A composition comprising cleavage-type interleukin-33 and interleukin-2

Abstract

A composition is disclosed herein that comprises interleukin 2 (IL-2) having at least 90% identity to SEQ ID NO: 13 and a truncated interleukin 33 (IL-33) having at least 90% identity to SEQ ID NO: 1. IL-2 and truncated IL-33 may be domains of a fusion protein. The composition or fusion protein can be used in a method for treating a disease or disorder, including administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising the composition or fusion protein. The disease or disorder may be an autoimmune disease or disorder, a disease or disorder characterized by inflammation, or cancer.

Description

Technical Field

[0001] 1. Related Applications This application claims priority to U.S. Provisional Application No. 63 / 340,777, filed May 11, 2022, the entire content of which is incorporated herein by reference.

[0002] 2. Sequence Listing This application includes a sequence listing that was electronically submitted in XML format, the entire content of which is incorporated herein by reference. The XML file was created on May 9, 2023, has the name 771185_000009_SL.xml, and is 18,768 bytes in size.

Background Art

[0003] 3. Background Interleukin 2 (IL-2) plays a role in maintaining immune tolerance, controlling helper T (Th) cell function, particularly Th2 function, and in the differentiation, survival, and function of Foxp3+CD4+ regulatory T (Treg) cells, and interacts with innate lymphoid cells (ILC).

[0004] Interleukin 33 (IL-33), in its wild-type form, is a cytokine that contains a nuclear localization signal at its N-terminus. Protease cleavage results in an 18 kDa C-terminal fragment, which may be referred to herein as cleaved IL-33. Cleaved IL-33 can stimulate innate lymphoid cells (ILC), Th2 cells, Treg cells, macrophages, and dendritic cells, thereby upregulating the expression of IL-2, IL-4, IL-5, and IL-13, and reducing inflammatory Th1 and Th17 responses.

[0005] Fusion proteins comprising an IL-2 domain and a truncated IL-33 domain have been described in the art. See, for example, WO2018 / 140504, US2019 / 0365714, US9,840,545; US10,851,145; and US20210261640, the disclosures of which are incorporated herein by reference. Such fusion proteins can treat, for example, diseases such as autoimmune diseases and disorders, and inflammation. However, many such fusion proteins have been observed to have one or more undesirable properties, such as aggregation, rapid clearance in vivo, or loss of IL-33 activity over time.

[0006] Accordingly, there is a need for fusion proteins comprising an IL-2 domain and a truncated IL-33 domain that do not have one or more undesirable properties. IL-33 loses its activity by spontaneous oxidation of certain residues. There is also a need for such fusion proteins to be readily expressed by recombinant organisms, readily purified, and to have both IL-2 activity and IL-33 activity equivalent to wild-type IL-2 and wild-type IL-33. SUMMARY OF THE INVENTION

[0007] 4. Summary The present disclosure provides a composition comprising interleukin 2 (IL-2) having at least 90% identity to SEQ ID NO: 13 and a truncated interleukin 33 (IL-33) comprising a sequence having at least 90% identity to SEQ ID NO: 1.

[0008] The present disclosure also provides 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 having at least 90% identity to SEQ ID NO: 1.

[0009] In the cleavage-type IL-33 polypeptide or domain, at least one of N60, C97, C116, C121, and C148 of SEQ ID NO: 1 may be substituted with a single amino acid. For example, N60 of SEQ ID NO: 1 may be substituted with Ser or Asp, C97 of SEQ ID NO: 1 may be substituted with Gly, and / or C116 of SEQ ID NO: 1 may be substituted with Phe. The cleavage-type IL-33 polypeptide or domain may have 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, and SEQ ID NO: 8; or may have a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 7.

[0010] The fusion protein may further include an optional linker such as an oligopeptide linker. For example, the linker may include GGGGS (SEQ ID NO: 9). The linker may have the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 10).

[0011] The IL-2 polypeptide or domain may have the sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

[0012] The fusion protein may further include an optional signal peptide. The signal peptide may have the sequence of SEQ ID NO: 11.

[0013] In the fusion protein, the domains may be, in order from the N-terminus to the C-terminus, an optional signal peptide; an interleukin-2 (IL-2) domain; an optional linker; and a cleavage-type interleukin-33 (IL-33) domain.

[0014] The present disclosure also includes a pharmaceutical composition comprising the composition or fusion protein described herein.

[0015] The pharmaceutical composition may further include a pharmaceutically acceptable carrier and, optionally, an additional therapeutic agent.

[0016] The present disclosure includes a polynucleotide comprising a nucleic acid sequence encoding at least a portion of the fusion protein described herein.

[0017] In another aspect, the present disclosure includes a vector comprising a polynucleotide operably linked to a promoter.

[0018] In yet another aspect, the present disclosure includes a recombinant host cell comprising the polynucleotide or the vector.

[0019] The present disclosure includes a method for treating a disease or disorder, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition described herein.

[0020] The disease or disorder may be acute kidney injury, ankylosing spondylitis, autoimmune myocarditis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune lymphoproliferative syndrome, autoimmune pancreatitis, polyglandular autoimmune 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), inflammatory demyelinating disease, inflammatory neuropathy, insulitis, interstitial cystitis, juvenile idiopathic arthritis, lupus, erythematosus, lupus glomerulonephritis, lupus nephritis, cutaneous lupus, IgA nephropathy, membranoproliferative glomerulonephritis (MPGN), microscopic colitis, multiple sclerosis, myasthenia gravis, obesity, pancreatitis, polymyositis, primary biliary cirrhosis (PBC), primary sclerosing cholangitis, progressive inflammatory neuropathy, renal ischemia-reperfusion injury, rheumatoid arthritis, Sjögren's syndrome, systemic lupus erythematosus, graft rejection, type 1 diabetes, type 2 diabetes, ulcerative colitis, vasculitis, psoriasis, atopic dermatitis, pemphigus vulgaris, or Wegener's granulomatosis.

[0021] The disease or disorder may be cancer. The cancer may be acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute myelogenous leukemia, adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, B-cell lymphoma, brain cancer, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, cancer of unknown primary, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, germ cell tumor, endometrial cancer, epithelioma, esophageal cancer, nasal neuroblastoma, fibrous histiocytoma, Ewing's sarcoma, eye cancer, embryonal cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi's 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 of unknown primary, midline carcinoma related to the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, polyposis fungosa, 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, papilloma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, phyllodes tumor, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureteral cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sézary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoblastic leukemia, T-cell lymphoma, teratoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0022] The cancer may 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 myeloid leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, T-cell lymphoblastic leukemia, bladder cancer, or lung cancer.

