorder is a cancer. For example, the cancer can be any one of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute myelogenous leukemia, adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, B-cell lymphoma, brain cancer, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, midline tract carcinomainvolving NUT gene, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal cavity and par nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphocytic leukemia, T-cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms tumor.

[0139] The cancer can be selected from the group consisting of melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, B-cell lymphoma, acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, bladder cancer, and lung cancer.

[0140] Though not wishing to be bound by theory, the method can either upregulate or down-regulate the expression of co-stimulatory molecules (including and not limited to CD80 (B7-1), CD86 (B7-2), CD40, ICOS, MHC-I, MHC-II, PD1, PD-L1, GITR, BAFF-R, 0x40, 41BB, DR3, CR2) on the antigen-presenting cells including and not limited to dendritic cells, macrophages, B-cells, innate lymphoid cells, NK cells, epithelial cells, endothelial cells or stromal cells.Modes of Administration and Dose

[0141] The compositions of this invention can be administered to the subject by any recognized methods, either systemically or at a localized site. The most convenient time to administer the alloactivated cells to prevent GVHD in a transplant patient or in a cancer patient is during the time of surgery. To keep the cells at the site until completion of the surgical procedure, it is convenient to administer the cells in a pharmaceutically compatible artificial gel, or in clotted plasma or by utilizing any other known controlled release mechanism.

[0142] When less invasive procedures are desired, the composition can be injected at a desired location through a needle. For deeper sites, the needle can be positioned using endoscopic ultrasound techniques, radioscintigraphy, or some other imaging technique, alone or in combination with the use of an appropriate scope or cannula. For such applications, the cell population is conveniently administered when suspended in isotonic saline or a neutral buffer to a volume of about 10 ml.

[0143] Similarly, prior to or simultaneously with an allogeneic transplant, an effective amount of isolated CD4+CD25+ cells, preferably modified to improve or culture-enhance their suppressive effect are administered as a cellular therapy. Implant to the transplant recipient (the host) in an amount sufficient to prevent or block the occurrence of GVHD. In an alternative, the cellular implant is administered following the allogeneic transplant to block, inhibit or reverse GVHD that may have already begun. The dose given is an amount “effective” in bringing about a desired therapeutic response, be it the stimulation of an immune response, or the treatment of cancer as defined elsewhere in this disclosure. For the pharmaceutical compositions of this invention, effective doses typically fall within the range of about 106to 1012cells, more preferably 108to 1011cells, including allogeneic stimulators and responders. Preferably, between about IxlO9to 5xlO10cells are used; more preferably between about 2xl09to 2xlO10. On average as many as IxlO9culture-expanded Treg cells will be needed for clinical trials in a human. Multiple doses when used in combination to achieve a desired effect each fall within the definition of an effective amount.

[0144] The various components of the implanted or injected composition are present in an “effective combination,” meaning that there are sufficient amounts of each of the components for the composition to be effective. Preferably, at least about IxlO8, more preferably between about IxlO9to 5xlO10, and more preferably between about 2xl09to 2xlO10responder cells are present. Preferably, at least about IxlO7, more preferably between about 5xl07to 5xl09and; more preferably between about IxlO8to 2xl09suppressor cells are present. Ratios of allogeneic lymphocytes to suppressor leukocytes are generally between 1: 1 and 100: 1, usually between about 5:1 and about 25:1, and typically about 10:1. However, any number of component cells or other constituents may be used, as long as the composition is effective as a whole. This will also depend upon culture conditions and other factors during preparation.

[0145] The pharmaceutical compositions of this invention may be given following, preceding, in lieu of, or in combination with, other therapies relating to generating an immune response or treating cancer in the subject or reducing the effect of GVHD. Forexample, the subject may have previously been or concurrently be in the process of treatment by chemotherapy, radiation therapy, and other forms of immunotherapy and adoptive transfer.

[0146] Where such modalities are used, they are preferably employed in a way or at a time that does not interfere with the immunogenicity of the compositions of this invention. The subject may also have been administered another composition, such as a vaccine, in order to stimulate an immune response. Such alternative compositions may include tumor antigen vaccines, nucleic acid vaccines encoding tumor antigens, anti-idiotype vaccines, and other types of cellular vaccines, including cytokine-expressing tumor cell lines. When the culture- expanded Treg cells are derived from a specific origin, such as a cancer or cancer cell, the term is intended to include, for example, not only a primary cancer cell, but any cell derived from a cancer cell ancestor, metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells.

[0147] Certain embodiments of this invention relate to combination therapies. In one combination therapy, the subject is infused with ex vivo culture-expanded autologous or allogeneic CD4+CD25+T cells, either before, during, or after tissue transplantation to promote engraftment and suppress or prevent GVHD responses. Although only a single infusion is disclosed, such infusions may be given weekly for a period of time (such as for 4- 6 weeks) following the cellular implant, to enhance the extent of the suppressive response in the host or the therapeutic effectiveness. The infusions can also be given after intervals of several months in order to replenish the response. Accordingly, certain embodiments of this disclosure relate to administering a cellular implant, and subsequently boosting the therapeutic effect or immunological response by administering to the patient a composition comprising alloactivated human CD4+CD25+T cells autologous or allogeneic to the patient, but treated ex vivo. Certain embodiments may further comprise an inactivated cell population of tumor cells or the progeny thereof when the patient is a cancer patient or progeny

[0148] In some embodiments, an allogeneic cell transplant would also provide the desired results, although it may take more cells than if autologous cells were used. Moreover, the method has a higher probability of success if the cells were purified at a high level of stringency and CD25 -depleted to reduce the volume of cells needed to achieve a therapeutic effect in the patient and to enhance the efficiency of the cellular implant.

[0149] Timing of administration of compositions of this disclosure is within the judgment of the managing physician, and depends on the clinical condition of the patient, the objectivesof treatment, and concurrent therapies also being administered. Suitable means of immunological monitoring include a one-way MLR using patient's PBL as responders and primary tumor cells as stimulators. An immunological reaction may also be manifest by a delayed inflammatory response at the injection site. Suitable means of monitoring of the effectiveness of the Treg cell treatment may include in vitro assays, such as MLR, or in vivo tracking, such as CT scan, magnetic resonance imaging (MRI), radioscintigraphy with a suitable imaging agent, monitoring of circulating tumor marker antigens, and the subject's clinical response. Additional doses may be given, such as on a monthly or weekly basis, until the desired effect is achieved. Thereafter, and particularly when the immunological or clinical benefit appears to subside, additional booster or maintenance doses may be given as required.

[0150] When multiple cellular implants or combinations of implants and cellular vaccines are given to the same patient, attention should be paid to the possibility that the allogeneic lymphocytes in the vaccine may generate an anti-allotype response. The use of a mixture of allogeneic cells from a plurality of donors, and the use of different allogeneic cell populations in each dose, are both strategies that can help minimize the occurrence of an anti-allotype response.

