Compositions comprising truncated interleukin-33 and interleukin-2

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 340,777, filed on May 11, 2022, the contents of which are incorporated herein in their entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy created on May 9, 2023 is named 771185_000009_SL.xml and is 18,768 bytes in size. Background Art

[0005] Interleukin-2 (IL-2) is a cytokine that plays a role in maintaining immune tolerance; it regulates T helper (Th) cell function, especially Th2 function; Foxp3 + CD4 + Differentiation, survival and function of regulatory T (Treg) cells; and interaction with innate lymphoid cells (ILCs).

[0006] Interleukin-33 (IL-33) is a cytokine whose wild type contains a nuclear localization signal at its N-terminus. Protease cleavage produces an 18kDa 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, and dendritic cells, thereby upregulating the expression of IL-2, IL-4, IL-5, and IL-13, and reducing proinflammatory Th1 and Th17 responses.

[0007] 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, US 9,840,545, US10,851,145, and US20210261640, the disclosures of which are hereby incorporated by reference. Such fusion proteins can treat diseases such as autoimmune diseases and disorders, inflammation, etc. However, it has been observed that many such fusion proteins have one or more undesirable properties, such as aggregation, rapid clearance in vivo, or loss of IL-33 activity over time.

[0008] Therefore, there is a need for a fusion protein comprising an IL-2 domain and a truncated IL-33 domain 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 a fusion protein that is easy to express from a recombinant organism, easy to purify, and has IL-2 and IL-33 activities comparable to wild-type IL-2 and wild-type IL-33, respectively. Summary of the invention

[0009] The present disclosure provides compositions comprising interleukin-2 (IL-2) having at least 90% identity to SEQ ID NO: 13 and a truncated interleukin-33 (IL-33), wherein the truncated IL-33 comprises a sequence having at least 90% identity to SEQ ID NO: 1.

[0010] 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.

[0011] In the truncated 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 truncated 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 a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 7.

[0012] The fusion protein may further comprise an optional linker, such as an oligopeptide linker. For example, the linker may comprise GGGGS (SEQ ID NO: 9). The linker may have the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 10).

[0013] 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.

[0014] The fusion protein may further comprise an optional signal peptide. The signal peptide may have a sequence of SEQ ID NO:11.

[0015] In the fusion protein, the domains may 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.

[0016] The present disclosure also encompasses a pharmaceutical composition comprising the composition or fusion protein as described herein.

[0017] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and optionally an additional therapeutic agent.

[0018] The present disclosure encompasses a polynucleotide comprising a nucleic acid sequence encoding at least a portion of a fusion protein described herein.

[0019] In another aspect, the present disclosure comprises a vector comprising a polynucleotide operably linked to a promoter.

[0020] In yet another aspect, the present disclosure comprises a recombinant host cell comprising a polynucleotide or a vector.

[0021] The present disclosure encompasses a method for treating a disease or disorder comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition described herein.

[0022] The disease or disorder can be 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 disease (GVH), Hashimoto's thyroiditis, thyroiditis, idiopathic inflammatory bowel disease (IBD), inflammatory demyelinating disease, inflammatory neuropathy, insulitis, interstitial cystitis, juvenile idiopathic arthritis, lupus, lupus erythematosus, lupus glomerulonephritis, lupus nephritis, cutaneous lupus, IgA nephropathy, membranous nephropathy (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, Sjogren's syndrome, systemic lupus erythematosus, transplant rejection, type 1 diabetes mellitus, type 2 diabetes mellitus, ulcerative colitis, vasculitis, psoriasis, atopic dermatitis, pemphigus vulgaris, or Wegener's granulomatosis.

[0023] The disease or disorder can be cancer. The cancer can be acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute myeloid leukemia, adrenocortical carcinoma, anal cancer, adnexal cancer, astrocytoma, basal cell carcinoma, B-cell lymphoma, brain cancer, biliary tract cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, primary unknown cancer, heart tumor, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, nasal glioma, 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 carcinoma, histiocytosis, Hodgkin lymphoma (Hodgkin lymphoma), hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi 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, occult primary metastatic squamous neck cancer, midline airway cancer involving NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, Myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, cancer of the nasal cavity and paranasal sinuses, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, 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, 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, teratoma, testicular cancer, laryngeal cancer, thymoma and thymic cancer, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms tumor.

[0024] The cancer can be melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, stomach 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, or lung cancer.

[0025] Depending on the IL-2 domain, the method can stimulate proliferation and / or activation of T regulatory (Treg) cells, T helper 2 (Th2) cells, innate lymphoid cells (ILCs), cytotoxic T cells, natural killer (NK) cells, NK-T cells, ST2+ cells, dendritic cells and / or macrophages.

[0026] Depending on the IL-2 domain, the method can promote anti-inflammatory M2 macrophages and inhibit pro-inflammatory M1 macrophages, or activate M1 macrophages to induce inflammation. M2 macrophages can inhibit inflammation in tissues or organs selected from the group consisting of: pancreas, liver, kidney, adipose tissue, salivary glands, central nervous system (CNS) and related organs. M1 macrophages can promote systemic or local anti-tumor immune responses.

[0027] Depending on the IL-2 domain, the method 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 lymphocytes, NK cells, epithelial cells, endothelial cells or stromal cells.

[0028] The method can further comprise administering to the subject an effective amount of a second therapy for the disease or condition, wherein the second therapy is not a fusion protein described herein.

[0029] The second therapy may be cell therapy or gene therapy.

[0030] In another aspect, the present disclosure comprises a method for stimulating proliferation and / or activation of T regulatory (Treg) cells in a subject, the method comprising: isolating T cells from the subject; and contacting the isolated T cells with an effective amount of a composition, a fusion protein, a recombinant host cell, or a cell expressing at least one of IL-2 or truncated IL-33, as described herein.

[0031] The present disclosure also encompasses kits comprising a pharmaceutical composition described herein; and instructions for performing a method of treating a disease or condition characterized by inflammation, the method comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1The sequences of wild-type human IL-2 (SEQ ID NO: 13) and truncated wild-type human IL-33 (SEQ ID NO: 1) are shown. In the IL-2 sequence, in some embodiments, two residues of interest, N88 and C125, are emphasized. In the truncated IL-33 sequence, in some embodiments, three residues of interest, N60, C97, and C116, are emphasized.