[0023] Depending on the IL-2 domain, the method can stimulate the proliferation and / or activation of regulatory T (Treg) cells, helper T2 (Th2) cells, innate lymphoid cells (ILC), cytotoxic T cells, natural killer (NK) cells, NK-T cells, ST2+ cells, dendritic cells, and / or macrophages.

[0024] Depending on the IL-2 domain, the method can promote anti-inflammatory M2 macrophages and inhibit inflammatory M1 macrophages, or activate M1 macrophages to induce inflammation. M2 macrophages can suppress inflammation in tissues or organs selected from the group consisting of the pancreas, liver, kidney, adipose tissue, salivary gland, central nervous system (CNS), and related organs. M1 macrophages can promote the anti-tumor immune response, either systemically or locally.

[0025] Depending on the IL-2 domain, the method can up-regulate or down-regulate the expression of co-stimulatory molecules (including, but not limited to, CD80 (B7-1), CD86 (B7-2), CD40, ICOS, MHC-I, MHC-II, PD1, PD-L1, GITR, BAFF-R, Ox40, 41BB, DR3, CR2) on antigen-presenting cells including, but not limited to, dendritic cells, macrophages, B cells, innate lymphoid cells, NK cells, epithelial cells, endothelial cells, or stromal cells.

[0026] The method may further comprise applying to the subject an effective amount of a second therapy for a disease or disorder, wherein the second therapy is not the fusion protein described herein.

[0027] The second therapy may be a cell therapy or a gene therapy.

[0028] In another aspect, the present disclosure includes isolating T cells from a subject; and contacting the isolated T cells with a cell expressing at least one of an effective amount of the compositions described herein, the fusion proteins described herein, the recombinant host cells described herein, or the IL-2 or cleaved IL-33 described herein, to stimulate the proliferation and / or activation of regulatory T (Treg) cells of the subject.

[0029] The present disclosure also includes a kit comprising the pharmaceutical composition described herein; and instructions for performing a method for treating a disease or disorder characterized by inflammation, the method comprising administering an effective amount of the pharmaceutical composition to a subject in need thereof.

[0030] 5. Brief Description of the Drawings

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0053] 6. Detailed Description The present disclosure relates to a composition or fusion protein comprising an IL-2 polypeptide or domain and a cleaved IL-33 polypeptide or domain. The fusion of IL-2 and IL-33 is the key to enabling improvements in efficacy, tissue targeting, and avidity. ST2 is a receptor for IL-33 and is expressed on the surface of a subset of regulatory T cells (Tregs). This subset is more persistent and active in the regulation of inflammation suppression in the tissues of the disease site. Since IL-33 is naturally released in the tissue, its function helps not only the proliferation of ST2+ Treg cells but also other ST2+ cells and promotes a local immunosuppressive tissue microenvironment. Thus, treatment with the fusion proteins and compositions described herein results in a sustained recovery from the disease.

[0054] In the fusion protein, the domains may be linked by a linker such as an oligopeptide linker. The composition or fusion protein may have one or more advantageous properties (including, but not limited to, reduction of aggregation, improvement of stability during storage, maintenance of IL-33 activity after storage, being more easily produced by recombinant organisms, IL-2 activity equivalent to wild-type IL-2, IL-33 activity equivalent to wild-type IL-33, and extension of IL-33 activity after administration). Without wishing to be bound by theory, such advantageous properties may result from, for example, site-specific mutations that reduce aggregation in the IL-2 domain and / or the truncated IL-33 domain by substituting cysteine residues with other residues that cannot form disulfide bonds.

[0055] All publications and patents cited in this disclosure are hereby incorporated by reference in their entirety. If the documents incorporated by reference conflict with or are contrary to this specification, this specification shall prevail over any such document. The citation of any reference in this specification does not admit that such reference is prior art to the present disclosure. When a range of values is indicated, embodiments using any specific value within that range are included. Further, reference to a value expressed as a range includes all values within that range. All ranges include their end values and can be combined. When a value is expressed as an approximation by use of the antecedent "about", that specific value is understood to form another embodiment. Reference to a specific numerical value includes at least that specific value, unless clearly contrary to the context. When "or" is used, it means "and / or" unless clearly contrary to the specific context in which it is used.

[0056] The various terms related to the aspects described herein are used throughout this specification and the claims. Such terms have their ordinary meanings in the art, unless otherwise indicated. Other specifically defined terms should be construed in a manner consistent with the definitions set forth herein. The techniques and procedures described or referenced herein are generally well understood by those of ordinary skill in the art and are generally utilized by using common methods, such as the widely used molecular cloning techniques described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where appropriate, procedures related to the use of commercially available kits and reagents are generally carried out according to the protocols and conditions defined by the manufacturer, unless otherwise described.

[0057] As used herein, the singular forms "a", "an", and "the" include the plural forms unless the context clearly dictates otherwise. Terms such as "include", "such as", etc. are intended to mean including without limitation, unless otherwise specifically indicated.

[0058] Unless otherwise indicated, terms such as "at least", "less than", and "about", or similar terms placed before a series of elements or a range, should be understood to mean all of the elements of that series or range. Those of ordinary skill in the art can recognize or confirm many equivalents of the specific embodiments of the invention described herein without performing experiments beyond routine experimentation. Such equivalents are intended to be encompassed by the following claims.

[0059] As used herein, the term "subject" refers to any animal, including, but not limited to, any mammal such as a human, non-human primate, rodent, mammal commonly kept as a pet (e.g., but not limited to, dogs and cats), livestock (e.g., but not limited to, cows, sheep, goats, pigs, horses, and camels). In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0060] Additional description of the method, and guidance for practicing the method, are provided herein.

[0061] A. Embodiments i. Compositions In some embodiments, the present disclosure relates to a composition. The composition may comprise interleukin 2 (IL-2) and cleaved interleukin 33 (IL-33). In the composition, interleukin 2 (IL-2) may comprise SEQ ID NO: 13, or may have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13. Alternatively, or in addition, cleaved interleukin 33 (IL-33) may comprise SEQ ID NO: 1, or may have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.

[0062] IL-2 and cleaved IL-33 are generally described herein in the context of a fusion protein. However, one of ordinary skill in the art will understand that the description of the IL-2 domain and the cleaved IL-33 domain will equally apply to the IL-2 polypeptide and the cleaved IL-33 polypeptide in the composition, except for descriptions specific to the fusion protein (e.g., the arrangement of domains in the fusion protein, etc.).

[0063] ii. Fusion proteins In some embodiments, the present disclosure relates to a fusion protein. The fusion protein may comprise an interleukin-2 (IL-2) domain and a cleaved interleukin-33 (IL-33) domain. The fusion protein may comprise an interleukin-2 (IL-2) domain comprising SEQ ID NO: 13, or an IL-2 domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13; and a cleaved interleukin-33 (IL-33) domain comprising SEQ ID NO: 1, or a cleaved IL-33 domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.

