WO2024145365A2 - Compositions for the diagnosis, treatment, prevention, and alleviation of neurodegenerative and autoimmune disorders - Google Patents

Compositions for the diagnosis, treatment, prevention, and alleviation of neurodegenerative and autoimmune disorders Download PDF

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WO2024145365A2
WO2024145365A2 PCT/US2023/086047 US2023086047W WO2024145365A2 WO 2024145365 A2 WO2024145365 A2 WO 2024145365A2 US 2023086047 W US2023086047 W US 2023086047W WO 2024145365 A2 WO2024145365 A2 WO 2024145365A2
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fragment
homolog
variant
seq
linker
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WO2024145365A3 (en
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Jack B. Cowie III
Timothy Riley
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Arete Discoveries, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Interleukin-33 is a tissue-derived nuclear cytokine discovered in 2005 that belongs to the IL-1 family and has roles in both homeostasis and inflammation (1).
  • Original studies defined IL-33 as a so-called “alarmin” protein released by damaged endothelial and epithelial cells that initially alerts the immune system of “danger”. Yet subsequent work has shown that it is expressed by a much wider variety of cells including glial cells of the nervous system, cardiomyocytes, osteoclasts, and several immune cell types including activated T cells, particularly Th2 cells, mast cells, basophils, macrophages, and dendritic cells.
  • IL-33 exerts pleiotropic effects on both immune and non-immune cells and is generally considered to have anti-inflammatory /protective activities. These functions arc a result of the strong ability of IL-33 to activate ILC2s, which in turn produce cytokines such as IL-5 and IL-13 that drive Th2 responses. Amphiregulin, also produced by IL-33-activated ILC2s promotes wound healing (6) and Th2 responses can block the pathogenic autoimmune Thl/Thl7 responses characteristic of Multiple Sclerosis (7).
  • IL-33 activates a population of ST2+ T regulatory cells that limit damaging effector CD4+ and CD8 cytotoxic T cell responses as shown in models of colitis, lung injury and Type 1 diabetes (8, 9, 10, 11).
  • IL-33 treatment ameliorates ongoing disease.
  • Studies in models of stroke, spinal cord injury and Alzheimer’s disease demonstrate that IL-33 reduces CNS tissue loss, reverses astrogliosis and demyelination, and improves cognition (3, 12, 13, 14, 15, 16).
  • IL- 33 also has a direct effect on oligodendrocyte gene expression and induces p38MAPK phosphorylation these cells, an event linked to myelination (12).
  • IL-33 therapy has the potential to prevent disease progression by blocking damaging inflammation as well as promoting myelin and neuronal repair in MS and other diseases of the CNS. While IL-33 drives Th2-mediated protection against bacterial infections such as Clostridium difficile as well as parasitic infections, it can also exacerbate allergic inflammation (17,18).
  • cytokines that have anti-inflammatory properties. Such cytokines used in combination may have the potential for downregulating the pathologic T cell and innate immune cell responses in chronic autoimmune, neurologic, and inflammatory diseases.
  • Antiinflammatory cytokine IL-37 has been used in some embodiments in combination with IL-33.
  • IL-37 like IL-33 is a member of IL-1 family cytokine.
  • IL-37 is expressed at low levels in a wide variety of tissues such as thymus, lung, colon, uterus, and bone marrow and is induced in inflammatory conditions (19).
  • IL-37 exists as a dimer and monomer form, with the monomer having potent anti-inflammatory activity.
  • This cytokine binds to two receptors that are expressed on a broad range of immune and non-immune cells: IL-18R, where it can block the proinflammatory actions of IL- 18, and IL-1R8 receptor (SIGRR).
  • IL-37 can also function as transcription factor, when and function via Smad3 dependent mechanism (20).
  • IL-37 broadly suppresses both innate and adaptive immune response (21, 22, 23).
  • IL-37 suppresses the secretion of pro-inflammatory cytokines such as TNF-a, IL-la, IL-ip, IL-6, G-CSF and GM-CSF from human blood LPS- stimulated monocytes as well as multiple chemokines (24).
  • IL-37 has been shown to suppress MHC and costimulatory molecule expression as well as the antigen presenting cell (APC) function, which reduces the ability of these cells to activate naive T cells, thereby impairing the effector T cell response and adaptive immunity (25, 26).
  • APC antigen presenting cell
  • IL-37 blocks Th2 differentiation and the subsequent recruitment of eosinophils to the airways in allergic asthma thereby preventing bronchial obstruction and mucus production characteristic of this disease. Also of relevance is the ability of IL-37 to suppress inflammatory actions in the Central Nervous System.
  • IL-37 inhibited infiltration of pro-inflammatory cells to sites of ischemic injury, blocked the activation of microglia, reduced infarct volume and significantly limited stroke induced mobility deficits (29).
  • IL- 37 is a potential therapeutic for the treatment of aortic valve disease, inflammatory arthritis, colitis, metabolic syndrome, aging and cancer genesis (19). When used together, these cytokines in combination offer immense immune modulatory possibilities for many diseases with inflammatory underpinnings.
  • the therapeutic proteins or cytokines can be delivered via a vehicle or payload directly to the desired target.
  • various delivery agents nanoparticles or liposome- mediated targeted delivery technologies have become popular.
  • Multiple studies have demonstrated that the delivery of proinflammatory cytokines such as IL-2, IL- 12 or TNF using nanoparticles with or without targeting moieties such as antibodies is possible (30, 31, 32, 33).
  • These types of cytokine nanoconjugates/immunoliposomes have the advantage of delivering the payload directly to the targets when used in association with antibodies directed against the target cell or tissues. Furthermore, these strategies have been proven to have less systemic toxicity (34).
  • the linker is a peptide linker, for example SEQ ID NO: 11 or a multiple thereof.
  • the second domain comprises a fragment of IL-37.
  • the fusion protein further comprises a third domain, optionally comprising a linker.
  • Also provided herein is a host cell comprising a nucleic acid molecule or a vector as disclosed herein.
  • linked is associated with the terms linked, linker, coupled, releasable, cleavable, water-soluble polymers, Fc fusion proteins, Fc albumin fusion proteins, a peptide sequence, a peptidase with a cleaving site, a bifunctional linker, and a polyglycine linker.
  • the term “linked” requires a covalent bond between the two proteins or polypeptides.
  • the term “linked” also includes the circumstances where two or more proteins may not be physically linked but nonetheless behave as if they are linked, for example, where two or more distinct proteins each use an Fc fusion protein and/or Fc albumen fusion protein that effectively links the two or more proteins.
  • two or more “linked” proteins may be introduced into a mammal or a human for a therapeutic treatment. Upon introduction into the body these proteins may dissociate completely or partially into their primary component parts once they enter the body to provide a full therapeutic benefit.
  • the mechanism of action in each scenario and under varying conditions could include, by way of example only, thermal, pH, chemical (blood), mechanical, electricity and so on.
  • drug delivery technologies will be affected by the therapeutics physical structure and properties.
  • IL-37 refers to any mammalian IL-37, such as human IL-37, mouse IL-37, or an active species or allelic variant, fragment, isoform or derivative thereof.
  • IL-37 refers to the IL-37 cytokine which is a member of the IL- 1 family and has an anti-inflammatory role.
  • a gene may thus comprise several operably linked sequences, such as a promoter, a 5' leader sequence comprising e.g., sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA), introns, and a 3' nontranslated sequence comprising e.g., transcription termination sites.
  • a promoter e.g., a promoter, a 5' leader sequence comprising e.g., sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA), introns, and a 3' nontranslated sequence comprising e.g., transcription termination sites.
  • a “3' UTR” or “3' non-translated sequence” refers to the nucleic acid sequence found downstream of the coding sequence of a gene, which comprises for example a transcription termination site and (in most, but not all eukaryotic mRNAs) a polyadenylation signal (such as e.g., AAUAAA or variants thereof).
  • a polyadenylation signal such as e.g., AAUAAA or variants thereof.
  • the mRNA transcript may be cleaved downstream of the polyadenylation signal and a poly(A) tail may be added, which is involved in the transport of the mRNA to the cytoplasm (where translation takes place).
  • RNA which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide (or active peptide fragment).
  • “Expression of a polypeptide” additionally refers to a process wherein an mRNA is translated into a protein product, which may or may not be secreted.
  • a “transcription regulatory sequence” is herein defined as a nucleic acid sequence that is capable of regulating the rate of transcription of a (coding) sequence operably linked to the transcription regulatory sequence.
  • a transcription regulatory sequence as herein defined will thus comprise all sequence elements necessary for initiation of transcription (promoter elements), for maintaining and for regulating transcription, including e.g. attenuators or enhancers.
  • promoter refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of ordinary skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter.
  • a “constitutive” promoter is a promoter that is active in most tissues under most physiological and developmental conditions.
  • An “inducible” promoter is a promoter that is physiologically (e.g. by external application of certain compounds) or developmentally regulated.
  • a “tissue specific” promoter is only active in specific types of tissues or cells.
  • sequence identity and “sequence similarity” can be determined by alignment of two peptides or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as “substantially identical” or “essentially similar” when they (when optimally aligned by for example the programs GAP or BEST FIT using default parameters) share at least a certain minimal percentage of sequence identity (as defined below). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps.
  • nucleic acid sequences and amino acid sequences corresponding to the fusion proteins disclosed herein are also provided herein. Certain fusion proteins disclosed herein display IL-33 activity. Certain fusion proteins disclosed herein display IL-37 activity.
  • a fusion protein comprising IL-33 or a variant or fragment or homolog thereof and IL-37 or a variant or fragment or homolog thereof is provided.
  • the fusion protein can comprise an IL-33 protein, or a variant or fragment or homolog thereof.
  • the IL-33 protein is preferably a mammalian IL-33 protein, such as a human IL-33, or mouse IL-33.
  • An amino acid sequence of IL-33 is set forth in SEQ ID NO: 1.
  • Variants of IL-33 include, for example, proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more, such as 100%, amino acid sequence identity to SEQ ID NO: 1, preferably over the entire length. Amino acid sequence identity is preferably determined by pairwise alignment using the Needleman and Wunsch algorithm and GAP default parameters as defined above. Variants also include proteins having IL-33 activity, which have been derived, by way of one or more amino acid substitutions, deletions or insertions, from the polypeptide having the amino acid sequence of SEQ ID NO: 1. Preferably, such proteins comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 amino acid substitutions, deletions or insertions.
  • the fusion protein can further comprise an IL-37 protein, or a variant or fragment or homolog thereof.
  • the IL-37 protein is preferably a mammalian IL-37 protein, such as a human IL-37, or mouse IL-37.
  • An amino acid sequence representing IL-37 is set forth in SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.
  • Variants of IL-37 include, for example, proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more, such as 100%, amino acid sequence identity to SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, preferably over the entire length. Amino acid sequence identity is preferably determined by pairwise alignment using the Nccdlcman and Wunsch algorithm and GAP default parameters as defined above.
  • Variants also include proteins having IL-37 activity, which have been derived, by way of one or more amino acid substitutions, deletions or insertions, from the polypeptide having the amino acid sequence of SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.
  • proteins comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, amino acid substitutions, deletions or insertions.
  • the IL-33 and IL-37 moieties in a fusion protein disclosed herein may or may not be connected by a linker. Additional amino acid sequences may be present at the N- and/or C- terminus of the fusion protein, e.g., to facilitate purification. For example, a histidine-tag may be present at the C- or N-terminus to facilitate purification.
  • a fusion protein disclosed herein may optionally comprise additional protein moieties, such as moieties capable of targeting, e.g., a protein moiety comprising one or more antibody Fe regions.
  • the IL-33 moiety may be located at the N-terminal of the IL-37 moiety, or may be located at the C-terminal of the IL-37 moiety. In a preferred embodiment, the IL-33 moiety is located at the N-terminal of the IL-37 moiety. It was found that the latter fusion protein displayed higher specific activity compared to an IL-33-IL-37 fusion protein in which the IL-33 moiety was located at the C-terminal of the IL-37 moiety (data not shown).
  • the IL-33-IL-37 fusion protein may be present in its monomeric form, in which case it has a formula weight of about 35 kDa, or it may be a dimeric IL-33-IL-37 fusion protein, i.e., two IL-33-IL-37 fusion proteins may be associated with one another non-covalently, in which case it has a formula weight of about 70 kDa.
  • a fusion protein disclosed herein consists essentially of IL-33 and IL-37, optionally linked by a linker.
  • both the IL-33 and the IL-37 moieties within certain fusion proteins disclosed herein are active and able to signal cells to downregulate the production of at least one inflammatory cytokine or mediator such as IL-1, IL-6, IL-8, TNFa.
  • at least TNFa, IL-6, and IL-8 are downregulated.
  • the fusion protein inhibits the generation of cytokines such as TNFa, IL-1, IL-6, IL-8 and other inflammatory cytokines while they induce secretion of inhibitory molecules such as IL-1 receptor antagonist and soluble TNF receptors by inflammatory and other cells stimulated by endotoxin, other Toll-like receptor (TLR) agonists, or other stimuli.
  • cytokines such as TNFa, IL-1, IL-6, IL-8 and other inflammatory cytokines while they induce secretion of inhibitory molecules such as IL-1 receptor antagonist and soluble TNF receptors by inflammatory and other cells stimulated by endotoxin, other Toll-like receptor (TLR) agonists, or other stimuli.
  • TLR Toll-like receptor
  • Certain fusion proteins disclosed herein inhibit the expression of adhesion molecules by inflammatory, endothelial, and other cells stimulated by agonists including endotoxin, other TLR agonists, and others.
  • the fusion protein inhibits the expression of tissue factor by endothelial, inflammatory, and other cells stimulated by endotoxin, other TLR agonists, or other stimuli.
  • Certain fusion proteins disclosed herein inhibit the generation of oxygen radicals by inflammatory and other cells stimulated by endotoxin, other TLR agonists, or other stimuli.
  • the fusion protein inhibits activity of IFN-y- and IL-17-secreting Thl and Th-17 cells and induce or sustain FoxP3-expressing suppressive T cells (CD25+), TGF- P-secreting Th2, Tri and Th3 cells generated in vitro in the presence or absence of antigen- presenting cells stimulated by self or non-self antigens, superantigens including Staphylococcus enterotoxin B (SEB) or mitogens such including CD3, CD28, phytohemagglutinin (PHA) or phorbol myristate acetate (PM A).
  • SEB Staphylococcus enterotoxin B
  • PHA phytohemagglutinin
  • PM A phorbol myristate acetate
  • a fusion protein disclosed herein is present in a homodimeric form. In an embodiment, a fusion protein disclosed herein is present in a heterodimeric form.
  • a fusion protein disclosed herein has a molecular weight of above 60 kDa.
  • Certain fusion proteins disclosed herein may be prepared by techniques known to the skilled person in the art. For example, certain fusion proteins may be prepared using a technique which produces recombinant fusion proteins by continuous cell lines in culture. For example, certain fusion proteins can be produced in a host cell transfectoma using a combination of recombinant DNA techniques and gene transfection methods.
  • nucleic acid sequences that encodes for a fusion protein disclosed herein, such as cDNA, genomic DNA, and RNA sequences. Due to the degeneracy of the genetic code, multiple nucleic acid sequences may encode the same amino acid sequence. Nucleic acid sequences encoding a fusion protein disclosed herein are herein referred to as “1L-33-1L-37 fusion protein encoding nucleic acid sequences”. The nucleic acid sequences provided include recombinant, artificial or synthetic nucleic acid sequences.
  • RNA sequences are depicted as DNA sequences while RNA is referred to, the actual base sequence of the RNA molecule is identical with the difference that thymine (T) is replaced by uracil (U).
  • T thymine
  • U uracil
  • Certain nucleic acid sequences disclosed herein are particularly useful for expression of a IL-33-IL-37 fusion protein disclosed herein, either for production of the protein or for gene therapy purposes.
  • a nucleic acid molecule whose sequence encodes the fusion protein can be prepared by standard molecular’ biology techniques.
  • the nucleic acid molecule is preferably operably linked to transcription regulatory sequences such as a promoter, and optionally a 3' untranslated region.
  • the nucleic acid molecule may be inserted into a vector, such as an expression vector, such that the genes are operatively linked to transcriptional and translational control sequences.
  • the expression vector and transcription regulatory sequences are selected to be compatible with the expression host cell used.
  • the nucleic acid molecule may be inserted into the expression vector by methods known in the art.
  • the nucleic acid molecule or vector may further include a nucleotide sequence encoding a signal peptide, which may facilitate secretion of the fusion protein from the host cell.
  • a nucleotide sequence encoding a signal peptide may be operably linked to the nucleic acid molecule encoding the fusion protein.
  • the signal peptide is located at the amino terminus of the fusion protein and as such, the nucleotide sequence encoding the signal peptide may be located 5' of the nucleic acid molecule encoding the fusion protein.
  • the signal peptide may be a cytokine signal peptide or a signal peptide from a noncytokine protein.
  • the promoter may be constitutive or inducible.
  • the vector may comprise a selectable marker for selection of a vector carrying host cell.
  • the vector may comprise an origin of replication when the vector is a replicable vector.
  • Certain fusion proteins disclosed herein may be synthesized de novo by chemical synthesis (using e.g. a peptide synthesizer such as supplied by Applied Biosystems), or may be produced by recombinant host cells by expressing a nucleic acid sequence encoding the fusion protein, fragment or variant.
  • Variants and fragments are preferably functional, i.e., have IL-33 and/or IL-37 activity, preferably IL-33 and IL-37 activity.
  • Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, ⁇ e.g., the adenovirus major late promoter (AdMLP)) and polyoma.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdMLP adenovirus major late promoter
  • nonviral regulatory sequences may be used, such as the ubiquitin promoter.
  • certain recombinant expression vectors disclosed herein may carry additional sequences, such as sequences that regulate replication of the vector in host cells ⁇ e.g., origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. No. 4,399,216, U.S. Pat. No. 4,634,665 and U.S. Pat. No. 5,179,017, all to Axel et al.).
  • the eukaryotic cells are mammalian host cells.
  • Preferred mammalian host cells for expressing recombinant IL-33-IL-37 fusion proteins include CHO cells (including dhfr-CHO cells, described in (Urlaub et al., 1980), used with a DHFR selectable marker, NS/0 myeloma cells, COS cells, HEK293 cells and SP2.0 cells.
  • nucleic acid molecule whose sequence encodes a fusion protein disclosed herein can be expressed in other expression systems, including prokaryotic cells, such as microorganisms, e.g. E. coli, algae, as well as insect cells.
  • prokaryotic cells such as microorganisms, e.g. E. coli, algae, as well as insect cells.
  • certain fusion proteins disclosed herein can be produced in transgenic non-human animals, such as in milk from sheep and rabbits or eggs from hens, or in transgenic plants.
  • nucleic acid sequences into a host cell may be carried out by any standard technique known in the art.
  • the expression vector(s) encoding the fusion protein may be transfected into a host cell by standard techniques.
  • the various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium phosphate precipitation, DEAE-dextran transfection, lipofectamine transfection, and freeze-dry method transfection, and the like.
  • Cell lines that secrete certain fusion proteins disclosed herein can be identified by assaying culture supernatants for the presence of the fusion protein.
  • the preferred screening procedure comprises two sequential steps, the first being identification of cell lines that secrete the fusion protein, the second being determination of the quality of the fusion protein such as the ability of the fusion protein to inhibit cytokine production by blood cells stimulated with LPS or other Toll-like receptor agonists, glycosylation patterns, and others.
  • a fusion protein encoding nucleic acid sequence can be codon optimized by adapting the codon usage to that most preferred in host cell genes.
  • codon usage Several techniques for modifying the codon usage to that preferred by the host cells are known in the art. The exact method of codon usage modification is not critical.
  • PCR primers and/or probes and kits for detecting the fusion protein encoding DNA or RNA sequences.
  • Degenerate or specific PCR primer pairs to amplify fusion protein encoding DNA from samples can be synthesized (see Dieffenbach and Dveksler (1995) PCR Primer; A Laboratory Manual, Cold Spring Harbor Laboratory Press, and McPherson at al. (2000) PCR-Basics: From Background to Bench, First Edition, Springer Verlag, Germany).
  • any stretch of 9, 10, 11, 12, 13, 14, 15, 16, 18 or more contiguous nucleotides of a fusion protein encoding nucleic acid sequence, or its complement strand may be used as primer or probe.
  • DNA fragments of a fusion protein encoding nucleic acid sequence can be used as hybridization probes.
  • a detection kit for a fusion protein encoding nucleic acid sequence may comprise primers specific for a fusion protein encoding nucleic acid sequence and/or probes specific for a fusion protein encoding nucleic acid sequence, and an associated protocol to use the primers or probes to detect specifically a fusion protein encoding nucleic acid sequence in a sample.
  • Such a detection kit may, for example, be used to determine whether a host cell has been transformed with a specific fusion protein encoding nucleic acid sequence. Because of the degeneracy of the genetic code, some amino acid codons can be replaced by others without changing the amino acid sequence of the protein.
  • a person skilled in the art will be capable of selecting conditions known in the art for production of a fusion protein as disclosed herein. Additionally, a person skilled in the art will be capable of recovering the fusion protein produced using methods known in the ail, which include, without limitation, chromatographic methods (including, without limitation, size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, metal binding, and the like), immunoprecipitation, HPLC, ultracentrifugation, precipitation and differential solubilisation, and extraction. Recovery or purification of a fusion protein may be facilitated by adding, for example, a Histidine-tag to the fusion protein.
  • composition comprising: a fusion protein or a recombinant protein as disclosed herein, and a pharmaceutically acceptable carrier.
  • the term “pharmaceutically acceptable carrier” relates to carriers or excipients which are inherently nontoxic and nontherapeutic. Examples of such excipients are, but are not limited to, saline, Ringer's solution, dextrose solution and Hank's solution. Non-aqueous excipients such as fixed oils and ethyl oleate may also be used. A preferred excipient is 5% dextrose in saline. The excipient may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, including buffers and preservatives.
  • a “pharmaceutically acceptable carrier” can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption delaying agents, and the like that are physiologically compatible.
  • the pharmaceutical composition may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques (e.g., as described in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995).
  • the pharmaceutically acceptable carrier includes physiologically acceptable diluents, excipients, solvents, or adjuvants.
  • suitable carriers include, but are not limited to, water, normal saline, dextrose, mannitol, lactose or other sugars, lecithin, albumin, sodium glutamate, cysteine hydrochloride, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), vegetable oils (such as olive oil), injectable organic esters such as ethyl oleate, ethoxylated isosteraryl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methahydroxide, bentonite, kaolin, agar- agar and tragacanth, or mixtures of these substances, and the like.
  • compositions disclosed herein further comprise a pharmaceutically acceptable carrier including physiologically acceptable diluents, excipients, solvents, adjuvants or formulating agents that can be used to prepare the composition into desired administrable formulations such as solid, tablets, capsules, semi-solid, gel, and so on.
  • a pharmaceutically acceptable carrier including physiologically acceptable diluents, excipients, solvents, adjuvants or formulating agents that can be used to prepare the composition into desired administrable formulations such as solid, tablets, capsules, semi-solid, gel, and so on.
  • the pharmaceutical composition may be administered by any suitable routes and modes. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.
  • the pharmaceutical composition may be formulated in accordance with routine procedures for administration by any routes, such as oral, topical, parenteral, sublingual, transdermal, or by inhalation.
  • the pharmaceutical composition may be in the form of tablets, capsules, powders, granules, lozenges, creams, or liquid preparations, such as oral or sterile parenteral solutions or suspensions, or in the form of sprays, aerosols or other conventional methods for inhalation.
  • the pharmaceutical composition further comprises at least one biologically active peptide.