[0033] Figure 2 Sequence alignments 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) are shown. In the human sequence, the N-glycosylation sites comprising N60 and four wild-type Cys residues C97, C116, C121, and C148 are highlighted. In the mouse and rat sequences, Cys residues that align with human Cys residues are emphasized.

[0034] Figure 3 A graph showing the IL-2 activity of a fusion protein as described in Example 1 comprising an IL-2 domain and a truncated IL-33 domain with a N60D point substitution.

[0035] Figure 4 A graph showing the IL-2 activity of a fusion protein as described in Example 2 comprising an IL-2 domain and a truncated IL-33 domain with a C97G point substitution.

[0036] Figure 5 A graph showing the IL-33 activity of a fusion protein as described in Example 2 comprising an IL-2 domain and a truncated IL-33 domain with a C97G point substitution.

[0037] Figure 6 The expression of a fusion protein comprising an IL-2 domain and a truncated IL-33 domain with a C116F point substitution in recombinant host cells as described in Example 3 is shown.

[0038] Figure 7 Shown is the purification of a fusion protein as described in Example 3 comprising an IL-2 domain and a truncated IL-33 domain with a C116F point substitution.

[0039] Figure 8 A graph showing the IL-2 activity of a fusion protein as described in Example 3 comprising an IL-2 domain and a truncated IL-33 domain with a C116F point substitution.

[0040] Fig. 9 A graph showing the IL-33 activity of a fusion protein as described in Example 3 comprising an IL-2 domain and a truncated IL-33 domain with a C116F point substitution.

[0041] Fig.10 A graph showing IL-33 activity of a fusion protein comprising an IL-2 domain and a truncated IL-33 domain with a C116F point substitution as described in Example 3 after storage at 4°C, 25°C or 37°C for 0, 3 or 7 days.

[0042] Fig.11 is a graph showing the IL2 and IL33 activities of wild-type IL2, wild-type fusion protein IL233 74291-1 (SLT1), and engineered IL233 fusion proteins 7429-2 (SLT2), 7429-3 (SLT3), 7429-4 (SLT4), 7429-5 (SLT5), and 7429-6 (SLT6).

[0043] Fig.12 is a graph showing the IL2 and IL33 activities of wild-type IL2, wild-type fusion protein IL233 74291-1 (SLT1), and engineered IL233 fusion proteins 7429-2 (SLT2), 7429-3 (SLT3), and 7429-4 (SLT4).

[0044] Fig.13 is a graph showing the IL2 and IL33 activities of wild-type IL2, wild-type fusion protein IL233 74291-1 (SLT1), and engineered IL233 fusion proteins 7429-5 (SLT5) and 7429-6 (SLT6).

[0045] Fig.14 is a graph showing the IL2 and IL33 activities of wild-type IL2 and engineered IL233 fusion proteins 7429-2 (SLT2) and 7429-4 (SLT4).

[0046] Fig.15 is a graph showing that IL233 fusion protein (SLT-518(h4)) promotes dose-dependent ST2+ Treg proliferation in fresh human PBMCs. IL-2 and SLT518(h4) were added at 25 ng / ml and 50 ng / ml, and the percentage of ST2 Tregs was measured.

[0047] Fig.16Results from a mouse model are presented, where mice received equimolar injections of SLT-518(h4) daily for five days. Blood ST2+Tregs were measured on day 6, ST2+Tregs in lymph nodes, kidneys, and lungs were measured on day 8, and cytokines were measured. The results demonstrate that human SLT-518(h4) (IL233 fusion protein) signals in vivo and drives Tregs into tissues.

[0048] Fig.17 It is a composite diagram schematically showing that SLT-518 (h4) (an IL233 fusion protein) drives the local tissue environment to a more tolerant phenotype in mice. Supporting data show that after administration of IL233 fusion protein, costimulatory molecules on natural killer (NK) cells and dendritic cells (DC) are reduced. After administration of IL233 fusion protein, the ratios of M2 macrophages, ST2+Treg, IL10 / TNFa, Treg / Th17, and ILC2 all increase.

[0049] Fig.18 is a graph showing that treatment with SLT-518(h4) (referred to as SLT4) promotes significant expansion of ST2+ Treg cells in healthy non-human primates.

[0050] Fig.19 is a graph showing the selective expansion of total ST2+ Tregs in healthy non-human primates (NHPs) following administration of SLT-518, without concomitant expansion of other pro-inflammatory T cells, cytotoxic T cells, B cells, or natural killer cells.

[0051] Fig. 20 Graph showing the percentage of Tregs over time (21 days) and the fold change over time (21 days) in non-human primates after administration of five daily doses of 100 ug / kg of wild-type fusion protein IL233 74291-1 (SLT1), engineered IL233 fusion protein 7429-2 (SLT2) or 7429-4 (SLT4).

[0052] Fig.21A and 21B is a graph showing that SLT-518(h4) induces superior expansion of ST2+ Tregs, thereby providing a wider therapeutic range relative to clinical doses of IL-2.

[0053] Fig. 22 is a graph showing that SLT-518(h4) exhibited significant expression of ST2+ Treg in blood and remained elevated in tissues for more than 28 days, far exceeding the levels observed in blood. DETAILED DESCRIPTION

[0054] The present disclosure relates to compositions or fusion proteins comprising IL-2 polypeptides or domains and truncated IL-33 polypeptides or domains. The fusion of IL-2 and IL-33 is the key to improving efficacy, tissue targeting and affinity. ST2 is a receptor for IL-33 and is expressed on the surface of a subset of regulatory T cells (Treg). This subset is more durable and active in regulating inflammatory suppression in tissues at disease sites. Because IL-33 is naturally released in tissues, its function not only helps to amplify ST2+Treg cells, but also helps to amplify other ST2+ cells and promote local tolerogenic tissue microenvironment. Therefore, treatment with the fusion proteins and compositions described herein produces lasting disease regression.

[0055] In a fusion protein, the domains may be joined 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 any one or more of the following: reduced aggregation, improved stability during storage, retention of IL-33 activity after storage, easier production of recombinant organisms, easier purification, IL-2 activity comparable to wild-type IL-2, IL-33 activity comparable to wild-type IL-33, and prolonged IL-33 activity after administration. Although not wishing to be bound by theory, such advantageous properties may be derived from site-specific mutations in the IL-2 domains and / or truncated IL-33 domains that reduce aggregation, such as by replacing cysteine ​​residues with other residues that cannot form disulfide bonds.