[0064] In some embodiments, the fusion protein may comprise an optional linker between the first domain and the second domain.

[0065] In some embodiments, the fusion protein may comprise two or more IL-2 domains, two or more cleaved IL-33 domains, or both.

[0066] IL-2 domain The IL-2 domain of the fusion protein may be identical to the wild-type IL-2 sequence of any organism, or a variant thereof. In one aspect, the IL-2 domain is at least 90% identical to the wild-type IL-2 sequence of the organism in which the fusion protein is intended to be administered in the methods described herein.

[0067] In one embodiment, the IL-2 domain has a sequence having at least 90% identity to SEQ ID NO: 13, for example, 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, and the like. The IL-2 domain of the fusion protein may differ from SEQ ID NO: 13 at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 positions.

[0068] The sequence of the IL-2 domain may include a number of variants of SEQ ID NO: 13. For example, the IL-2 domain may include a point mutation of Asn at position 88 of SEQ ID NO: 13 (N88). The IL-2 domain containing the N88 point mutation may exhibit one or more advantageous properties, including but not limited to, higher stability during storage and / or higher activity compared to the corresponding IL-2 domain lacking the N88 point mutation.

[0069] As a specific example, the IL-2 domain may include a point substitution of Asn to Asp at position 110 of SEQ ID NO: 13 (N88D). A specific IL-2 domain containing the N88D substitution has the sequence of SEQ ID NO: 14.

[0070] In another embodiment, the IL-2 domain may include a point mutation of Cys at position 125 of SEQ ID NO: 13 (C125). The IL-2 domain containing the C125 point mutation is less likely to form disulfide bonds with Cys residues in the IL-2 domain and / or in the IL-33 domain, and may exhibit one or more advantageous properties, including but not limited to, less aggregation, slower clearance in vivo, or higher IL-2 activity compared to the corresponding IL-2 domain lacking the C125 point mutation.

[0071] In certain embodiments, the IL-2 domain may include a point substitution of Cys to Ser at position 125 of SEQ ID NO: 13 (C125S). A particular IL-2 domain that includes the C125S substitution has the sequence of SEQ ID NO: 15.

[0072] Different IL-2 mutations can reduce the interaction of IL-2 with IL2Rα (CD25) and / or increase the interaction of IL-2 with IL2Rβ (CD122), thereby resulting in the activation of NK and CD8 T cells, which is different from that of Tregs.

[0073] Any two or more deletions or substitutions among the above IL-2 domains may be combined. In one embodiment, the IL-2 domain may include point mutations at both Asn at position 88 and Cys at position 125 of SEQ ID NO: 13. As a specific example, the IL-2 domain may have both the point substitution N88D and the point substitution C125S of SEQ ID NO: 13. A particular IL-2 domain that includes both the N88D substitution and the C125S substitution has the sequence of SEQ ID NO: 16.

[0074] Linker The optional linker can be at the N-terminus of the polypeptide, at the C-terminus of the polypeptide, or any portion capable of forming at least two covalent bonds with the amino acid side chains of the polypeptide. Preferably, the linker is an oligopeptide sequence containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. The oligopeptide can form a covalent bond with the C-terminus of one polypeptide and (typically another) the N-terminus of the polypeptide.

[0075] Preferably, the amino acids are encoded by the codons of the standard genetic code. Thereby, the fusion protein can be encoded by an entire single polynucleotide and transcribed and translated as one unit, thus simplifying the production of the fusion protein.

[0076] When the linker is an oligopeptide, the amino acids thereof may be selected such that the linker is flexible, generally unlikely to form a secondary structure, generally unlikely to interfere with the tertiary structure of the domain linked by the linker, generally unlikely to bind to any domain of the fusion protein other than the above-mentioned C-terminus and N-terminus, and generally non-immunogenic. An oligopeptide linker containing at least about 75% Gly residues can be used.

[0077] The specific amino acid residues of the oligopeptide linker may be selected to adjust the isoelectric point (pI) of the fusion protein, which may be useful for improving the yield, purity, storage stability, or efficacy of the fusion protein in production or use.

[0078] For example, the linker may contain GGGGS (SEQ ID NO: 9). In another embodiment, the linker may contain the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 10).

[0079] In some embodiments, the fusion protein may contain an optional additional linker, whereby the fusion protein can be dimerized or multimerized. The additional linker may be an oligopeptide linker such as the linkers described herein.

[0080] IL-33 domain The IL-33 domain may have the wild-type sequence, variant sequence, or truncated variant sequence of IL-33 of any species. In one embodiment, the IL-33 domain comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the wild-type IL-33 sequence of the organism in which the fusion protein is intended to be administered by the method described herein.

[0081] Wild-type cleaved human IL-33 has the sequence of SEQ ID NO: 1. Other organisms have cleaved IL-33 containing residues homologous to human cleaved IL-33 of SEQ ID NO: 1.

[0082] In one aspect, the cleaved IL-33 domain has a sequence having at least 90% identity to SEQ ID NO: 1, such as, for example, 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 cleaved IL-33 domain of the fusion protein may differ from SEQ ID NO: 1 at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 positions.

[0083] In some embodiments, the cleaved IL-33 domain may comprise a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1, wherein one or more of the sites where glycosylation or disulfide bonding is possible (e.g., N60, C97, C116, C121, and C148) are deleted or substituted with one or more amino acids.

[0084] A number of variants of SEQ ID NO: 1 can be specifically contemplated for the sequence of the cleaved IL-33 domain.

[0085] In one aspect, the cleaved IL-33 domain may contain a point mutation of Asn (N60) at position 60 of SEQ ID NO: 1. As shown in Figure 3, N60 is present in human cleaved IL-33 but not at the homologous position of mouse or rat cleaved IL-33. The cleaved IL-33 domain containing the point mutation of N60 is less likely to be glycosylated and may exhibit one or more advantageous properties (including, but not limited to, less aggregation, slower clearance in vivo, or higher activity compared to the corresponding cleaved IL-33 domain lacking the point mutation of N60). In certain examples, the cleaved IL-33 domain may contain a point substitution of Asn to Ser (N60S) or Asn to Asp (N60D) at position 60. A specific cleaved IL-33 domain containing the N60S point substitution may have the sequence of SEQ ID NO: 2.

[0086] In one embodiment, the cleaved IL-33 domain may contain a point mutation of Cys (C116) at position 116 of SEQ ID NO: 2. The cleaved IL-33 domain containing the point mutation of C116 is less likely to form disulfide bonds with Cys residues in the cleaved IL-33 domain and / or in the IL-2 domain and may exhibit one or more advantageous properties (including, but not limited to, less aggregation, slower clearance in vivo, or higher activity compared to the corresponding cleaved IL-33 domain lacking the point mutation of C116).