  • Certain fusion proteins disclosed herein may be prepared with carriers that can protect against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • a controlled release formulation including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, poly anhydrides, polyglycolic acid, collagen, polyorthoesters, and poly(lactic acid). Methods for the preparation of such formulations are generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • a fusion protein in a certain pharmaceutical composition as disclosed herein may be varied so as to obtain an amount of the fusion protein which is effective (“effective amount”) to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors, including the activity of the particular composition employed, the route of administration, the time of administration, the rate of excretion, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • At least one of the one or more additional anti-inflammatory agents is a steroidal chemical anti-inflammatory therapeutic agent.
  • the condition is characterized by pain and may be selected from inflammatory pain and neuropathic pain.
  • condition, disease, or disorder is an inflammatory disease mediated by the production of proinflammatory cytokines such as TNF, IL-1, IL-6, and/or other inflammatory mediators.
  • proinflammatory cytokines such as TNF, IL-1, IL-6, and/or other inflammatory mediators.
  • a fusion protein as disclosed herein, or a pharmaceutical composition thereof for use in the treatment or prevention of a clinical condition in a mammal, such as a human, for which administration of IL-37 is indicated.
  • the neurodegenerative disease is selected from the group consisting of Alexander's disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis, Ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, chronic fatigue syndrome, Chronic Traumatic Encephalopathy, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt- Jakob disease, frontotemporal dementia, Gerstmann-Straussler-Scheinker syndrome, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe's disease, Kuru, Lewy body dementia, Machado-Joseph disease (Spinocerebellar ataxia type 3), Multiple sclerosis, Multiple System Atrophy, myalgic encephalomyelitis, Narcolepsy, Neuroborreliosis, Parkinson'
  • the condition is selected from the group consisting of: sepsis, adult respiratory distress syndrome, allo- and xenotransplantation, dermatitis, inflammatory bowel disease, sarcoidosis, allergies, psoriasis, ankylosing spondylarthitis, autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, glomerolonephritis, immune complex- induced and other forms of vasculitis, multiple sclerosis, Sjogren’s disease, gout, lymphoproliferatieve diseases such as non-Hodgkin lymphoma and B cell chronic lymphocytic leukemia, bum injuries, multiple trauma, stroke, myocardial infarction, atherosclerosis, diabetes mellitus, extracorporeal dialysis and blood oxygenation, ischemia-reperfusion injury (IRI), toxicity induced by the in vivo administration of cytokines or therapeutic mono
  • the condition is characterized by pain and may be selected from inflammatory pain and neuropathic pain.
  • the condition is characterized by local or systemic inflammation, immune activation, and/or lymphoproliferation.
  • inhibition of the production of pro-inflammatory cytokines and other inflammatory mediators has a beneficial effect on the condition, disease, or disorder.
  • the condition, disease, or disorder is an inflammatory disease mediated by the production of proinflammatory cytokines such as TNF, IL- 1 , IL-6, and/or other inflammatory mediators.
  • administration of IL-33 is indicated for treatment or prevention of the condition, disease, or disorder.
  • administration of IL-37 is indicated for treatment or prevention of the condition, disease, or disorder.
  • Also provided herein is a method for the treatment or prevention of osteoarthritis or chronic pain, the method comprising the step of administration of an effective amount of a fusion protein as disclosed herein, or a pharmaceutical composition thereof, to a person in need thereof.
  • Also provided herein is a method for the treatment or prevention of osteoarthritis, the method comprising the step of administration of an effective amount of a fusion protein as disclosed herein, or a pharmaceutical composition thereof, to a person in need thereof.
  • Also provided herein is a method for the treatment or prevention of a neurodegenerative disease, the method comprising the step of administration of an effective amount of a fusion protein as disclosed herein, or a pharmaceutical composition thereof, to a person in need thereof.
  • the neurodegenerative disease is selected from the group consisting of Alexander's disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis, Ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, chronic fatigue syndrome, Chronic Traumatic Encephalopathy, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, frontotemporal dementia, Gerstmann-Straussler-Scheinker syndrome, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe's disease, Kuru, Lewy body dementia, Machado-Joseph disease (Spinocerebellar ataxia type 3), Multiple sclerosis, Multiple System Atrophy, myalgic encephalomyelitis, Narcolepsy, Neuroborreliosis, Parkinson'
  • Also provided is a method for the treatment of a condition, disease, or disorder comprising the steps of: administering a fusion protein as disclosed herein, and administering an additional pharmaceutical agent.
  • Treatment may consist of parenteral administration of a fusion protein disclosed herein, or a pharmaceutical composition thereof.
  • the fusion protein or pharmaceutical composition is administered intravenously, intramuscularly, intrathecally, epidurally, spinally, or subcutaneously.
  • other administration routes as set forth above with respect to pharmaceutical compositions comprising the fusion proteins recited above may also be employed.
  • the dose and administration regimen may depend on the extent of inhibition of the production and release of inflammatory cytokines aimed at.
  • the amount of the fusion protein given will be in the range of about 0.5 pg to about 1 mg per kg of body weight.
  • the dosage is from about 10 pg/kg to about 500 pg/kg body weight. In another aspect, the dosage is from about 20 pg/kg to about 100 pg/kg body weight. In a further aspect, the disage is from about 30 pg/kg to about 50 pg/kg body weight.
  • the dosage can be determined or adjusted by measuring the amount of circulating cytokine (IL-37, IL-33) upon administration in a biological sample.
  • compositions comprising fusion proteins disclosed herein [0247] Further administration details are set forth above in the section relating to pharmaceutical compositions comprising fusion proteins disclosed herein.
  • a method for attenuating an inflammatory reaction in a cell comprising the step of contacting the cell the cell with a fusion protein as disclosed herein, thereby inhibiting the release of cytokines and other inflammatory mediators by the cell.
  • the method is performed on a cell in vivo.
  • Also provided is a method for inhibiting the production and/or release of cytokines or other inflammatory mediators by a cell comprising the step of contacting the cell with a fusion protein as disclosed herein.
  • the cell is chosen from a macrophage, a monocyte, and a T- lymphocyte.
  • the gene therapy agent can be used for the treatment or prevention of an osteoarthritis or chronic pain.
  • the gene therapy agent can be used for the treatment or prevention of a condition characterized by local or systemic inflammation, immune activation, and/or lymphoproliferation, preferably wherein the condition characterized by local or systemic inflammation, immune activation, and/or lymphoproliferation is selected from the group consisting of: sepsis, adult respiratory distress syndrome, allo- and xenotransplantation, dermatitis, inflammatory bowel disease, sarcoidosis, allergies, psoriasis, ankylosing spondylarthitis, autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, glomerolonephritis, immune complex-induced and other forms of vasculitis, multiple sclerosis, Sjogren's disease, gout, lymphoproliferatieve diseases such as non-Hodgkin lymphoma and B cell chronic lymphocytic leukemia, bum injuries, multiple trauma, stroke,
  • SEQ ID NO: 2. represents amino acid sequence for IL-33 (for 95-270 fragment)
  • SEQ ID NO: 3. represents amino acid sequence for IL-33 fragment (for 107-270 fragment)
  • SEQ ID NO: 5 represents amino acid sequence for IL-33 fragment (for 112-270 fragment)
  • SEQ ID NO: 6. represents amino acid sequence for full length IL-37 (1-218).
  • SEQ ID NO: 7. represents amino acid sequence for IL-37 fragment having length of
  • the recombinant protein further comprises a third domain, comprising a linker.
  • the recombinant protein comprises at least one of the amino acid sequences from SEQ ID NO: 1-5 for first domain or a biologically active substantially homologous sequence.
  • nucleic acid comprising a nucleic acid sequence encoding a recombinant peptide having the nucleotide sequence of IL-33, or a fragment, variant or homolog thereof, or IL-37, or a fragment, variant or homolog thereof.
  • Also provided herein is a method for producing a protein comprising the steps of: introducing a target isolated nucleic acid sequence of IL-33 into one or more vectors along with a promoter introduced in one or more recombinant host cell to express a full length IL-33 protein, wherein the full length IL-33 may be cleaved into one or more fragments selected from the group consisting of amino acid residues 95-270 (SEQ ID NO: 2), 107- 270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4) or 112-270 (SEQ ID NO: 5) of IL-33, by mast cell enzymes like chymase and tryptases; introducing a target sequence of an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, into one or more vectors along with a promoter introduced in one or more recombinant host cell to express the antiinflammatory cytokine, or a fragment, variant or homolog thereof, optionally linking the
  • Also provided herein is a recombinant host cell comprising: an isolated nucleic acid as disclosed herein and a vector that contains the isolated nucleic acid.
  • the kit further comprises one or more containers. In some embodiments, the kit further comprises one or more applicators. In some embodiments, the kit further comprises instructional material for the use of the kit.
  • compositions and related methods for producing a recombinant protein for treating, preventing, and alleviating a neurodegenerative and autoimmune disorders comprise poly(alkylene glycol), poly(olcfinic alcohol), poly(vinylpyrrolidonc), poly(hydroxylalkyl-mcthacrylamidc), poly(hydroxyalkyl-methacrylate), poly(a-hydroxy acid), poly(acrylic acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), and copolymers or terpolymers thereof.
  • a pharmaceutical composition for treating, preventing, and alleviating neurodegenerative and autoimmune disorders comprising: a first domain comprising interleukin-33 (IL-33), or a fragment, variant or homolog thereof, a second domain comprising an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, and an optional linker that creates a linkage between the IL-33, or a fragment, variant or homolog thereof, and the anti-inflammatory cytokine, or a fragment, variant or homolog thereof.
  • IL-33 interleukin-33
  • second domain comprising an anti-inflammatory cytokine, or a fragment, variant or homolog thereof
  • an optional linker that creates a linkage between the IL-33, or a fragment, variant or homolog thereof, and the anti-inflammatory cytokine, or a fragment, variant or homolog thereof.
  • the IL-33 fragment comprises any one of the amino acid sequences selected from the group consisting of residues 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4) and 112-270 (SEQ ID NO: 5) of IL-33.
  • the fragment, variant or homolog of IL-33 is processed and obtained using one or more mast cell protease enzymes, including but not limited to chymase and tryptase.
  • the second domain comprises a fragment of IL-37.
  • the sequence of the fragment of IL-37 comprises SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.
  • the IL-33 is linked with the anti-inflammatory cytokine, or a fragment, variant or homolog thereof.
  • the composition comprising IL-33, or a fragment, variant or homolog thereof is linked with the anti-inflammatory cytokine, or a fragment, variant or homolog thereof, through a linker (X).
  • the IL-33, or fragment, variant or homolog thereof, and the anti-inflammatory cytokine, or fragment, variant or homolog thereof are directly linked or fused without using a linker.
  • the IL-33, or fragment, variant or homolog thereof is coadministered with the anti-inflammatory cytokine, or a fragment, variant or homolog thereof.
  • a recombinant protein comprising: a first domain comprising IL-33, or a fragment, variant or homolog thereof, and a second domain comprising an anti-inflammatory cytokine, or a fragment, variant or homolog thereof.
  • the link may be formed via a condensation reaction, including but not limited to condensation of an aldehyde with an amine, condensation of a carboxylic acid, or derivative thereof, with an alcohol, and condensation of a carboxylic acid, or derivative thereof, with an amine.
  • the resulting heterodimer comprising IL-33, or fragment, variant or homolog thereof, IL-37, or fragment, variant or homolog thereof, and water-soluble polymer, remains intact over time, thus improving the pharmacokinetic property of the composition within the human system.
  • a mixture comprising the first domain and the second domain, both having unprotected amino groups, can be reacted with aldehyde groups of the linker.
  • aldehyde groups of the linker A person of skill will appreciate that a mixture of the desired heterodimer and the two homodimers can be formed under these conditions.
  • a stepwise reaction sequence may facilitate synthesis of the desired heterodimer, comprising the first domain, the second domain, and a linker.
  • a linker precursor is provided which has two termini, one of which is linked to a protecting group, and one of which remains unprotected and therefore available for reaction.
  • the unprotected terminus may consist of e.g. an aldehyde.
  • a method for producing a conjugated protein is also provided herein.
  • the IL-33, or a fragment, variant or homolog thereof, is covalently attached with an anti-inflammatory cytokine, or a fragment or homofragment, variant or homolog log thereof, to produce a conjugated protein.
  • the covalent attachment is non-hydrolysable in nature and the primary covalent attachment is established by a covalent linkage, including but not limited to a disulfide linkage, and other covalent linkages that are known in the art.
  • the method comprises oral administration.
  • the dosage of the pharmaceutical composition administered to the animal is about 0.01 pg to about 1000 pg per kg of body weight of the animal depending upon the factors such as age, frequency of administration, route of administration etc.
  • the dosage is about 200 pg or less per kg of body weight, optionally about 100 pg or less per kg of body weight, optionally about 25 pg or less per kg of body weight, optionally about 10 pg or less per kg of body weight.
  • the dosage is about 0.05 pg or greater per kg of body weight, optionally about 0.2 pg or greater per kg of body weight, optionally about 0.5 pg or greater per kg of body weight.
  • Embodiment 10A The composition of Embodiment 1A, wherein the IL-33, or fragment, variant or homolog thereof, is PEGylated at one or more specific sites to improve stability and half-life of the molecule.
  • Embodiment 12A A composition comprising an interleukin-33 (IL-33) (SEQ ID NO: 1), or a fragment, variant or homolog thereof comprising at least one amino acid sequence selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109- 270 (SEQ ID NO: 4), and 112-270 (SEQ ID NO: 5).
  • IL-33 interleukin-33
  • Embodiment IB A method for producing a protein comprising the steps of: a) introducing a target isolated nucleic acid sequence encoding IL-33 into one or more vectors along with a promoter expressed in one or more recombinant host cells to express full-length IL-33 proteins, wherein the IL-33 may optionally be cleaved into one or more fragments thereof by mast cell enzymes; b) introducing a target sequence of an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, into one or more vectors along with a promoter expressed in one or more recombinant host cells to express the antiinflammatory cytokine, or a fragment, variant or homolog thereof, and optionally linking the IL-33, or fragment, variant or homolog thereof, with the anti-inflammatory cytokine, or a fragment, variant or homolog thereof, using an optional linker; c) culturing the host cell under suitable conditions to allow the production of a desired recombin
  • Embodiment 2B The method of Embodiment IB, wherein the protein comprises: a first domain comprising IL-33, or a fragment, variant or homolog thereof, a second domain comprising an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, and a third domain comprising a linker.
  • Embodiment 3B The method of Embodiment 2B, wherein the recombinant protein further comprises a fourth domain having a cytokine receptor targeting moiety or a cellular internalization moiety.
  • Embodiment 4B The method of Embodiment IB, wherein the linker is a non- hydrolysable covalent linker used to produce IL-33, or a fragment, variant or homolog thereof, comprising at least one amino acid residue fragment of IL-33 selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4) or 112-270 (SEQ ID NO: 5) conjugated with the anti-inflammatory cytokine, or a fragment, variant or homolog thereof.
  • the linker is a non- hydrolysable covalent linker used to produce IL-33, or a fragment, variant or homolog thereof, comprising at least one amino acid residue fragment of IL-33 selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4) or 112-270 (SEQ ID NO: 5) conjugated with the anti
  • Embodiment 5B The method of Embodiment IB, wherein any one of the sequences encoding IL-33 or a fragment, variant or homolog thereof is directly fused with the target sequence of the anti-inflammatory cytokine, or a fragment, variant or homolog thereof, to produce a fusion protein.
  • Embodiment 6B The method of Embodiment IB, wherein the linker is a peptide linker or a non-peptide linker selected from the group consisting of direct linkage of two proteins, covalent linkers preferably non hydrolysable, PEGylated, polyethylene glycol (PEG), organic linkers, water-soluble organic linkers, water-soluble polymers, Fc fusion proteins, Fc albumin fusion proteins, a peptide sequence, a peptidase with a cleaving site, a bifunctional linker, a polyglycine linker, carbohydrates, bridging factors, biostable and biodegradable polymers, diamino and or diacid units, natural or unnatural amino acids or derivatives thereof, as well as aliphatic moieties, including alkyl, aryl, heteroalkyl, heteroaryl, alkoxy and combinations thereof.
  • the linker is a peptide linker or a non-peptide linker selected from the group consisting of direct
  • Embodiment 8B The method of Embodiment IB, wherein the fragment of IL-33 is derived from mast cells comprising the step of expressing the full-length IL-33 polypeptide and processing the full-length IL-33 polypeptide using mast cell enzymes to cleave it into one or more amino acid residue fragments of IL-33 selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), or 112-270 (SEQ ID NO: 5) of IL-33.
  • SEQ ID NO: 2 The method of Embodiment IB, wherein the fragment of IL-33 is derived from mast cells comprising the step of expressing the full-length IL-33 polypeptide and processing the full-length IL-33 polypeptide using mast cell enzymes to cleave it into one or more amino acid residue fragments of IL-33 selected from the group consisting of 95-270 (SEQ ID NO: 2),
  • Embodiment 9B The method of Embodiment IB, wherein the IL-33, or fragment, variant or homolog thereof, is PEGylated at one or more specific sites to enhance stability and half-life of the molecule.
  • Embodiment 1C Use of a pharmaceutical composition comprising IL-33, or a fragment, variant or homolog thereof, comprising at least one amino acid residue fragment selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109- 270 (SEQ ID NO: 4) or 112-270 (SEQ ID NO: 5), an optional linker and an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, for treating, preventing, or alleviating neurodegenerative and auto-immune disorders.
  • Embodiment 2C The composition of Embodiment 1C, wherein the linker is a peptide linker or a non-peptide linker selected from the group consisting of direct linkage of two proteins, covalent linkers preferably non hydrolysable, PEGylated, polyethylene glycol (PEG), organic linkers, water-soluble organic linkers, water-soluble polymers, Fc fusion proteins, Fc albumin fusion proteins, a peptide sequence, a peptidase with a cleaving site, a bifunctional linker, a polyglycine linker, carbohydrates, bridging factors, biostable and biodegradable polymers, diamino and or diacid units, natural or unnatural amino acids or derivatives thereof, as well as aliphatic moieties, including alkyl, aryl, heteroalkyl, heteroaryl, alkoxy and combinations thereof.
  • the linker is a peptide linker or a non-peptide linker selected from the group consisting of direct
  • Embodiment ID A compound for treating, preventing, or alleviating neurodegenerative and auto-immune disorders, comprising interleukin-33 (IL-33), or a fragment thereof, comprising at least one amino acid sequence selected from 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), or 112-270 (SEQ ID NO: 5) wherein the IL-33, or fragment thereof, is PEGylated at one or more specific sites.
  • IL-33 interleukin-33
  • a fragment thereof comprising at least one amino acid sequence selected from 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), or 112-270 (SEQ ID NO: 5) wherein the IL-33, or fragment thereof, is PEGylated at one or more specific sites.
  • Embodiment IE A method for the treatment of a condition, disease, or disorder, the method comprising the step of administration of an effective amount of a composition comprising:
  • IL-33 or a variant, fragment, or homolog thereof,
  • IL-37 or a variant, fragment, or homolog thereof, and an optional linker for creating linkage between the IL-33, or a fragment, variant or homolog thereof, and IL-37, or fragment, variant or homolog thereof, to a patient in need thereof.
  • Embodiment 2E The method of Embodiment IE, wherein the IL-33, or variant, fragment, or homolog thereof, has at least one amino acid sequence selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), and 112-270 (SEQ ID NO: 5).
  • Embodiment 3E The method of Embodiment IE, wherein the inteiieukin-37 (IL-37), or a fragment, variant or homolog thereof, has amino acid sequences selected from SEQ ID NO: 6-10.
  • Embodiment 4E The method of Embodiment IE, wherein the composition comprises an optional linker for creating linkage between the IL-33, or fragment, variant or homolog thereof, and IL-37, or fragment, variant or homolog thereof.
  • Embodiment 5E The method of Embodiment 4E: , wherein the linkage is at least one selected from: a) the IL-33 or at least one fragment of IL-33 selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4) or 112-270 (SEQ ID NO: 5) is fused with the anti-inflammatory cytokine, or a fragment, variant or homolog thereof; b) the IL-33 or at least one fragment of IL-33 selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), or 112-270 (SEQ ID NO: 5) is conjugated with the anti-inflammatory cytokine, or a fragment, variant or homolog thereof; c) the IL-33 or at least one amino acid residue fragment of IL-33 selected from the group consisting of
  • Embodiment 6E The method of Embodiment IE, wherein the condition, disease, or disorder is an inflammatory disease mediated by the production of proinflammatory cytokines such as TNF, IL-1, IL-6, and/or other inflammatory mediators.
  • proinflammatory cytokines such as TNF, IL-1, IL-6, and/or other inflammatory mediators.
  • Embodiment 7E The method of Embodiment IE, wherein administration of IL-33 is indicated for treatment or prevention of the condition, disease, or disorder.
  • Embodiment 8E The method of Embodiment IE, wherein administration of IL-37 is indicated for treatment or prevention of the condition, disease, or disorder.
  • Embodiment 10E The method of claim 36, wherein the pain is selected from inflammatory pain and neuropathic pain.
  • Embodiment 1 IE The method of Embodiment IE, wherein the condition is osteoarthritis or chronic pain.
  • Embodiment 13E The method of Embodiment IE, wherein the condition is characterized by local or systemic inflammation, immune activation, and/or lymphoproliferation.
  • Embodiment 2F The method of Embodiment IF, wherein the composition comprising IL-33, or a variant, fragment, or homolog thereof and the composition comprising IL-37, or a variant, fragment, or homolog thereof are administered simultaneously or sequentially.
  • Embodiment 3F The method of Embodiment IF, wherein the IL-33, or variant, fragment, or homolog thereof, comprises at least one amino acid sequence selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), and 112-270 (SEQ ID NO: 5).
  • Embodiment 6F The method of Embodiment IF, wherein administration of IL-33 is indicated for treatment or prevention of the condition, disease, or disorder.
  • Embodiment 9F The method of Embodiment 8F, wherein the pain is selected from inflammatory pain and neuropathic pain.
  • Embodiment 10F The method of Embodiment IF, wherein the condition is osteoarthritis or chronic pain.
  • the “survival cohort” included 12 mice monitored for grip strength (Ix/week), body weights (5x/week), NeuroScores (5x/week), disease onset and survival. This cohort was sacrificed when mice reached the humane endpoint, tissues (brain, spinal cord, plasma) were collected, flash frozen with liquid nitrogen and stored at -80 C; 2)
  • the “pharmacodynamic (PD) cohort” included 8 mice monitored for body weights (5x/week) and NeuroScores (5x/week) and were harvested at approximately age 105 ⁇ l days (i.e. when 25% of the PD cohort presented with symptoms of the disease), were perfused with saline, brains and spinal cords were collected, submerged in RPMI buffer and were sent on ice for study.
  • SOD1G93A mice underwent grip strength testing on a weekly basis for eleven consecutive weeks, using a Bioseb grip strength apparatus. Each grip strength test consisted of three trials, and the average of the three trials was used for the analysis. For each trial, the experimenter gently pulled the mouse away from a platform and the machine recorded the force that the mouse exerted on the platform sensors, with all its limbs simultaneously, to resist the force of the pull. In the event where a mouse was unable to complete the test, either because it was severely progressed or diseased, the last recorded grip strength value was progressed forward for purpose of the analyses. The data are presented as a ratio of the baseline grip strength for each group (shown in FIG. 8). Each point represents the average force for 12 mice per group ⁇ SEM.
  • a novel fusion protein comprising an IL-33 fragment fused to an IL-37 fragment via an intervening peptide linker is prepared.
  • the IL-33 fragment is selected from the group consisting of amino acid sequence 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), and 112-270 (SEQ ID NO: 5).