[0056] All publications and patents cited in this disclosure are incorporated by reference as a whole. To the extent that the material incorporated by reference is contradictory or inconsistent with this specification, this specification will replace any such material. Any reference cited herein is not an admission that such reference is the prior art of the present disclosure. When a numerical range is expressed, it includes an embodiment using any specific value within the range. Further, a reference to the value described in the range includes each value within the range. All ranges include their endpoints and are combinable. When a value is expressed as an approximation by using the antecedent "about", it should be understood that a specific value forms another embodiment. Unless the context clearly states otherwise, a reference to a specific numerical value at least includes the specific value. The use of "or" will mean "and / or", unless the specific context of its use indicates otherwise.

[0057] Various terms related to the various aspects of the description are used throughout the specification and claims. Unless otherwise noted, such terms have their ordinary meanings in the art. Other specifically defined terms will be interpreted in a manner consistent with the definitions provided herein. The techniques and procedures described or cited herein are generally fully understood by those skilled in the art and are generally used to use conventional methods, for example, the widely used molecular cloning methods described below: Sambrook et al., "Molecular Cloning: A Laboratory Manual" 4th edition, 2012, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where appropriate, unless otherwise noted, the procedures involving the use of commercially available kits and reagents are generally performed according to the schemes and / or conditions defined by the manufacturer.

[0058] As used herein, the singular forms "a", "an", and "the" include the plural forms unless the context clearly indicates otherwise. Unless specifically stated otherwise, the terms "including", "such as", etc. are intended to express inclusion rather than limitation.

[0059] Unless otherwise indicated, the terms "at least," "less than," and "about," or similar terms preceding a series of elements or ranges should be understood to refer to each element in the series or range. Those skilled in the art will recognize, or be able to ascertain using only routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the following claims.

[0060] As used herein, the term "subject" refers to any animal, such as any mammal, including but not limited to humans, non-human primates, rodents, mammals commonly kept as pets (e.g., dogs and cats, etc.), livestock (e.g., cattle, sheep, goats, pigs, horses, camels, etc.), etc. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0061] This article provides additional description of the method and guidance for practicing the method.

[0062] AImplementation Plan

[0063] i. Composition

[0064] In some embodiments, the present disclosure relates to a composition. The composition may include interleukin-2 (IL-2) and truncated interleukin-33 (IL-33). In the composition, interleukin-2 (IL-2) may include SEQ ID NO: 13, or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 13. Alternatively or additionally, the truncated interleukin-33 (IL-33) may include SEQ ID NO: 1, or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1.

[0065] IL-2 and truncated IL-33 are generally described herein in the context of fusion proteins. However, one of ordinary skill in the art will understand that, in addition to the specific description of the fusion protein (e.g., the arrangement of the domains in the fusion protein, etc.), the description of the IL-2 domain and the truncated IL-33 domain will also apply to the IL-2 and truncated IL-33 polypeptides in the composition.

[0066] ii. Fusion Protein

[0067] In some embodiments, the present disclosure relates to a fusion protein. The fusion protein may include an interleukin-2 (IL-2) domain and a truncated interleukin-33 (IL-33) domain. The fusion protein may include an interleukin-2 (IL-2) domain containing 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 with SEQ ID NO: 13; and an interleukin-33 (IL-33) domain containing a truncated IL-33 domain containing SEQ ID NO: 1, or a truncated IL-33 domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1.

[0068] In some embodiments, the fusion protein can include an optional linker between the first and second domains.

[0069] In some embodiments, the fusion protein may comprise more than one IL-2 domain, more than one truncated IL-33 domain, or both.

[0070] IL-2 domain

[0071] The IL-2 domain of the fusion protein can be identical to the wild-type IL-2 sequence of any organism or a variant thereof. On the one hand, the IL-2 domain is at least 90% identical to the wild-type IL-2 sequence of the organism, and in the methods described herein, the fusion protein is intended to be administered to the organism.

[0072] In one embodiment, the IL-2 domain comprises a sequence that is at least 90% identical to SEQ ID NO: 13, such as at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 13. The IL-2 domain of the fusion protein may differ from SEQ ID NO: 13 in zero, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen positions.

[0073] The sequence of the IL-2 domain may comprise many variants of SEQ ID NO: 13. For example, the IL-2 domain may comprise a point mutation of Asn at position 88 (N88) of SEQ ID NO: 13. An IL-2 domain comprising a point mutation of N88 may exhibit one or more advantageous properties, including, but not limited to, greater stability and / or greater activity during storage than a corresponding IL-2 domain lacking the point mutation of N88.

[0074] For a specific example, the IL-2 domain can comprise a point substitution from Asn to Asp at position 110 (N88D) of SEQ ID NO: 13. A specific IL-2 domain containing the N88D substitution has the sequence of SEQ ID NO:14.

[0075] In another embodiment, the IL-2 domain may comprise a point mutation of Cys at position 125 (C125) of SEQ ID NO: 13. An IL-2 domain comprising a point mutation of C125 is less likely to form disulfide bonds on Cys residues within the IL-2 domain and / or within the IL-33 domain and may exhibit one or more advantageous properties, including, but not limited to, reduced aggregation, slower in vivo clearance, or higher IL-2 activity compared to a corresponding IL-2 domain lacking a point mutation of C125.

[0076] In a specific embodiment, the IL-2 domain may comprise a point substitution from Cys to Ser at position 125 (C125S) of SEQ ID NO: 13. A specific IL-2 domain containing the C125S substitution has the sequence of SEQ ID NO:15.

[0077] 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 allowing differential activation of NK and CD8 T cells compared with Tregs.

[0078] Any two or more deletions or substitutions within the IL-2 domain as described above can be combined. In one embodiment, the IL-2 domain can include a point mutation of both Asn at position 88 and Cys at position 125 of SEQ ID NO:13. For a specific example, the IL-2 domain can include both point substitutions N88D and C125S of SEQ ID NO:13. A specific IL-2 domain containing both N88D and C125S substitutions has a sequence of SEQ ID NO:16.

[0079] Connectors

[0080] The optional linker can be any part that can form at least two covalent bonds with the N-terminus of a polypeptide, the C-terminus of a polypeptide, or the amino acid side chains 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. The oligopeptide can form a covalent bond with the C-terminus of a polypeptide and the N-terminus of (usually another) polypeptide.