[0087] In one embodiment, the cleaved IL-33 domain may contain a point substitution of Cys to Phe (C116F) at position 116. The C116F point substitution was based on the observation that mouse and rat cleaved IL-33 contain Phe at a position homologous to human wild-type C116, as shown in Figure 3.

[0088] The C116F point substitution results in improvements in the expression and purification of a fusion protein containing both an IL-2 domain and a truncated IL-33 domain, and the activities of its truncated IL-33 domain and IL-2 domain are substantially the same as those of wild-type IL-33 alone and wild-type IL-2 alone, respectively.

[0089] A specific truncated IL-33 domain containing the C116F point substitution may have the sequence of SEQ ID NO: 3.

[0090] In one aspect, the truncated IL-33 domain may contain a point mutation of Cys at position 97 (C97) of SEQ ID NO: 2. The truncated IL-33 domain containing the point mutation of C97 is less likely to form disulfide bonds and may exhibit one or more of the advantageous properties described for C116.

[0091] In another aspect, the truncated IL-33 domain may contain a point substitution of Cys to Gly at position 97 (C97G). The C97G point substitution was based on the observation that mouse and rat truncated IL-33 contain Gly at a position homologous to human wild-type C97, as shown in Figure 3.

[0092] In the context of the C97G point substitution, the activities of the truncated IL-33 domain and the IL-2 domain are both substantially the same as those of wild-type IL-33 alone and wild-type IL-2 alone, respectively.

[0093] A specific truncated IL-33 domain containing the C97G point substitution may have the sequence of SEQ ID NO: 4.

[0094] Any two or more deletions or substitutions among the above-described truncated IL-33 domains may be combined. For example, the truncated IL-33 domain may contain both N60S and C116F (SEQ ID NO: 5); both N60S and C97G (SEQ ID NO: 6); both C116F and C97G (SEQ ID NO: 7); or all three of N60S, C116F, and C97G (SEQ ID NO: 8).

[0095] Thus, in some embodiments, the cleaved 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, and SEQ ID NO: 8.

[0096] In some embodiments, the cleaved IL-33 domain has a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 7.

[0097] Signal peptide The fusion protein may include a signal peptide, for example, at the N-terminus of the fusion protein. The signal peptide may be selected from signal peptides known to be used with the N-terminal domain of the fusion protein, or may be selected based on theoretical design or routine experiments.

[0098] In one embodiment, when the N-terminal domain of the fusion protein is the IL-2 domain, the signal peptide may have the sequence of SEQ ID NO: 11.

[0099] Linkage of domains The various domains of the fusion protein may be linked directly or via the reactive site of an optional linker by any desired reactive site of the various domains. In some embodiments where the linker is an oligopeptide, the various domains may be linked to the corresponding end of the linker by its N-terminus and / or C-terminus. The fusion protein may comprise one continuous polypeptide sequence. Such a fusion protein can be prepared by well-known transcription and translation processes from one polynucleotide coding region encoding the entire sequence of the fusion protein.

[0100] A fusion protein comprising a single continuous polypeptide sequence may contain various domains in any desired order. For example, the fusion protein may contain either an IL-2 domain or a cleaved IL-33 domain at the N-terminus; a linker; and the remaining domains at the C-terminus.

[0101] In one particular embodiment, the fusion protein may contain, in order from the N-terminus to the C-terminus, a signal peptide; an interleukin-2 (IL-2) domain; a linker; and a cleaved interleukin-33 (IL-33) domain.

[0102] In one particular embodiment, the fusion protein may contain, in order from the N-terminus to the C-terminus, a signal peptide; a cleaved IL-33 domain; a linker; and an IL-2 domain.

[0103] In one particular embodiment, the fusion protein may contain, in order from the N-terminus to the C-terminus, a signal peptide; an IL-2 domain; and a cleaved IL-33 domain.

[0104] In one particular embodiment, the fusion protein may contain, in order from the N-terminus to the C-terminus, a signal peptide; a cleaved IL-33 domain; and an IL-2 domain.

[0105] In one particular embodiment, the fusion protein may contain, in order from the N-terminus to the C-terminus, an IL-2 domain; a linker; and a cleaved IL-33 domain.

[0106] In one particular embodiment, the fusion protein may contain, in order from the N-terminus to the C-terminus, a cleaved IL-33 domain; a linker; and an IL-2 domain.

[0107] In one particular embodiment, the fusion protein may contain, in order from the N-terminus to the C-terminus, a cleaved IL-33 domain and an IL-2 domain.

[0108] In one particular embodiment, the fusion protein may comprise an IL-2 domain and a cleaved IL-33 domain, in order from the N-terminus to the C-terminus.

[0109] In another particular embodiment, the fusion protein may comprise two polypeptides linked by an additional linker between the amino acid side chains. The first polypeptide may comprise an optional signal peptide and an IL-2 domain, in order from the N-terminus to the C-terminus; the second polypeptide may comprise a cleaved IL-33 domain; and the first polypeptide and the second polypeptide may be linked by an additional linker between the amino acid side chain in the IL-2 domain of the first polypeptide and the amino acid side chain in the cleaved IL-33 domain of the second polypeptide.

[0110] Production of the fusion protein The fusion proteins described herein can be produced 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 those skilled in the art.

[0111] In biochemical methods of polypeptide synthesis, a polynucleotide comprising a nucleic acid sequence encoding a polypeptide of interest (e.g., a fusion protein or a portion thereof) is incorporated into a vector, where the polynucleotide is operably linked to a promoter. The vector may contain other sequences, such as non-promoter control sequences, an origin of replication, sequences engineered for inserting the polynucleotide into the vector, and the like. The promoter is selected such that when the vector is introduced into a host cell by recombinant DNA technology, the promoter is recognized by the host cell's endogenous RNA polymerase and produces an RNA transcript either constitutively or by induction due to one or more changes in processing conditions. The RNA transcript is then translated by the host cell's ribosomes to produce the polypeptide of interest (e.g., a fusion protein or a portion thereof).

[0112] Another route of polypeptide synthesis is to introduce a polynucleotide that expresses the polypeptide into the cells of a subject in need thereof. The polynucleotide can contain mRNA or DNA. The cells of the subject are capable of transcribing the DNA and / or translating the mRNA into the polypeptide of interest.

[0113] In embodiments where the fusion protein contains multiple polypeptides, each polypeptide is produced individually and the fusion protein may be constructed by a peptide synthase reaction or by non-amino acid backbone linkages, such as by one or more non-oligopeptide linkers, by polypeptide-to-polypeptide linkages through the amino acid side chains of an oligopeptide linker, and / or by chemical bonds.