  • the IL-37 fragment is an amino acid sequence selected from SEQ ID NO: 6-10.
  • the linker is SEQ ID NO: 11.
  • the recombinant coding sequence for this fusion protein is subcloned into a eukaryotic expression vector.
  • the expression construct is stably transfected into mammalian cells.
  • the fusion protein is detected in cell media.
  • a western blot is used to detect the size and to confirm the identity of the fusion protein.
  • the fusion protein may be purified by chromatography .
  • mice receive IL-33-IL-37 fusion protein twice per week i.p. at a dosage of 0.1 pg/mousc to 1 pg/mousc. Treatment is continued for 50 days or more, for example 100 days or more. Treatment may begin before or after onset of ALS symptoms. The onset of the disease in the SOD1G93A model is defined as the age when the mice first exhibit signs of paresis in their hindlimbs. A wire-hang test may be conducted, for example as described in Example 1 . Mice receiving PBS or untreated mice arc used as a control.
  • Binding of the fragment to IL-33 receptor is analyzed by flow cytometry.
  • the recombinant coding sequence for this IL-37 is subcloned into a eukaryotic expression vector.
  • the expression construct is stably transfected into mammalian cells.
  • the protein is detected in cell media.
  • a western blot is used to detect the size and to confirm the identity of the IL-37.
  • the IL-37 may be purified by chromatography.
  • mice receive TL-33 fragment and/or IL-37 twice or more per week i.p. at a dosage of 0.1 pg/mouse to 1 pg/mouse. Treatment is continued for 50 days or more, for example 100 days or more. Treatment may begin before or after onset of AD symptoms. Contextual fear conditioning tests or open field tests are conducted. Mice receiving vehicle or PBS or untreated mice are used as a control.
  • SEQ ID NO: 2 represents amino acid sequence for IL-33 (for 95-270 fragment)
  • SEQ ID NO: 3 represents amino acid sequence for IL-33 fragment (for 107-270 fragment)
  • SEQ ID NO: 4 represents amino acid sequence for IL-33 fragment (for 109-270 fragment)
  • SEQ ID NO: 5 represents amino acid sequence for IL-33 fragment (for 112-270 fragment)
  • SEQ ID NO: 9 represents amino acid sequence for human IL-37 fragment having length of 197 residues (Isoform 2; NCBI Accession NP_775294.1).
  • SEQ ID NO: 11 represents the amino acid sequence of a peptide linker GGGGS.
  • the linker may be a multiple of SEQ ID NO: 11, for example a 2- or 3- or 4- time repeat.
  • Nanoparticle anchoring targets immune agonists to tumors enabling anti-cancer immunity without systemic toxicity, Nature Communications, 9(1), Article Number 6

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Abstract

Disclosed herein are compositions comprising IL-33, or a fragment, variant or homolog thereof, and an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, for treating, preventing, and alleviating neurodegenerative and autoimmune disorders. The IL-33, or a fragment, variant or homolog thereof, and the anti-inflammatory cytokine, or a fragment, variant or homolog thereof, are in combination or may be fused or attached by means of various linkers. In some embodiments, the composition comprises a recombinant protein having three domains, where the first domain comprises IL-33, or a fragment, variant or homolog thereof, a second domain comprises an anti-inflammatory cytokine, or fragment, variant or homolog thereof, and a third domain comprises a linker. In some embodiments, the anti-inflammatory cytokine, or a fragment, variant or homolog thereof, is interleukin-37 (IL-37).

Description

COMPOSITIONS FOR THE DIAGNOSIS, TREATMENT, PREVENTION, AND ALLEVIATION OF NEURODEGENERATIVE AND AUTOIMMUNE DISORDERS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/435,396, filed December 27, 2022, which is hereby incorporated by reference in its entirety.
SEQUENCE LISTING
[0002] The contents of the electronic sequence listing (318293-00003. xml; Size: 12,348 bytes; and Date of Creation: December 26, 2023) is herein incorporated by reference in its entirety.
FIELD
[0003] Disclosed herein are compositions and methods comprising cytokines, cytokine fragments and combinations of cytokines, interleukins, and fragments of interleukins for the diagnosis, treatment, prevention, and alleviation of neurodegenerative and autoimmune disorders, including related pharmaceutical products and devices used with the compositions and methods.
BACKGROUND
[0004] Autoimmune inflammatory diseases, their related conditions, such as local or systemic inflammation, immune activation, and/or lymphoproliferation is selected from the group consisting of sepsis, adult respiratory distress syndrome, allo- and xenotransplantation, dermatitis, inflammatory bowel disease, sarcoidosis, allergies, psoriasis, ankylosing spondylarthritis, autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, glomerulonephritis, immune complex-induced and other forms of vasculitis, Sjogren's disease, gout, lymphoproliferative diseases such as non-Hodgkin lymphoma and B cell chronic lymphocytic leukemia, burn injuries, multiple trauma, stroke, myocardial infarction, atherosclerosis, diabetes mellitus, extracorporeal dialysis and blood oxygenation, ischemiareperfusion injury (IRI), toxicity induced by the in vivo administration of cytokines or therapeutic monoclonal antibodies, chronic pain syndrome, and neuropathic and/ or inflammatory pain, are among the most debilitating conditions observed in clinical practice. The inflammation process is central to these medical conditions, where both pro-inflammatory cytokines and anti-inflammatory cytokines play important roles as mediators of the inflammatory processes.
[0005] Interleukin-33 (IL-33) is a tissue-derived nuclear cytokine discovered in 2005 that belongs to the IL-1 family and has roles in both homeostasis and inflammation (1). Original studies defined IL-33 as a so-called “alarmin” protein released by damaged endothelial and epithelial cells that initially alerts the immune system of “danger”. Yet subsequent work has shown that it is expressed by a much wider variety of cells including glial cells of the nervous system, cardiomyocytes, osteoclasts, and several immune cell types including activated T cells, particularly Th2 cells, mast cells, basophils, macrophages, and dendritic cells. ST2 (IL1RL1) is the receptor for IL-33 and is expressed on several immune target cells that include IL-33- expressing cells as well as Type 2 innate lymphoid cells (ILC2s), eosinophils, T regulatory cells, Thl, Thl7 and CD8 T cells, Natural Killer (NK) cells, NK T cells, B cells and neutrophils (2). Thus IL-33 has a potential for influencing multiple arms of the immune response. ST2 expression by microglia, oligodendrocytes, astrocytes and neurons within the CNS indicates the IL-33/ST2 signaling pathway also has a role in modulating neurologic functioning (3,4,5).
[0006] Pro-inflammatory cytokines promote local and systemic inflammation. Among the large groups of pro-inflammatory cytokines, two are particularly prominent, i.e. the tumor necrosis factor (TNFa) and interleukin 1 (IL- 1 P). A great deal of effort has been devoted toward developing therapeutic strategies aimed at inhibiting TNFa and IL-ip. Specifically, reducing the biological activities of TNFa and IL-i has been accomplished by several strategies such as neutralizing antibodies, soluble receptors, receptor antagonists, and inhibitors of proteases that convert inactive precursors to active molecules. For example, inhibitors of TNFa, such as Infliximab® (anti-TNFa antibody), Humira® (fully human anti-TNFa antibody), Enbrel® (TNF- receptor-Fc-fusion protein), and Anakinra (Kineret®; IL-1 receptor antagonist, IL- Ira) have been tested in clinical trials. Although blocking TNFa and/or IL-i was successful in many patients suffering from rheumatoid arthritis and inflammatory bowel diseases, not all patients respond well to such types of therapeutic interventions. Therefore, there is still a great need for alternative and effective therapeutic strategies for the treatment of inflammatory diseases, chronic pain, and related conditions. [0007] IL-33 exerts pleiotropic effects on both immune and non-immune cells and is generally considered to have anti-inflammatory /protective activities. These functions arc a result of the strong ability of IL-33 to activate ILC2s, which in turn produce cytokines such as IL-5 and IL-13 that drive Th2 responses. Amphiregulin, also produced by IL-33-activated ILC2s promotes wound healing (6) and Th2 responses can block the pathogenic autoimmune Thl/Thl7 responses characteristic of Multiple Sclerosis (7). Importantly, IL-33 activates a population of ST2+ T regulatory cells that limit damaging effector CD4+ and CD8 cytotoxic T cell responses as shown in models of colitis, lung injury and Type 1 diabetes (8, 9, 10, 11). In mouse models of Multiple Sclerosis and type 1 diabetes, IL-33 treatment ameliorates ongoing disease. Studies in models of stroke, spinal cord injury and Alzheimer’s disease demonstrate that IL-33 reduces CNS tissue loss, reverses astrogliosis and demyelination, and improves cognition (3, 12, 13, 14, 15, 16). IL- 33 also has a direct effect on oligodendrocyte gene expression and induces p38MAPK phosphorylation these cells, an event linked to myelination (12). Thus, IL-33 therapy has the potential to prevent disease progression by blocking damaging inflammation as well as promoting myelin and neuronal repair in MS and other diseases of the CNS. While IL-33 drives Th2-mediated protection against bacterial infections such as Clostridium difficile as well as parasitic infections, it can also exacerbate allergic inflammation (17,18).
[0008] There are multiple cytokines that have anti-inflammatory properties. Such cytokines used in combination may have the potential for downregulating the pathologic T cell and innate immune cell responses in chronic autoimmune, neurologic, and inflammatory diseases. Antiinflammatory cytokine IL-37 has been used in some embodiments in combination with IL-33.
[0009] IL-37, like IL-33 is a member of IL-1 family cytokine. IL-37 is expressed at low levels in a wide variety of tissues such as thymus, lung, colon, uterus, and bone marrow and is induced in inflammatory conditions (19). IL-37 exists as a dimer and monomer form, with the monomer having potent anti-inflammatory activity. This cytokine binds to two receptors that are expressed on a broad range of immune and non-immune cells: IL-18R, where it can block the proinflammatory actions of IL- 18, and IL-1R8 receptor (SIGRR). Unlike conventional cytokines but similar to IL-33, IL-37 can also function as transcription factor, when and function via Smad3 dependent mechanism (20). IL-37 broadly suppresses both innate and adaptive immune response (21, 22, 23). IL-37 suppresses the secretion of pro-inflammatory cytokines such as TNF-a, IL-la, IL-ip, IL-6, G-CSF and GM-CSF from human blood LPS- stimulated monocytes as well as multiple chemokines (24). Moreover, IL-37 has been shown to suppress MHC and costimulatory molecule expression as well as the antigen presenting cell (APC) function, which reduces the ability of these cells to activate naive T cells, thereby impairing the effector T cell response and adaptive immunity (25, 26).
[0010] Many studies indicate that IL-37 is a candidate for a therapeutic role in autoimmune disorders. Indeed, in mice models of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), IL-37 inhibited the pathogenesis of the disease significantly (27). For example, dendritic cells (DCs) and macrophages play a pivotal role in Thl7 differentiation via secreting proinflammatory cytokines such as IL-6, IL-ip and IL-23, inhibition of these APCs by IL-37 indirectly suppresses Th 17 cell mediated immune response in autoimmune diseases (28). Through its actions in limiting Th2 response, IL-37 blocks Th2 differentiation and the subsequent recruitment of eosinophils to the airways in allergic asthma thereby preventing bronchial obstruction and mucus production characteristic of this disease. Also of relevance is the ability of IL-37 to suppress inflammatory actions in the Central Nervous System. In a mouse model of stroke using IL-37 transgenic mice, IL-37 inhibited infiltration of pro-inflammatory cells to sites of ischemic injury, blocked the activation of microglia, reduced infarct volume and significantly limited stroke induced mobility deficits (29). Based on studies in mouse models, IL- 37 is a potential therapeutic for the treatment of aortic valve disease, inflammatory arthritis, colitis, metabolic syndrome, aging and cancer genesis (19). When used together, these cytokines in combination offer immense immune modulatory possibilities for many diseases with inflammatory underpinnings.
[0011] The therapeutic proteins or cytokines can be delivered via a vehicle or payload directly to the desired target. Among various delivery agents, nanoparticles or liposome- mediated targeted delivery technologies have become popular. Multiple studies have demonstrated that the delivery of proinflammatory cytokines such as IL-2, IL- 12 or TNF using nanoparticles with or without targeting moieties such as antibodies is possible (30, 31, 32, 33). These types of cytokine nanoconjugates/immunoliposomes have the advantage of delivering the payload directly to the targets when used in association with antibodies directed against the target cell or tissues. Furthermore, these strategies have been proven to have less systemic toxicity (34). [0012] Therapeutic effects related to IL-33 have been discussed in US 10851 145B2, US10130705B2, US20200339674A1, US20190216898A1 and US20180171405A1. These patents and patent application publications disclose compositions comprising IL-33 and fragments thereof, and antibodies targeting IL-33. Therapeutic effects of IL-37 have been described in CN108948178A, WO2015160795A1 and EP3058082A1.
[0013] IL-4 and IL-10 fusion proteins and related methods are described in U.S. Patent Pub. No. 20180094037A1 which issued as U.S. Patent No. 10,851,143.
[0014] Combination therapies using cytokines are described in W02005014642A2, which discloses novel fusion proteins with X-Y or Y-X configurations, wherein X represents a first immunoregulating polypeptide and Y represents a second immunoregulating polypeptide different from X. US10851145B2 discloses a novel IL-2 and IL-33 fusion peptide, and its use for preventing, treating, or alleviating diseases and disorders such as autoimmune diseases and disorders, and inflammation. Also disclosed are the nucleic acids encoding these fusion peptides, the vector comprising such peptides and the host comprising such nucleic acids.
[0015] There remains a need for therapeutic compositions comprising IL-33 or a fragment, variant or homolog thereof and at least one anti-inflammatory cytokine such as IL-37 or a fragment, variant or homolog thereof. There also remains a need for fusion or recombinant proteins comprising IL-33 or a fragment, variant or homolog thereof for treating, preventing and alleviating nerodegenerative and autoimmune diseases.
SUMMARY
[0016] An objective of the disclosure is to develop a novel composition based on antiinflammatory cytokines, or a fragment, variant or homolog thereof, for treating, preventing, and alleviating neurodegenerative and autoimmune disorders.
[0017] Another objective of the disclosure is to provide a recombinant protein and method of preparing a recombinant protein for treating, preventing, and alleviating neurodegenerative and autoimmune disorders.
[0018] Yet another objective of the disclosure is to provide multiple ways of linking two or more anti-inflammatory cytokines, or a fragment, variant or homolog thereof.
[0019] Accordingly, provided herein is a fusion protein comprising: a first domain, comprising interleukin-33 (IL-33), or a fragment, variant or homolog thereof, and a second domain, comprising an anti-inflammatory cytokine, or a fragment, variant or homolog thereof.
[0020] In some embodiments, the first domain and the second domain are connected via an optional linker. In further embodiments, the first domain and the second domain are connected via a linker. In yet further embodiments, the linker comprises a protein linker, for example SEQ ID NO: 11 or a multiple thereof.
[0021] In some embodiments, the fusion protein is a recombinant protein.
[0022] In some embodiments, the IL-33 is a mammalian IL-33. In some embodiments, the IL-33 is human IL-33 or mouse IL-33. In further embodiments, the IL-33 comprises the amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.
[0023] In some embodiments, the fusion protein further comprises a third domain, optionally comprising a linker. In some embodiments, the fusion protein comprising a third domain is a recombinant protein.
[0024] In some embodiments, the IL-33, or fragment, variant or homolog thereof, is fused with the N-tcrminal of the anti-inflammatory cytokine, or fragment, variant or homolog thereof.
[0025] In some embodiments, the anti-inflammatory cytokine, or fragment, variant or homolog thereof, is fused with the N-terminal of the IL-33, or fragment, variant or homolog thereof.
[0026] In some embodiments, the linker is an organic linker. In some embodiments, the linker is a water-soluble organic linker. In some embodiments, the linker is a cleavable linker.
[0027] In some embodiments, the linker is a peptide linker, for example SEQ ID NO: 11 or a multiple thereof.
[0028] In some embodiments, the linker is a peptide linker that comprises cleavage site to be recognized by human serum or human cellular enzymes like protease or esterases. In some embodiments, the linker is a non-peptide linker that is cleaved by thermal energy, pH, salt concentration, water dissolution and by other means.
[0029] In some embodiments, the recombinant protein comprises a fourth domain having a cytokine receptor targeting moiety.
[0030] In some embodiments, the recombinant protein comprises a fourth domain having cellular internalization moiety.
[0031] In some embodiments, the first domain comprises a fragment of IL-33. In some embodiments, the fragment of IL-33 comprises residues 95-270 (SEQ ID NO: 2) of IL-33. In some embodiments, the fragment of IL-33 comprises residues 107-270 (SEQ ID NO: 3) of IL- 33. In some embodiments, the fragment of IL-33 comprises residues 109-270 (SEQ ID NO: 4) of IL-33. In some embodiments, the fragment of IL-33 comprises residues of 112-270 (SEQ ID NO: 5) of IL-33.
[0032] In some embodiments, the fragment, variant or homolog of IL-33 is biologically active.
[0033] In some embodiments, the first domain binds with an IL-33 receptor. In further embodiments, the IL-33 receptor is IL1RL1 (also known as ST2).
[0034] In some embodiments, the second domain binds with an anti-inflammatory cytokine receptor.
[0035] In some embodiments, the second domain comprises a biologically active fragment, variant or homolog of an anti-inflammatory cytokine selected from the group consisting of IL- 37, IL-lra, IL-4, IL-6, IL-10, IL-11, IL-13, and TGF-P, or a fragment, variant or homolog thereof.
[0036] In one embodiment the second domain comprises an anti-inflammatory cytokine selected from the group consisting of IL-37, IL-lra, IL-4, IL-6, IL-10, IL-11, IL-13, and TGF-p, or a fragment, variant or homolog thereof.
[0037] In some embodiments, the second domain comprises a biologically active fragment, variant or homolog of interleukin-37 (IL-37). In further embodiments, the IL-37, or fragment, variant or homolog thereof comprises SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10. [0038] In some embodiments, the second domain comprises interieukin-37 (IL-37), or a fragment, variant or homolog thereof. In some embodiments, the IL-37 is mammalian IL-37. In some embodiments, the IL-37 is human IL-37 or mouse IL-37.
[0039] In some embodiments, the second domain comprises a fragment of IL-37.
[0040] In some embodiments, the IL-33, or fragment, variant or homolog thereof, is fused with the N-terminal of the IL-37, or fragment, variant or homolog thereof.
[0041] In some embodiments, the IL-37 or fragment, variant or homolog thereof, is fused with the N-terminal of the IL-33, or fragment, variant or homolog thereof.
[0042] Also provided herein is a fusion protein as disclosed herein, for use as a medicament.
[0043] Also provided herein is a fusion protein as disclosed herein, for use in the treatment or prevention of a condition, disease, or disorder.
[0044] Also provided herein is a fusion protein as disclosed herein, for use in the treatment or prevention of a neurodegenerative or autoimmune disorder.
[0045] Also provided herein is the use of a fusion protein as disclosed herein as a medicament.
[0046] Also provided herein is the use of a fusion protein as disclosed herein for the treatment or prevention of a condition, disease, or disorder.
[0047] Also provided herein is the use of a fusion protein as disclosed herein for the treatment or prevention of a neurodegenerative or autoimmune disorder.
[0048] Also provided herein is the use of a fusion protein as disclosed herein as a medicament, the fusion protein comprising: a first domain, comprising interleukin- 33 (IL-33), or a fragment, variant or homolog thereof, and a second domain, comprising an anti-inflammatory cytokine or a fragment, variant or homolog thereof.
[0049] In some embodiments, the fusion protein further comprises a third domain, comprising a linker. [0050] Also provided herein is the use of a fusion protein as disclosed herein for treating, alleviating, or preventing a ncurodcgcncrativc or autoimmune disorder, the fusion protein comprising: a first domain, comprising interleukin- 33 (IL-33), or a fragment, variant or homolog thereof, a second domain, comprising an anti-inflammatory cytokine or a fragment, variant or homolog thereof, and an optional linker.
[0051] In some embodiments, the fusion protein further comprises a third domain, optionally comprising a linker.
[0052] Also provided herein is a pharmaceutical composition comprising: a fusion protein as disclosed herein, and a pharmaceutically acceptable carrier.
[0053] Also provided herein is a pharmaceutical composition comprising:
IL-33, or a fragment, variant or homolog thereof, and an anti-inflammatory cytokine, or a fragment, variant or homolog thereof.
[0054] Also provided herein is a pharmaceutical composition comprising:
IL-33, or a fragment, variant or homolog thereof, and an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, wherein IL-33, or a fragment, variant or homolog thereof, is fused to, conjugated to, or co-administered with the anti-inflammatory cytokine or fragment, variant or homolog thereof.
[0055] Also provided herein is a pharmaceutical composition comprising IL-33 or a fragment, variant or homolog thereof. In some embodiments, the IL-33 or a fragment, variant or homolog thereof comprises an amino acid sequence selected from the group consisting of residues 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), and 112-270 (SEQ ID NO: 5) of IL-33. In some embodiments, the IL-33 comprises SEQ ID NO: 1. In some embodiments, the pharmaceutical composition further comprises an anti-inflammatory cytokine, or fragment, variant or homolog thereof. In some embodiments, the anti-inflammatory cytokine is IL-37. In some embodiments, the pharmaceutical composition further comprises IL- 37, or fragment, variant or homolog thereof. In further embodiments, the IL-37, or fragment, variant or homolog thereof comprises SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.
[0056] In some embodiments, the pharmaceutical composition further comprises one or more additional anti-inflammatory agents.
[0057] Also provided herein is a pharmaceutical composition as disclosed herein, for use as a medicament.
[0058] Also provided herein is a pharmaceutical composition comprising IL-33 or any one of the peptides selected from the group consisting of residues 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), and 112-270 (SEQ ID NO: 5) of IL-33, for use as a medicament. In some embodiments, the IL-33 comprises SEQ ID NO: 1. In some embodiments, the pharmaceutical composition further comprises an anti-inflammatory cytokine, or fragment, variant or homolog thereof. In some embodiments, the anti-inflammatory cytokine is IL-37. In some embodiments, the pharmaceutical composition further comprises IL-37, or a fragment, variant or homolog thereof. In further embodiments, the IL-37, or fragment, variant or homolog thereof comprises SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.
[0059] Also provided herein is a pharmaceutical composition as disclosed herein, for use in the treatment or prevention of a condition, disease, or disorder.
[0060] Also provided herein is a pharmaceutical composition as disclosed herein, for use in the treatment or prevention of a neurodegenerative or autoimmune disorder.
[0061] Also provided herein is the use of a pharmaceutical composition as disclosed herein for treating, alleviating, or preventing a condition, disease, or disorder.
[0062] Also provided herein is the use of a pharmaceutical composition as disclosed herein for treating, alleviating, or preventing a neurodegenerative or autoimmune disorder. [0063] Also provided herein is the use of a pharmaceutical composition as for treating, alleviating, or preventing condition, disease, or disorder, the composition comprising:
IL-33, or a fragment, variant or homolog thereof, an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, and an optional linker.
[0064] In some embodiments, the IL-33 comprises SEQ ID NO: 1 or any one of the peptides selected from the group consisting of residues 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), and 112-270 (SEQ ID NO: 5) of IL-33. In some embodiments, the anti-inflammatory cytokine is IL-37. In some embodiments, the pharmaceutical composition further comprises a fragment of IL-37, or a fragment, variant or homolog thereof. In further embodiments, the IL-37, or fragment, variant or homolog thereof comprises SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.