[0081] Preferably, the amino acids are encoded by codons of the typical genetic code. This allows the fusion protein as a whole to be encoded by a single polynucleotide and transcribed and translated as a unit, thereby simplifying the preparation of the fusion protein.

[0082] When the linker is an oligopeptide, its amino acids can be selected to allow the linker to be flexible, generally less likely to form secondary structure, generally less likely to interfere with the tertiary structure of the domains connected by the linker, generally less likely to be connected to any domain of the fusion protein other than 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.

[0083] Specific amino acid residues of the oligopeptide linker can be selected to adjust the isoelectric point (pi) of the fusion protein, which can be used to increase the yield, purity, storage or efficacy of the fusion protein during preparation or use.

[0084] For example, the linker can comprise GGGGS (SEQ ID NO: 9). In another aspect, the linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 10).

[0085] In some embodiments, the fusion protein may comprise an optional additional linker, whereby the fusion protein may dimerize or multimerize. The additional linker may be an oligopeptide linker such as those described herein.

[0086] IL-33 domain

[0087] 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 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 to be administered in the methods described herein.

[0088] Wild-type human truncated IL-33 has the sequence of SEQ ID NO: 1. Other organisms have truncated IL-33 that contain residues homologous to the human truncated IL-33 of SEQ ID NO:1.

[0089] In one aspect, the truncated IL-33 domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1, such as at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 1. The truncated IL-33 domain of the fusion protein may differ from SEQ ID NO: 1 at zero, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen positions.

[0090] In some embodiments, the truncated 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 possible glycosylation or disulfide bond sites (e.g., N60, C97, C116, C121, and C148) are deleted or substituted with one or more amino acids.

[0091] With regard to the sequence of truncated IL-33 domains, a number of variants of SEQ ID NO: 1 are particularly contemplated.

[0092] In one aspect, the truncated IL-33 domain may comprise a point mutation of Asn at position 60 (N60) of SEQ ID NO: 1. Figure 3As shown, N60 is present in human truncated IL-33, but not in the homologous position of mouse or rat truncated IL-33. Truncated IL-33 domains comprising N60 point mutations are less sensitive to glycosylation and may exhibit one or more advantageous properties, including, but not limited to, reduced aggregation, slower in vivo clearance, or higher activity compared to corresponding truncated IL-33 domains lacking N60 point mutations. In a specific example, the truncated IL-33 domain may comprise a point substitution at position 60 from Asn to Ser (N60S) or from Asn to Asp (N60D). A specific truncated IL-33 domain comprising an N60S point substitution may have a sequence of SEQ ID NO: 2.

[0093] In one embodiment, the truncated IL-33 domain may comprise a point mutation of Cys at position 116 (C116) of SEQ ID NO: 2. A truncated IL-33 domain comprising a point mutation of C116 is less likely to form disulfide bonds on Cys residues within the truncated IL-33 domain and / or within the IL-2 domain, and may exhibit one or more advantageous properties, including, but not limited to, reduced aggregation, slower in vivo clearance, or higher activity, compared to a corresponding truncated IL-33 domain lacking the point mutation of C116.

[0094] In one embodiment, the truncated IL-33 domain may comprise a point substitution from Cys to Phe at position 116 (C116F). Figure 3 As shown, the C116F point substitution was based on the observation that mouse and rat truncated IL-33 contain a Phe at a position homologous to human wild-type C116.

[0095] The C116F point substitution improved the expression and purification of the fusion protein comprising both the IL-2 domain and the truncated IL-33 domain, and the activities of the truncated IL-33 domain and the IL-2 domain were essentially the same as those of wild-type IL-33 alone and IL-2 alone, respectively.

[0096] A specific truncated IL-33 domain comprising a C116F point substitution may have the sequence of SEQ ID NO:3.

[0097] In one aspect, the truncated IL-33 domain may comprise a point mutation of Cys at position 97 (C97) of SEQ ID NO: 2. A truncated IL-33 domain comprising a C97 point mutation is less likely to form disulfide bonds and may exhibit one or more desirable properties discussed with reference to C116.

[0098] Alternatively, the truncated IL-33 domain may comprise a point substitution from Cys to Gly at position 97 (C97G). Figure 3 As shown, the C97G point substitution was based on the observation that mouse and rat truncated IL-33 contain a Gly at a position homologous to human wild-type C97.

[0099] In the context of the C97G point substitution, the activities of both the truncated IL-33 domain and the IL-2 domain were essentially the same as wild-type IL-33 alone and IL-2 alone, respectively.

[0100] A specific truncated IL-33 domain comprising a C97G point substitution may have the sequence of SEQ ID NO:4.

[0101] Any two or more deletions or substitutions within the truncated IL-33 domains as described above can be combined. For example, the truncated IL-33 domain can include 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 N60S, C116F and C97G (SEQ ID NO: 8).

[0102] Thus, 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, and SEQ ID NO:8.

[0103] 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, or SEQ ID NO:7.

[0104] Signal peptide

[0105] The fusion protein may also include a signal peptide, such as 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 rational design or routine experimentation.

[0106] In one embodiment, if the N-terminal domain of the fusion protein is an IL-2 domain, the signal peptide may have the sequence of SEQ ID NO:11.

[0107] Connection of domains

[0108] The various domains of fusion protein can be connected by any desired reaction site of various domains, or directly connected, or connected by the reactive part of optional joint.In some embodiments, wherein joint is oligopeptide, various domains can be connected to the complementary end of joint by their N-terminus and / or C-terminus.Fusion protein can comprise single continuous polypeptide sequence.This type of fusion protein can be prepared by the single polynucleotide coding region of the complete sequence of encoding fusion protein by well-known transcription and translation process.

[0109] A fusion protein comprising a single continuous polypeptide sequence may comprise the various domains in any desired order. For example, a fusion protein may comprise an IL-2 domain or a truncated IL-33 domain at its N-terminus; a linker; and any of the remaining domains at the C-terminus.

[0110] In a specific embodiment, the fusion protein may 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.

[0111] In a specific embodiment, the fusion protein may comprise, in order from N-terminus to C-terminus, a signal peptide; a truncated IL-33 domain; a linker; and an IL-2 domain.

[0112] In a specific embodiment, the fusion protein may comprise, in order from N-terminus to C-terminus, a signal peptide; an IL-2 domain; and a truncated IL-33 domain.