[0114] In some embodiments, a composition comprising an IL-2 polypeptide and a cleaved IL-33 polypeptide can be an intermediate step in the preparation of a fusion protein comprising an IL-2 domain, a cleaved IL-33 domain, and optionally a linker.

[0115] iii. Pharmaceutical composition In some embodiments, the present disclosure relates to a pharmaceutical composition. The pharmaceutical composition may comprise any of the above compositions or fusion proteins. In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. Alternatively, or in addition, the pharmaceutical composition may further comprise an additional therapeutic agent.

[0116] Pharmaceutically acceptable carrier The pharmaceutical composition may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include physiologically acceptable or tolerable diluents, excipients, solvents, or adjuvants. The pharmaceutical composition is preferably sterile and pyrogen-free.

[0117] Additional therapeutic agent In embodiments, the pharmaceutical composition may comprise an additional therapeutic agent (which means one or more therapeutic agents different from the above fusion protein). Examples of additional therapeutic agents include, but are not limited to, cytotoxic agents, anti-angiogenic agents, apoptosis promoters, antibiotics, hormones, hormone antagonists, chemokines, drugs, prodrugs, toxins, or enzymes.

[0118] The specific therapeutic agent(s) may be selected, among other factors known to those skilled in the art, in particular considering the disease or disorder intended to be treated by the pharmaceutical composition.

[0119] The pharmaceutical composition may also contain small amounts of non-toxic auxiliary pharmaceutical substances, excipients, and / or additives (such as, among others known to those skilled in the art, wetting agents, emulsifying agents, and / or pH buffering agents).

[0120] If necessary, absorption promoters or retardants (such as liposomes, aluminum monostearate, or gelatin) may be used. The composition may be prepared in conventional forms, as a solution or suspension, in a solid form suitable for dissolving or suspending in a liquid prior to injection, or as an emulsion.

[0121] iv. Treatment of diseases and disorders In some embodiments, the present disclosure relates to the treatment of diseases and disorders by a method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a composition or fusion protein described herein. The disease or disorder may be characterized by an inflammatory condition. In embodiments, the inflammatory condition may be present in a tissue or organ selected from the group consisting of the pancreas, liver, kidney, adipose tissue, salivary gland, central nervous system (CNS), and associated organs.

[0122] Without wishing to be bound by theory, the fusion protein can stimulate the proliferation and / or activation of regulatory T (Treg) cells, helper T2 (Th2) cells, innate lymphoid cells (ILC), cytotoxic T cells, natural killer (NK) cells, NK-T cells, ST2+ cells, dendritic cells, and / or macrophages; can promote anti-inflammatory M2 macrophages; and / or can inhibit inflammatory M1 macrophages.

[0123] Without wishing to be bound by theory, the fusion protein can upregulate or downregulate the expression of co-stimulatory molecules (including, but not limited to, CD80 (B7-1), CD86 (B7-2), CD40, ICOS, MHC-I, MHC-II, PD1, PD-L1, GITR, BAFF-R, Ox40, 41BB, DR3, CR2) on antigen-presenting cells including, but not limited to, dendritic cells, macrophages, B cells, innate lymphoid cells, NK cells, epithelial cells, endothelial cells, or stromal cells, depending on the IL-2 domain.

[0124] Diseases and disorders In some embodiments, the disease or disorder may be an autoimmune disorder. In some embodiments, the disease or disorder is acute kidney injury, ankylosing spondylitis, autoimmune myocarditis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune lymphoproliferative syndrome, autoimmune pancreatitis, polyglandular autoimmune 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), inflammatory demyelinating disease, inflammatory neuropathy, insulitis, interstitial cystitis, juvenile idiopathic arthritis, lupus, systemic lupus erythematosus, lupus glomerulonephritis, lupus nephritis, IgA nephropathy, membranoproliferative glomerulonephritis (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, Sjögren's syndrome, systemic lupus erythematosus, transplant rejection, type 1 diabetes, type 2 diabetes, ulcerative colitis, vasculitis, and Wegener's granulomatosis.

[0125] In some embodiments, the disease or disorder is cancer. For example, the cancer can be acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute myelogenous leukemia, adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, B-cell lymphoma, brain cancer, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, cancer of unknown primary, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, germ cell tumor, endometrial cancer, epithelioma, esophageal cancer, nasal neuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, embryonal cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular carcinoma, 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 of unknown primary, midline carcinoma related to the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, polyposis, 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, papilloma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, chromaffin cell tumor, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sézary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoblastic leukemia, T-cell lymphoma, teratoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms tumor, any one of which may be present.

[0126] The cancer may 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 myeloid leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, T-cell lymphoblastic leukemia, bladder cancer, and lung cancer.

[0127] Administration Technique The pharmaceutical composition may be delivered to a subject by any route by which the fusion protein can reach a target site, such as a tissue or organ where inflammation, cancerous cells, or other phenomena associated with a disease or disorder are present. The route may be local or systemic. For example, the pharmaceutical composition may be delivered systemically, among other routes, but in particular, by intravenous or intra-arterial administration, or locally, among other routes, but in particular, by subcutaneous, intramuscular, intra-organ, or intratumoral administration. Another route of administration is the introduction of mRNA expressing the polypeptide into the cells of the subject. The cells of the subject can translate the mRNA into the polypeptide of interest (similar to the use of mRNA in vaccines against SARS / CoV2).

[0128] Particular routes of administration may also be considered, among others, but in particular, may be selected based on the target site, disease or disorder, effective amount of the fusion protein, other components of the pharmaceutical composition, and / or a second therapy (if any) included in the pharmaceutical composition. The particular route of administration can be selected by one of ordinary skill in the art who will benefit from the present disclosure as part of their routine business.

[0129] Adjuvant Therapy For any disease or disorder, the method may further include the application of adjuvant therapy, where the adjuvant therapy does not include the fusion protein.

[0130] For example, if the disease or disorder is cancer, the adjuvant therapy may include surgical resection, radiation therapy, chemotherapy, checkpoint inhibitor therapy, or other therapies known to those of skill in the art.

[0131] If the adjuvant therapy has a mode suitable for administration by the same route as the fusion protein and substantially simultaneously with the fusion protein, the pharmaceutical composition may include the adjuvant therapy.

[0132] In some embodiments, the adjuvant therapy is cell therapy or gene therapy.

[0133] Medical Use of Fusion Proteins In some embodiments, the present disclosure relates to the compositions or fusion proteins described herein for use in the treatment of the diseases or disorders described herein.

[0134] In some embodiments, the present disclosure relates to the use of the compositions or fusion proteins described herein in the treatment of the diseases or disorders described herein.

[0135] In some embodiments, the present disclosure relates to the use of the compositions or fusion proteins described herein in the manufacture of a medicament. The medicament may be for the treatment of the diseases or disorders described herein.