[0151] During the course of therapy, the subject is evaluated on a regular basis for general side effects such as a febrile response. Side effects are managed with appropriate supportive clinical care. iii. Compositions

[0152] The methods described herein can utilize compositions that comprise an interleukin- 2 (IL-2) and a truncated interleukin- 33 (IL-33). In the composition, the interleukin-2 (IL-2) can comprise SEQ ID NO: 13, or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13. Alternatively or in addition, the truncated interleukin- 33 (IL-33) can comprise SEQ ID NO: 1, or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.

[0153] The IL-2 and the truncated IL-33 will generally be described herein in the context of fusion proteins. However, the person of ordinary skill in the art will understand that the descriptions of IL-2 and truncated IL-33 domains will apply equally to IL-2 and truncated IL- 33 polypeptides in the compositions, except for description specific to fusion proteins (e.g., arrangement of domains in a fusion protein or the like). iv. Fusion Proteins

[0154] In some embodiments, the methods of the present disclosure utilize a fusion protein. The fusion protein can comprise an interleukin-2 (IL-2) domain and a truncated interleukin- 33 (IL-33) domain. The fusion protein can comprise an interleukin-2 (IL-2) domain comprising SEQ ID NO: 13, or an IL-2 domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13; and a truncated interleukin-33 (IL-33) domain comprising SEQ ID NO:1, or a truncated IL-33 domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.

[0155] In some embodiments, the fusion protein can comprise an optional linker between a first and a second domain.

[0156] In some embodiments, the fusion protein can comprise more than one IL-2 domain, more than one truncated IL-33 domain, or both.

[0157] In the fusion proteins, the domains can be joined by a linker, such as an oligopeptide linker. The compositions or fusion proteins can have one or more advantageous properties, including but not limited to any one or more of reduced aggregation, improved stability during storage, retention of IL-33 activity after storage, easier production by recombinant organisms, easier purification, IL-2 activity comparable to wild type IL-2, IL-33 activity comparable to wild-type IL-33, and prolongation of IL-33 activity post administration. Though not wishing to be bound by theory, such advantageous properties can arise from sitespecific mutations in IL-2 domains and / or truncated IL-33 domains that reduce aggregation, such as by replacing cysteine residues with other residues that are not capable of forming disulfide linkages.IL-2 domains

[0158] The IL-2 domain of the fusion protein can be identical to or a variant of the wild type IL-2 sequence of any organism. In one aspect, the IL-2 domain is at least 80%, 85%, or 90% identical to the wild type IL-2 sequence of the organism to which the fusion protein is intended for administration in the methods described herein.

[0159] In one embodiment, the IL-2 domain comprises a sequence having at least 80%, at least 85%, at least 90% identity to SEQ ID NO: 13, such as at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 13. The IL-2 domain of the fusion protein can differ from SEQ ID NO: 13 byzero, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or more positions.

[0160] The sequence of the IL-2 domain can comprise a number of variants of SEQ ID NO: 13. For example, the IL-2 domain can comprise a point mutation of the Asn at position 88 (N88) of SEQ ID NO: 13. IL-2 domains comprising a point mutation of N88 can exhibit one or more advantageous properties, including, but not limited to, higher stability during storage and / or higher activity than a corresponding IL-2 domain lacking the point mutation of N88.

[0161] For a particular example, the IL-2 domain can comprise a point substitution from Asn to Asp at position 110 (N88D) of SEQ ID NO: 13. A particular IL-2 domain containing the N88D substitution has the sequence of SEQ ID NO: 14.

[0162] In another embodiment, the IL-2 domain can comprise a point mutation of the Cys at position 125 (Cl 25) of SEQ ID NO: 13. IL-2 domains comprising a point mutation of Cl 25 are less likely to form disulfide linkages within the IL-2 domain and / or to Cys residues in the IL-33 domain, and can exhibit one or more advantageous properties, including but not limited to, reduced aggregation, slower clearance in vivo, or higher IL-2 activity than a corresponding IL-2 domain lacking the point mutation of Cl 25.

[0163] In a specific embodiment, the IL-2 domain can comprise a point substitution from Cys to Ser at position 125 (C125S) of SEQ ID NO: 13. A particular IL-2 domain containing the C125S substitution has the sequence of SEQ ID NO: 15.

[0164] Different IL-2 mutations can decrease the interaction of IL-2 with IL2Ra (CD25) and / or increase the interaction of IL-2 with IL2R0 (CD122), allowing differential activation of NK and CD8 T cells compared to Tregs.

[0165] Any two or more of the deletions or substitutions within the IL-2 domain as described above can be combined. In one embodiment, the IL-2 domain can comprise point mutations of both the Asn at position 88 and the Cys at position 125 of SEQ ID NO: 13. For a particular example, the IL-2 domain can comprise both point substitutions N88D and C125S of SEQ ID NO: 13. A particular IL-2 domain containing both the N88D and the C125S substitution has the sequence of SEQ ID NO: 16.

[0166] The IL-2 domain may comprise mutations that yield increased affinity for IL-2R0, known in the art as “super-2” or “IL-2 superkines.” For example, the IL-2 domain may comprise any of the point mutation combinations provided in Table X below:Table X

[0167] In one embodiment, the IL-2 domain may include the following mutations: G27M, I28L, K32D, M39L, E52S, V69A, L72A, Q74G, S75D, K76D, N77P, F78K, H79T, L80I, deletion of R81, deletion of P82, L85V, I86V, and I92F. In another embodiment, the IL-2 domain may include the following mutations: G27M, I28L, K32D, V69A, L72Q, R81 D, F42A and E62A.Linkers

[0168] The optional linker can be any moiety capable of forming at least two covalent bonds to the N-terminus of a polypeptide, the C-terminus of a polypeptide, or an amino acid side chain of a polypeptide. Preferably, the linker is an oligopeptide sequence, comprising one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. An oligopeptide can form a covalent bond to the C-terminus of a polypeptide and the N-terminus of (typically another) a polypeptide.

[0169] Preferably, the amino acids are encoded by codons of the canonical genetic code. This can allow a fusion protein as a whole to be encoded by a single polynucleotide and be transcribed and translated as a unit, thereby simplifying manufacture of the fusion protein.

[0170] When the linker is an oligopeptide, the amino acids thereof can be chosen to allow the linker to be flexible, generally unlikely to form secondary structures, generally unlikely to interfere with the tertiary structures of the domains linked by the linker, generally unlikely tolink with any domain of the fusion protein other than at the C-terminus and N-terminus described above, and generally non-immunogenic. An oligopeptide linker comprising at least about 75% Gly residues can be used.

[0171] Particular amino acid residues of an oligopeptide linker can be chosen to adjust the isoelectric point (pl) of the fusion protein, which can be useful in increasing the yield, purity, storage, or efficacy of the fusion protein in manufacture or use.

[0172] For example, the linker can comprise GGGGS (SEQ ID NO: 9). In another aspect, the linker can comprise the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 10).

[0173] In some embodiments, the fusion protein can comprise an optional additional linker, whereby the fusion protein can dimerize or multimerize. The additional linker can be an oligopeptide linker, such as those described herein.IL-33 domains

[0174] The IL-33 domain can have a wild type, variant, truncated, or truncated variant sequence of IL-33 from any species. In one aspect, the IL-33 domain comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the wild type IL-33 sequence of the organism to which the fusion protein is intended for administration in the methods described herein.