[0113] In a specific embodiment, the fusion protein may comprise, in order from N-terminus to C-terminus, a signal peptide; a truncated IL-33 domain; and an IL-2 domain.

[0114] In a specific embodiment, the fusion protein may comprise, in order from N-terminus to C-terminus, an IL-2 domain; a linker; and a truncated IL-33 domain.

[0115] In a specific embodiment, the fusion protein may comprise, in order from N-terminus to C-terminus, a truncated IL-33 domain; a linker; and an IL-2 domain.

[0116] In a specific embodiment, the fusion protein may comprise a truncated IL-33 domain and an IL-2 domain in order from N-terminus to C-terminus.

[0117] In a specific embodiment, the fusion protein may comprise an IL-2 domain and a truncated IL-33 domain in order from N-terminus to C-terminus.

[0118] In another specific embodiment, the fusion protein may comprise two polypeptides connected 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 truncated IL-33 domain; and the first polypeptide and the second polypeptide may be connected by an additional linker between the side chains of the amino acids in the IL-2 domain of the first polypeptide and the amino acids in the truncated IL-33 domain of the second polypeptide.

[0119] Preparation of fusion proteins

[0120] The fusion proteins described herein can be prepared by any known method. Generally, any polypeptide component of the fusion protein can be synthesized by chemical or biochemical methods, both of which are well known to those of ordinary skill in the art.

[0121] In the biochemical method 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, wherein the polynucleotide is operably linked to a promoter. The vector may contain other sequences, such as non-promoter control sequences, replication origins, sequences engineered to insert the polynucleotide into the vector, and the like. The promoter may be selected so 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 constitutively or under the induction of one or more changes in process conditions, an RNA transcript is produced. The RNA transcript is then translated by the host cell's ribosomes to produce a polypeptide of interest (e.g., a fusion protein or a portion thereof).

[0122] Another approach to polypeptide synthesis is to introduce a polynucleotide expressing the polypeptide into the cells of a subject in need. The polynucleotide may comprise mRNA or DNA. The subject's cells may transcribe the DNA and / or translate the mRNA into the polypeptide of interest.

[0123] In embodiments where the fusion protein comprises multiple polypeptides, each polypeptide can be produced separately and the fusion protein assembled by a peptide synthase reaction or by non-amino acid backbone linkages, such as by one or more non-oligopeptide linkers, by cross-polypeptide linkages of amino acid side chains of oligopeptide linkers, and / or by chemical conjugation.

[0124] 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.

[0125] iii. Pharmaceutical Compositions

[0126] In some embodiments, the present disclosure relates to a pharmaceutical composition. The pharmaceutical composition may include any of the compositions or fusion proteins described above. In some embodiments, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. Alternatively or additionally, the pharmaceutical composition may further include another therapeutic agent.

[0127] Pharmaceutically acceptable carrier

[0128] The pharmaceutical composition may include a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier includes a physiologically tolerable or acceptable diluent, excipient, solvent or adjuvant. The composition is preferably sterile and non-pyrogenic.

[0129] Additional therapeutic agents

[0130] In an embodiment, additional therapeutic agents may be included in the pharmaceutical composition, which means one or more therapeutic agents in addition to the fusion protein described above. Additional therapeutic agents may include, but are not limited to, cytotoxic agents, anti-angiogenic agents, pro-apoptotic agents, antibiotics, hormones, hormone antagonists, chemokines, drugs, prodrugs, toxins or enzymes.

[0131] The particular therapeutic agent may be selected based on the disease or condition that the pharmaceutical composition is intended to treat, among other parameters known to those of ordinary skill in the art.

[0132] The pharmaceutical compositions may also contain minor amounts of non-toxic auxiliary pharmaceutical substances, excipients and / or additives, such as wetting agents, emulsifiers and / or pH buffering agents, as well as other substances known to those of ordinary skill in the art.

[0133] If desired, absorption enhancers or delays (such as liposomes, aluminum monostearate or gelatin) may be used. 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.

[0134] iv. Treatment of diseases and conditions

[0135] In some embodiments, the disclosure relates to treating 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 as described herein. The disease or disorder is characterized by inflammation. In embodiments, the inflammatory condition may be present in a tissue or organ selected from the group consisting of: pancreas, liver, kidney, adipose tissue, salivary gland, central nervous system (CNS), and related organs.

[0136] Although not wishing to be bound by theory, the fusion protein can stimulate proliferation and / or activation of T regulatory (Treg) cells, T helper 2 (Th2) cells, innate lymphoid cells (ILCs), cytotoxic T cells, natural killer (NK) cells, NK-T cells, ST2+ cells, dendritic cells and / or macrophages; promote anti-inflammatory M2 macrophages; and / or inhibit pro-inflammatory M1 macrophages.

[0137] Although not wishing to be bound by theory, the fusion protein depends on the IL-2 domain and the method 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 lymphocytes, NK cells, epithelial cells, endothelial cells or stromal cells.

[0138] Diseases and conditions

[0139] In some embodiments, the disease or disorder can be an autoimmune disorder. In some embodiments, the disease or disorder can be 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 diseases, inflammatory neuropathy, islet dysplasia, inflammatory bowel disease ... inflammation, 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, Sjögren's syndrome, systemic lupus erythematosus, transplant rejection, type 1 diabetes mellitus, type 2 diabetes mellitus, ulcerative colitis, vasculitis, and Wegener's granulomatosis.

[0140] In some embodiments, the disease or disorder is cancer. For example, the cancer can be any of the following: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute myeloid leukemia, adrenocortical carcinoma, anal cancer, adnexal cancer, astrocytoma, basal cell carcinoma, B-cell lymphoma, brain cancer, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, primary unknown cancer, heart tumor, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma , embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, nasal glioma, fibrous histiocytoma, Ewing's sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, 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, giant glomerulonephritis Leukemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, occult primary metastatic squamous neck cancer, midline respiratory tract cancer involving the NUT gene, oral cancer, multiple endocrine neoplasms syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penis carcinoma, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, colorectal cancer, renal cell carcinoma, 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, gastric cancer, T-cell lymphocytic leukemia, T-cell lymphoma, teratoma, testicular cancer, laryngeal cancer, thymoma and thymic cancer, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer and Wilms' tumor.

[0141] The cancer may be selected from the group consisting of melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, stomach 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.