[0136] Method for Stimulating Proliferation and / or Activation of v.Treg Cells In some embodiments, the present disclosure relates to a method for stimulating the proliferation and / or activation of regulatory T (Treg) cells of a subject. The stimulation can be performed in vivo or in vitro.

[0137] For example, one in vitro method includes isolating T cells from a subject; and contacting the isolated T cells with an effective amount of the compositions or fusion proteins described herein.

[0138] vi. Kit In some embodiments, the present disclosure relates to a kit comprising the compositions or fusion proteins described herein and instructions for performing the methods described herein.

[0139] For example, the kit may include the pharmaceutical composition described herein; and instructions for performing a method for treating a disease or disorder characterized by inflammation, the method including administering an effective amount of the pharmaceutical composition to a subject in need thereof.

[0140] The instructions may be included in the kit in the form of a printed instruction, a printed material indicating a URL (Uniform Resource Locator) where detailed instructions can be viewed or downloaded, a printed material indicating a QR (Quick Response) code that can lead a smartphone or tablet computer to the URL by scanning, etc.

[0141] The kit will generally include one or more instruments or containers so that some or all of the individual components and reagents (e.g., pharmaceutical compositions) can be individually contained. The kit may also include means (e.g., a plastic box) for sealing and storing the individual containers to some extent, and may contain instructions, packing materials such as styrofoam, etc. Identifiers such as barcodes, radio frequency identification (RFID) tags, etc. may be present in the kit or on the kit, or on one or more instruments or containers included in the kit. The identifier can be used to identify each individual kit, for purposes such as quality control, inventory management, tracking, movement between workplaces, etc. B. Array TIFF2025517661000002.tif236166TIFF2025517661000003.tif251164TIFF2025517661000004.tif184165

[0142] 7. Equivalents Other suitable modifications and adaptations to the methods of the present invention described herein will be apparent and can be implemented without departing from the scope of the present disclosure or embodiments, and will readily become apparent to those skilled in the art. Specific compositions and methods have been described in detail up to this point, but they will be more clearly understood by referring to the following examples (which are added for illustrative purposes only and are not intended to be limiting).

Examples

[0143] 8. Examples The following are examples of the methods and compositions of the present invention. Given the general descriptions provided herein, it will be understood that various other embodiments can be implemented. [Table 1]

[0144] Example 1: Effect of the N60D Mutation on Activity The effect of the N60D point substitution in the truncated IL-33 domain on the IL-2 activity of a fusion protein comprising an IL-2 domain and a truncated IL-33 domain was examined. Specifically, the fusion protein was synthesized and purified by recombinant cells as one polypeptide. Samples were evaluated for IL-2 activity using a known analytical method that measures activity at OD620nm. IL-2 alone was used as a positive control. IL-33 alone was used as a negative control.

[0145] The results are shown in Figure 3. The fusion protein containing the N60D substitution in the truncated IL-33 domain retained substantially the same IL-2 activity as IL-2 alone.

[0146] Example 2: Effect of the C97G Mutation on Activity The effect of the C97G point substitution in the truncated IL-33 domain on the IL-2 activity and IL-33 activity of a fusion protein comprising an IL-2 domain and a truncated IL-33 domain was examined. Specifically, the fusion protein was synthesized and purified by recombinant cells as one polypeptide. Samples were evaluated for IL-2 activity or IL-33 activity using a known analytical method that measures activity at OD620nm. In the IL-2 activity assay, IL-2 alone was used as a positive control and IL-33 alone was used as a negative control. In the IL-33 activity assay, IL-33 alone was used as a positive control and IL-2 alone was used as a negative control.

[0147] The results of the IL-2 activity assay are shown in Figure 4. The fusion protein containing the C97G point substitution in the truncated IL-33 domain retained substantially the same IL-2 activity as IL-2 alone.

[0148] The results of the IL-33 activity assay are shown in Figure 5. The fusion protein containing the C97G point substitution in the truncated IL-33 domain showed IL-33 activity equivalent to that of IL-33 alone.

[0149] Example 3: Effects of the C116F Mutation on Expression, Activity, and Stability The effects of the C116F point substitution in the truncated IL-33 domain on the expression, purification, IL-2 activity, IL-33 activity, and stability of a fusion protein containing both the IL-2 domain and the truncated IL-33 domain were examined. Specifically, the fusion protein was synthesized by recombinant cells as a single polypeptide and purified. Samples were stored at 4 °C, 25 °C, or 37 °C and evaluated for IL-2 activity and IL-33 activity after 0, 3, or 7 days. IL-2 activity and IL-33 activity were measured as described above.

[0150] Figure 6 shows that the fusion protein containing the C116F substitution in the truncated IL-33 domain was sufficiently expressed in recombinant cells. The thick bands in the middle and right columns correspond to the molecular weight of the fusion protein.

[0151] Figure 7 shows that the fusion protein containing the C116F substitution in the truncated IL-33 domain was easily purified by standard chromatography techniques.

[0152] Figure 8 shows that the fusion protein containing the C116F substitution in the truncated IL-33 domain retained substantially the same IL-2 activity as IL-2 alone.

[0153] Figure 9 shows that the fusion protein containing the C116F substitution in the truncated IL-33 domain retained substantially the same IL-33 activity as IL-33 alone.

[0154] Figure 10 shows that the fusion protein containing the C116F substitution in the cleavage-type IL-33 domain retained at least about 90% or more of the original IL-33 activity even after storage at 37°C for 7 days.

[0155] Example 4: Activity of constructs with multiple genes manipulated [Table 2] Nine genetically engineered constructs (SLT-518(h1), 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)) having various mutations in IL2 and / or IL33 were created. Details of the mutations contained in each construct are shown in Table 2. Variants were generated that were theoretically designed to optimize expression, activity, and stability, while retaining the wild-type sequence to reduce immunogenic risk.

[0156] For six genetically engineered variants (SLT1, SLT2, SLT3, SLT4, SLT5, SLT6), IL2 activity and IL33 activity were measured (Figure 35). In Figures 11 - 14, the SLT-518 constructs are represented by 7429-(variant number) and correspond to SLT-518(h1), SLT-518(h2), SLT-518(h3), SLT-518(h4), SLT-518(h5), or SLT-518(h6). SLT1 and SLT2 have minimal IL33 activity and are compared to SLT3, SLT4, SLT5, and SLT6.

[0157] SLT3 (IL233 fusion protein containing IL2 (N88D) mutation and IL33 (N60S) mutation) and SLT4 (IL233 fusion protein containing IL2 (N88D) mutation and IL33 (C116F) mutation) were compared with SLT1 (wild-type fusion protein IL233) and SLT2 (IL233 fusion protein containing IL2 (N88D) mutation) (Figure 12). As shown in Figure 11, the IL33 activities of SLT1 and SLT2 were barely detectable. SLT3 and SLT4 have significantly higher IL33 activities compared to SLT1 and SLT2.