[0175] Wild type human truncated IL-33 has the sequence of SEQ ID NO: 1. Other organisms have truncated IL-33 comprising residues homologous to the human truncated IL- 33 of SEQ ID NO: 1.

[0176] In one aspect, the truncated IL-33 domain comprises a sequence having at least 90% identity to SEQ ID NO: 1, such as at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 1. The truncated IL-33 domain of the fusion protein can differ from SEQ ID NO: 1 at zero, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more positions.

[0177] In some embodiments, the truncated IL-33 domain can comprise a sequence with at least 90%, / 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1, wherein one or more possible sites of glycosylation or disulfide linkage (e.g., N60, C97, Cl 16, C121, and C148) are deleted or substituted by one or more amino acids.

[0178] In some embodiments, the truncated IL-33 domain comprises one or more amino acid substitutions at N60, C97, Cl 16, C121, and C148 of SEQ ID NO: 1. In some embodiments, the N60 of SEQ ID NO: 1 is substituted. In some embodiments, the C97 of SEQ ID NO:1 is substituted. In some embodiments, the Cl 16 of SEQ ID NO: 1 is substituted. In some embodiments, the C 121 of SEQ ID NO: 1 is substituted. In some embodiments, the C148 of SEQ ID NO: 1 is substituted. In some embodiments, the N60 and C97 of SEQ ID NO: 1 are substituted. In some embodiments, the N60 and Cl 16 of SEQ ID NO: 1 are substituted. In some embodiments, the N60 and C121 of SEQ ID NO: 1 are substituted. In some embodiments, the N60 and C148 of SEQ ID NO: 1 are substituted. In some embodiments, the C97 and Cl 16 of SEQ ID NO: 1 are substituted. In some embodiments, the C97 and C121 of SEQ ID NO: 1 are substituted. In some embodiments, the C97 and C148 of SEQ ID NO:1 are substituted. In some embodiments, the Cl 16 andC121 of SEQ ID NO: 1 are substituted. In some embodiments, the Cl 16 and C148 of SEQID NO:1 are substituted. In some embodiments, the C121 and C148 of SEQ ID NO: 1 are substituted. In some embodiments, the N60, C97 and Cl 16 of SEQ ID NO: 1 are substituted. In some embodiments, the N60, C97 and C121 of SEQ ID NO: 1 are substituted.In some embodiments, the N60, C97 and C148 of SEQ ID NO: 1 are substituted. In some embodiments, the N60, Cl 16 and C121 of SEQ ID NO: 1 are substituted. In some embodiments, the N60, Cl 16 and C148 of SEQ ID NO: 1 are substituted. In some embodiments, the N60, C121 and C148 of SEQ ID NO: 1 are substituted. In some embodiments, the C97, C116 and C121 of SEQ ID NO: 1 are substituted. In some embodiments, the C97, Cl 16 and C148 of SEQ ID NO: 1 are substituted. In some embodiments, the C97, C121 and Cl 48 of SEQ ID NO: 1 are substituted. In some embodiments, the N60, C97, Cl 16 and C121 of SEQ ID NO: 1 are substituted. In some embodiments, the N60, C97, Cl 16 and C148 of SEQ ID NO:1 are substituted. In some embodiments, the N60, Cl 16, C121 and C148 of SEQ ID NO: 1 are substituted. In some embodiments, the C97, C116, C121 and C148 of SEQ ID NO:1 are substituted. In some embodiments, the N60, C97, Cl 16, C121 and C 148 of SEQ ID NO. 1 are substituted.

[0179] A number of variants of SEQ ID NO: 1 can be particularly considered for the sequence of the truncated IL-33 domain.

[0180] In one aspect, the truncated IL-33 domain can comprise a point mutation of the Asn at position 60 (N60) of SEQ ID NO: 1. As shown in FIG. 3, N60 is present in human truncated IL-33, but is not present in the homologous position in mouse or rat truncated IL-33. Truncated IL-33 domains comprising a point mutation of N60 are less susceptible to glycosylation, and can exhibit one or more advantageous properties including, but not limited to, reduced aggregation, slower clearance in vivo, or higher activity than a corresponding truncated IL-33 domain lacking the point mutation of N60. In a particular example, the truncated IL-33 domain can comprise a point substitution at position 60 from Asn to Ser (N60S), or from Asn to Asp (N60D). A particular truncated IL-33 domain comprising the N60S point substitution can have the sequence of SEQ ID NO: 2.

[0181] In one embodiment the truncated IL-33 domain can comprise a point mutation of the Cys at position 116 (Cl 16) of SEQ ID NO: 2. Truncated IL-33 domains comprising a point mutation of Cl 16 are less likely to form disulfide linkages within the truncated IL-33 domain and / or to Cys residues in the IL-2 domain, and can exhibit one or more advantageous properties including, but not limited to, reduced aggregation, slower clearance in vivo, or higher activity than a corresponding truncated IL-33 domain lacking the point mutation of C116.

[0182] In one embodiment, the truncated IL-33 domain can comprise a point substitution from Cys to Phe at position 116 (C116F). The Cl 16F point substitution was based on the observation that mouse and rat truncated IL-33 comprise a Phe at the position homologous to human wild type Cl 16, as shown in FIG. 3.

[0183] The C116F point substitution provides improvement to expression and purification of fusion proteins comprising both IL-2 domains and truncated IL-33 domains, and the activities of the truncated IL-33 domains and the IL-2 domains are essentially the same as wild type IL-33 alone and IL-2 alone, respectively.

[0184] A particular truncated IL-33 domain comprising the C116F point substitution can have the sequence of SEQ ID NO: 3.

[0185] In one aspect, the truncated IL-33 domain can comprise a point mutation of the Cys at position 97 (C97) of SEQ ID NO: 2. Truncated IL-33 domains comprising a point mutation of C97 are less likely to form disulfide linkages and can exhibit one or more of the desirable properties discussed with reference to Cl 16.

[0186] In another aspect, the truncated IL-33 domain can comprise a point substitution from Cys to Gly at position 97 (C97G). The C97G point substitution was based on the observation that mouse and rat truncated IL-33 comprise a Gly at the position homologous to human wild type C97, as shown in FIG. 3.

[0187] In the context of the C97G point substitution, the activities of both the truncated IL- 33 domains and the IL-2 domains are essentially the same as wild type IL-33 alone and IL-2 alone, respectively.

[0188] A particular truncated IL-33 domain comprising the C97G point substitution can have the sequence of SEQ ID NO: 4.

[0189] Any two or more of the deletions or substitutions within the truncated IL-33 domain as described above can be combined. For example, the truncated IL-33 domain can comprise both N60S and Cl 16F (SEQ ID NO: 5); both N60S and C97G (SEQ ID NO: 6); both Cl 16F and C97G (SEQ ID NO: 7); or all three of N60S, Cl 16F, and C97G (SEQ ID NO: 8), or three of C97G, Cl 16F and C148G (SEQ ID NO: 17).Accordingly, in some embodiments, the truncated IL-33 domain has a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 17.