[0065] Also provided herein is the use of a pharmaceutical composition comprising IL-33 or any one of the peptides selected from the group consisting of residues 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), and 112-270 (SEQ ID NO: 5) of IL-33, for use as a medicament. In some embodiments, the pharmaceutical composition further comprises an anti-inflammatory cytokine, or fragment, valiant or homolog thereof. In some embodiments, the anti-inflammatory cytokine is IL-37. In some embodiments, the pharmaceutical composition further comprises a fragment of IL-37, or a fragment, variant or homolog thereof. In further embodiments, the IL-37, or fragment, variant or homolog thereof comprises SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.
[0066] Also provided herein is a method for the treatment or prevention of a condition, disease, or disorder, the method comprising the step of administration of an effective amount of a fusion protein as disclosed herein, to a person in need thereof.
[0067] Also provided herein is a method for the treatment or prevention of a condition, disease, or disorder, the method comprising the step of administration of an effective amount of a pharmaceutical composition as disclosed herein, to a person in need thereof.
[0068] In an embodiment, the condition is characterized by pain and may be selected from inflammatory pain and neuropathic pain. [0069] In an embodiment, the condition is osteoarthritis or chronic pain.
[0070] In an embodiment, the condition is a neurodegenerative disease.
[0071] In an embodiment, the condition is characterized by local or systemic inflammation, immune activation, and/or lymphoproliferation.
[0072] In an embodiment, the condition, disease, or disorder is an inflammatory disease mediated by the production of proinflammatory cytokines such as TNF, IL- 1 , IL-6, and/or other inflammatory mediators.
[0073] In an embodiment, administration of IL-33 is indicated for treatment or prevention of the condition, disease, or disorder.
[0074] In an embodiment, administration of IL-37 is indicated for treatment or prevention of the condition, disease, or disorder.
[0075] Also disclosed herein are methods to link IL-33 and an anti-inflammatory cytokine, such as IL-37.
[0076] Also provided herein is a pharmaceutical composition comprising IL-33, or a fragment, variant or homolog thereof, wherein the IL-33, or fragment, variant or homolog thereof is PEGylated at one or more specific sites, thereby enhancing stability and half-life, or otherwise modified to achieve similar outcomes.
[0077] In some embodiments, the IL-33 or its fragment, variant or homolog thereof is PEGylated at one or more specific sites to improve stability and half-life, or otherwise modified to achieve similar results.
[0078] In some embodiments, the anti-inflammatory cytokine, or a fragment, variant or homolog thereof, is IL-37, or a fragment, variant or homolog thereof. In one embodiment, the IL- 37 is a full-length IL-37. In another embodiment, the IL-37 comprises more than one fragment of IL-37, each having different fragmental lengths, for example, 192, 178, 197, 157 and so on. In further embodiments, the IL-37, or fragment, variant or homolog thereof comprises SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10. [0079] Also provided is a pharmaceutical composition for treating, preventing, and alleviating ncurodcgcncrativc and autoimmune disorders that comprises a recombinant peptide as disclosed herein.
[0080] The sequence detail of the human version of the recombinant protein and nucleic acid comprising IL-33, or a fragment, variant or homolog thereof, linker and an anti-inflammatory cytokine have been provided below.
[0081] Provided herein is a compound comprising an interleukin-33 (IL-33) (SEQ ID NO: 1), or any one of the peptides selected from the group consisting of residues 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), and 112-270 (SEQ ID NO: 5) of IL-33.
[0082] In some embodiments, the compound further comprises an anti-inflammatory cytokine, or fragment, variant or homolog thereof. In some embodiments, the anti-inflammatory cytokine is IL-37, or a fragment, variant or homolog thereof. In further embodiments, the IL-37, or fragment, variant or homolog thereof comprises SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.
[0083] Provided herein is a compound comprising: a first domain, comprising an interleukin-33 (IL-33) (SEQ ID NO: 1) or a fragment selected from the group consisting of residues 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), and 112-270 (SEQ ID NO: 5) of IL-33; and a second domain, comprising an anti-inflammatory cytokine or fragment, variant or homolog thereof.
[0084] In some embodiments, the compound further comprises a third domain, comprising a linker for creating a linkage between IL-33, or a fragment, variant or homolog thereof, with the anti-inflammatory cytokine or fragment, variant or homolog thereof.
[0085] Also provided herein is a method for the treatment or prevention of a condition, disease, or disorder, the method comprising the step of: administering a pharmaceutical composition comprising an interleukin-33 (IL-33), or a fragment, variant or homolog thereof, and an anti-inflammatory, or a fragment, variant or homolog thereof. [0086] In some embodiments, the pharmaceutical composition comprises an interleukin-33 (IL-33) (SEQ ID NO: 1), or a peptide comprising an amino acid sequence selected from the group consisting of residues 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), and 112-270 (SEQ ID NO: 5) of IL-33.
[0087] In some embodiments, the anti-inflammatory is interleukin-37 (IL-37), or a fragment, valiant or homolog thereof. In further embodiments, the IL-37, or fragment, valiant or homolog thereof comprises SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.
[0088] In some embodiments, the fusion protein further comprises one or more chemical modifications. In some embodiments, the one or more chemical modifications is selected from the group comprising glycosylation, fucosylation, sialylation, and PEGylation.
[0089] Also provided herein is a nucleic acid molecule comprising a polynucleotide encoding a fusion protein as disclosed herein.
[0090] Also provided herein is a vector comprising a nucleic acid molecule as disclosed herein.
[0091] Also provided herein is a host cell comprising a nucleic acid molecule or a vector as disclosed herein.
[0092] Also provided herein is a method for producing a fusion protein as disclosed herein, the method comprising the steps of: culturing a host cell as disclosed herein under conditions permitting the production of the fusion protein as disclosed herein, and optionally recovering the fusion protein.
[0093] Also provided herein is a pharmaceutical composition comprising a fusion protein as disclosed herein, and a pharmaceutically acceptable carrier.
[0094] Also provided herein is a fusion protein as disclosed herein for use as a medicament. In some embodiments, the medicament can be used for the treatment or prevention of a condition characterized by local or systemic inflammation, immune activation, lymphoproliferation and/or pain. [0095] Also disclosed herein is a vector for use in the treatment or prevention of a condition characterized by local or systemic inflammation, immune activation, lymphoproliferation and/or chronic pain.
[0096] In any one of the embodiments or sequences disclosed herein, the IL-33 and/or the IL-37 fragment, variant or homolog thereof may optionally lack an N-terminus Methionine.
BRIEF DESCRIPTION OF THE DRAWINGS
[0097] The foregoing and other features and advantages of the materials and methods disclosed herein will be more fully understood from the following description made with reference to the drawings.
[0098] FIG. 1 illustrates an overview of a composition as disclosed herein, according to an exemplary embodiment of the present disclosure.
[0099] FIG. 2 illustrates a composition as disclosed herein, according to an exemplary embodiment of the present disclosure.
[0100] FIG. 3 illustrates water-soluble conjugates comprising water-soluble polymers for reaction with IL-33, or a fragment, variant or homolog thereof, and/or an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, according to an exemplary embodiment of the present disclosure.
[0101] FIG. 4 illustrates a releasable water-soluble conjugate comprising water-soluble polymers with NHS group for reaction with amino terminus of IL-33, or a fragment, variant or homolog thereof, and/or an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, according to an exemplary embodiment of the present disclosure.
[0102] FIG. 5 illustrates an asymmetric conjugate for coupling IL-33, or a fragment, variant or homolog thereof, and an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, according to an exemplary embodiment of the present disclosure.
[0103] FIG. 6 illustrates reaction between aldehyde and amino terminus of IL-33, or a fragment, variant or homolog thereof, and an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, according to an exemplary embodiment of the present disclosure. [0104] FIG. 7 illustrates non-specific cleavage of ester linkers by esterase, according an exemplary embodiment of the present disclosure.
[0105] FIG. 8 illustrates grip strength (% baseline), as described in Example 1.
[0106] FIG. 9 illustrates onset K-M curves, as described in Example 1.
[0107] FIG. 10 illustrates survival K-M curves, as described in Example 1.
[0108] FIG. 11 illustrates % baseline weight, as described in Example 1.
DETAILED DESCRIPTION
General Definitions
[01091 Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the disclosed materials and methods belong. All publications and patents referred to herein are incorporated by reference.
[0110] As used herein, the articles “a” and “an” may refer to one or to more than one (e.g. to at least one) of the grammatical object of the article.
[0111] As used herein, “about” may generally refer to an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Example degrees of error are within 5% of a given value or range of values.
[0112] Concentrations, amounts, volumes, percentages and other numerical values may be presented herein in a range format. It is also to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.
[0113] The term “cytokine” as used herein refers to intercellular signaling molecules which after binding with their receptors regulate mammalian cells. Cytokines have been further characterized into interleukins, interferons, chemokines, protein, or peptide hormones, and transforming growth factors depending upon their stimulatory or inhibitory roles. [0114] As used herein, the term “derivative” of a protein or peptide refers to a variant of the protein or peptide in which one or more of the residues has been chemically modified.
[0115] The term “IL-33” as used herein refers to the IL-33 cytokine which is a member of the IL-1 family and has a pleiotropic role.
[0116] The term “anti-inflammatory cytokine” as used herein refers to cytokine which suppresses the immune response.
[0117] The term “expression” as used herein refers to a gene or protein, which is intended to encompass transcription of a gene and/or translation of the transcript into a protein.
[0118] The term “fragment” as used herein refers to a segment of an amino acid sequence, comprising at least one amino acid.
[0119] The term “co-administered” as used herein refers to two or more active agents, such as cytokines, delivered together in a pharmaceutical composition. The term may also refer to simultaneous or sequential delivery of two or more active agents, each in its own composition.
[0120] The term “homology” as used herein refers to similarity in amino acid or nucleic acid sequence.
[0121] The term “transgenic mammal” as used herein refers to the mammal whose cell comprises exogenous nucleic acid.
[0122] The term “vector” as used herein refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid within the cell.
[0123] The term “encoding” refers to the ability of a nucleic acid to serve as a template for the synthesis of other polymers and macromolecules in biological such as polypeptide or protein.
[0124] The term “nucleic acid molecule” (or “nucleic acid sequence”, “polynucleotide”, or “nucleotide sequence”) refers to a DNA or RNA molecule, in single or double stranded form, particularly a DNA encoding a protein as disclosed herein. An “isolated nucleic acid sequence” refers to a nucleic acid sequence which is no longer in the natural environment from which it was isolated, e.g., the nucleic acid sequence in a bacterial host cell or in the plant nuclear or plastid genome. [0125] The term “recombinant polypeptide” (otherwise known as recombinant peptide or recombinant protein) as used herein refers to a peptide which is produced upon expression of a recombinant polynucleotide. The terms “protein” and “polypeptide” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3-dimensional structure or origin. An “isolated protein” is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.
[0126] The term “fusion protein” refers to a protein or polypeptide that has an amino acid sequence derived from two or more proteins. The fusion protein optionally may also include one or more linkers, linking regions, or one or more amino acid linkers between polypeptide portions derived from separate proteins. A fusion protein can be synthesized chemically, or it may be expressed from one or more recombinant polynucleotide(s).
[0127] The term “IL-33-IL-37 fusion protein” refers to a fusion polypeptide comprising at least IL-33, or a fragment, variant or homolog thereof, and IL-37, or a fragment, variant or homolog thereof, optionally coupled to one another via a linker. The IL-33-IL-37 fusion protein may comprise additional polypeptide sequences, for example and without limitation, a signal sequence, a Histidine-tag, an antibody Fe fragment, and the like.
[0128] As used herein, a “linker” refers to an organic moiety or polypeptide used to couple two proteins or polypeptides, for example IL-33, or a fragment, variant or homolog thereof, and IL-37, or a fragment, variant or homolog thereof. An IL-33-IL-37 fusion protein may therefore comprise an IL-33, or a fragment, variant or homolog thereof, an IL-37, or a fragment, variant or homolog thereof, and a linker. The linker typically is a stretch of amino acids, e.g., predominantly glycine and/or serine. In an embodiment, the linker is a stretch of amino acids having a length of up to 100 nucleotides, such as from about 2, 5, 7, 10, 15 amino acids up to about 15, 20, 25, 30, 35, 50, 75, or 100 amino acids, preferably comprising predominantly serine and glycine residues. The term “linked” as used herein is intended to broadly include the technologies used for linking, de-linking, connecting, or releasing two or more distinct proteins, for use in therapeutic medicines. The term “linked” is associated with the terms linked, linker, coupled, releasable, cleavable, water-soluble polymers, Fc fusion proteins, Fc albumin fusion proteins, a peptide sequence, a peptidase with a cleaving site, a bifunctional linker, and a polyglycine linker. In some embodiments, the term “linked” requires a covalent bond between the two proteins or polypeptides. In some embodiments, the term “linked” also includes the circumstances where two or more proteins may not be physically linked but nonetheless behave as if they are linked, for example, where two or more distinct proteins each use an Fc fusion protein and/or Fc albumen fusion protein that effectively links the two or more proteins. By way of example only, two or more “linked” proteins may be introduced into a mammal or a human for a therapeutic treatment. Upon introduction into the body these proteins may dissociate completely or partially into their primary component parts once they enter the body to provide a full therapeutic benefit. The mechanism of action in each scenario and under varying conditions could include, by way of example only, thermal, pH, chemical (blood), mechanical, electricity and so on. Finally, drug delivery technologies will be affected by the therapeutics physical structure and properties.
[0129] As used herein, “interleukin-33” (IL-33) refers to any mammalian IL-33, such as human IL-33, mouse IL-33, or an active species or allelic variant, fragment, isoform or derivative thereof.
[0130] As used herein, “interleukin-37” (IL-37) refers to any mammalian IL-37, such as human IL-37, mouse IL-37, or an active species or allelic variant, fragment, isoform or derivative thereof. The term “IL-37” as used herein refers to the IL-37 cytokine which is a member of the IL- 1 family and has an anti-inflammatory role.
[0131] As used herein, the term “dimeric” refers to a molecule wherein two polypeptides are associated stably through covalent or non-covalent interactions. The term “homodimeric” refers to a molecule wherein two identical polypeptides are associated stably through covalent or noncovalent interactions. In some embodiments, they are associated stably through non-covalent interactions. The term “heterodimeric” refers to a molecule wherein two nonidentical polypeptides are associated stably through covalent or noncovalent interactions. In some embodiments, they are associated stably through non-covalent interactions.
[0132] The term “functional”, in relation to the fusion proteins as disclosed herein (or variants, or a fragment or homolog thereof,), refers to the capability to display both IL-33 and IL-37 functionality. A functional assay for IL-33 and IL-37 is the lipopolysaccharide (LPS)- induced cytokine release (e.g., IL-1, IL-6, ILS, TNFa) in whole blood. [0133] The term “gene” refers to a DNA sequence comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. a mRNA) in a cell, operably linked to suitable regulatory regions (e.g., a promoter). A gene may thus comprise several operably linked sequences, such as a promoter, a 5' leader sequence comprising e.g., sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA), introns, and a 3' nontranslated sequence comprising e.g., transcription termination sites.
[0134] A “3' UTR” or “3' non-translated sequence” (also often referred to as 3' untranslated region, or 3' end) refers to the nucleic acid sequence found downstream of the coding sequence of a gene, which comprises for example a transcription termination site and (in most, but not all eukaryotic mRNAs) a polyadenylation signal (such as e.g., AAUAAA or variants thereof). After termination of transcription, the mRNA transcript may be cleaved downstream of the polyadenylation signal and a poly(A) tail may be added, which is involved in the transport of the mRNA to the cytoplasm (where translation takes place).
[0135] The term “expression of a gene” refers to the process wherein a DNA region, which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide (or active peptide fragment). “Expression of a polypeptide” additionally refers to a process wherein an mRNA is translated into a protein product, which may or may not be secreted.
[0136] A “transcription regulatory sequence” is herein defined as a nucleic acid sequence that is capable of regulating the rate of transcription of a (coding) sequence operably linked to the transcription regulatory sequence. A transcription regulatory sequence as herein defined will thus comprise all sequence elements necessary for initiation of transcription (promoter elements), for maintaining and for regulating transcription, including e.g. attenuators or enhancers.
Although this term usually refers to upstream (5') transcription regulatory sequences of a coding sequence, regulatory sequences found downstream (3') of a coding sequence are also encompassed by this definition.
[0137] As used herein, the term “promoter” refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of ordinary skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A “constitutive” promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An “inducible” promoter is a promoter that is physiologically (e.g. by external application of certain compounds) or developmentally regulated. A “tissue specific” promoter is only active in specific types of tissues or cells.
[0138] As used herein, the term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid sequence is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter, or rather a transcription regulatory sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous.
[0139] A “nucleic acid construct” or “vector” is herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology and which is used to deliver exogenous DNA into a host cell. Vectors usually comprise further genetic elements to facilitate their use in molecular cloning, such as e.g., selectable markers, multiple cloning sites and the like (see below).
[0140] “Stringent hybridization conditions” can be used to identify nucleotide sequences, which are substantially identical to a given nucleotide sequence. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequences at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be chosen in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least 60° C. Lowering the salt concentration and/or increasing the temperature increases stringency. Stringent conditions for RNA-DNA hybridization (Northern blots using a probe of e.g. 100 nt) are for example those which include at least one wash in 0.2xSSC at 63 °C for 20 min, or equivalent conditions. Stringent conditions for DNA-DNA hybridization (Southern blots using a probe of e.g. 100 nt) are for example those which include at least one wash (usually 2) in 0.2xSSC at a temperature of at least 50 °C, usually about 55 °C, for 20 min, or equivalent conditions. See also Sambrook et al. (1989) and Sambrook and Russell (2001).
[0141] “Sequence identity” and “sequence similarity” can be determined by alignment of two peptides or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as “substantially identical” or “essentially similar” when they (when optimally aligned by for example the programs GAP or BEST FIT using default parameters) share at least a certain minimal percentage of sequence identity (as defined below). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Generally, the GAP default parameters are used, with a gap creation penalty=50 (nucleotides)/8 (proteins) and gap extension penalty=3 (nucleotides)/2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif. 92121-3752 USA, or Emboss Win version 2.10.0 (using the program “needle”). Alternatively, percent similarity or identity may be determined by searching against databases, using algorithms such as PASTA, BEAST, etc. Preferably, the sequence identity refers to the sequence identity over the entire length of the sequence.
[0142] A “host cell” or a “recombinant host cell” or “transformed cell” are terms referring to a new individual cell (or organism) arising as a result of at least one nucleic acid molecule, especially comprising a nucleic acid molecule encoding a desired protein. The host cell is preferably a plant cell or a bacterial cell. The host cell may contain a nucleic acid molecule or a vector as disclosed herein as an extra-chromosomally (episomal) replicating molecule, or more preferably, comprises a nucleic acid molecule or a vector, as disclosed herein, integrated in the genome of the host cell.
[0143] The term “selectable marker” is used herein to describe any genetic entity which, when expressed, can be used to select for a cell or cells containing the selectable marker. Selectable marker gene products confer for example antibiotic resistance or nutritional requirements.
[0144] The term “biological half-life” refers to the time required for the amount of a substance, e.g., protein, in a biological system, e.g., the circulation within the animal or human body, to be reduced to one half of its value by biological processes such as renal clearance and degradation.
[0145] Embodiments described herein as “comprising” one or more features may also be considered as disclosure of the corresponding embodiments “consisting of’ and/or “consisting essentially of’ such features. In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. It also encompasses the more limiting verb “to consist of’. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”. It is further understood that, when referring to “sequences” herein, generally the actual physical molecules with a certain sequence of subunits (e.g. amino acids) are referred to.
[0146] As used herein, “and/or” means that either or both of the items separated by such terminology are involved. For example, the phrase “A and/or B” would mean A alone, B alone, or both A and B .
[0147] The present inventors have now devised a novel “single molecule therapy”. Specifically, the inventors provide a fusion protein comprising an IL-33 protein and an IL-37 protein, optionally physically fused together via a linker. Particularly, certain fusion proteins were found to have a superior biological activity (e.g. inhibits TNFa and IL-1) over their individual counterparts, i.e., IL-33 and IL-37 separately. Specifically, it was found that the certain fusion proteins were significantly larger (-35 kD) than the individual cytokines (both <20 kD). The present inventors also found that the fusion protein itself forms dimers (via the IL-37 portion of the fusion protein), thus providing a fusion protein with an even larger molecular weight (-70 kD). Such increases in molecular weight, and consequently the molecular radius, not only significantly prolongs the biological half-life of the IL-33-IL-37 fusion protein in the circulation compared to the individual cytokines, but also increases its bioavailability at the site of inflammation to an unprecedented level. Certain fusion proteins can also greatly lengthen the therapeutic time window for synergetic effects between IL-33 and IL-37 to occur, since the fusion protein delivers both cytokines at the site of inflammation, where they can both exert their actions for an equal amount of time in each other's presence. Furthermore, it was found that certain fusion proteins also exert a dual therapeutic action. Specifically, the IL-33-IL-37 fusion protein was shown to act as an anti-inflammatory agent when administered systematically while it acted as an anti-hyperalgesia agent when administered intrathecally.
[0148] Also provided herein are nucleic acid sequences and amino acid sequences corresponding to the fusion proteins disclosed herein. Certain fusion proteins disclosed herein display IL-33 activity. Certain fusion proteins disclosed herein display IL-37 activity.
[0149] In one aspect, a fusion protein comprising IL-33 or a variant or fragment or homolog thereof and IL-37 or a variant or fragment or homolog thereof is provided. The fusion protein can comprise an IL-33 protein, or a variant or fragment or homolog thereof. The IL-33 protein is preferably a mammalian IL-33 protein, such as a human IL-33, or mouse IL-33. An amino acid sequence of IL-33 is set forth in SEQ ID NO: 1. Variants of IL-33 include, for example, proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more, such as 100%, amino acid sequence identity to SEQ ID NO: 1, preferably over the entire length. Amino acid sequence identity is preferably determined by pairwise alignment using the Needleman and Wunsch algorithm and GAP default parameters as defined above. Variants also include proteins having IL-33 activity, which have been derived, by way of one or more amino acid substitutions, deletions or insertions, from the polypeptide having the amino acid sequence of SEQ ID NO: 1. Preferably, such proteins comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 amino acid substitutions, deletions or insertions.
[0150] The fusion protein can further comprise an IL-37 protein, or a variant or fragment or homolog thereof. The IL-37 protein is preferably a mammalian IL-37 protein, such as a human IL-37, or mouse IL-37. An amino acid sequence representing IL-37 is set forth in SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10. Variants of IL-37 include, for example, proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more, such as 100%, amino acid sequence identity to SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, preferably over the entire length. Amino acid sequence identity is preferably determined by pairwise alignment using the Nccdlcman and Wunsch algorithm and GAP default parameters as defined above. Variants also include proteins having IL-37 activity, which have been derived, by way of one or more amino acid substitutions, deletions or insertions, from the polypeptide having the amino acid sequence of SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10. Preferably, such proteins comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, amino acid substitutions, deletions or insertions.
[0151] The IL-33 and IL-37 moieties in a fusion protein disclosed herein may or may not be connected by a linker. Additional amino acid sequences may be present at the N- and/or C- terminus of the fusion protein, e.g., to facilitate purification. For example, a histidine-tag may be present at the C- or N-terminus to facilitate purification. Alternatively, a fusion protein disclosed herein may optionally comprise additional protein moieties, such as moieties capable of targeting, e.g., a protein moiety comprising one or more antibody Fe regions.
[0152] The IL-33 moiety may be located at the N-terminal of the IL-37 moiety, or may be located at the C-terminal of the IL-37 moiety. In a preferred embodiment, the IL-33 moiety is located at the N-terminal of the IL-37 moiety. It was found that the latter fusion protein displayed higher specific activity compared to an IL-33-IL-37 fusion protein in which the IL-33 moiety was located at the C-terminal of the IL-37 moiety (data not shown).