[0158] SLT5 (IL233 fusion protein containing IL2 (N88D) mutation and IL33 (C97G) mutation) and SLT6 (IL233 fusion protein containing mutations of IL2 (N88D) and IL33 (N60S, C97G, C116F)) were compared with SLT1 (wild-type fusion protein IL233) and SLT2 (IL233 fusion protein containing IL2 (N88D) mutation) (Figure 13). As described above, the IL33 activities of SLT1 and LST2 are minimal. SLT5 and SLT6 have significantly higher IL33 activities compared to SLT1 and SLT2.

[0159] SLT4 (IL233 fusion protein containing IL2 (N88D) mutation and IL33 (C116F) mutation) was compared with SLT1 and SLT2 (Figure 14). SLT4 achieved approximately the same level of IL2 activity compared to wild-type IL2, while also achieving approximately the same level of IL33 activity as wild-type IL33.

[0160] Example 5: Tissue Targeting and ST2+ Treg Cells Healthy mice were treated with 66 pmol of IL233 fusion protein (SLT-518(h4)) per day for 5 days. Tissues were collected on the 8th day. In Tregs, the average ST2 expression level was increased. In the spleen, the total number of ST2+ Tregs was increased. In the Treg population, the proportion of ST2+ cells was increased. Therefore, in the tissue microenvironment, SLT-518(h4) upregulates highly suppressive ST2+ Tregs (Figure 16).

[0161] In an ischemic kidney injury model, SLT-518(h4) stimulated the active mobilization of Tregs to the injured tissue by IL-33-dependent signals in the tissue microenvironment.

[0162] In summary, it was shown that SLT518(h4) promotes the expression of ST2+ Treg cells in healthy mice. SLT518(h4) can accelerate the mobilization of ST2+ Treg cells to the injury site, which means that systemic administration of this therapeutic agent can cause a local effect. Tregs from SLT518(h4) more potently suppress T cell proliferation in vitro and diseases in vivo on a per cell basis.

[0163] Data using SLT518(h4) presented herein show that newly discovered mutations improve the molecular function and stability compared to previously published mouse proteins.

[0164] Example 6: Tissue Microenvironment and Persistence In a sustained immune response, not only Tregs but also others are required. Overlooking other cells in the tissue microenvironment may result in a transient and weaker effect, and the underlying cause of the disease is not addressed. Previous studies have shown that low-dose IL-2 targets mainly Tregs, which results in a mere transient response. However, low-dose IL-2 + IL33 targets many types of cells, including immune-tolerogenic dendritic cells (tolDCs) that produce IL-2 (Figure 17). Therefore, the IL233 fusion cytokine restores the tissue microenvironment and establishes a long-term immunotolerogenic and reparative state. The effect of the restored microenvironment is a sustained effect that does not require additional drug administration.

[0165] Example 7: Cynomolgus Monkey Test The healthy non-human primate test was conducted at WuXi AppTec (Shanghai, China) using SLT-518(h4) derived from a CHO manufacturing process developed by WuXi Biologics (Shanghai, China). All comparisons of SLT-518(h4) and IL-2 were performed with equimolar once-daily dosing for 5 times, adjusted to Proleukin as the reference.

[0166] In an extensive cynomolgus monkey test, more than 35 animal individuals were evaluated with gradually increasing doses. Systemic pharmacokinetics (as Tregs and Treg subsets), local pharmacokinetics (in lymph nodes, tissue undetermined), tolerability, safety and selectivity index (as Treg / Teff subsets), and pharmacodynamics (single and repeated dosing) were evaluated. Different routes of administration were tested, and multiple control drugs were also tested in the same way.

[0167] Treatment with SLT-518(h4) caused a dramatic increase in ST2+ Treg cells in healthy non-human primates (Figure 18). Over 21 days, two monkeys were administered SLT518(h4) and two monkeys were administered IL-2 alone. When the percentage of ST2+ Tregs was measured, 3000-fold more ST2+ Tregs were measured on day 6 in the monkeys administered the engineered IL233 fusion protein (SLT-518(h4)).

[0168] SLT-518(h4) also caused a marked expansion of ST2+ Tregs, thereby providing a wider therapeutic margin compared to the clinical dose of IL-2 (Figures 21A and 21B). SLT-518(h4) was administered to monkeys three times (Figure 21A), and IL-2 alone was administered to monkeys three times (Figure 21B). The percentage of the Treg subset was measured and tracked against the targeted clinical range.

[0169] SLT-518(h4) also resulted in a dramatic expression of ST2+ Tregs in the blood and remained elevated well above the levels observed in the blood over 28 days in the tissues (Figure 19).

[0170] In summary, the C116F mutation resulted in a humanized form of IL233 (SLT518(h4)) with superior activity compared to the wild-type, as shown by comparative data comparing SLT 518(h1) and SLT518(h2) with SLT518(h4) in both in vitro and non-human primate tests.

[0171] Example 8: Non-Human Primate Tregs

[0172] Non-human primates were administered any one of SLT1 (wild-type fusion protein IL233), SLT2 (IL233 fusion protein containing the IL2(N88D) mutation), or SLT4 (IL233 fusion protein containing the IL2(N88D) and IL33(C116F) mutations) at a dose of 100 μg / kg per day for 5 days. The proportion of Tregs was measured over 21 days. Figure 20 shows that SLT4 brought about a dramatic increase in the proportion and change (fold) of Tregs relative to day 0 in non-human primates, with day 6 being the peak.

[0173] Similar to the mouse data above, SLT518(h4) (shown as SLT4 in Figure 20) was shown to promote the expression of ST2+ Treg cells in non-human primates and acted significantly better than SLT518(h1) (shown as SLT1) and SLT518(h2) (shown as SLT2). This data indicates that the key mutation for increasing the expression of ST2+ Treg cells is due to the IL33 C116F mutation.

Claims

1. Interleukin-2 (IL-2) having at least 90% identity with SEQ ID NO: 13; and A cleaved interleukin 33 (IL-33) containing a sequence having at least 90% identity with SEQ ID NO:

1. A composition containing the following:

2. Interleukin-2 (IL-2) having the amino acid sequence of SEQ ID NO: 14: and A cleaved interleukin 33 (IL-33) having the amino acid sequence of SEQ ID NO: 3 A composition containing the following:

3. Interleukin-2 (IL-2) domains having at least 90% identity with SEQ ID NO: 13, A cleaved interleukin 33 (IL-33) domain containing a sequence that is at least 90% identical to Sequence ID No.