[0190] In some embodiments, the truncated IL-33 domain has a sequence from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 7.Signal peptides

[0191] The fusion protein can also comprise a signal peptide, such as at the N-terminus of the fusion protein. The signal peptide can be chosen from signal peptides known for use with the N-terminal domain of the fusion protein, or can be selected based on rational design or routine experimentation.

[0192] In one embodiment, if the N-terminal domain of the fusion protein is the IL-2 domain, the signal peptide can have the sequence of SEQ ID NO: 11.Linkage of domains

[0193] The various domains of a fusion protein can be linked through any desired reactive sites of the various domains, either directly or via the reactive moieties of the optional linker. In some embodiments, wherein the linker is an oligopeptide, the various domains can be linked through their N- and / or C-termini to the complementary terminus of the linker. A fusion protein can comprise a single continuous polypeptide sequence. Such a fusion protein can be prepared from a single polynucleotide coding region encoding the entire sequence of the fusion protein, by well-known transcriptional and translational processes.

[0194] A fusion protein comprising a single continuous polypeptide sequence can comprise the various domains in any desired order. For example, the fusion protein can comprise anyof the IL-2 domain or the truncated IL-33 domain at its N-terminus; the linker; and the remaining domain at its C-terminus.

[0195] In one particular embodiment, the fusion protein can comprise, in order from N- terminus to C-terminus, a signal peptide; an interleukin-2 (IL-2) domain; a linker; and a truncated interleukin-33 (IL-33) domain.

[0196] In one particular embodiment, the fusion protein can comprise, in order from N- terminus to C-terminus, a signal peptide; a truncated IL-33 domain; a linker; and an IL-2 domain.

[0197] In one particular embodiment, the fusion protein can comprise, in order from N- terminus to C-terminus, a signal peptide; an IL-2 domain; and a truncated IL-33 domain.

[0198] In one particular embodiment, the fusion protein can comprise, in order from N- terminus to C-terminus, a signal peptide; a truncated IL-33 domain; and an IL-2 domain.

[0199] In one particular embodiment, the fusion protein can comprise, in order from N- terminus to C-terminus, an IL-2 domain; a linker; and a truncated IL-33 domain.

[0200] In one particular embodiment, the fusion protein can comprise, in order from N- terminus to C-terminus, a truncated IL-33 domain; a linker; and an IL-2 domain.

[0201] In one particular embodiment, the fusion protein can comprise, in order from N- terminus to C-terminus, a truncated IL-33 domain and an IL-2 domain.

[0202] In one particular embodiment, the fusion protein can comprise, in order from N- terminus to C-terminus, an IL -2 domain and a truncated IL-33 domain.

[0203] In another particular embodiment, the fusion protein can comprise two polypeptides linked by an additional linker between amino acid side chains. The first polypeptide can comprise, in order from N-terminus to C-terminus, an optional signal peptide and an IL-2 domain; the second polypeptide can comprise a truncated IL-33 domain; and the first and second polypeptides can be linked by an additional linker between side chains of an amino acid in the IL-2 domain of the first polypeptide and an amino acid in the truncated IL-33 domain of the second polypeptide.

[0204] Manufacture of fusion proteins

[0205] The fusion proteins described herein can be manufactured by any known method. Generally, any polypeptide component of the fusion protein can be synthesized by chemical or biochemical methods, both of which are generally known to a person of ordinary skill in the art.

[0206] In biochemical methods of polypeptide synthesis, a polynucleotide comprising a nucleic acid sequence encoding the polypeptide of interest (e.g., the fusion protein or a portion thereof) is incorporated into a vector, wherein the polynucleotide is operatively coupled to a promoter. The vector can comprise other sequences, such as non-promoter control sequences, an origin of replication, sequences engineered for insertion of the polynucleotide into the vector, etc. The promoter can be chosen such that when the vector is introduced into a host cell by recombinant DNA techniques, the promoter is recognized by the host cell’s endogenous RNA polymerase and, either constitutively or upon induction by one or more changes in process conditions, produces RNA transcripts. The RNA transcripts are then translated by the host cell’s ribosomes to produce the polypeptide of interest (e.g., the fusion protein or the portion thereof).

[0207] Another route of polypeptide synthesis is the introduction of a polynucleotide expressing the polypeptide into cells of a subject in need thereof. The polynucleotide can comprise mRNA or DNA. The subject’s cells can transcribe the DNA and / or translate the mRNA into the polypeptide of interest.

[0208] In embodiments wherein the fusion protein comprises multiple polypeptides, each polypeptide can be produced separately and the fusion protein assembled by peptide synthase reactions or by non-amino acid backbone linkages, such as by one or more non-oligopeptide linkers, by cross-polypeptide linkages through amino acid side chains of oligopeptide linkers, and / or by chemical conjugation.

[0209] In some embodiments, a composition comprising an IL-2 polypeptide and a truncated IL-33 polypeptide can be an intermediate stage in the preparation of a fusion protein comprising an IL-2 domain and a truncated IL-33 domain, and optionally a linker. v. Pharmaceutical compositions

[0210] In some embodiments, the present disclosure relates to a pharmaceutical composition. The pharmaceutical composition can comprise regulatory cells that have been stimulated with any composition or fusion protein described above. In some embodiments, the pharmaceutical composition can further comprise a pharmaceutically-acceptable carrier. Alternatively, or in addition, the pharmaceutical composition can further comprise an additional therapeutic agent.Pharmaceutically acceptable carriers

[0211] The pharmaceutical compositions can comprise a pharmaceutically-acceptable carrier. Pharmaceutically-acceptable carriers include physiologically tolerable or acceptablediluents, excipients, solvents, or adjuvants. The compositions are preferably sterile and nonpyrogenic.Additional therapeutic agents

[0212] In embodiments, additional therapeutic agents, by which is meant one or more therapeutic agents other than the fusion proteins described above, can be included in the pharmaceutical compositions. The additional therapeutic agents can include, but are not limited to, cytotoxic agents, anti-angiogenic agents, pro-apoptotic agents, antibiotics, hormones, hormone antagonists, chemokines, drugs, prodrugs, toxins, or enzymes.

[0213] Particular therapeutic agent(s) can be chosen in view of a disease or disorder intended to be treated by the pharmaceutical composition, among other parameters that will be known to a person of ordinary skill in the art.

[0214] The pharmaceutical compositions can also contain minor amounts of nontoxic auxiliary pharmaceutical substances, excipients, and / or additives, such as wetting agents, emulsifying agents, and / or pH buffering agents, among others known to a person of ordinary skill in the art.

[0215] If desired, absorption enhancing or delaying agents (such as liposomes, aluminum monostearate, or gelatin) can be used. The compositions can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.Adjunct therapies

[0216] For any disease or disorder, the method can further comprise administering adjunct therapy, wherein the adjunct therapy does not comprise the cell therapy.

[0217] For example, when the disease or disorder is cancer, the adjunct therapy can comprise surgical resection, radiation, chemotherapy, checkpoint inhibitor therapy, or other therapies known to a person of ordinary skill in the art.