[0142] Application technology

[0143] The pharmaceutical composition can be delivered to the subject by any route by which the fusion protein can reach the target site, such as a tissue or organ where inflammation, cancer cells, or other phenomena associated with a disease or condition exist. The route can be local or systemic. For example, the pharmaceutical composition can be delivered systemically by intravenous or intraarterial, as well as other routes; or locally by subcutaneous, intramuscular, intraorgan or intratumoral, as well as other routes. Another route of administration is to introduce 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.

[0144] A specific route of administration can be selected based on the target site, disease or condition, the effective amount of the fusion protein, other components of the pharmaceutical composition and / or the second therapy (if any) included in the pharmaceutical composition, and other considerations. A person of ordinary skill in the art having the benefit of this disclosure can select a specific route of administration as a routine matter.

[0145] Adjunctive therapy

[0146] For any disease or condition, the method can further comprise administering an adjunctive therapy, wherein the adjunctive therapy does not comprise a fusion protein.

[0147] For example, when the disease or condition is cancer, adjuvant therapy may include surgical resection, radiation, chemotherapy, checkpoint inhibitor therapy, or other therapies known to those of ordinary skill in the art.

[0148] The pharmaceutical composition may include the adjunctive therapy if it is in a form suitable for administration by the same route as the fusion protein and is administered substantially simultaneously with the fusion protein.

[0149] In some embodiments, the adjunctive therapy is cell therapy or gene therapy.

[0150] Medical uses of fusion proteins

[0151] In some embodiments, the disclosure relates to a composition or fusion protein described herein for use in treating a disease or disorder described herein.

[0152] In some embodiments, the disclosure relates to the use of a composition or fusion protein described herein for treating a disease or disorder described herein.

[0153] In some embodiments, the present disclosure relates to the use of the composition or fusion protein described herein in the preparation of a medicament. The medicament can be used to treat the diseases or conditions described herein.

[0154] v. Methods for stimulating Treg cell proliferation and / or activation

[0155] In some embodiments, the present disclosure relates to methods for stimulating proliferation and / or activation of T regulatory (Treg) cells in a subject. The stimulation can be performed in vivo or in vitro.

[0156] For example, an in vitro method includes isolating T cells from a subject; and contacting the isolated T cells with an effective amount of a composition or fusion protein as described herein.

[0157] vi. Kit

[0158] In some embodiments, the disclosure relates to kits comprising a composition or fusion protein as described herein and instructions for performing a method as described herein.

[0159] For example, the kit can include a pharmaceutical composition as described herein; and instructions for performing a method of treating a disease or condition characterized by inflammation, the method comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition.

[0160] The instructions may be packaged in the kit in the form of printed instructions, a printed document providing a uniform resource locator (URL), a printed document providing a quick response (QR) code, etc., from which detailed instructions can be browsed or downloaded, and the QR code can be scanned to direct the browser of a smart phone or tablet computer to the URL.

[0161] The test kit typically includes one or more vessels or containers so that some or all of the individual components and reagents, such as pharmaceutical compositions, can be separately contained. The test kit may also include a device for enclosing the individual containers in a relatively closed space for commercial sale, such as a plastic box, in which instructions, packaging materials such as polystyrene foam, etc. may be loaded. Identifiers, such as bar codes, radio frequency identification (ID) tags, etc., may be present in or on the test kit, or in one or more vessels or containers included in the test kit. The identifier may be used, for example, to uniquely identify the test kit for purposes such as quality control, inventory control, tracking, movement between workstations, etc.

[0162] B. Sequence

[0163]

[0164]

[0165] Equivalent

[0166] It will be apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the invention described herein are apparent and can be made using suitable equivalents without departing from the scope of the present disclosure or embodiments. Having now described certain compositions and methods in detail, they will be more clearly understood by reference to the following examples, which are intended to be illustrative only and are not intended to be limiting.

[0167] Example

[0168] The following are examples of methods and compositions of the present invention.It is understood that various other embodiments may be practiced, given the general description provided herein.

[0169]

[0170]

[0171] Table 1

[0172] Example 1: Effect of N60D mutation on activity

[0173] 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 studied. Specifically, the fusion protein was synthesized as a single polypeptide by recombinant cells, purified, and the samples were evaluated for IL-2 activity using a known assay, wherein the activity was measured by OD 620 nm IL-2 alone was used as a positive control. IL-33 alone was used as a negative control.

[0174] Figure 3 Results are shown. The fusion protein containing the N60D substitution in the truncated IL-33 domain retained essentially the same IL-2 activity as IL-2 alone.

[0175] Example 2: Effect of C97G mutation on activity

[0176] 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 studied. Specifically, the fusion protein was synthesized as a single polypeptide by recombinant cells, purified, and the samples were evaluated for IL-2 activity or IL-33 activity using known assays, wherein the activity was measured by OD 620 nmMeasurement. 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.

[0177] Figure 4 Results of IL-2 activity assays are shown. The fusion protein containing the C97G point substitution in the truncated IL-33 domain retained essentially the same IL-2 activity as IL-2 alone.

[0178] Figure 5 Results of IL-33 activity assays are shown. Fusion proteins containing a C97G point substitution in the truncated IL-33 domain showed IL-33 activity comparable to that of IL-33 alone.

[0179] Example 3: Effect of C116F mutation on expression, activity and stability

[0180] The effect 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 an IL-2 domain and a truncated IL-33 domain was studied. Specifically, the fusion protein was synthesized as a single polypeptide by recombinant cells, purified, and the samples were evaluated for IL-2 activity and IL-33 activity after storage at 4°C, 25°C or 37°C for 0 days, 3 days or 7 days. IL-2 and IL-33 activities were measured as described above.

[0181] Figure 6 It shows that the fusion protein containing the C116F substitution in the truncated IL-33 domain is well expressed in recombinant cells. The thick bands in the middle and right columns correspond to the molecular weight of the fusion protein.

[0182] Figure 7 It was shown that the fusion protein containing the C116F substitution in the truncated IL-33 domain was easily purified by standard chromatographic techniques.

[0183] Figure 8 It was shown that the fusion protein containing the C116F substitution in the truncated IL-33 domain retained essentially the same IL-2 activity as IL-2 alone.

[0184] Fig. 9 It was shown that the fusion protein containing the C116F substitution in the truncated IL-33 domain retained essentially the same IL-33 activity as IL-33 alone.

[0185] Fig.10It was shown that the fusion protein containing the C116F substitution in the truncated IL-33 domain retained at least about 90% or more of its original IL-33 activity, even after storage at 37°C for 7 days.