1. A fusion protein containing [the specified ingredient].

4. Interleukin-2 (IL-2) having the amino acid sequence of SEQ ID NO: 14; and A cleaved interleukin 33 (IL-33) having the amino acid sequence of SEQ ID NO: 3 A fusion protein containing [the specified ingredient].

5. The composition according to claim 1, wherein in the cleaved IL-33, at least one of N60, C97, C116, C121, and C148 of SEQ ID NO: 1 is substituted with a single amino acid.

6. The composition according to claim 1, wherein N60 of SEQ ID NO: 1 is substituted with Ser or Asp, C97 of SEQ ID NO: 1 is substituted with Gly, or C116 of SEQ ID NO: 1 is substituted with Phe.

7. The composition according to claim 1, wherein the cleavage-type IL-33 has an sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO:

7.

8. The fusion protein according to claim 3, wherein in the cleaved IL-33 domain, at least one of N60, C97, C116, C121, and C148 of SEQ ID NO: 1 is substituted with a single amino acid.

9. The fusion protein according to claim 3, wherein N60 of SEQ ID NO: 1 is replaced with Ser or Asp, C97 of SEQ ID NO: 1 is replaced with Gly, or C116 of SEQ ID NO: 1 is replaced with Phe.

10. The fusion protein according to claim 3, wherein the cleaved 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.

11. A fusion protein according to any one of claims 3, 8 to 10, further comprising a linker.

12. The fusion protein according to claim 11, wherein the linker comprises GGGGS (SEQ ID NO: 9).

13. The fusion protein according to claim 11, wherein the linker has the sequence GGGGSGGGGSGGGGS (Sequence ID 10).

14. The composition according to claim 1, wherein IL-2 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.

15. The fusion protein according to any one of claims 3, 8 to 10, wherein 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.

16. A fusion protein according to any one of claims 3, 8 to 10, further comprising a signal peptide.

17. The fusion protein according to claim 16, wherein the signal peptide has the sequence of SEQ ID NO:

11.

18. The fusion protein according to any one of claims 3, 8 to 10, wherein the domains are, in order from the N-terminus to the C-terminus, an optional signal peptide; the interleukin-2 (IL-2) domain; an optional linker; and the cleaved interleukin-33 (IL-33) domain.

19. A pharmaceutical composition comprising the composition according to claim 1 or 2 or the fusion protein according to any one of claims 3, 8 to 10.

20. The pharmaceutical composition according to claim 19, further comprising a pharmaceutically acceptable carrier and, optionally, an additional therapeutic agent.

21. A polynucleotide comprising a nucleic acid sequence encoding at least a portion of the fusion protein described in claim 3.

22. A vector comprising the polynucleotide according to claim 21, which is operably linked to a promoter.

23. Recombinant host cells comprising the polynucleotide described in claim 21.

24. A pharmaceutical composition according to claim 19 for use in a method for treating a disease or disorder, wherein the method comprises administering an effective amount of the pharmaceutical composition to a subject in need thereof.

25. The aforementioned diseases or disorders include acute kidney injury, ankylosing spondylitis, autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune lymphoproliferative syndrome, autoimmune pancreatitis, polyglandular autoimmune 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), inflammatory demyelinating disease, inflammatory neuropathy, isletitis, interstitial cystitis, juvenile idiopathic arthritis, lupus. The pharmaceutical composition according to claim 24, selected from the group consisting of lupus erythematosus, lupus glomerulonephritis, 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, Sjögren's syndrome, systemic lupus erythematosus, graft rejection, type 1 diabetes, type 2 diabetes, ulcerative colitis, vasculitis, and Wegener's granulomatosis.

26. The pharmaceutical composition according to claim 24, wherein the disease or disorder is cancer.

27. The aforementioned cancers include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute myeloid leukemia, adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, B-cell lymphoma, brain cancer, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, cancer of unknown primary origin, cardiac tumor, cervical cancer, chordoma, chronic lymphoblastic leukemia, chronic myeloid leukemia, colon cancer, colorectal cancer, craniopharyngioma, adenoid carcinoma, germ blastoma, endometrial cancer, ependymoma, esophageal cancer, nasal neuroblastoma, fibrous histiocytoma, Ewing's sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, and gestational trophoblastic tumor. Diseases, glioma, head and neck cancer, hepatocellular carcinoma, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma of unknown primary origin, median duct cancer related to the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal cavity and paranasal sinus cancer (nasal The pharmaceutical composition according to claim 26, selected from the group consisting of cavity and par nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, chromaffin cell tumor, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureteral cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sézary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, gastric cancer, T-cell lymphocytic leukemia, T-cell lymphoma, teratoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor.

28. The pharmaceutical composition according to claim 27, wherein the cancer is 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 myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, bladder cancer, and lung cancer.

29. The pharmaceutical composition according to claim 24, wherein the method stimulates the proliferation and / or activation of regulatory T (Treg) cells, helper T2 (Th2) cells, innate lymphoid cells (ILCs), cytotoxic T cells, natural killer (NK) cells, NK-T cells, ST2+ cells, dendritic cells, and / or macrophages.

30. The pharmaceutical composition according to claim 24, wherein the method promotes anti-inflammatory M2 macrophages and inhibits inflammatory M1 macrophages.

31. The pharmaceutical composition according to claim 30, wherein the M2 macrophages suppress inflammation in a tissue or organ selected from the group consisting of the pancreas, liver, kidney, adipose tissue, salivary gland, central nervous system (CNS), and related organs.

32. The pharmaceutical composition according to claim 24, wherein the method can upregulate or downregulate the expression of costimulatory molecules (including but not limited to CD80 (B7-1), CD86 (B7-2), CD40, ICOS, MHC-I, MHC-II, PD1, PD-L1, GITR, BAFF-R, Ox40, 41BB, DR3, CR2) on antigen-presenting cells, including but not limited to dendritic cells, macrophages, B cells, innate lymphoid cells, NK cells, epithelial cells, endothelial cells, or stromal cells, in a manner dependent on the IL-2 domain.

33. The pharmaceutical composition according to claim 24, wherein the method further comprises applying an effective amount of a second therapy for the disease or disorder to the subject, the second therapy comprising neither the composition nor the fusion protein.

34. The pharmaceutical composition according to claim 33, wherein the second therapy is cell therapy or gene therapy.

35. A method for stimulating the proliferation and / or activation of target regulatory T (Treg) cells, The T cells isolated from the subject are brought into contact with an effective amount of the composition according to claim 1; the fusion protein according to any one of claims 3, 8 to 10; cells expressing at least one of IL-2 or cleaved IL-33; or the recombinant host cell according to claim 23. Methods that include...

36. The pharmaceutical composition according to claim 19; and Instructions for implementing methods to treat diseases or disorders characterized by inflammation. A kit comprising the method of administering an effective amount of the pharmaceutical composition to a subject requiring it.

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