[0218] If the adjunct therapy is of a modality suitable for administration by the same route as the cell therapy and essentially simultaneously with the cell therapy, the pharmaceutical composition can comprise the adjunct therapy.

[0219] In some embodiments, the adjunct therapy is a gene therapy.Medical uses of the fusion proteins

[0220] In some embodiments, the present disclosure relates to a cell therapy described herein for use in the treatment of a disease or disorder described herein.

[0221] In some embodiments, the present disclosure relates to the use of a cell therapy described herein in the treatment of a disease or disorder described herein.

[0222] In some embodiments, the present disclosure relates to the use of a cell therapy described herein in the manufacture of a medicament. The medicament can be for the treatment of a disease or disorder described herein. vi. Kits

[0223] In some embodiments, the present disclosure relates to kits, comprising a cell therapy as described herein and instructions for performing a method as described herein.

[0224] For example, a kit can comprise a pharmaceutical composition as described herein; and instructions for performing a method for treating a disease or disorder characterized by inflammation, the method comprising administering to a subject in need thereof an effective amount of the cell therapy.

[0225] The instructions can be packaged in the kit in the form of printed instructions, a printed document providing a uniform resource locator (URL) from which detailed instructions can be browsed or downloaded, a printed document providing a Quick Response (QR) code which can be scanned to direct a smartphone or tablet computer’ s browser to a URL, or the like.

[0226] Kits will generally include one or more vessels or containers so that some or all of the individual components and reagents, e.g., the pharmaceutical composition, can be separately housed. Kits can also include a means for enclosing individual containers in relatively close confinement for commercial sale, e.g., a plastic box, in which instructions, packaging materials such as Styrofoam, etc., can be enclosed. An identifier, e.g., a bar code, radio frequency identification (ID) tag, etc., can be present in or on the kit or in or one or more of the vessels or containers included in the kit. An identifier can be used, e.g., to uniquely identify the kit for purposes of quality control, inventory control, tracking, movement between workstations, etc.B. SequencesEQUIVALENTS

[0227] It will be readily apparent to those skilled in the art that other suitable modifications and adaptions of the methods of the invention described herein are obvious and can be made using suitable equivalents without departing from the scope of the disclosure or the embodiments. Having now described certain compositions and methods in detail, the same will be more clearly understood by reference to the following examples, which are introduced for illustration only and not intended to be limiting.EXAMPLES

[0228] The following are examples of methods and compositions of the invention. It is understood that various other embodiments can be practiced, given the general description provided herein.Table 1Example 1: Human Ex Vivo Idiopathic Nephritis Proof of Concept Study

[0229] The objective of the study was to investigate whether SLT-518(h4) can be used to expand ST2+ Tregs in PBMC from patients with autoimmune glomerulopathies as an ex vivo proof of concept. As shown in FIGs 1A and IB, SLT-518(h4) promotes dose dependent ST2+ Treg proliferation in fresh human PBMCs.Example 2: ST2+ Tregs in Patients with Autoimmune Glomerulopathies

[0230] This study identifies whether a deficiency exists in ST2+ Tregs in patients with autoimmune glomerulopathies, such as lupus nephritis, IgA nephropathy, membranous glomerulonephritis, and healthy controls. Five to ten donors from each group are analyzed.Example 3: Mechanisms Involving the effect on Tregs

[0231] scRNA transcriptomics on healthy PBMC samples treated with or without SLT-518 will be performed to identify the mechanisms involving the effect on Tregs and beyond.Example 4: Multiple Engineered ConstructsTable 2

[0232] Nine engineered constructs were made with various mutations in IL2 and / or IL33 (SLT-518(hl), SLT-518(h2), SLT-518(h3), SLT-518(h4), SLT-518(h5), SLT-518(h6), SLT- 518(h7), SLT-518(h8), and SLT-518(h9). The details of the mutations contained within each construct are provided in the above Table 2. The rationally designed variants were produced to optimize expression, activity, and stability, while retaining wild type sequences to limit immunogenicity risk.Example 5: Ex- vivo ST2+ Treg cell expansion

[0233] This example tested expansion of ST2+Tregs ex vivo using human PBMC from diseased donors by treating cells with SLT-518(h4), IL-2, IL-33, or IL-2 and IL-33.

[0234] PBMCs from Autoimmune Lupus Nephritis, Membranous Nephropathy, IgA Nephropathy and non-autoimmune CKD donors were prepared and treated with IL-233 fusion protein (SLT-518(h4)) or equimolar concentrations of IL-33 and IL-2(N88D)-Fc for overnight in an ex vivo assay. The expansion of ST2+Treg cells was quantified using flow cytometry. Live lymphocytes were first isolated from each cell expansion sample. CD4+ and CD3+ T cells were further analyzed for ST2+ Treg cells.

[0235] The results were shown in FIG. 5 and FIG. 6. PBMC samples treated with SLT518(h4) fusion protein showed greater ST2+ Treg cell expansion, as compared to the Treg expansion treated with IL-33 alone, IL-2 alone, or IL-2 and IL-33. For example, the PBMC samples from IgA Nephropathy donors have a strong response to the SLT-518(h4) stimulation. 33.2% ST2+ Treg cells were covered from PBMCs treated with SLT518(h4). 23.4% ST2+ Treg cells from IL-2 alone, 19.8% ST2+ Treg cells from IL-33 alone, 21.5% ST2+ Treg cells from IL-2&IL-33 and 17.2% ST2+ Treg cells from IL-2 (N88D)-Fc, were covered. A very low number of ST2+ Treg cells (2.6%) was present in the control PBMC sample which was cultured without any stimulation.

[0236] Similarly, the PBMC samples from Membranous Nephropathy donors have a strong response to the SLT-518(h4) stimulation. 40.1% ST2+ Treg cells were covered from PBMCstreated with SLT518(h4). 26.2% ST2+ Treg cells from IL-2 alone, 6.1% ST2+ Treg cells from IL-33 alone, 26.8% ST2+ Treg cells from IL-2&IL-33 and 5.0% ST2+ Treg cells from IL-2 (N88D)-Fc, were covered. A low number of ST2+ Treg cells (5.3%) was present in the control PBMC sample which was cultured without any stimulation.

Claims

CLAIMSWHAT IS CLAIMED:

1. A method of suppressing inflammation in a patient in need thereof comprising administering to the patient activated, expanded regulatory cells, wherein the culture- expanded regulatory cells are stimulated with IL-33, a combination of IL-2 and IL-33, or an IL233 fusion protein or variants thereof.

2. A method of treating autoimmune disease in a patient in need thereof comprising administering to the patient activated, culture-expanded regulatory cells, wherein the culture- expanded regulatory cells are stimulated with IL-33, a combination of IL-2 and IL-33, or an IL233 fusion protein or variants thereof.

3. The method of claim 1 or 2, wherein the expanded regulatory cells are ex vivo culture- expanded regulatory cells.

4. The method of claim 1 or 2, wherein the expanded regulatory cells are expanded in vitro.

5. The method of claim 4, wherein the autoimmune disease is selected from the group consisting of type-1 diabetes, SLE (systemic lupus erythematosus), lupus nephritis, inflammatory bowel disease, and multiple sclerosis.