[0186] Example 4: Activity of multiple engineered constructs

[0187] SLT-518 IL2N88D IL2 C125S IL33 N60S IL33 C116F IL33 C97G h1(SLT1) h2(SLT2) X h3(SLT3) X X h4(SLT4) X X h5(SLT5) X X h6(SLT6) X X X X h7(SLT7) X X X h8*(SLT8) X X X X h9(SLT9) X X X

[0188] Table 2

[0189] Nine engineered constructs were prepared with various mutations in IL2 and / or IL33 (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). Details of the mutations included in each construct are provided in Table 2 above. Rationally designed variants were generated to optimize expression, activity, and stability while retaining the wild-type sequence to limit the risk of immunogenicity.

[0190] In Figure 35, the IL2 and IL33 activities of six engineered variants (SLT1, SLT2, SLT3, SLT4, SLT5, SLT6) were measured. Figure 11-14 In the present invention, the SLT-518 construct is referred to as 7429-(variant #), corresponding to SLT-518(h1), SLT-518(h2), SLT-518(h3), SLT-518(h4), SLT-518(h5) or SLT-518(h6). Compared with SLT3, SLT4, SLT5 and SLT6, SLT1 and SLT2 have minimal IL33 activity.

[0191] SLT3 (IL233 fusion protein containing IL2(N88D) and IL33(N60S) mutations) and SLT4 (IL233 fusion protein containing IL2(N88D) and IL33(C116F) mutations) were compared with SLT1 (wild-type fusion protein IL233) and SLT2 (IL233 fusion protein containing IL2(N88D) mutation) ( Fig.12 ).like Fig.11 As can be seen, SLT1 and SLT2 IL33 activity was barely detectable. SLT3 and SLT4 had significantly higher IL33 activity than SLT1 and SLT2.

[0192] SLT5 (IL233 fusion protein containing IL2(N88D) and IL33(C97G) mutations) and SLT6 (IL233 fusion protein containing IL2(N88D) and IL33(N60S, C97G, C116F) mutations) were compared with SLT1 (wild-type fusion protein IL233) and SLT2 (IL233 fusion protein containing IL2(N88D) mutation) ( Fig.13 ). As seen above, SLT1 and SLT2 have the least IL33 activity. SLT5 and SLT6 have significantly higher IL33 activity than SLT1 and SLT2.

[0193] SLT4 (IL233 fusion protein containing IL2(N88D) and IL33(C116F) mutations) was compared with SLT1 and SLT2 ( Fig.14 ). SLT4 achieved approximately the same level of IL2 activity as wild-type IL2, and also achieved approximately the same level of IL33 activity as wild-type IL33.

[0194] Example 5: Tissue Targeting and ST2+ Treg Cells

[0195] Healthy mice were treated with 66 pmol of IL233 fusion protein (SLT-518(h4)) daily for five days. Tissues were harvested on day 8. Average ST2 expression in Tregs increased. The total number of ST2+Tregs in the spleen increased. The proportion of ST2+ cells in the Treg population increased. Therefore, in the tissue microenvironment, SLT-518(h4) upregulated the highly suppressive ST2+Tregs ( Fig.16 ).

[0196] In the ischemic renal injury model, SLT-518(h4) stimulated the active mobilization of Tregs to the injured tissue through IL-33-dependent signals in the tissue microenvironment.

[0197] In summary, we show that SLT518(h4) drives the expression of ST2+ Treg cells in healthy mice. SLT518(h4) can accelerate the mobilization of ST2+ Treg cells to sites of injury, implying that systemic administration of this therapeutic agent can drive local effects. Tregs from SLT518(h4) more effectively suppress T cell proliferation in vitro and disease in vivo on a per-cell basis.

[0198] The data presented here for SLT518(h4) show that the newly identified mutations improve the function and stability of the molecule compared to the previously published mouse protein.

[0199] Example 6: Tissue Microenvironment and Persistence

[0200] Sustained immune responses require more than just Tregs. Ignoring other cells in the tissue microenvironment results in temporary, less potent effects and does not address the underlying cause of the disease. Previous studies have shown that low-dose IL-2 targets primarily Tregs, which can only produce a temporary response. However, low-dose IL-2+IL33 targets multiple cell types, including tolerogenic dendritic cells (tolDCs) that produce IL-2 ( Fig.17 ). Therefore, IL233 fusion cytokines restore the tissue microenvironment to establish a long-term tolerance and repair state. The effect of the restored microenvironment is a lasting effect and does not require additional treatment input.

[0201] Example 7: Cynomolgus Monkey Study

[0202] Healthy nonhuman primate studies were conducted at WuXi AppTec (Shanghai, China) using SLT-518(h4) derived from a CHO production process developed by WuXi Biologics (Shanghai, China). All comparisons of SLT-518(h4) and IL-2 were done at equimolar doses five times daily to match the benchmark aldesleukin (Proleukin).

[0203] The comprehensive study in cynomolgus monkeys evaluated more than 35 individual animals with increasing doses. Systemic pharmacodynamics (as Treg and Treg subsets), local pharmacodynamics (as Treg and Treg subsets in lymph nodes, tissues TBD), tolerability, safety and selectivity index (as Treg / Teff subsets), pharmacokinetics (single and repeated doses) were evaluated. Different routes of administration were tested, as well as multiple control products.

[0204] Treatment with SLT-518(h4) promoted a significant expansion of ST2+ Treg cells in healthy nonhuman primates ( Fig.18 ). Two monkeys were administered SLT518 (h4), and two monkeys were administered IL-2 alone for 21 days. The percentage of ST2 + Tregs was measured, and on day 6, more than 3000 times more ST2 + Tregs were measured in monkeys administered the engineered IL233 fusion protein (SLT-518 (h4)).

[0205] SLT-518(h4) also induced superior expansion of ST2+ Tregs, thereby providing a wider therapeutic range relative to clinical doses of IL-2 ( Fig.21A and 21B Monkeys were given three doses of SLT-518(h4) ( Fig.21A ), and monkeys were given three doses of IL-2 alone ( Fig.21B ). Measure and track the percentage of Treg subsets according to target clinical ranges.

[0206] SLT-518(h4) also showed a significant expansion of ST2+ Treg in the blood and remained elevated in tissues for more than 28 days, far exceeding the levels observed in the blood ( Fig.19 ).