[0153] The IL-33-IL-37 fusion protein may be present in its monomeric form, in which case it has a formula weight of about 35 kDa, or it may be a dimeric IL-33-IL-37 fusion protein, i.e., two IL-33-IL-37 fusion proteins may be associated with one another non-covalently, in which case it has a formula weight of about 70 kDa.
[0154] In an embodiment, a fusion protein disclosed herein consists essentially of IL-33 and IL-37, optionally linked by a linker.
[0155] In an embodiment, both the IL-33 and the IL-37 moieties within certain fusion proteins disclosed herein are active and able to signal cells to downregulate the production of at least one inflammatory cytokine or mediator such as IL-1, IL-6, IL-8, TNFa. Preferably, at least TNFa, IL-6, and IL-8 are downregulated. [0156] In an embodiment, the fusion protein inhibits the generation of cytokines such as TNFa, IL-1, IL-6, IL-8 and other inflammatory cytokines while they induce secretion of inhibitory molecules such as IL-1 receptor antagonist and soluble TNF receptors by inflammatory and other cells stimulated by endotoxin, other Toll-like receptor (TLR) agonists, or other stimuli.
[0157] Certain fusion proteins disclosed herein inhibit the expression of adhesion molecules by inflammatory, endothelial, and other cells stimulated by agonists including endotoxin, other TLR agonists, and others.
[0158] In an embodiment, the fusion protein inhibits the expression of tissue factor by endothelial, inflammatory, and other cells stimulated by endotoxin, other TLR agonists, or other stimuli.
[0159] Certain fusion proteins disclosed herein inhibit the generation of oxygen radicals by inflammatory and other cells stimulated by endotoxin, other TLR agonists, or other stimuli.
[0160] In an embodiment, the fusion protein inhibits activity of IFN-y- and IL-17-secreting Thl and Th-17 cells and induce or sustain FoxP3-expressing suppressive T cells (CD25+), TGF- P-secreting Th2, Tri and Th3 cells generated in vitro in the presence or absence of antigen- presenting cells stimulated by self or non-self antigens, superantigens including Staphylococcus enterotoxin B (SEB) or mitogens such including CD3, CD28, phytohemagglutinin (PHA) or phorbol myristate acetate (PM A).
[0161] In an embodiment, the fusion proteins inhibit the expression of activating Fey receptors while inducing the expression of inhibitory Fey receptors preventing activation of cells such as monocytes, macrophages, and dendritic cells by IgG-containing immune complexes.
[0162] In an embodiment, a fusion protein disclosed herein is present in a homodimeric form. In an embodiment, a fusion protein disclosed herein is present in a heterodimeric form.
[0163] In one embodiment, a fusion protein disclosed herein has a molecular weight of above 60 kDa.
[0164] Certain fusion proteins disclosed herein may be prepared by techniques known to the skilled person in the art. For example, certain fusion proteins may be prepared using a technique which produces recombinant fusion proteins by continuous cell lines in culture. For example, certain fusion proteins can be produced in a host cell transfectoma using a combination of recombinant DNA techniques and gene transfection methods.
[0165] Also provided herein is an isolated nucleic acid sequence that encodes for a fusion protein disclosed herein, such as cDNA, genomic DNA, and RNA sequences. Due to the degeneracy of the genetic code, multiple nucleic acid sequences may encode the same amino acid sequence. Nucleic acid sequences encoding a fusion protein disclosed herein are herein referred to as “1L-33-1L-37 fusion protein encoding nucleic acid sequences”. The nucleic acid sequences provided include recombinant, artificial or synthetic nucleic acid sequences. It is understood that when sequences are depicted as DNA sequences while RNA is referred to, the actual base sequence of the RNA molecule is identical with the difference that thymine (T) is replaced by uracil (U). Certain nucleic acid sequences disclosed herein are particularly useful for expression of a IL-33-IL-37 fusion protein disclosed herein, either for production of the protein or for gene therapy purposes.
[0166] For example, to express a fusion protein disclosed herein, a nucleic acid molecule whose sequence encodes the fusion protein can be prepared by standard molecular’ biology techniques. In some embodiments, the nucleic acid molecule is preferably operably linked to transcription regulatory sequences such as a promoter, and optionally a 3' untranslated region. The nucleic acid molecule may be inserted into a vector, such as an expression vector, such that the genes are operatively linked to transcriptional and translational control sequences. The expression vector and transcription regulatory sequences are selected to be compatible with the expression host cell used. The nucleic acid molecule may be inserted into the expression vector by methods known in the art. The nucleic acid molecule or vector may further include a nucleotide sequence encoding a signal peptide, which may facilitate secretion of the fusion protein from the host cell. A nucleotide sequence encoding a signal peptide may be operably linked to the nucleic acid molecule encoding the fusion protein. Preferably, the signal peptide is located at the amino terminus of the fusion protein and as such, the nucleotide sequence encoding the signal peptide may be located 5' of the nucleic acid molecule encoding the fusion protein. The signal peptide may be a cytokine signal peptide or a signal peptide from a noncytokine protein. The promoter may be constitutive or inducible. The vector may comprise a selectable marker for selection of a vector carrying host cell. The vector may comprise an origin of replication when the vector is a replicable vector. [0167] Certain fusion proteins disclosed herein may be synthesized de novo by chemical synthesis (using e.g. a peptide synthesizer such as supplied by Applied Biosystems), or may be produced by recombinant host cells by expressing a nucleic acid sequence encoding the fusion protein, fragment or variant. Variants and fragments are preferably functional, i.e., have IL-33 and/or IL-37 activity, preferably IL-33 and IL-37 activity.
[0168] The anti-inflammatory activity and thus functionality of IL-33 and IL-37, as well as the 1L-33-1L-37 fusion protein can be determined using routine methods. For example, a suitable assay for functionality of IL-33 and IL-37, as well as the IL-33-IL-37 fusion protein, is the lipopolysaccharide (LPS) induced cytokine release (IL- 10, IL-6, ILS, TNFa) in whole blood.
[0169] A nucleic acid sequence, particularly a DNA sequence, encoding a fusion protein disclosed herein, can be inserted in expression vectors to produce (high amounts of) the fusion protein. Suitable vectors include, without limitation, linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and the like. Non-limiting examples of a viral vector include a retrovirus, an adenovirus, and an adeno-associated virus. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, {e.g., the adenovirus major late promoter (AdMLP)) and polyoma. Alternatively, nonviral regulatory sequences may be used, such as the ubiquitin promoter.
[0170] In addition to the nucleic acid molecules encoding IL-33-IL-37 fusion proteins and regulatory sequences, certain recombinant expression vectors disclosed herein may carry additional sequences, such as sequences that regulate replication of the vector in host cells {e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. No. 4,399,216, U.S. Pat. No. 4,634,665 and U.S. Pat. No. 5,179,017, all to Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, in a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection). Finally, the recombinant expression vector may contain a gene that codes for a glycosyl transferase in addition to a nucleic acid sequence encoding a fusion protein disclosed herein.
[0171] Also provided herein is a host cell comprising a nucleic acid sequence, a nucleic acid construct, or vector comprising a nucleic acid sequence disclosed herein. The host cell may be any host cell. The host cell may be selected from prokaryotic and eukaryotic cells. The host cell may also be a cell line, such as a prokaryotic or eukaryotic cell line. The host cell is preferably an animal cell or cell line, such as a mammalian cell or cell line.
[0172] Also provided is a method for expression in eukaryotic cells of a fusion protein disclosed herein. In certain embodiments, the eukaryotic cells are mammalian host cells. Preferred mammalian host cells for expressing recombinant IL-33-IL-37 fusion proteins include CHO cells (including dhfr-CHO cells, described in (Urlaub et al., 1980), used with a DHFR selectable marker, NS/0 myeloma cells, COS cells, HEK293 cells and SP2.0 cells. When recombinant expression vectors comprising nucleic acid sequences encoding IL-33-IL-37 fusion proteins are introduced into mammalian host cells, certain fusion proteins as disclosed herein may be produced by culturing the host cells for a period of time sufficient to allow for expression of the fusion proteins in the host cells or, more preferably, secretion of the fusion proteins into the culture medium in which the host cells are grown. Fusion proteins may be recovered from the culture medium in which the host cells are grown and/or may be purified from the culture medium using standard protein purification methods.
[0173] Alternatively, a nucleic acid molecule whose sequence encodes a fusion protein disclosed herein can be expressed in other expression systems, including prokaryotic cells, such as microorganisms, e.g. E. coli, algae, as well as insect cells. Furthermore, certain fusion proteins disclosed herein can be produced in transgenic non-human animals, such as in milk from sheep and rabbits or eggs from hens, or in transgenic plants.
[0174] Introduction of nucleic acid sequences into a host cell may be carried out by any standard technique known in the art. For expression of fusion proteins, the expression vector(s) encoding the fusion protein may be transfected into a host cell by standard techniques. The various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium phosphate precipitation, DEAE-dextran transfection, lipofectamine transfection, and freeze-dry method transfection, and the like. Cell lines that secrete certain fusion proteins disclosed herein can be identified by assaying culture supernatants for the presence of the fusion protein. The preferred screening procedure comprises two sequential steps, the first being identification of cell lines that secrete the fusion protein, the second being determination of the quality of the fusion protein such as the ability of the fusion protein to inhibit cytokine production by blood cells stimulated with LPS or other Toll-like receptor agonists, glycosylation patterns, and others.
[0175] For optimal expression in a host cell, a fusion protein encoding nucleic acid sequence can be codon optimized by adapting the codon usage to that most preferred in host cell genes. Several techniques for modifying the codon usage to that preferred by the host cells are known in the art. The exact method of codon usage modification is not critical.
[0176] Also provided herein are PCR primers and/or probes and kits for detecting the fusion protein encoding DNA or RNA sequences. Degenerate or specific PCR primer pairs to amplify fusion protein encoding DNA from samples can be synthesized (see Dieffenbach and Dveksler (1995) PCR Primer; A Laboratory Manual, Cold Spring Harbor Laboratory Press, and McPherson at al. (2000) PCR-Basics: From Background to Bench, First Edition, Springer Verlag, Germany). For example, any stretch of 9, 10, 11, 12, 13, 14, 15, 16, 18 or more contiguous nucleotides of a fusion protein encoding nucleic acid sequence, or its complement strand, may be used as primer or probe. Likewise, DNA fragments of a fusion protein encoding nucleic acid sequence can be used as hybridization probes. A detection kit for a fusion protein encoding nucleic acid sequence may comprise primers specific for a fusion protein encoding nucleic acid sequence and/or probes specific for a fusion protein encoding nucleic acid sequence, and an associated protocol to use the primers or probes to detect specifically a fusion protein encoding nucleic acid sequence in a sample. Such a detection kit may, for example, be used to determine whether a host cell has been transformed with a specific fusion protein encoding nucleic acid sequence. Because of the degeneracy of the genetic code, some amino acid codons can be replaced by others without changing the amino acid sequence of the protein.
[0177] Also disclosed herein is a method for producing a fusion protein as disclosed herein, the method comprising the steps of: culturing a host cell under conditions permitting the production of the fusion protein, and optionally, recovering the fusion protein.
[0178] A person skilled in the art will be capable of selecting conditions known in the art for production of a fusion protein as disclosed herein. Additionally, a person skilled in the art will be capable of recovering the fusion protein produced using methods known in the ail, which include, without limitation, chromatographic methods (including, without limitation, size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, metal binding, and the like), immunoprecipitation, HPLC, ultracentrifugation, precipitation and differential solubilisation, and extraction. Recovery or purification of a fusion protein may be facilitated by adding, for example, a Histidine-tag to the fusion protein.
Pharmaceutical Compositions
[0179] Provided is a pharmaceutical composition comprising: a fusion protein or a recombinant protein as disclosed herein, and a pharmaceutically acceptable carrier.
[0180] The term “pharmaceutically acceptable carrier” relates to carriers or excipients which are inherently nontoxic and nontherapeutic. Examples of such excipients are, but are not limited to, saline, Ringer's solution, dextrose solution and Hank's solution. Non-aqueous excipients such as fixed oils and ethyl oleate may also be used. A preferred excipient is 5% dextrose in saline. The excipient may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, including buffers and preservatives. A “pharmaceutically acceptable carrier” can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption delaying agents, and the like that are physiologically compatible.
[0181] The pharmaceutical composition may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques (e.g., as described in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995). [0182] In some embodiments the pharmaceutically acceptable carrier includes physiologically acceptable diluents, excipients, solvents, or adjuvants. Examples of suitable carriers include, but are not limited to, water, normal saline, dextrose, mannitol, lactose or other sugars, lecithin, albumin, sodium glutamate, cysteine hydrochloride, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), vegetable oils (such as olive oil), injectable organic esters such as ethyl oleate, ethoxylated isosteraryl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methahydroxide, bentonite, kaolin, agar- agar and tragacanth, or mixtures of these substances, and the like.
[0183] Certain pharmaceutical compositions disclosed herein further comprise a pharmaceutically acceptable carrier including physiologically acceptable diluents, excipients, solvents, adjuvants or formulating agents that can be used to prepare the composition into desired administrable formulations such as solid, tablets, capsules, semi-solid, gel, and so on.
[0184] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the ail. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in a pharmaceutical composition as disclosed herein is contemplated. Preferably, the carrier is suitable for parenteral administration, e.g. intravenous or subcutaneous injection or infusion.
[0185] The pharmaceutical composition may be administered by any suitable routes and modes. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.
[0186] The pharmaceutical composition may be formulated in accordance with routine procedures for administration by any routes, such as oral, topical, parenteral, sublingual, transdermal, or by inhalation. The pharmaceutical composition may be in the form of tablets, capsules, powders, granules, lozenges, creams, or liquid preparations, such as oral or sterile parenteral solutions or suspensions, or in the form of sprays, aerosols or other conventional methods for inhalation.
[0187] The pharmaceutical composition may be suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. [0188] In an embodiment, the pharmaceutical composition is administered parenterally.
[0189] The phrases “parenteral administration” and “administered parenterally” as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular-, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
[0190] In an embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion. In another embodiment, the pharmaceutical composition is administered by slow continuous infusion over a long period, such as more than 24 hours, thereby reducing deleterious or toxic side effects.
[0191] In an embodiment, a fusion protein disclosed herein is administered in crystalline form by subcutaneous injection.
[0192] Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions disclosed herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
[0193] In some embodiments, the pharmaceutical composition further comprises at least one biologically active peptide.
[0194] In some embodiments, the pharmaceutical composition further comprises another therapeutic agent.
[0195] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable salt. [0196] In some embodiments, the pharmaceutical composition further comprises an inorganic acid, or a salt thereof. In some embodiments, the inorganic acid is chosen from hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid.
[0197] In some embodiments, the pharmaceutical composition further comprises an organic acid, or a salt thereof. In some embodiments, the organic acid is chosen from formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, anthranilic acid, cinnamic acid, naphthalenesulfonic acid, and sulfanilic acid.
[0198] In another embodiment the pharmaceutical composition may contains additional supporting pharmaceutical agents which includes, but not limited to one or more of the following excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. In yet another embodiment the composition can be made either as liquid solutions or suspensions, solid forms.
[0199] Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
[0200] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients e.g. as enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients e.g. from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. [0201] Depending on the route of administration, the active compound, z.e., the fusion protein, may be coated in a material to protect it from the action of acids and other natural conditions that may inactivate the compound. For example, the compound may be administered to a subject in an appropriate carrier, for example, liposomes. Liposomes include water-in-oil-in- water CGF emulsions as well as conventional liposomes (Strejan et al., 1984).
[0202] Certain fusion proteins disclosed herein may be prepared with carriers that can protect against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, poly anhydrides, polyglycolic acid, collagen, polyorthoesters, and poly(lactic acid). Methods for the preparation of such formulations are generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
[0203] Dosage regimens may be adjusted to provide the optimum desired response e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage regiment for a pharmaceutical composition disclosed herein are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular’ therapeutic effect to be achieved, and (b) the limitations inherent in the ait of compounding such an active compound for the treatment of sensitivity in individuals.
[0204] Actual dosage levels of a fusion protein in a certain pharmaceutical composition as disclosed herein may be varied so as to obtain an amount of the fusion protein which is effective (“effective amount”) to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors, including the activity of the particular composition employed, the route of administration, the time of administration, the rate of excretion, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
[0205] In one embodiment, a fusion protein disclosed herein can be administered via intravenous injection or infusion. In another embodiment, the fusion protein is administered by slow continuous infusion over a long period, such as more than 24 hours, thereby reducing deleterious or toxic side effects.
[0206] In yet another embodiment, a fusion protein disclosed herein can be administered as maintenance therapy, such as, e.g., once a week for a period of 6 months or more.
[0207] Also provided herein is a pharmaceutical composition comprising: a fusion protein as disclosed herein, one or more additional anti-inflammatory agents, and a pharmaceutically acceptable carrier.
[0208] In some embodiments, at least one of the one or more additional anti-inflammatory agents is a protein. In some embodiments, at least one of the one or more additional antiinflammatory agents is a monoclonal antibody. In some embodiments, the monoclonal antibody is chosen from Actemra, Adakveo, Alzumab, Antova, Avastin, Benlysta, Beovu, Bosatria, Cablivi, Campath, Cimzia, Cinqair, Cosentyx, Dupixent, Enspryng, Entyvio, Evenity, Fasenra, Gamifant, Hemlibra, Humira, Haris, Ilumya, Kevzara, Lemtrada, Lucentis, MabThera, Ocrevus, Prolia, Remicade, Rituxan, RoActemra, Siliq, Simponi, Skyrizi, Soliris, Stelara, Taltz, Tepezza, Tremfya, Trogarzo, Tysabri, Ultomiris, Uplizna, Xolair and Zenapax.
[0209] In some embodiments, at least one of the one or more additional anti-inflammatory agents is a natural anti-inflammatory therapeutic agent. In some embodiments, the natural antiinflammatory therapeutic agent is chosen from the group consisting of apigenin, luteolin, baicalein, resveratrol, rutin, catechin, and curcumin, or a combination thereof.
[0210] In some embodiments, at least one of the one or more additional anti-inflammatory agents is a chemical anti-inflammatory therapeutic agent. In some embodiments, the chemical anti-inflammatory therapeutic agents is chosen from the group consisting of of diclofenac, piroxicam, nimcsulidc, kctoprofcn, ibuprofen, sulindac, oxaprozin, indomethacin, mcloxicam, fenoprofen, flurbiprofen, mefenamic acid, ketorolac, and methyl salicylate, or a pharmaceutically acceptable salt thereof.
[0211] In some embodiments, at least one of the one or more additional anti-inflammatory agents is a steroidal chemical anti-inflammatory therapeutic agent.
[0212] In some embodiments, at least one of the one or more additional anti-inflammatory agents is a non-steroidal chemical anti-inflammatory therapeutic agent.
[0213] In some embodiments, the pharmaceutical composition comprises two or more additional anti-inflammatory agents.
[0214] Also provided herein is a pharmaceutical composition comprising: a fusion protein as disclosed herein, one or more anti-inflammatory proteins, and a pharmaceutically acceptable carrier.
[0215] In some embodiments, the pharmaceutical composition comprises two or more antiinflammatory proteins.
Therapeutic Uses
[0216] Also provided herein is a fusion protein as disclosed herein, or a pharmaceutical composition thereof, for use as a medicament.
[0217] Also provided herein is a fusion protein as disclosed herein, for use in the manufacture of a medicament.
[0218] Also provided herein is a fusion protein as disclosed herein, or a pharmaceutical composition thereof, for use in the treatment or prevention of a condition, disease, or disorder.
[0219] In an embodiment, the condition is selected from the group consisting of: sepsis, adult respiratory distress syndrome, allo- and xenotransplantation, dermatitis, inflammatory bowel disease, sarcoidosis, allergies, psoriasis, ankylosing spondylarthritis, autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, glomerolonephritis, immune complex- induced and other forms of vasculitis, multiple sclerosis, Sjogren’s disease, gout, lymphoprolifcraticvc diseases such as non-Hodgkin lymphoma and B cell chronic lymphocytic leukemia, bum injuries, multiple trauma, stroke, myocardial infarction, atherosclerosis, diabetes mellitus, extracorporeal dialysis and blood oxygenation, ischemia-reperfusion injury (IRI), toxicity induced by the in vivo administration of cytokines or therapeutic monoclonal antibodies, chronic pain syndrome, and neuropathic and/or inflammatory pain.
[0220] In an embodiment, the condition is characterized by pain and may be selected from inflammatory pain and neuropathic pain.
[0221] In an embodiment, the condition is characterized by local or systemic inflammation, immune activation, and/or lymphoproliferation.
[0222] In an embodiment, inhibition of the production of pro-inflammatory cytokines and other inflammatory mediators has a beneficial effect on the condition, disease, or disorder.
[0223] In an embodiment, the condition, disease, or disorder is an inflammatory disease mediated by the production of proinflammatory cytokines such as TNF, IL-1, IL-6, and/or other inflammatory mediators.
[0224] Also provided herein is a fusion protein as disclosed herein, or a pharmaceutical composition thereof, for use in the treatment or prevention of a clinical condition in a mammal, such as a human, for which administration of IL-33 is indicated.
[0225] Also provided herein is a fusion protein as disclosed herein, or a pharmaceutical composition thereof, for use in the treatment or prevention of a clinical condition in a mammal, such as a human, for which administration of IL-37 is indicated.
[0226] Also provided herein is a fusion protein as disclosed herein, or a pharmaceutical composition thereof, for use in the treatment or prevention of osteoarthritis or chronic pain.
[0227] Also provided herein is a fusion protein as disclosed herein, or a pharmaceutical composition thereof, for use in the treatment or prevention of osteoarthritis. Particularly, it was found that certain fusion proteins disclosed herein have a cartilage protective activity. Therefore, certain fusion proteins disclosed herein may be used for prevention and treatment of cartilage breakdown, particularly in osteoarthritis. Additionally, it was found in a canine osteoarthritis model that dogs given a certain fusion protein disclosed herein experienced significantly less pain as compared to dogs not given the fusion protein. As such, certain fusion proteins disclosed herein may be particularly useful for treatment or prevention of osteoarthritis (treatment or prevention of cartilage degradation) with its associated chronic pain.
[0228] Also provided herein is a fusion protein as disclosed herein, or a pharmaceutical composition thereof, for use in the treatment or prevention of a neurodegenerative disease.
[0229] In some embodiments, the neurodegenerative disease is selected from the group consisting of Alexander's disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis, Ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, chronic fatigue syndrome, Chronic Traumatic Encephalopathy, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt- Jakob disease, frontotemporal dementia, Gerstmann-Straussler-Scheinker syndrome, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe's disease, Kuru, Lewy body dementia, Machado-Joseph disease (Spinocerebellar ataxia type 3), Multiple sclerosis, Multiple System Atrophy, myalgic encephalomyelitis, Narcolepsy, Neuroborreliosis, Parkinson's disease, Pelizaeus-Merzbacher Disease, Pick's disease, Primary lateral sclerosis, Prion diseases, Refsum's disease, Sandhoffs disease, Schilder's disease, Subacute combined degeneration of spinal cord secondary to Pernicious Anaemia, Schizophrenia, Spinocerebellar ataxia (multiple types with varying characteristics), Spinal muscular atrophy, Steele-Richardson- Olszewski disease, progressive supranuclear palsy, and Tabes dorsalis.
[0230] Also provided herein is a fusion protein as disclosed herein, or a pharmaceutical composition thereof, for use in the treatment or prevention of an autoimmune disease.