6. A method of suppressing allograft rejection in a patient in need thereof comprising administering to the patient activated, culture-expanded regulatory cells, wherein the culture- expanded regulatory cells are stimulated with IL-33, a combination of IL-2 and IL-33, or an IL233 fusion protein or variants thereof.

7. The method of claim 6, wherein the allograph rejection is acute allograph rejection.

8. The method of claim 6, wherein the allograph rejection is chronic allograph rejection.

9. A method of treating or preventing GvHD in a patient in need thereof comprising administering to the patient activated, ex vivo culture-expanded regulatory cells, wherein the culture-expanded regulatory cells are stimulated with IL-33, a combination of IL-2 and IL-33, or an IL233 fusion protein or variants thereof.

10. The method of any one of the preceding claims, wherein said treating, preventing, or suppression includes inhibiting proliferation of auto-reactive T cells, auto-reactive B cells, alloreactive T cells, alloreactive B cells, syngeneic natural killer cells, allogeneic natural killer cells, innate lymphoid cells (ILC), macrophages, mast cells, ST2+ Tregs, and / or gamma-delta T cells.

11. The method of any one of the preceding claims, wherein said regulatory cells are selected from the group consisting of autologous CAR-Tregs, allogeneic CAR-Tregs, engineered CAR-Tregs, and CD4+ Tregs.

12. The method of claim 11, wherein said CD4+ Tregs are ST2+ Tregs.

13. The method of claim 6, wherein said regulatory cells are selected from the group consisting of Foxp3+ Tregs, Foxp3-negative IL- 10+ (Tri Tregs), TGF-b+ (Th3 Tregs), IL- 35+ (Tr35 Tregs), CD8 Tregs, regulatory macrophages, regulatory dendritic cells, B- regulatory cells (Bregs), regulatory mast cells, regulatory basophils, regulatory eosinophils, regulatory NK cells (NK-regs), regulatory NK-T cells, and regulatory innate lymphoid cells (ILC).

14. The method of any one of claims 11-13, wherein said regulatory cells are modified to express IL-33 receptor (ST2) by genetic manipulation selected from the group consisting of transfection, transduction, electroporation, CRISPR / Cas9 mediated gene-insertion, and synthetic mRNA delivery.

15. A method for generating tolerogenic dendritic cells or reparative macrophages comprising contacting one or more cells with activated, ex vivo culture-expanded regulatory cells, wherein the culture-expanded regulatory cells are stimulated with IL-33, a combination of IL-2 and IL-33, or an IL233 fusion protein or variants thereof.

16. The method of any one of the preceding claims, wherein the culture-expanded regulatory cells are stimulated with: a. an interleukin-2 (IL-2) having at least 90% identity to SEQ ID NO: 13; and b. a truncated interleukin- 33 (IL-33), comprising a sequence with at least 90% identity to SEQ ID NO: 1.

17. The method of any one of claims 1-15, wherein the culture-expanded regulatory cells are stimulated with a. an interleukin-2 (IL-2) having the amino acid sequence of SEQ ID NO: 14; and b. a truncated interleukin- 33 (IL-33), having the amino acid sequence of SEQ ID NO: 3.

18. The method of any one of claims 1-15, wherein the culture-expanded regulatory cells are stimulated with a fusion protein, comprising: a. an interleukin-2 (IL- 2) domain having at least 90% identity to SEQ ID NO: 13; and b. a truncated interleukin-33 (IL-33) domain, comprising a sequence with at least 90% identity to SEQ ID NO:

119. The method of any one of claims 1-15, wherein the culture-expanded regulatory cells are stimulated with a fusion protein, comprising: a. an interleukin-2 (IL- 2) having the amino acid sequence of SEQ ID NO: 14; and b. a truncated interleukin-33 (IL-33), having the amino acid sequence of SEQ ID NO:3.

20. The method of any one of claims 16-19, wherein in the truncated IL-33 or the truncated IL-33 domain, at least one of N60, C97, Cl 16, C121, and C148 of SEQ ID NO: 1 is substituted by a single amino acid.

21. The method of claim 16 or 18, wherein N60 of SEQ ID NO: 1 is substituted by Ser or Asp; C97 of SEQ ID NO: 1 is substituted by Gly; and / or Cl 16 of SEQ ID NO: 1 is substituted by Phe.

22. The method of any one of claims 18 to 21, wherein the truncated IL-33 or the truncated IL-33 domain has a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 7.

23. The method of any one of claims 18 to 22, wherein the fusion protein further comprising a linker.

24. The method of claim 23, wherein the linker comprises GGGGS (SEQ ID NO: 9).

25. The method of claim 23 or 24, wherein the linker has the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 10).

26. The method of any one of claims 18 to 25, wherein the IL-2 or the IL-2 domain has a sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14; SEQ ID NO: 15; and SEQ ID NO: 16.

27. The method of any one of claims 18 to 26, wherein the fusion protein further comprises a signal peptide.

28. The method of claim 27, wherein the fusion protein comprises a signal peptide that has the sequence of SEQ ID NO: 11.

29. The method of any one of claims 18 to 28, wherein the domains are, in order from N- terminus to C-terminus, an optional signal peptide; the interleukin-2 (IL-2) domain; an optional linker; and the truncated interleukin- 33 (IL-33) domain.

30. A method for achieving an immunosuppressive effect in a patient with an alloresponse or autoimmune response comprising administering to said patient, an effective amount of activated long-term, ex vivo culture-expanded regulatory cells to achieve therapeuticsuppression of said response, wherein the culture-expanded regulatory cells are stimulated with a. an interleukin-2 (IL-2) having at least 90% identity to SEQ ID NO: 13; and b. a truncated interleukin- 33 (IL-33), comprising a sequence with at least 90% identity to SEQ ID NO: 1, and wherein the regulatory cells are capable of inhibiting proliferation of the alloreactive T cells in a mixed lymphocyte reaction (MLR) assay by at least 90%.

31. A method for achieving an immunosuppressive effect in a patient with an alloresponse or autoimmune response comprising administering to said patient, an effective amount of activated long-term, ex vivo culture-expanded regulatory cells to achieve therapeutic suppression of said response, wherein the culture-expanded regulatory cells are stimulated with a. an interleukin-2 (IL- 2) having the amino acid sequence of SEQ ID NO: 14; and b. a truncated interleukin-33 (IL-33), having the amino acid sequence of SEQ ID NO: 3 and wherein the regulatory cells are capable of inhibiting proliferation of the alloreactive T cells in a mixed lymphocyte reaction (MLR) assay by at least 90%.

32. A method for achieving an immunosuppressive effect in a patient with an alloresponse or autoimmune response comprising administering to said patient, an effective amount of activated long-term, ex vivo culture-expanded regulatory cells to achieve therapeutic suppression of said response, wherein the culture-expanded regulatory cells are stimulated with a fusion protein comprising: a. an interleukin-2 (IL-2) domain having at least 90% identity to SEQ ID NO: 13; and b. a truncated interleukin- 33 (IL-33) domain, comprising a sequence with at least 90% identity to SEQ ID NO: 1, and wherein the regulatory cells are capable of inhibiting proliferation of the alloreactive T cells in a mixed lymphocyte reaction (MLR) assay by at least 90%.