[0207] In conclusion, the C116F mutation generated a humanized form of IL233 (SLT518(h4)) that was more active than the wild type, as shown by comparative data in which SLT518(h1) and SLT518(h2) were compared with SLT518(h4) in in vitro and non-human primate studies.

[0208] Example 8: Non-human primate Tregs

[0209] Nonhuman primates were given 100ug / kg daily doses of SLT1 (wild-type fusion protein IL233), SLT2 (IL233 fusion protein containing IL2(N88D) mutation), or SLT4 (IL233 fusion protein containing IL2(N88D) and IL33(C116F) mutations) for five days. The percentage of Tregs was measured for 21 days. Fig. 20 It is shown that SLT4 produced a significant increase in Treg percentage and fold change at day 0, peaking at day 6 in non-human primates.

[0210] Similar to the mouse data shown above, SLT518(h4) (in Fig. 20 Indicated as SLT4 in (indicated as SLT4) showed driving the expression of ST2+Treg cells in non-human primates, and performed significantly better than SLT518 (h1) (indicated as SLT1) and SLT518 (h2) (indicated as SLT2). This data indicates that the key mutation for increased expression of ST2+Treg cells is attributed to the IL33C116F mutation.

Claims

1. 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.

2. A composition comprising: 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.

3. 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.

4. A fusion protein comprising: 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.

5. The composition of claim 1 or the fusion protein of claim 3, 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.

6. The composition according to claim 1 or the fusion protein according to claim 3 or 5, wherein N60 of SEQ ID NO: 1 is substituted by Ser or Asp, C97 of SEQ ID NO: 1 is substituted by Gly, or C116 of SEQ ID NO: 1 is substituted by Phe.

7. The composition of claim 1 or the fusion protein of any one of claims 3 to 6, 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. The fusion protein according to any one of claims 3 to 7 , further comprising a linker.

9. The fusion protein of claim 8, wherein the linker comprises GGGGS (SEQ ID NO: 9). 10 . The fusion protein according to claim 8 , wherein the linker has the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 10).

11. The composition of claim 1 or the fusion protein of any one of claims 3 to 10, 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.

12. The fusion protein according to any one of claims 3 to 11, further comprising a signal peptide. The fusion protein according to claim 12 , wherein the signal peptide has the sequence of SEQ ID NO:

11.

14. A fusion protein according to any one of claims 3 to 13, 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.

15. A pharmaceutical composition comprising the composition according to claim 1 or 2 or the fusion protein according to any one of claims 3 to 14.

16. The pharmaceutical composition of claim 15, further comprising a pharmaceutically acceptable carrier and optionally an additional therapeutic agent.

17. A polynucleotide comprising a nucleic acid sequence encoding at least a portion of the fusion protein according to any one of claims 3 to 14.

18. A vector comprising the polynucleotide according to claim 17, wherein the polynucleotide is operably linked to a promoter.

19. A recombinant host cell comprising the polynucleotide according to claim 17 or the vector according to claim 18.

20. A method for treating a disease or condition, the method comprising: An effective amount of the pharmaceutical composition according to claim 15 or 16 is administered to a subject in need thereof.

21. The method of claim 20, wherein the disease or condition is selected from the group consisting of 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), inflammatory demyelination sheath disease, inflammatory neuropathy, insulitis, interstitial cystitis, juvenile idiopathic arthritis, lupus, 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, transplant rejection, type 1 diabetes mellitus, type 2 diabetes mellitus, ulcerative colitis, vasculitis, and Wegener's granulomatosis.

22. The method of claim 20, wherein the disease or condition is cancer.

23. The method of claim 22, wherein the cancer is selected from the group consisting of acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute myeloid leukemia, adrenocortical carcinoma, anal cancer, adnexal cancer, astrocytoma, basal cell carcinoma, B-cell lymphoma, brain cancer, biliary tract cancer, bladder cancer, bone cancer, breast cancer, bronchial tumors, cancer of unknown primary cause, heart tumor, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, colorectal cancer, Intestinal cancer, Craniopharyngioma, Ductal carcinoma, Embryonal tumor, Endometrial cancer, Ependymoma, Esophageal cancer, Nasal glioma, Fibrous histiocytoma, Ewing's 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 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 in situ cancer, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, occult primary metastatic squamous neck cancer, midline airway cancer involving the NUT gene, oral cancer, multiple endocrine neoplasms syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, 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, Sezary 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, laryngeal cancer, thymoma and thymic cancer, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer and Wilms' tumor.

24. The method of claim 23, wherein the cancer is selected from the group consisting of melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, stomach 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.

25. The method according to any one of claims 20 to 24, wherein the method stimulates proliferation and / or activation of T regulatory (Treg) cells, T helper 2 (Th2) cells, innate lymphoid cells (ILCs), cytotoxic T cells, natural killer (NK) cells, NK-T cells, ST2+ cells, dendritic cells and / or macrophages.

26. The method of any one of claims 20 to 25, wherein the method promotes anti-inflammatory M2 macrophages and inhibits pro-inflammatory M1 macrophages.

27. The method of claim 26, wherein the M2 macrophages inhibit inflammation in a tissue or organ selected from the group consisting of: pancreas, liver, kidney, adipose tissue, salivary gland, central nervous system (CNS), and related organs.

28. The method according to any one of claims 20 to 24, wherein the method is capable of upregulating or downregulating 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, wherein the antigen presenting cells include but are not limited to dendritic cells, macrophages, B cells, innate lymphocytes, NK cells, epithelial cells, endothelial cells or stromal cells, depending on the IL-2 domain.

29. The method according to any one of claims 20 to 27, further comprising: An effective amount of a second therapy for the disease or condition is administered to the subject, wherein the second therapy does not include the composition of claim 1 or 2 or the fusion protein of any one of claims 3 to 14.

30. The method of claim 29, wherein the second therapy is cell therapy or gene therapy.

31. A method for stimulating proliferation and / or activation of T regulatory (Treg) cells in a subject, the method comprising: isolating T cells from the subject; as well as The separated T cells are mixed with an effective amount of the following touch: The composition of claim 1; the fusion protein of any one of claims 3 to 14; a cell expressing at least one of IL-2 or truncated IL-33; or the recombinant host cell of claim 19.

32. A kit comprising: The pharmaceutical composition according to claim 15 or 16; and Instructions for performing a method for treating a disease or condition characterized by inflammation, the method comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition.

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