[0231] In some embodiments, the autoimmune disease is selected from the group consisting of Acute Disseminated Encephalomyelitis (ADEM), Achalasia, Acquired hemophilia, Acute motor axonal neuropathy, Addison's Disease, Adiposis Dolorosa, Adult-Onset Still's Disease, Agammaglobulinemia, Alopecia Areata, Amyloidosis, Amyotrophic Lateral Sclerosis (ALS), Angioedema, Ankylosing spondylitis, Anti- Glomerular Basement Membrane Nephritis, Antineutrophil cytoplasmic antibody-associated vasculitis, Anti-N-Methyl-D-Aspartate Receptor Encephalitis, Antiphospholipid syndrome, Antisynthetase Syndrome, Anti-Tubular Basement Membrane Nephritis, Aplastic Anemia, Atopic Allergy, Atopic Dermatitis, Autoimmune angioedema, Autoimmune dysautonomia, Autoimmune encephalitis, Autoimmune encephalomyelitis, Autoimmune enteropathy, Autoimmune hemolytic anemia, Autoimmune hemophilia, Autoimmune hepatitis. Autoimmune inner car disease (AIED), Autoimmune lymphoproliferative syndrome, Autoimmune myocarditis, Autoimmune neutropenia, Autoimmune oophoritis, Autoimmune orchitis, Autoimmune pancreatitis, Autoimmune poly endocrine syndrome type 1, Autoimmune poly endocrine syndrome type 2, Autoimmune polyendocrine syndrome type 3, Autoimmune progesterone dermatitis, Autoimmune retinopathy, Autoimmune thrombocytopenic purpura, Autoimmune thyroiditis, Autoimmune urticaria, Autoimmune uveitis, Axonal & neuronal neuropathy (AMAN), Balo Concentric Sclerosis, Bald disease, Behcet's Disease, Benign mucosal pemphigoid, Bickerstaffs Encephalitis, Blau Syndrome, Bullous pemphigoid, Cancer, Castleman Disease (CD), Celiac disease, Chagas disease, Chronic Fatigue Syndrome, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic Obstructive Pulmonary Disease, Chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss syndrome, Churg-Strauss Syndrome (CSS) or Eosinophilic Granulomatosis (EGPA), Cicatricial pemphigoid, Clostridium difficile, Coeliac Disease, Cogan syndrome, Cogan’s syndrome, Cold Agglutinin Disease, Complement Component 2 Deficiency, Complex Regional Pain Syndrome, Congenital Heart Block, Contact Dermatitis, Coxsackie myocarditis, CREST syndrome, Crohn's Disease, Cushing's Syndrome, Cutaneous Leukocytoclastic Angiitis, Dego's Disease, Dercum’s disease, Dermatitis Herpetiformis, Dermatomyositis, Devic’s disease (neuromyelitis optica), Diabetes Mellitus Type 1, Diffuse interstitial keratitis, Discoid lupus, Discoid Lupus Erythematosus, Dressier’s syndrome, Drug- Induced Lupus, Eczema, Emia, Endometriosis, Enteropathy, Enthesitis-Related Arthritis, Eosinophilic Esophagitis, Eosinophilic fasciitis, Eosinophilic Gastroenteritis, Eosinophilic Granulomatosis With Polyangiitis (Egpa), Eosinophilic Pneumonia, Epidermolysis Bullosa Acquisita, Erythema Nodosum, Erythroblastosis Fetalis, Essential Mixed Cryoglobulinemia, Evans Syndrome, Felty Syndrome, Fibromyalgia, Fibrosing alveolitis, Gastritis, Gastrointestinal Pemphigoid, Gestational Pemphigoid, Giant Cell Arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture’s syndrome, Granulomatosis With Polyangiitis (Gpa), Graves' Disease, Graves Ophthalmopathy, Guillain-Barre Syndrome, Hashimoto's Thyroiditis, Hashimoto's Encephalopathy, Hemolytic Anemia, Henoch-Schonlein purpura (HSP), Hepatitis, Herpes gestationis or pemphigoid gestationis (PG), Hidradenitis Suppurativa (HS) (Acne Inversa), Hypogammalglobulinemia, Idiopathic dilated cardiomyopathy, Idiopathic Giant-Cell Myocarditis, Idiopathic Inflammatory Demyelinating Diseases, Idiopathic Pulmonary Fibrosis, Iga Nephropathy, Iga Vasculitis (Igav), Igg4-Rclatcd Disease, IgG4-rclatcd sclerosing disease, IgG4-related systemic disease, Immune thrombocytopenic purpura (ITP), Inclusion Body Myositis, Inflammatory bowel disease (IBD), Inner Ear Disease (IED), Intermediate Uveitis, Interstitial Cystitis, Interstitial Lung Disease, IPEX Syndrome, Juvenile Arthritis, Juvenile diabetes (Type 1 diabetes), Juvenile myositis (JM), Kawasaki's Disease, Lambert-Eaton Myasthenic Syndrome, Lambert-Eaton syndrome, Leukocytoclastic Vasculitis, Lichen Planus, Lichen Sclerosus, Ligneous Conjunctivitis, Lineal’ IgA disease (LAD), Lupus, Lupus Nephritis, Lupus Vasculitis, Lyme Disease (Chronic), Lymphoproliferative Syndrome, Majeed Syndrome, Meniere's Disease, Microscopic Colitis, Microscopic polyangiitis (MPA), Mixed connective tissue disease (MCTD), Mooren's Ulcer, Morphea, Mucha-Habermann Disease, Multifocal Motor Neuropathy (MMN) or MMNCB, Multiple sclerosis, Myasthenia Gravis, Myelin oligodendrocyte glycoprotein disease (MOG), Myocarditis, Myositis, Narcolepsy, Neonatal Lupus, Neuromyelitis Optica, Neuromyotonia, Neutropenia, Ocular cicatricial pemphigoid, Oophoritis, Opsoclonus Myoclonus Syndrome, Optic Neuritis, Orchitis, Ord's Thyroiditis, Oshtoran Syndrome, Palindromic Rheumatism, Pancreatitis (AIP), PANDAS, Paraneoplastic cerebellar degeneration, Paraneoplastic cerebellar degeneration (PCD), Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg Syndrome, Pars planitis (peripheral uveitis), Parsonage- Turner syndrome, Pediatric Autoimmune Neuropsychiatric Disorder Associated with Streptococcus, Pediatric Neuropsychiatric Disorder Associated With Streptococcus (Pandas), Pemphigus, Pemphigus Vulgaris, Peripheral Neuropathy, Perivenous encephalomyelitis, Pernicious Anemia, Pityriasis Lichenoides Et Varioliformis Acuta, POEMS syndrome, Polyarteritis Nodosa (Pan), Polyendocrine Syndrome (Aps) Type 1, Polyendocrine Syndrome (Aps) Type 2, Polyendocrine Syndrome (Aps) Type 3, Polymyalgia Rheumatica, Polymyositis, Postmyocardial Infarction Syndrome, Postpericardiotomy Syndrome, Primary Biliary Cirrhosis (Pbc), Primary Sclerosing Cholangitis, Progesterone dermatitis, Progressive Inflammatory Neuropathy, Psoriasis, Psoriatic arthritis, Pure red cell aplasia (PRCA), Pyoderma Gangrenosum, Rasmussen's Encephalitis, Raynaud's Phenomenon, Reactive Arthritis, Reflex sympathetic dystrophy, Relapsing polychondritis, Restless Leg Syndrome, Retinopathy, Retroperitoneal Fibrosis, Rheumatic Fever, Rheumatoid Arthritis, Rheumatoid Vasculitis, Sarcoidosis, Schizophrenia, Schnitzler Syndrome, Scleritis, Scleroderma, Serum Sickness, Sjogren's Syndrome, Sperm & testicular autoimmunity, Spondyloarthropathy, Stiff person syndrome (SPS), Subacute bacterial endocarditis (SBE), Susac's Syndrome, Sweet's Syndrome, Sydenham Chorea, Sympathetic Ophthalmia, Systemic Lupus Erythematosus (SLE), Systemic Scleroderma, Takayasu's Arteritis, Temporal arteritis/Giant cell arteritis, Thrombocytopenia, Thrombocytopenic Purpura, Thyroiditis, Tolosa-Hunt syndrome (THS), Transverse Myelitis, Type 1 diabetes, Ulcerative Colitis, Undifferentiated connective tissue disease (UCTD), Undifferentiated Spondyloarthropathy, Urticaria, Urticarial Vasculitis, Uveitis, Vasculitis, Vitiligo, Vogt-Koyanagi-Harada Disease, Warm autoimmune, and hemolytic anemia
Methods of treatment
[0232] Also provided herein is a method for the treatment or prevention of a condition, disease, or disorder, the method comprising the step of administration of an effective amount of a fusion protein as disclosed herein, or a pharmaceutical composition thereof, to a person in need thereof.
[0233] In an embodiment, the condition is selected from the group consisting of: sepsis, adult respiratory distress syndrome, allo- and xenotransplantation, dermatitis, inflammatory bowel disease, sarcoidosis, allergies, psoriasis, ankylosing spondylarthitis, autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, glomerolonephritis, immune complex- induced and other forms of vasculitis, multiple sclerosis, Sjogren’s disease, gout, lymphoproliferatieve diseases such as non-Hodgkin lymphoma and B cell chronic lymphocytic leukemia, bum injuries, multiple trauma, stroke, myocardial infarction, atherosclerosis, diabetes mellitus, extracorporeal dialysis and blood oxygenation, ischemia-reperfusion injury (IRI), toxicity induced by the in vivo administration of cytokines or therapeutic monoclonal antibodies, chronic pain syndrome, and neuropathic and/or inflammatory pain.
[0234] In an embodiment, the condition is characterized by pain and may be selected from inflammatory pain and neuropathic pain.
[0235] In an embodiment, the condition is characterized by local or systemic inflammation, immune activation, and/or lymphoproliferation.
[0236] In an embodiment, inhibition of the production of pro-inflammatory cytokines and other inflammatory mediators has a beneficial effect on the condition, disease, or disorder. [0237] In an embodiment, the condition, disease, or disorder is an inflammatory disease mediated by the production of proinflammatory cytokines such as TNF, IL- 1 , IL-6, and/or other inflammatory mediators.
[0238] In an embodiment, administration of IL-33 is indicated for treatment or prevention of the condition, disease, or disorder.
[0239] In an embodiment, administration of IL-37 is indicated for treatment or prevention of the condition, disease, or disorder.
[0240] Also provided herein is a method for the treatment or prevention of osteoarthritis or chronic pain, the method comprising the step of administration of an effective amount of a fusion protein as disclosed herein, or a pharmaceutical composition thereof, to a person in need thereof.
[0241] Also provided herein is a method for the treatment or prevention of osteoarthritis, the method comprising the step of administration of an effective amount of a fusion protein as disclosed herein, or a pharmaceutical composition thereof, to a person in need thereof.
[0242] Also provided herein is a method for the treatment or prevention of a neurodegenerative disease, the method comprising the step of administration of an effective amount of a fusion protein as disclosed herein, or a pharmaceutical composition thereof, to a person in need thereof.
[0243] In some embodiments, the neurodegenerative disease is selected from the group consisting of Alexander's disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis, Ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, chronic fatigue syndrome, Chronic Traumatic Encephalopathy, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, frontotemporal dementia, Gerstmann-Straussler-Scheinker syndrome, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe's disease, Kuru, Lewy body dementia, Machado-Joseph disease (Spinocerebellar ataxia type 3), Multiple sclerosis, Multiple System Atrophy, myalgic encephalomyelitis, Narcolepsy, Neuroborreliosis, Parkinson's disease, Pelizaeus-Merzbacher Disease, Pick's disease, Primary lateral sclerosis, Prion diseases, Refsum's disease, Sandhoffs disease, Schilder's disease, Subacute combined degeneration of spinal cord secondary to Pernicious Anaemia, Schizophrenia, Spinocerebellar ataxia (multiple types with varying characteristics), Spinal muscular atrophy, Steele-Richardson- Olszcwski disease, progressive supranuclear palsy, and Tabes dorsalis.
[0244] Also provided is a method for the treatment of a condition, disease, or disorder, the method comprising the steps of: administering a fusion protein as disclosed herein, and administering an additional pharmaceutical agent.
[0245] Treatment (prophylactic or therapeutic) may consist of parenteral administration of a fusion protein disclosed herein, or a pharmaceutical composition thereof. In some embodiments, the the fusion protein or pharmaceutical composition is administered intravenously, intramuscularly, intrathecally, epidurally, spinally, or subcutaneously. However, other administration routes as set forth above with respect to pharmaceutical compositions comprising the fusion proteins recited above may also be employed. The dose and administration regimen may depend on the extent of inhibition of the production and release of inflammatory cytokines aimed at. Typically, the amount of the fusion protein given will be in the range of about 0.5 pg to about 1 mg per kg of body weight. In one aspect, the dosage is from about 10 pg/kg to about 500 pg/kg body weight. In another aspect, the dosage is from about 20 pg/kg to about 100 pg/kg body weight. In a further aspect, the disage is from about 30 pg/kg to about 50 pg/kg body weight. The dosage can be determined or adjusted by measuring the amount of circulating cytokine (IL-37, IL-33) upon administration in a biological sample.
[0246] For parenteral administration, the fusion protein is preferably formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle. Such vehicles are well-known in the art and examples include saline, dextrose solution, Ringer’s solution, and solutions containing small amounts of human serum albumin. Typically, the fusion proteins of disclosed herein may be formulated in such vehicles at a concentration of from about 50 pg to about 100 mg per mL. In some embodiments, the fusion protein is administered by intravenous injection.
[0247] Further administration details are set forth above in the section relating to pharmaceutical compositions comprising fusion proteins disclosed herein. [0248] Also provided herein is a method for attenuating an inflammatory reaction in a cell, the method comprising the step of contacting the cell the cell with a fusion protein as disclosed herein, thereby inhibiting the release of cytokines and other inflammatory mediators by the cell. In some embodiments, the method is performed on a cell in vivo.
[0249] Also provided herein is a method for the preparation of a pharmaceutical composition for attenuating an inflammatory reaction in a cell, the method comprising the step of combining a fusion protein as disclosed herein with a pharmaceutically acceptable carrier.
[0250] Also provided is a method for inhibiting the production and/or release of cytokines or other inflammatory mediators by a cell, the method comprising the step of contacting the cell with a fusion protein as disclosed herein.
[0251] In some embodiments, the cell is chosen from a macrophage, a monocyte, and a T- lymphocyte.
Gene Therapy
[0252] Also provided herein is a gene therapy agent comprising a nucleic acid construct or vector as disclosed herein, for the treatment of a condition, disease, or disorder set forth herein. In one embodiment, adeno-associated viruses are used as gene therapy vectors.
[0253] In one embodiment, the gene therapy agent can be used for the treatment or prevention of an osteoarthritis or chronic pain.
[0254] In one embodiment, the gene therapy agent can be used for the treatment or prevention of a condition characterized by local or systemic inflammation, immune activation, and/or lymphoproliferation, preferably wherein the condition characterized by local or systemic inflammation, immune activation, and/or lymphoproliferation is selected from the group consisting of: sepsis, adult respiratory distress syndrome, allo- and xenotransplantation, dermatitis, inflammatory bowel disease, sarcoidosis, allergies, psoriasis, ankylosing spondylarthitis, autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, glomerolonephritis, immune complex-induced and other forms of vasculitis, multiple sclerosis, Sjogren's disease, gout, lymphoproliferatieve diseases such as non-Hodgkin lymphoma and B cell chronic lymphocytic leukemia, bum injuries, multiple trauma, stroke, myocardial infarction, atherosclerosis, diabetes mellitus, extracorporeal dialysis and blood oxygenation, ischemia-reperfusion injury (TRI), and toxicity induced by the in vivo administration of cytokines or therapeutic monoclonal antibodies, chronic pain syndrome, and neuropathic and/or inflammatory pain.
Summary of Sequences
[0255] SEQ ID NO: 1. represents amino acid sequence for full length IL-33 (for 1-270)
[0256] SEQ ID NO: 2. represents amino acid sequence for IL-33 (for 95-270 fragment)
[0257] SEQ ID NO: 3. represents amino acid sequence for IL-33 fragment (for 107-270 fragment)
[0258] SEQ ID NO: 4. represents amino acid sequence for IL-33 fragment (for 109-270 fragment)
[0259] SEQ ID NO: 5. represents amino acid sequence for IL-33 fragment (for 112-270 fragment)
[0260] SEQ ID NO: 6. represents amino acid sequence for full length IL-37 (1-218).
[0261] SEQ ID NO: 7. represents amino acid sequence for IL-37 fragment having length of
192 residues.
[0262] SEQ ID NO: 8 represents amino acid sequence IL-37 fragment having length of 178 residues.
[0263] SEQ ID NO: 9 represents amino acid sequence for IL-37 fragment having length of 197 residues.
[0264] SEQ ID NO: 10. represents amino acid sequence for IL-37 fragment having length of 157 residues.
[0265] SEQ ID NO: 11 represents amino acid sequence of the peptide linker.
[0266] Also provided herein is a recombinant protein comprising: a first domain, comprising interleukin-33 (IL-33) or a fragment, variant or homolog thereof, and a second domain, comprising an anti-inflammatory cytokine or a fragment, variant or homolog thereof. [0267] In some embodiments, the first domain binds with the anti-inflammatory cytokine receptor.
[0268] In some embodiments, the second domain binds with the IL-33 receptor.
[0269] In some embodiments, the recombinant protein further comprises a third domain, comprising a linker.
[0270] In some embodiments, the recombinant protein comprises at least one of the amino acid sequences from SEQ ID NO: 1-5 for first domain or a biologically active substantially homologous sequence.
[0271] Also provided herein is a nucleic acid encoding a recombinant protein disclosed herein, wherein the recombinant protein is a fusion of full-length IL-33, or a fragment, variant or homolog thereof, and an anti-inflammatory cytokine, or a fragment, variant or homolog thereof.
[0272] Also provided herein is a recombinant protein encoded by an isolated nucleic acid as disclosed herein.
[0273] Also provided herein is an isolated nucleic acid comprising a nucleic acid sequence encoding a recombinant peptide having the nucleotide sequence of IL-33, or a fragment, variant or homolog thereof, or IL-37, or a fragment, variant or homolog thereof.
[0274] In some embodiments the isolated nucleic acid comprises the a nucleic acid encoding the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.
[0275] Also provided herein is a method for producing a protein comprising the steps of: introducing a target isolated nucleic acid sequence of IL-33 into one or more vectors along with a promoter introduced in one or more recombinant host cell to express a full length IL-33 protein, wherein the full length IL-33 may be cleaved into one or more fragments selected from the group consisting of amino acid residues 95-270 (SEQ ID NO: 2), 107- 270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4) or 112-270 (SEQ ID NO: 5) of IL-33, by mast cell enzymes like chymase and tryptases; introducing a target sequence of an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, into one or more vectors along with a promoter introduced in one or more recombinant host cell to express the antiinflammatory cytokine, or a fragment, variant or homolog thereof, optionally linking the full length IL-33, or a fragment, variant or homolog thereof, with another anti-inflammatory cytokine, or a fragment, variant or homolog thereof, using an optional linker, culturing the host cell under suitable conditions to allow the production of a desired recombinant protein, recovering the produced proteins from the culture of the host cell, and optionally purifying the recovered protein particles.
[0276] Also provided herein is a vector that comprises: an isolated nucleic acid sequence encoding a recombinant protein as disclosed herein, and a promoter sequence for initiation of transcription.
[0277] In some embodiments, the vector is selected from the group consisting of a bacterial vector, a viral vector, and a mammalian vector.
[0278] Also provided herein is a recombinant host cell comprising: an isolated nucleic acid as disclosed herein and a vector that contains the isolated nucleic acid.
[0279] Also provided herein is an antibody which is capable of binding a recombinant IL-33 fragment attached to an anti-inflammatory cytokine or fragment, variant or homolog thereof (IL- 33/anti-inflammatory cytokine). The antibody will be used for detection and purification of IL- 33/anti-inflammatory cytokine, or a fragment, variant or homolog thereof, via a cleavable linker.
[0280] Also provided herein is a transgenic non-human mammal comprising an isolated nucleic acid encoding IL-33/ anti-inflammatory cytokine recombinant protein, or a fragment, variant or homolog thereof. [0281] Also provided herein is a method of administering recombinant protein IL-33/anti- inflammatory cytokine, or a fragment, variant or homolog thereof, or a combination of interleukin- 33 (IL-33), or a fragment, variant or homolog thereof, and anti-inflammatory cytokine proteins, that is administered to a subject in need thereof at a dosage ranging from about 0.01 pg to 1000 pg per kg of body weight.
[0282] Also provided herein are compositions and methods for making recombinant peptides and isolated nucleic acids comprising sequences encoding the peptides.
[0283] Also provided is a pharmaceutical composition comprising a recombinant protein IL- 33/anti-inflammatory cytokine or a combination of IL-33 and an anti-inflammatory cytokine protein, or a fragment, variant or homolog thereof, which is administered at least twice daily. In yet another embodiment, the pharmaceutical composition is administered five times daily.
[0284] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.
[0285] In some embodiments, the composition further comprises an additional therapeutic agent.
[0286] Also provided herein is a kit, comprising one or more of the following: one or more of the recombinant proteins as disclosed herein, an isolated nucleic acid comprising a sequence encoding a recombinant protein as disclosed herein, one or more cytokines (IL-33 and an anti-inflammatory cytokine), and a pharmaceutical composition optionally comprising a pharmaceutically acceptable carrier.
[0287] In some embodiments, the kit further comprises one or more containers. In some embodiments, the kit further comprises one or more applicators. In some embodiments, the kit further comprises instructional material for the use of the kit.
[0288] As disclosed herein, the use of IL-33, or a fragment, variant or homolog thereof, and an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, which includes but not limited to IL-37 has potential to skew the immune response towards Th2 type and have ability to turn the microenvironment towards anti-inflammatory side, which ultimately can help in treating, preventing, and alleviating ncurodcgcncrativc and autoimmune disorders.
[0289] Aspects of the materials and methods disclosed herein are best understood by reference to the description set forth herein. All the aspects described herein will be better appreciated and understood when considered in conjunction with the following descriptions. It should be understood, however, that the following descriptions, while indicating preferred aspects and numerous specific details thereof, are given by way of illustration only and should not be treated as limitations. Changes and modifications may be made within the scope herein without departing from the spirit and scope thereof, and the materials and methods disclosed herein include all such modifications.
[0290] Several aspects of materials and methods contemplated with this disclosure are disclosed herein. It is to be understood that these aspects may or may not overlap with one another. Thus, part of one aspect may fall within the scope of another aspect, and vice versa. Each aspect is illustrated by a number of embodiments, each of which in turn, can include one or more specific embodiments. It is to be understood that the embodiments may or may not overlap with each other. Thus, part of one embodiment, or specific embodiments thereof, may or may not fall within the ambit of another, or specific embodiments thereof, and vice versa.
[0291] A broad framework of the principles will be presented by describing various embodiments of the disclosure using exemplary aspects. The terms “one embodiment” or “an embodiment” means that a particular feature, structure, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. For clarity and ease of description, each aspect includes only a few embodiments. Different embodiments from different aspects may be combined or practiced separately, to design a customized process or product depending upon application requirements. Many different combinations and sub-combinations of a few representative processes or structures shown within the broad framework of this disclosure, that may be apparent to those skilled in the art but not explicitly shown or described, should not be construed as precluded.
[0292] Provided herein are compositions and related methods for producing a recombinant protein for treating, preventing, and alleviating a neurodegenerative and autoimmune disorders. [0293] In one embodiment, the water-soluble polymers comprise poly(alkylene glycol), poly(olcfinic alcohol), poly(vinylpyrrolidonc), poly(hydroxylalkyl-mcthacrylamidc), poly(hydroxyalkyl-methacrylate), poly(a-hydroxy acid), poly(acrylic acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), and copolymers or terpolymers thereof.