33. A method for achieving an immunosuppressive effect in a patient with an alloresponse or autoimmune response comprising administering to said patient, an effective amount of activated long-term, ex vivo culture-expanded regulatory cells to achieve therapeutic suppression of said response, wherein the culture-expanded regulatory cells are stimulated with a fusion protein comprising: a. an interleukin-2 (IL-2) having the amino acid sequence of SEQ ID NO: 14; andb. a truncated interleukin- 33 (IL-33), having the amino acid sequence of SEQ ID NO:3, and wherein the regulatory cells are capable of inhibiting proliferation of the alloreactive T cells in a mixed lymphocyte reaction (MLR) assay by at least 90%.

34. The method of any one of claims 30-33, wherein in the truncated IL-33 or the truncated IL-33 domain, at least one of N60, C97, Cl 16, C121, and C148 of SEQ ID NO: 1 is substituted by a single amino acid residue.

35. The method of claim 30 or 32, wherein N60 of SEQ ID NO: 1 is substituted by Ser or Asp; C97 of SEQ ID NO: 1 is substituted by Gly; and / or Cl 16 of SEQ ID NO: 1 is substituted by Phe.

36. The method of any one of claims 32 to 35, wherein the truncated IL-33 or the truncated IL-33 domain has a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 7.

37. The method of any one of claims 32 to 36, wherein the fusion protein further comprising a linker.

38. The method of claim 37, wherein the linker comprises GGGGS (SEQ ID NO: 9).

39. The method of claim 37 or 38, wherein the linker has the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 10).

40. The method of any one of claims 32 to 39, wherein the IL-2 or the IL-2 domain has a sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14; SEQ ID NO: 15; and SEQ ID NO: 16.

41. The method of any one of claims 32 to 40, wherein the fusion protein further comprises a signal peptide.

42. The method of claim 41, wherein the fusion protein comprises a signal peptide that has the sequence of SEQ ID NO: 11.

43. The method of any one of claims 32 to 42, wherein the domains are, in order from N- terminus to C-terminus, an optional signal peptide; the interleukin-2 (IL-2) domain; an optional linker; and the truncated interleukin- 33 (IL-33) domain.

44. The method of any one of claims 30-33, wherein administering to said patient with said alloresponse or autoimmune response suppresses, blocks or inhibits in vivo alloresponses or autoimmune responses in the patient.

45. The method of any one of claims 30-33, wherein the alloresponse or autoimmune response follows tissue transplantation, and wherein the method suppresses, blocks or inhibits graft-vs-host disease in the patient.

46. A method for achieving a preventative therapeutic effect in a patient comprising administering to said patient, prior to onset of an alloresponse or autoimmune response, an effective amount of activated long-term, ex vivo culture-expanded Treg cells to prevent said response, wherein the culture-expanded regulatory cells are stimulated with a. an interleukin-2 (IL-2) having at least 90% identity to SEQ ID NO: 13; and b. a truncated interleukin- 33 (IL-33) comprising a sequence with at least 90% identity to SEQ ID NO: 1, and wherein the regulatory cells are capable of inhibiting proliferation of the alloreactive T cells in a mixed lymphocyte reaction (MLR) assay by at least 90%.

47. A method for achieving a preventative therapeutic effect in a patient comprising administering to said patient, prior to onset of an alloresponse or autoimmune response, an effective amount of activated long-term, ex vivo culture-expanded regulatory cells to prevent said response, wherein the culture-expanded regulatory cells are stimulated with a. an interleukin-2 (IL-2) having the amino acid sequence of SEQ ID NO: 14; and b. a truncated interleukin- 33 (IL-33) having the amino acid sequence of SEQ ID NO: 3, and wherein the regulatory cells are capable of inhibiting proliferation of the alloreactive T cells in a mixed lymphocyte reaction (MLR) assay by at least 90%.

48. A method for achieving a preventative therapeutic effect in a patient comprising administering to said patient, prior to onset of an alloresponse or autoimmune response, an effective amount of activated long-term, ex vivo culture-expanded regulatory cells to prevent said response, wherein the culture-expanded regulatory cells are stimulated with a fusion protein comprising: a. an interleukin-2 (IL-2) domain having at least 90% identity to SEQ ID NO: 13; and b. a truncated interleukin- 33 (IL-33) domain, comprising a sequence with at least 90% identity to SEQ ID NO: 1, and wherein the regulatory cells are capable of inhibiting proliferation of the alloreactive T cells in a mixed lymphocyte reaction (MLR) assay by at least 90%.

49. A method for achieving a preventative therapeutic effect in a patient comprising administering to said patient, prior to onset of an alloresponse or autoimmune response, an effective amount of activated long-term, ex vivo culture-expanded regulatory cells to prevent said response, wherein the culture-expanded Treg cells are stimulated with a fusion protein comprising: a. an interleukin-2 (IL-2) having the amino acid sequence of SEQ ID NO: 14; andb. a truncated interleukin- 33 (IL-33), having the amino acid sequence of SEQ ID NO: 3, and wherein the Treg cells are capable of inhibiting proliferation of the alloreactive T cells in a mixed lymphocyte reaction (MLR) assay by at least 90%.

50. The method of any one of claims 46-49, wherein in the truncated IL-33 or the truncated IL-33 domain, at least one of N60, C97, Cl 16, C121, and C148 of SEQ ID NO: 1 is substituted by a single amino acid residue.

51. The method of claim 46 or 48, wherein N60 of SEQ ID NO: 1 is substituted by Ser or Asp; C97 of SEQ ID NO: 1 is substituted by Gly; and / or Cl 16 of SEQ ID NO: 1 is substituted by Phe.

52. The method of any one of claims 48 to 51, wherein the truncated IL-33 or the truncated IL-33 domain has a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 7.

53. The method of any one of claims 48 to 52, wherein the fusion protein further comprises a linker.

54. The method of claim 53, wherein the linker comprises GGGGS (SEQ ID NO: 9).

55. The method of claim 53 or 54, wherein the linker has the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 10).

56. The method of any one of claims 48 to 55, wherein the IL-2 or the IL-2 domain has a sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14; SEQ ID NO: 15; and SEQ ID NO: 16.

57. The method of any one of claims 48 to 56, wherein the fusion protein further comprises a signal peptide.

58. The method of claim 57, wherein the fusion protein comprises a signal peptide that has the sequence of SEQ ID NO: 11.

59. The method of any one of claims 48 to 58, wherein the domains are, in order from N- terminus to C-terminus, an optional signal peptide; the interleukin-2 (IL-2) domain; an optional linker; and the truncated interleukin- 33 (IL-33) domain.

60. The method of any one of claims 46-49, wherein the patient is treated prior to, at the time of, or immediately after tissue transplantation, and wherein the method further comprises preventing onset of graft-vs-host disease in the patient.

61. The method of any one of claims 46-49, wherein the patient is treated prior to, at the time of, or immediately after tissue transplantation, and wherein the method further comprises blocking rejection of the transplanted tissue in the patient.

62. The method of any one of claims 1-61, wherein said regulatory cells comprise CD4+, CD25+ T cells.

63. The method of any one of claims 1-61, wherein said regulatory cells inhibit activation of cytotoxic T cells.

64. A method of treating or preventing cancer in a patient in need thereof comprising administering to the patient activated, culture-expanded effector cells, wherein the culture- expanded regulatory cells are stimulated with IL-33, a combination of IL-2 and IL-33, or an IL233 fusion or variants thereof.

65. The method of claim 64, wherein said treating or preventing includes promoting the production of effector cells.

66. The method of claim 65, wherein said effector cells include auto-reactive T cells, auto- reactive B cells, alloreactive T cells, alloreactive B cells, syngeneic natural killer cells, allogeneic natural killer cells, innate lymphoid cells (ILC), macrophages, dendritic cells, mast cells, ST2+ T cells, CD8 T cells, natural killer gamma cells, and / or gamma-delta T cells.

67. The method of any one of claims 64-66, wherein said effector cells are selected from the group consisting of autologous CAR-T cells, allogeneic CAR-T cells, engineered CAR-T cells, and CD4+ T cells.

68. The method of claim 67, wherein said CD4+ T cells are ST2+ T cells.

69. The method of claim 68, wherein said effector cells are selected from the group consisting of Foxp3 negative T cells, IFNg+ T cells (Thl T cells), IL-4+ (Th2 T cells), IL-9+ T cells (Th9 T cells), IL-21+ T cells (Th21 T cells), TNFa-i- (Inflammatory T cells), CD8 Tregs.

70. The method of any one of claims 64-69, wherein said effector cells are modified to express IL-33 receptor (ST2) by genetic manipulation selected from the group consisting of transfection, transduction, electroporation, CRISPR / Cas9 mediated gene-insertion, and synthetic mRNA delivery.

71. The method of any one of claims 64-70, wherein said cancer is lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute myelogenous leukemia, adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, B-cell lymphoma, brain cancer, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrialcancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, midline tract carcinoma involving NUT gene, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal cavity and par nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, pl europul mon ary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphocytic leukemia, T-cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, or Wilms tumor.

72. The method of any one of claims 64-70, wherein said cancer is melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, B-cell lymphoma, acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, T- cell lymphocytic leukemia, bladder cancer, or lung cancer.

73. The method of any one of claims 64-72, wherein T-regulatory (Treg) cells, T-helper2 (Th2) cells, innate lymphoid cells (ILC), cytotoxic T cells, natural killer (NK) cells, NK-T cells, ST2+ cells, dendritic cells, and / or macrophages are proliferated or stimulated.

74. The method of any one of claims 64-72, wherein can either promote anti-inflammatory M2 macrophages are promoted and pro-inflammatory Ml macrophages are inhibited, or wherein Ml macrophages are activated to induce inflammation.

75. The method of claim 74, wherein the M2 macrophages inhibit inflammation in a tissue or organ selected from the group consisting of pancreas, liver, kidneys, adipose tissue, salivary glands, central nervous system (CNS), and related organs.

76. The method of claim 74, wherein the Ml macrophages promote anti-tumor immune response either systemically or locally.

77. The method of any one of claims 64-76, wherein expression of co-stimulatory molecules (including and not limited to CD80 (B7-1), CD86 (B7-2), CD40, ICOS, MHC-I, MHC-II, PD1, PD-L1, GITR, BAFF-R, 0x40, 41BB, DR3, CR2) is upregulated or down- regulated on antigen-presenting cells.

78. The method of any one of claims 64-77, further comprising administering an effective amount of a second therapy to the subject, wherein the second therapy is not a fusion protein described herein.

79. The method of claim 78, wherein the second therapy can be a cell therapy or a gene therapy.

80. A method of making activated, culture-expanded Treg cells comprising the step of stimulating Treg cells with a. an interleukin-2 (IL-2) having at least 90% identity to SEQ ID NO: 13; and b. a truncated interleukin- 33 (IL-33), comprising a sequence with at least 90% identity to SEQ ID NO: 1.

81. A method of making activated, culture-expanded Treg cells comprising the step of stimulating Treg cells with a. an interleukin-2 (IL-2) having the amino acid sequence of SEQ ID NO: 14; and b. a truncated interleukin-33 (IL-33) having the amino acid sequence of SEQ ID NO:3.

82. A method of making activated, culture-expanded regulatory cells comprising the step of stimulating regulatory cells with a. an interleukin-2 (IL-2) domain having at least 90% identity to SEQ ID NO: 13; and b. a truncated interleukin- 33 (IL-33) domain, comprising a sequence with at least 90% identity to SEQ ID NO: 1.

83. A method of making activated, culture-expanded regulatory cells comprising the step of stimulating regulatory cells with a. an interleukin-2 (IL-2) having the amino acid sequence of SEQ ID NO: 14; andb. a truncated interleukin- 33 (IL-33), having the amino acid sequence of SEQ ID NO: 3.

84. A method of making activated, effector cells comprising the step of stimulating effector cells with a. an interleukin-2 (IL-2) having at least 90% identity to SEQ ID NO: 13; and b. a truncated interleukin- 33 (IL-33), comprising a sequence with at least 90% identity toSEQ ID NO: 1.

85. A method of making activated, effector cells comprising the step of stimulating effector cells with a. an interleukin-2 (IL-2) having the amino acid sequence of SEQ ID NO: 14; and b. a truncated interleukin- 33 (IL-33), having the amino acid sequence of SEQ ID NO: 3.

86. The method of any one of claims 80-85, wherein in the truncated IL-33 or the truncated IL-33 domain, at least one of N60, C97, C116, C121, and C148 of SEQ ID NO: 1 is substituted by a single amino acid residue.

87. The method of claim 86, wherein N60 of SEQ ID NO: 1 is substituted by Ser or Asp; C97 of SEQ ID NO: 1 is substituted by Gly; and / or Cl 16 of SEQ ID NO: 1 is substituted by Phe.

88. The method of any one of claims 80 to 87, wherein the truncated IL-33 or the truncated IL-33 domain has a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 7.

89. The method of any one of claims 80 to 88, wherein the fusion protein further comprises a linker.

90. The method of claim 89, wherein the linker comprises GGGGS (SEQ ID NO: 9).

91. The method of claim 89 or 90, wherein the linker has the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 10).

92. The method of any one of claims 80 to 91, wherein the IL-2 or the IL-2 domain has a sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14; SEQ ID NO: 15; and SEQ ID NO: 16.

93. The method of any one of claims 80 to 92, wherein the fusion protein further comprises a signal peptide.

94. The method of claim 93, wherein the fusion protein comprises a signal peptide that has the sequence of SEQ ID NO: 11.

95. The method of any one of claims 80 to 94, wherein the domains are, in order from N- terminus to C-terminus, an optional signal peptide; the interleukin-2 (IL-2) domain; an optional linker; and the truncated interleukin- 33 (IL-33) domain.

96. A pharmaceutical composition, comprising the culture-expanded regulatory cells made by any one of claims 80 to 95.

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