[0294] Also provided herein is a pharmaceutical composition for treating, preventing, and alleviating neurodegenerative and autoimmune disorders comprising: a first domain comprising interleukin-33 (IL-33), or a fragment, variant or homolog thereof, a second domain comprising an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, and an optional linker that creates a linkage between the IL-33, or a fragment, variant or homolog thereof, and the anti-inflammatory cytokine, or a fragment, variant or homolog thereof.
[0295] In a preferrable embodiment, the IL-33 fragment comprises any one of the amino acid sequences selected from the group consisting of residues 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4) and 112-270 (SEQ ID NO: 5) of IL-33. In some embodiments, the fragment, variant or homolog of IL-33 is processed and obtained using one or more mast cell protease enzymes, including but not limited to chymase and tryptase.
[0296] In some embodiments, the second domain comprises a fragment of IL-37. In some embodiments, the sequence of the fragment of IL-37 comprises SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.
[0297] According to one embodiment, the first domain is directly linked with the antiinflammatory cytokine, or fragment, variant or homolog thereof, without an intervening nonpeptide linker. According to another embodiment, the first domain is linked with the second domain, via one or more linkers.
[0298] In one embodiment, the IL-33 is linked with the anti-inflammatory cytokine, or a fragment, variant or homolog thereof. Referring to FIG. 1, the composition comprising IL-33, or a fragment, variant or homolog thereof, is linked with the anti-inflammatory cytokine, or a fragment, variant or homolog thereof, through a linker (X). In one embodiment, the IL-33, or fragment, variant or homolog thereof, and the anti-inflammatory cytokine, or fragment, variant or homolog thereof, are directly linked or fused without using a linker.
[0299] In another embodiment, the IL-33, or fragment, variant or homolog thereof, is coadministered with the anti-inflammatory cytokine, or a fragment, variant or homolog thereof.
[0300] In another embodiment, the IL-33, or fragment, variant or homolog thereof, and the anti-inflammatory cytokine, or fragment, valiant or homolog thereof, are linked with a water- soluble polymer. FIG. 2 illustrates an exemplary embodiment depicting IL-33, or fragment, variant or homolog thereof, which is modified with the attachment of a water-soluble polymer through an -NH- bond. The IL-37, or fragment, variant or homolog thereof, is modified with the attachment of a water-soluble polymer through an -NH- bond. The modified IL-33, or fragment, variant or homolog thereof, is further attached with the modified IL-37, or fragment, variant or homolog thereof, through one or more linkers.
[0301] In another embodiment, the IL-33 (SEQ ID NO: 1), or a fragment of IL-33 selected from the group consisting of residues 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3) 109- 270 (SEQ ID NO: 4), and 112-270 (SEQ ID NO: 5) of IL-33, is fused with the anti-inflammatory cytokine, or a fragment, variant or homolog thereof. In case of fusion, the sequences of two cytokines are directly fused together without linkers.
[0302] In another embodiment the IL-33 (SEQ ID NO: 1) or a fragment of IL-33 selected from the group consisting of residues 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109- 270 (SEQ ID NO: 4) or 112-270 (SEQ ID NO: 5) of IL-33, is conjugated with the antiinflammatory cytokine, or a fragment, variant or homolog thereof. In case of conjugation, the sequences of two cytokines are fused together with peptide or non-peptide linkers.
[0303] In another embodiment, the IL-33 (SEQ ID NO: 1), or a fragment of IL-33 selected from the group consisting of residues 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109- 270 (SEQ ID NO: 4) or 112-270 (SEQ ID NO: 5) of IL-33, is co-administered with the antiinflammatory cytokine, or a fragment, variant or homolog thereof.
[0304] Also provided is a recombinant protein comprising: a first domain comprising IL-33, or a fragment, variant or homolog thereof, and a second domain comprising an anti-inflammatory cytokine, or a fragment, variant or homolog thereof.
[0305] In some embodiments, the recombinant protein further comprises a third domain, comprising a linker.
[0306] In some embodiments, the first domain comprises a fragment having a sequence chosen from SEQ ID NO: 1-5.
[0307] In some embodiments, the anti-inflammatory cytokine is IL-37, or a fragment, variant or homolog thereof. In some embodiments, the second domain comprises a fragment, variant or homolog of IL-37. In some embodiments, the sequence of the fragment of IL-37, or a fragment, variant or homolog thereof, is given by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.
[0308] In some embodiments, the second domain comprises a fragment having a sequence chosen from SEQ ID NO: 6-10.
[0309] In some embodiments, the recombinant protein further comprises a fourth domain having a cytokine receptor targeting moiety or a cellular internalization moiety.
[0310] In one embodiment, the IL-33, or fragment, variant or homolog thereof, is PEGylated at specific sites to enhance stability and half-life of the anti-inflammatory cytokine.
[0311] The linker can be chosen from the group consisting of a peptide linker, direct linkage of two proteins, covalent linkers preferably non hydrolysable, PEGylated, polyethylene glycol (PEG), organic linkers, water-soluble organic linkers, water-soluble polymers, Fc fusion proteins, Fc albumin fusion proteins, a peptide sequence, a peptidase with a cleaving site, a bifunctional linker, a polyglycine linker, carbohydrates, bridging factors, biostable and biodegradable polymers, diamino and or diacid units, natural or unnatural amino acids or derivatives thereof, as well as aliphatic moieties, including alkyl, aryl, heteroalkyl, heteroaryl, alkoxy, and other possible linkers that are well known in the ait. In one embodiment, the linker comprises a cleavage site which can be cleaved by human serum. In another embodiment, the linker comprises a cleavage site that can be cleaved by human cellular enzymes, preferably proteases and esterases. [0312] In some embodiments, the linker is a peptide linker. In a further embodiment, the peptide linker has the sequence of SEQ ID NO: 11, or a multiple thereof. In some embodiments, the linker is a non-peptide linker.
[0313] In some embodiments, the linker is a water-soluble polymer selected from the group consisting of poly(alkylene glycol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxylalkyl-methacrylamide), poly(hydroxyalkyl-methacrylate), poly(a-hydroxy acid), poly(acrylic acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, and poly(N- acryloylmorpholine), and copolymers and terpolymers thereof.In a preferred embodiment, the linker is poly(ethylene glycol). Other water-soluble polymers such as other poly (alkylene glycols), such as poly(propylene glycol) (“PPG”), copolymers of ethylene glycol and propylene glycol and the like, poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly (hydroxy alkylmethacrylate), poly(saccharides), polyphydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, poly(N- acryloylmorpholine) can also be used. The water-soluble polymer can be a homopolymer, copolymer, terpolymer, nonrandom block, and random block polymer of any of the foregoing.
[0314] In a preferrable embodiment, the linker is a Fc fusion Protein, where the Fc region of the protein (i.e., antibody) is directly linked to the anti-inflammatory protein.
[0315] In another preferrable embodiment, the linker is a glycosylated linker, where a carbohydrate is attached with anti-inflammatory proteins.
[0316] In another preferrable embodiment, the linker is an albumin, where the albumin is considered as a non-glycosylated and water-soluble protein. These proteins are soluble in concentrated salt solution.
[0317] In some embodiments, the linker is a PEGylated linker, where the linker is the covalent attachment of polyethylene glycol to the anti-inflammatory proteins by an incubation process.
[0318] In one embodiment, only one linker is used to link the two proteins, or a fragment, variant or homolog thereof. In another embodiment, two or more linkers are used to link two or more proteins, or a fragment, variant or homolog thereof. In another embodiment, multi-arm linkers are used to link multiple proteins, or a fragment, variant or homolog thereof, together. [0319] In one embodiment, a single IL-33, or fragment, variant or homolog thereof, is linked with multiple IL-37, or fragment, valiant or homolog thereof, through multi-arm polymer linkers. In another embodiment, the single IL-37, or fragment, variant or homolog thereof, is linked with multiple IL-33, or fragment, variant or homolog thereof, through multi-arm polymer linkers.
[0320] In one embodiment, multiple moieties are attached to provide synergism between the molecules.
[0321] The linkage and activity of certain pharmaceutical compositions disclosed herein is explained below in detail. This information is provided for illustrative purposes only, and is not intended to limit the scope of the claimed subject-matter.
[0322] Referring to FIG. 3, the composition comprising the glycosylated or non-glycosylatcd IL-33, or fragment, variant or homolog thereof, and the glycosylated or non-glycosylated antiinflammatory cytokine, or fragment, variant or homolog thereof, are linked using a water-soluble polymer. In one embodiment, the linker is a water-soluble organic linker (e.g., PEG) which forms a water-soluble conjugate between the IL-33, or fragment, variant or homolog thereof, and the anti-inflammatory cytokine, or fragment or homolofragment, variant or homolog g thereof. The link may be formed via a condensation reaction, including but not limited to condensation of an aldehyde with an amine, condensation of a carboxylic acid, or derivative thereof, with an alcohol, and condensation of a carboxylic acid, or derivative thereof, with an amine. The resulting heterodimer, comprising IL-33, or fragment, variant or homolog thereof, IL-37, or fragment, variant or homolog thereof, and water-soluble polymer, remains intact over time, thus improving the pharmacokinetic property of the composition within the human system.
[0323] In some embodiments, a precursor to the water-soluble linker comprises an aldehyde group which reacts with an amino terminal of a cytokine, or a fragment, variant or homolog thereof, as disclosed herein (i.e., IL-33 or IL-37) to form an imine bond, which further undergoes reduction to form an amine bond. The reduction reaction takes place in the presence of compounds like sodium cyanoborohydride. The value of n in FIG. 3 can range between 1-10.
[0324] In some embodiments, the water-soluble polymer is selected from the group consisting of poly(alkylene glycol), poly(olefinic alcohol), poly (vinylpyrrolidone), poly(hydroxylalkyl-methacrylamide), poly(hydroxyalkyl-methacrylate), poly(a-hydroxy acid), poly(acrylic acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, poly(N- acryloylmorpholinc), and copolymers or terpolymers thereof.
[0325] In a preferred embodiment, poly(ethylene glycol), a water-soluble polymer is used. Other water-soluble polymers such as other polyfalkylene glycols), such as polypropylene glycol) (“PPG”), copolymers of ethylene glycol and propylene glycol and the like, poly(olefinic alcohol), poly (vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine) can also be used. The water- soluble polymer can be a homopolymer, copolymer, terpolymer, nonrandom block, and random block polymer of any of the foregoing.
[0326] Referring to FIG. 4, the linker for attaching IL-33, or a fragment, variant or homolog thereof, and an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, (IL-37) comprises water-soluble conjugate/polymer having multiple moieties. The water-soluble polymers with multiple moieties provide synergism of two moieties. In an embodiment, the water-soluble polymer comprises an N-hydroxy succinimide (NHS) group, which reacts with the amino terminus of a glycosylated or non-glycosylated cytokine (i.e., IL-33 or IL-37), or a fragment, valiant or homolog thereof. According to this exemplary embodiment, the water- soluble polymer additionally comprises an acidic group, such as a carboxyl group (A) attached to the NHS group, which undergoes reaction with the amino terminal to form a stable conjugate having IL-33, or a fragment, variant or homolog thereof, or IL-37, or a fragment, variant or homolog thereof, attached to the water-soluble polymer.
[0327] In some embodiments, the conjugate undergoes a further reaction to liberate the IL- 33, or a fragment, variant or homolog thereof, or IL-37, or a fragment, variant or homolog thereof. In this reaction, the acidic group (A) is labile and is also liberated separately from the water-soluble polymer, and the liberated acidic group (A) follows a prodrug approach. In some embodiments, the conjugate undergoes a reaction to liberate only IL-33, or a fragment, variant or homolog thereof, or IL-37, or a fragment, variant or homolog thereof, where the water-soluble polymer is attached with a stable acidic group (A). The acidic group (A) that is attached with the water-soluble polymer has a different type of CO group than that of the acidic group (A) which is liberated from the water-soluble polymer. [0328] Referring to FIG. 5, the linkage of two cytokines is depicted. Water-soluble polymer- I can be used as a linker between two cytokines, which can be the same or different.
[0329] In an embodiment, water-soluble polymer I, having different functional groups at its termini can be used to provide sequential coupling with two different cytokines. In an embodiment is provided a molecule having an IL-33, or a fragment, variant or homolog thereof, at one end of polymer I, and an IL-37, or a fragment, variant or homolog thereof, at the other end of polymer 1.
[0330] In some embodiments, polymer I has a molecular weight ranging between 500 to 1000 or less than 500. In some embodiments, polymer I is linked to the cytokines via cleavable linkers.
[0331] In some embodiments, one or more water-soluble organic polymers II can be incorporated into a molecule. The one or more polymers II can be incorporated into the first domain, comprising IL-33, or fragment, variant or homolog thereof. The one or more polymers II can be incorporated into the second domain, comprising the anti-inflammatory cytokine, or fragment, variant or homolog thereof.
[0332] The one or more polymers II can be incorporated before linkage between the first domain and the second domain.
[0333] Alternatively, as shown in FIG. 5, polymers II can be incorporated into either or both the first domain and the second domain subsequent to linkage. This can be accomplished by employing cytokines having one or more protected amino acid side chains, such as aminoprotected lysines. Subsequent to linking of the cytokines with polymer I, the side chains can be deprotected, using techniques known in the art, followed by reaction with a suitably activated derivative of polymer II. In some embodiments, the water-soluble polymer-I that links the first domain and the second domain, is smaller than water-soluble polymer-11 that is attached to either or both of the first domain and the second domain. In some embodiments, the molecular weight of the water-soluble polymer-II can range between 10 kDa to 20kDa. Water-soluble polymer-II can act as a stabilizing agent, thereby further regulating the bio distribution and/or pKa value.
[0334] FIG. 5 depicts a heterodimer comprising two different groups (z.e., X and Y) attached to the water-soluble polymer-I and the first and second domains, which can assist in the sequential release of the second domain and the first domain. For example, the initial release of the second domain from the conjugate can provide greater access to tissue due to the reduction and oxidative stress caused by the second domain, so that the first domain remains attached to the water-soluble polymer longer, thereby maintaining the second domain in an inactive form for a longer period.
[0335] Referring to FIG. 6, a mixture comprising the first domain and the second domain, both having unprotected amino groups, can be reacted with aldehyde groups of the linker. A person of skill will appreciate that a mixture of the desired heterodimer and the two homodimers can be formed under these conditions.
[0336] The values of the integers in FIG. 6 can range as follows.
[0337] In some embodiments, 1 is from 1 to 5000, preferably from 5 to 1000, more preferably from 5 to 200, yet more preferably from 5 to 100, even yet more preferably from 5 to 20. In some embodiments, m is between 1 and 20, preferably between 1 and 10, more preferably between 1 and 5. In even more preferred embodiments, m is chosen from 1 and 2. In some embodiments, n is between 1 and 20, preferably between 1 and 10, more preferably between 1 and 5. In even more preferred embodiments, n is chosen from 1 and 2.
[0338] A stepwise reaction sequence may facilitate synthesis of the desired heterodimer, comprising the first domain, the second domain, and a linker. A linker precursor is provided which has two termini, one of which is linked to a protecting group, and one of which remains unprotected and therefore available for reaction. The unprotected terminus may consist of e.g. an aldehyde.
[0339] In a first step, the linker precursor, is allowed to react with a first molecule intended for combination in the heterodimer (/'.<?., either the IL-33, or fragment, variant or homolog thereof, or the anti-inflammatory protein, or fragment, variant or homolog thereof), or a suitably activated and/or protected derivative. The first molecule reacts with the unprotected terminus of the linker precursor, such as by a reductive amination process known in the art.
[0340] In a second step, the resulting first molecule / linker precursor conjugate is reacted with a reagent suitable for removing the protecting group on the linker precursor. [0341] Optionally, intervening steps are performed to activate this terminus for chemical reaction. These steps will be familial' to those skilled in the art.
[0342] In a third step, the first molecule / linker precursor conjugate is allowed to react with a second molecule intended for combination in the heterodimer (z.e., either the anti-inflammatory protein, or fragment, valiant or homolog thereof, or the IL-33, or fragment, variant or homolog thereof), or a suitably activated and/or protected derivative.
[0343] Optional subsequent steps, known to those skilled in the art, may be performed to remove any remaining protecting groups on the first molecule and/or second molecule.
[0344] The linker between the first domain and the second domain may undergo non-specific cleavage in vivo to release either or both domains from the linker. In some embodiments, the non-specific cleavage is accomplished with an esterase enzyme.
[0345] FIG. 7 depicts an alternative mechanism for selective release of one or both of the first domain and the second domain. A heterodimer containing an ester linkage is prepared, using techniques disclosed herein and known in the art. One or both of the esterase linkages can be cleaved in vivo, severing the linkage between the first and second domains. The remaining imine moiety can be hydrolyzed in vivo, affording the free amine.
[0346] The values of the integers in FIG. 7 can range as follows.
[0347] In some embodiments, 1 is from 1 to 5000, preferably from 5 to 1000, more preferably from 5 to 200, yet more preferably from 5 to 100, even yet more preferably from 5 to 20. In some embodiments, m is between 1 and 20, preferably between 1 and 10, more preferably between 1 and 5. In even more preferred embodiments, m is chosen from 1 and 2. In some embodiments, n is between 1 and 20, preferably between 1 and 10, more preferably between 1 and 5. In even more preferred embodiments, n is chosen from 1 and 2.
[0348] In one embodiment, the composition for treating, preventing, and alleviating neurodegenerative and autoimmune disorders comprises a recombinant peptide comprising: a first domain, comprising IL-33, or a fragment, variant or homolog thereof, and a second domain, comprising an anti-inflammatory cytokine, or a fragment, variant or homolog thereof. [0349] In some embodiments, the anti-inflammatory cytokine is IL-37 or a fragment, variant or homolog thereof.
[0350] In some embodiments, the recombinant peptide further comprises a third domain, comprising a linker.
[0351] In some embodiments, the recombinant peptide further comprises a fourth domain comprising a cytokine receptor targeting moiety.
[0352] In some embodiments, the recombinant peptide further comprises a fourth domain comprising a cellular internalization moiety.
[0353] Also provided herein is a method for producing a protein comprising the steps of: (A) introducing a target isolated nucleic acid sequence of IL-33 having SEQ ID NO: 11-15 into one or more vectors along with a promoter expressed in one or more recombinant host cell to express a full length IL-33 proteins and cleaving the full length IL-33 into its fragments using mast cell enzymes; (b) introducing a target sequence of an anti-inflammatory cytokine, or a fragment, valiant or homolog thereof, into one or more vectors along with a promoter expressed in one or more recombinant host cell to express an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, and optionally linking the full length IL-33, or a fragment, variant or homolog thereof, with the an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, using an optional linker; (c) culturing the host cell under suitable conditions to allow the production of a desired recombinant protein; (d) recovering the produced proteins from the culture of the host cell; and (e) optionally purifying the recovered protein particles.
[0354] In some embodiments, the anti-inflammatory cytokine, or a fragment, variant or homolog thereof, is IL-37, or a fragment, variant or homolog thereof, which has a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 6, 7, 8, 9 or 10.
[0355] According to one embodiment, the IL-33, or a fragment, variant or homolog thereof, is PEGylated at one or more specific site to increase the stability and half-life of the molecule. According to one embodiment, the full-length IL-33 is pre-cleaved to produce IL-33 fragments before linking with an anti-inflammatory cytokine, or a fragment, variant or homolog thereof.
[0356] Also provided herein is an isolated nucleic acid that comprises a sequence encoding a protein or peptide as disclosed herein. [0357] Also provided herein is a vector comprising a nucleic acid that encodes a protein or peptide as disclosed herein.
[0358] Also provided herein is a recombinant host cell comprising: an isolated nucleic acid as disclosed herein, and a vector as disclosed herein.
[0359] In yet another embodiment, the vector comprises a single copy of gene encoding IL- 33, or a fragment, variant or homolog thereof, and one or more copies of gene encoding the antiinflammatory cytokine, or a fragment, variant or homolog thereof, or vice versa. In one embodiment, the ratio of gene encoding IL-33 and an anti-inflammatory cytokine is 4:1 respectively or vice versa, especially 3:1 or vice versa, especially 2:1 or vice versa. In another embodiment, the IL-33 cell, and an anti-inflammatory cytokine are produced from different host cells, in which the two proteins are chemically linked or bridged using several linkers as disclosed herein.
[0360] In some embodiments, the method of obtaining an IL-33 fragment from full length IL-33 comprises the steps of: incubating the IL-33 with a human mast cell protease enzyme, thereby generating an IL-33 fragment
[0361] In some embodiments, the human mast cell protease enzyme is chymase. In some embodiments, the human mast cell protease enzyme is tryptase. In some embodiments, the IL-33 fragment consists of residues 109-270 of IL-33. In some embodiments, the IL-33 fragment consists of residues 107-270 of IL-33. In some embodiments, the IL-33 fragment consists of residues 95-270 of IL-33. In some embodiments, the IL-33 fragment consists of residues 72-270 of IL-33.
[0362] The IL-33, or fragment, variant or homolog thereof, can be further incubated with an anti-inflammatory cytokine and optional linkers to produce desired fusion protein.
[0363] The IL-33, or fragment, variant or homolog thereof, activates the group-2 innate lymphoid cells (ILC2s), which are involved in the initiation of allergic inflammation, in vivo. More specifically, the fragment of IL-33 having amino acid residues 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), and 112-270 (SEQ ID NO: 5) has 30-fold more potential than that of the full-length IL-33 for activation of ILC2s. The IL-33 fragments induce a strong expansion of ILC2s and eosinophils in vivo and associated with elevated concentrations of IL-5 and IL- 13.
[0364] In some embodiments, the IL-33, or fragment, variant or homolog thereof, is then PEGylated or linked with linkers to an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, thus creating a new molecular’ entity (NME), increasing stability and half-life.
[0365] In another embodiment, a peptide disclosed herein is modified with conservative amino acid changes, including insertions, deletions, and substitutions as long as it remains effective. The changes in amino acid can be made independently in each of IL-33m or fragment, variant or homolog thereof, and anti-inflammatory cytokine, or fragment, variant or homolog thereof, when they are being used in combination therapy. In yet another embodiment, a peptide disclosed herein is modified to improve its resistance to proteolytic cleavage, increase its stability or to optimize its solubility properties or to render it more suitable as a therapeutic agent. It will be readily apparent to the person skilled in the art how to make changes in a peptide without affecting its effectiveness.
[0366] In another embodiment, the half-life or longevity of a peptide as disclosed herein may be enhanced by adding adducts such as polyethylene glycol, peptide-IgG fusion proteins, cyclization, etc.
[0367] In some embodiments, IL-33, or a fragment, variant or homolog thereof, is attached to an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, with a peptide linker sequence. The peptide linker is cleavable by human serum proteases. In another embodiment, there is provided an antibody which binds recombinant IL-33 attached to an antiinflammatory cytokine via a cleavable linker. The antibody may be monoclonal or polyclonal. In an embodiment, the antibody may be used in affinity column for purification of a recombinant protein (IL-33/anti-inflammatory cytokines). Methods for the affinity purification of recombinant proteins using antibodies are well-known to those skilled in the art. The antibodies for detection of recombinant protein may be conjugated with reporter molecules which may be based on but not limited to radioisotope, chemiluminescent or bioluminescent molecules. It will be readily apparent to the person skilled in the art how to vary the procedure to suit the required purpose. [0368] Also provided herein is a pharmaceutical composition comprising recombinant protein IL-33/anti-inflammatory cytokine, or a fragment, variant or homolog thereof, attached to a vehicle via a cleavable linker to form a nanoconjugate. In one embodiment, the vehicle includes but not limited to nanoparticles, liposomes or any nanobots. In yet another embodiment, the vehicle comprises at least one targeting moiety for directing the cytokine nanoconjugates to the desired target tissues. The targeting moiety includes an antibody and antibody fragment. In some embodiments, the targeting moiety is an antibody fragment selected from the group consisting of scFv, a Fv, a Fab, a Fab', a F(ab)2, a bis-scFv, heavy-light chains. In another embodiment, the targeting moiety is a monoclonal antibody. In yet another embodiment, the targeting moiety is a polyclonal antibody. In yet another embodiment, the antibody is a nanobody, a single domain antibody, a minibody, a diabody, a triabody, or a tetrabody.
[0369] Also provided herein is a method for producing a recombinant protein comprising: a first domain, comprising IL-33, or a fragment, variant or homolog thereof, a second domain comprising an anti-inflammatory cytokine, or a fragment, variant or homolog thereof.
[0370] In some embodiments, the recombinant protein further comprises a third domain, comprising a linker.
[0371] In some embodiments, the recombinant protein further comprises a fourth domain comprising a cytokine receptor targeting moiety.
[0372] In some embodiments, the recombinant protein further comprises a fourth domain comprising a cellular internalization moiety.
[0373] Also provided herein is a method for producing a conjugated protein. The IL-33, or a fragment, variant or homolog thereof, is covalently attached with an anti-inflammatory cytokine, or a fragment or homofragment, variant or homolog log thereof, to produce a conjugated protein. The covalent attachment is non-hydrolysable in nature and the primary covalent attachment is established by a covalent linkage, including but not limited to a disulfide linkage, and other covalent linkages that are known in the art. [0374] Also provided herein is a fusion protein, where at least one amino acid sequence selected from SEQ ID NO: 1-5 is directly fused with an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, to produce a fusion protein.
[0375] Also provided herein is a method for producing individually IL-33, or a fragment, variant or homolog thereof, and anti-inflammatory cytokines, or a fragment, variant or homolog thereof, in which the two or more cytokines or its fragments are administered by co-administered means.
[0376] Also provided herein is a method for treating, preventing, and alleviating a neurodegenerative or autoimmune disorder the method comprising the step of administering an effective dose of a pharmaceutical composition comprising:
IL-33, or a fragment, variant or homolog thereof, and an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, to a person in need thereof.
[0377] In some embodiments, the anti-inflammatory cytokine is IL-37, or a fragment, variant or homolog thereof.
[0378] In some embodiments, the method comprises administration of IL-33 or antiinflammatory cytokine, or a fragment, variant or homolog thereof, at a dosage of about 0.01 pg to about 1000 pg per kg of body weight of a subject in need thereof. In general, the dosage and concentration depends upon the degree of severity and progression of the disease. In some embodiments, the dosage is about 200 pg or less per kg of body weight, optionally about 100 pg or less per kg of body weight, optionally about 25 pg or less per kg of body weight, optionally about 10 pg or less per kg of body weight. In some embodiments, the dosage is about 0.05 pg or greater per kg of body weight, optionally about 0.2 pg or greater per kg of body weight, optionally about 0.5 pg or greater per kg of body weight
[0379] Also provided herein is a method for treating, preventing, and alleviating a neurodegenerative or autoimmune disorder, the method comprising the step of administering an effective dose of a recombinant protein comprising: a first domain, comprising IL-33, or a fragment, variant or homolog thereof, and a second domain, comprising an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, to a person in need thereof.
[0380] In some embodiments, the method comprises administration of recombinant peptide IL-33/anti-inflammatory cytokine, or a fragment, variant or homolog thereof, at a dosage of about 0.01 pg to about 1000 pg per kg of body weight of a subject in need thereof. In general, the dosage and concentration of the recombinant peptide IL-33/anti-inflammatory cytokine, or fragment, variant or homolog thereof, depends upon the degree of severity and progression of the disease. In some embodiments, the dosage is about 200 pg or less per kg of body weight, optionally about about 100 pg or less per kg of body weight, optionally about 25 pg or less per kg of body weight, optionally about 10 pg or less per kg of body weight. In some embodiments, the dosage is about 0.05 pg or greater per kg of body weight, optionally about 0.2 pg or greater per kg of body weight, optionally about 0.5 pg or greater per kg of body weight.
[0381] In another embodiment, the method comprises administration of a recombinant peptide having multiple domains relating to IL-33/anti-inflammatory cytokine, or one or more fragments or homologs as disclosed herein, or a pharmaceutical composition thereof, to a person in need.
[0382] In some embodiments, the method comprises oral administration.
[0383] In some embodiments, the method comprises parenteral administration. In some embodiments, the parenteral administration is chosen from the group consisting of, intravenous, intrathecal, local, intramuscular, topical, intra-arterial, and intranasally.
[0384] In another embodiment, the method comprises administration of a fusion protein as disclosed herein at least once a day, or at least once a week, or at least once a month. In one embodiment, the method comprises administration of a fusion protein as disclosed herein at least twice a day, at least twice a week, or at least twice a month.
[0385] Also provided herein is a pharmaceutical composition comprising a fusion protein that is disclosed herein that is suitable for in vivo administration. The pharmaceutical composition comprises IL-33, or a fragment, variant or homolog thereof, and an antiinflammatory cytokine, or a fragment, variant or homolog thereof, or a recombinant peptide having multiple domains relating to IL-33/anti-inflammatory cytokine, or a fragment, variant or homolog thereof, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The recombinant peptide may be present in a pharmaceutical composition in an amount from 0.001 to 99.9 wt %, preferably from about 0.1 to 99.0 wt %, more preferably from about 1.0 to 90.0 wt %, yet more preferably from about 1.0 to 50.0 wt %, even yet more preferably from about 1.0 to 20.0 wt %.
[0386] In yet another embodiment the pharmaceutical composition comprises a nanoconjugate which comprises recombinant peptide IL-33/anti-inflammatory cytokine, or a fragment, variant or homolog thereof, is attached to the nanoparticle via a cleavable linker for administration.
[0387] In another embodiment the pharmaceutical composition comprises administration via but not limited to, intravenously, intrathecally, locally, intramuscularly, topically, orally, intraarterially, parenterally, intranasally etc.
[0388] In another embodiment, the pharmaceutical composition may be administered to an animal, more preferably human several times daily, as once a day, once a week, once every two weeks, once a every four week, or even less frequently, such as once every several months or even once a year or less.
[0389] In another embodiment, the dosage of the pharmaceutical composition administered to the animal, more preferably human in the range of about 0.01 pg to about 1000 pg per kg of body weight of the animal depending upon the factors such as age, frequency of administration, route of administration etc. In some embodiments, the dosage is about 200 pg or less per kg of body weight, optionally about 100 pg or less per kg of body weight, optionally about 25 pg or less per kg of body weight, optionally about 10 pg or less per kg of body weight. In some embodiments, the dosage is about 0.05 pg or greater per kg of body weight, optionally about 0.2 pg or greater per kg of body weight, optionally about 0.5 pg or greater per kg of body weight.
[0390] Also provided herein is a kit comprising one or more of the following: one or more peptides, isolated nucleic acids, expression vectors, a pharmaceutical composition and instruction material for administering the composition.
[0391] The kit may further comprise suitable solvent for suspending a pharmaceutical composition prior to administration. The kit may further comprise one or more applicator devices for administration of the composition. In one embodiment the applicator device can be a syringe. In yet another embodiment, the applicator is vibrating mesh nebulizer.
[0392] In some embodiments, the kit further comprises PCR primers for detecting the IL-33- IL-37 fusion protein encoding DNA or RNA sequences are provided. In some embodiments, the kits comprising probes for detecting the IL-33-IL-37 fusion protein encoding DNA or RNA sequences arc provided. For example, any stretch of 9, 10, 11, 12, 13, 14, 15, 16, 18 or more contiguous nucleotides of an IL-33-IL-37 fusion protein encoding nucleic acid sequence (or the complement strand) may be used as primer or probe. Likewise, DNA fragments of an IL-33-IL- 37 fusion protein encoding nucleic acid sequence can be used as hybridization probes. A detection kit for an IL-33-IL-37 fusion protein encoding nucleic acid sequence may comprise primers specific for an IL-33-IL-37 fusion protein encoding nucleic acid sequence and/or probes specific for an IL-33-IL-37 fusion protein encoding nucleic acid sequences, and an associated protocol to use the primers or probes to detect specifically IL-33-IL-37 fusion protein encoding nucleic acid sequence in a sample. Such a detection kit may, for example, be used to determine whether a host cell has been transformed with a specific IL-33-IL-37 fusion protein encoding nucleic acid sequence. Because of the degeneracy of the genetic code, some amino acid codons can be replaced by others without changing the amino acid sequence of the protein.
[0393] Also provided is a compound for treating, preventing, or alleviating neurodegenerative and auto-immune disorders, comprising interleukin-33 (IL-33) or a fragment, variant or homolog thereof. In some embodiments, the compound comprises IL-33, or a fragment, variant or homolog thereof. In some embodiments, the fragment of IL-33 is any one of the peptides selected from the group consisting of residues 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), or 112-270 (SEQ ID NO: 5) of IL-33. In some embodiments, the IL-33, or fragment, variant or homolog thereof, is PEGylated at one or more specific sites. [0394] Thus, the advantages of the invention include but are not limited to a novel composition, a compound, comprising PEGylatcd IL-33, or a fragment, variant or homolog thereof, for treating, preventing, and alleviating neurodegenerative and auto-immune disorders. Further, the materials and methods disclosed herein are generally related to a pharmaceutical composition comprising IL-33, or a fragment, variant or homolog thereof, and anti-inflammatory cytokines, or a fragment, variant or homolog thereof, with or without likers which are administered to a subject for treating, preventing, and alleviating neurodegenerative and autoimmune disorders.
[0395] In some embodiments, the novel compositions, IL-33, or fragment, variant or homolog thereof may be used to generate antibodies that may be used in diagnostic methods, such as ELISA tests or Western blots. In further embodiments, IL-33, or fragment, variant or homolog thereof may be used as a control.
[0396] In some embodiments, the novel compositions, IL-37, or fragment, variant or homolog thereof may be used to generate antibodies that may be used in diagnostic methods, such as ELISA tests or Western blots. In further embodiments, IL-37, or fragment, variant or homolog thereof may be used as a control.
[0397] In some embodiments, fusion proteins described herein may be used to generate antibodies that may be used in diagnostic methods, such as ELISA tests or Western blots. In further embodiments, fusion proteins described herein may be used as a control.
[0398] The following exemplary embodiments further describe optional aspects of the invention and are part of this Specification. These exemplary embodiments are set forth in a format substantially akin to claims (each with a numerical designation followed by a letter designation), although they are not technically claims of the present application. The following exemplary embodiments refer to each other in dependent relationships as “embodiments” instead of “claims.”
[0399] Embodiment 1A: A composition comprising an interleukin-33 (IL-33) (SEQ ID NO: 1), or a fragment, variant or homolog thereof, comprising at least one amino acid sequence selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109- 270 (SEQ ID NO: 4), and 112-270 (SEQ ID NO: 5); an anti-inflammatory cytokine, or fragment, variant or homolog thereof; and an optional linker for creating linkage between the IL-33, or a fragment, variant or homolog thereof, and the anti-inflammatory cytokine or fragment, variant or homolog thereof.
[0400] Embodiment 2A: The composition of Embodiment 1 A, wherein the linkage is at least one selected from the group comprising: a) the IL-33 or a fragment, variant or homolog thereof, is fused with the antiinflammatory cytokine, or a fragment, variant or homolog thereof; b) the IL-33 or a fragment, variant or homolog thereof, is conjugated with the antiinflammatory cytokine, or a fragment, variant or homolog thereof; c) the IL-33 or a fragment, variant or homolog thereof, is co-administered with the anti-inflammatory cytokine, or a fragment, variant or homolog thereof; and d) a recombinant protein comprising: a first domain comprising IL-33, or a fragment, variant or homolog thereof, comprising any one sequence from SEQ ID NO: 1-5, a second domain comprising the anti-inflammatory cytokine, or fragment, variant or homolog thereof, and a third domain comprising a linker.
[0401] Embodiment 3A: The composition of Embodiment 2A, wherein the recombinant protein further comprises a fourth domain having a cytokine receptor targeting moiety or cellular' internalization moiety.
[0402] Embodiment 4A: The composition of Embodiment 1 A, wherein the fragment of IL-
33 is derived from mast cells.
[0403] Embodiment 5A: The composition of Embodiment 1 A, wherein the linker is a peptide or a non-peptide linker selected from the group consisting of direct linkage of two proteins, covalent linkers preferably non hydrolysable, PEGylated, Polyethylene glycol (PEG), organic linkers, water-soluble organic linkers, water-soluble polymers, Fc fusion proteins, Fc albumin fusion proteins, a peptide sequence, a peptidase with a cleaving site, a bifunctional linker, a polyglycine linker, carbohydrates, bridging factors, biostable and biodegradable polymers, diamino and or diacid units, natural or unnatural amino acids or derivatives thereof, as well as aliphatic moieties, including alkyl, aryl, heteroalkyl, heteroaryl, alkoxy and combinations thereof.
[0404] Embodiment 6A: The composition of Embodiment 1 A or 5A, wherein the linker comprises a cleavage site recognized by human serum or human cellular enzymes.
[0405] Embodiment 7 A: The composition of Embodiment 1 A, further comprising two or more anti-inflammatory therapeutic agents.
[0406] Embodiment 8A: The composition of Embodiment 1A, further comprising two or more anti-inflammatory proteins.
[0407] Embodiment 9A: The composition of Embodiment 1 A, further comprising one or more pharmaceutically acceptable carriers selected from the group of diluents, excipients, solvents, adjuvants, or formulating agents.
[0408] Embodiment 10A: The composition of Embodiment 1A, wherein the IL-33, or fragment, variant or homolog thereof, is PEGylated at one or more specific sites to improve stability and half-life of the molecule.
[0409] Embodiment 11 A: The composition of any one of Embodiments 1A - 10A, wherein the anti-inflammatory cytokine is interleukin-37 (IL-37), or a fragment, variant or homolog thereof, and comprising an amino acid sequence selected from SEQ ID NO: 6- 10.
[0410] Embodiment 12A: A composition comprising an interleukin-33 (IL-33) (SEQ ID NO: 1), or a fragment, variant or homolog thereof comprising at least one amino acid sequence selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109- 270 (SEQ ID NO: 4), and 112-270 (SEQ ID NO: 5).
[0411] Embodiment 13A: The composition of Embodiment 1A, wherein the IL-33 fragment, variant or homolog thereof is modified at one or more sites.
[0412] Embodiment 14A: The composition of Embodiment 2A, wherein the IL-33 fragment, variant or homolog thereof is modified at one or more sites by PEGylation.
[0413] Embodiment IB: A method for producing a protein comprising the steps of: a) introducing a target isolated nucleic acid sequence encoding IL-33 into one or more vectors along with a promoter expressed in one or more recombinant host cells to express full-length IL-33 proteins, wherein the IL-33 may optionally be cleaved into one or more fragments thereof by mast cell enzymes; b) introducing a target sequence of an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, into one or more vectors along with a promoter expressed in one or more recombinant host cells to express the antiinflammatory cytokine, or a fragment, variant or homolog thereof, and optionally linking the IL-33, or fragment, variant or homolog thereof, with the anti-inflammatory cytokine, or a fragment, variant or homolog thereof, using an optional linker; c) culturing the host cell under suitable conditions to allow the production of a desired recombinant protein; d) recovering the protein from the culture of the host cell; and e) optionally purifying the recovered protein.
[0414] Embodiment 2B: The method of Embodiment IB, wherein the protein comprises: a first domain comprising IL-33, or a fragment, variant or homolog thereof, a second domain comprising an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, and a third domain comprising a linker.
[0415] Embodiment 3B: The method of Embodiment 2B, wherein the recombinant protein further comprises a fourth domain having a cytokine receptor targeting moiety or a cellular internalization moiety.
[0416] Embodiment 4B: The method of Embodiment IB, wherein the linker is a non- hydrolysable covalent linker used to produce IL-33, or a fragment, variant or homolog thereof, comprising at least one amino acid residue fragment of IL-33 selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4) or 112-270 (SEQ ID NO: 5) conjugated with the anti-inflammatory cytokine, or a fragment, variant or homolog thereof. [0417] Embodiment 5B: The method of Embodiment IB, wherein any one of the sequences encoding IL-33 or a fragment, variant or homolog thereof is directly fused with the target sequence of the anti-inflammatory cytokine, or a fragment, variant or homolog thereof, to produce a fusion protein.
[0418] Embodiment 6B: The method of Embodiment IB, wherein the linker is a peptide linker or a non-peptide linker selected from the group consisting of direct linkage of two proteins, covalent linkers preferably non hydrolysable, PEGylated, polyethylene glycol (PEG), organic linkers, water-soluble organic linkers, water-soluble polymers, Fc fusion proteins, Fc albumin fusion proteins, a peptide sequence, a peptidase with a cleaving site, a bifunctional linker, a polyglycine linker, carbohydrates, bridging factors, biostable and biodegradable polymers, diamino and or diacid units, natural or unnatural amino acids or derivatives thereof, as well as aliphatic moieties, including alkyl, aryl, heteroalkyl, heteroaryl, alkoxy and combinations thereof.
[0419] Embodiment 7B: The method of either one of Embodiments IB and 6B, wherein the linker comprises a cleavage site recognized by human serum or human cellular' enzymes.
[0420] Embodiment 8B: The method of Embodiment IB, wherein the fragment of IL-33 is derived from mast cells comprising the step of expressing the full-length IL-33 polypeptide and processing the full-length IL-33 polypeptide using mast cell enzymes to cleave it into one or more amino acid residue fragments of IL-33 selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), or 112-270 (SEQ ID NO: 5) of IL-33.
[0421] Embodiment 9B: The method of Embodiment IB, wherein the IL-33, or fragment, variant or homolog thereof, is PEGylated at one or more specific sites to enhance stability and half-life of the molecule.
[0422] Embodiment 10B: The method of Embodiment IB, wherein the anti-inflammatory cytokine, or a fragment, variant or homolog thereof, is an interleukin-37 (IL-37), or a fragment, variant or homolog thereof.
[0423] Embodiment 1C: Use of a pharmaceutical composition comprising IL-33, or a fragment, variant or homolog thereof, comprising at least one amino acid residue fragment selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109- 270 (SEQ ID NO: 4) or 112-270 (SEQ ID NO: 5), an optional linker and an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, for treating, preventing, or alleviating neurodegenerative and auto-immune disorders.
[0424] Embodiment 2C: The composition of Embodiment 1C, wherein the linker is a peptide linker or a non-peptide linker selected from the group consisting of direct linkage of two proteins, covalent linkers preferably non hydrolysable, PEGylated, polyethylene glycol (PEG), organic linkers, water-soluble organic linkers, water-soluble polymers, Fc fusion proteins, Fc albumin fusion proteins, a peptide sequence, a peptidase with a cleaving site, a bifunctional linker, a polyglycine linker, carbohydrates, bridging factors, biostable and biodegradable polymers, diamino and or diacid units, natural or unnatural amino acids or derivatives thereof, as well as aliphatic moieties, including alkyl, aryl, heteroalkyl, heteroaryl, alkoxy and combinations thereof.
[0425] Embodiment ID: A compound for treating, preventing, or alleviating neurodegenerative and auto-immune disorders, comprising interleukin-33 (IL-33), or a fragment thereof, comprising at least one amino acid sequence selected from 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), or 112-270 (SEQ ID NO: 5) wherein the IL-33, or fragment thereof, is PEGylated at one or more specific sites.
[0426] Embodiment IE: A method for the treatment of a condition, disease, or disorder, the method comprising the step of administration of an effective amount of a composition comprising:
IL-33, or a variant, fragment, or homolog thereof,
IL-37, or a variant, fragment, or homolog thereof, and an optional linker for creating linkage between the IL-33, or a fragment, variant or homolog thereof, and IL-37, or fragment, variant or homolog thereof, to a patient in need thereof.
[0427] Embodiment 2E: The method of Embodiment IE, wherein the IL-33, or variant, fragment, or homolog thereof, has at least one amino acid sequence selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), and 112-270 (SEQ ID NO: 5).
[0428] Embodiment 3E: The method of Embodiment IE, wherein the inteiieukin-37 (IL-37), or a fragment, variant or homolog thereof, has amino acid sequences selected from SEQ ID NO: 6-10.
[0429] Embodiment 4E: The method of Embodiment IE, wherein the composition comprises an optional linker for creating linkage between the IL-33, or fragment, variant or homolog thereof, and IL-37, or fragment, variant or homolog thereof.
[0430] Embodiment 5E: The method of Embodiment 4E: , wherein the linkage is at least one selected from: a) the IL-33 or at least one fragment of IL-33 selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4) or 112-270 (SEQ ID NO: 5) is fused with the anti-inflammatory cytokine, or a fragment, variant or homolog thereof; b) the IL-33 or at least one fragment of IL-33 selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), or 112-270 (SEQ ID NO: 5) is conjugated with the anti-inflammatory cytokine, or a fragment, variant or homolog thereof; c) the IL-33 or at least one amino acid residue fragment of IL-33 selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), or 112-270 (SEQ ID NO: 5) is co-administered with the antiinflammatory cytokine, or a fragment, variant or homolog thereof; and d) a recombinant protein comprising: a first domain comprising IL-33, or a fragment, variant or homolog thereof, comprising any one sequence from SEQ ID NO: 1-5, a second domain comprising the anti-inflammatory cytokine, or fragment, variant or homolog thereof. [0431] Embodiment 6E: The method of Embodiment IE, wherein the condition, disease, or disorder is an inflammatory disease mediated by the production of proinflammatory cytokines such as TNF, IL-1, IL-6, and/or other inflammatory mediators.
[0432] Embodiment 7E: The method of Embodiment IE, wherein administration of IL-33 is indicated for treatment or prevention of the condition, disease, or disorder.
[0433] Embodiment 8E: The method of Embodiment IE, wherein administration of IL-37 is indicated for treatment or prevention of the condition, disease, or disorder.
[0434] Embodiment 9E: The method of Embodiment IE, wherein the condition, disease, or disorder is characterized by pain.
[0435] Embodiment 10E: The method of claim 36, wherein the pain is selected from inflammatory pain and neuropathic pain.
[0436] Embodiment 1 IE: The method of Embodiment IE, wherein the condition is osteoarthritis or chronic pain.
[0437] Embodiment 12E: The method of Embodiment IE, wherein the condition is a neurodegenerative disease.
[0438] Embodiment 13E: The method of Embodiment IE, wherein the condition is characterized by local or systemic inflammation, immune activation, and/or lymphoproliferation.
[0439] Embodiment IF: A method for the treatment of a condition, disease, or disorder, the method comprising administering an effective amount of: a composition comprising IL-33, or a variant, fragment, or homolog thereof, and a composition comprising IL-37, or a variant, fragment, or homolog thereof, to a patient in need thereof.
[0440] Embodiment 2F: The method of Embodiment IF, wherein the composition comprising IL-33, or a variant, fragment, or homolog thereof and the composition comprising IL-37, or a variant, fragment, or homolog thereof are administered simultaneously or sequentially. [0441] Embodiment 3F: The method of Embodiment IF, wherein the IL-33, or variant, fragment, or homolog thereof, comprises at least one amino acid sequence selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), and 112-270 (SEQ ID NO: 5).
[0442] Embodiment 4F: The method of Embodiment IF, wherein the interleukin-37 (IL-37), or a fragment, variant or homolog thereof, comprises an amino acid sequence selected from SEQ ID NO: 6-10.
[0443] Embodiment 5F: The method of Embodiment IF, wherein the condition, disease, or disorder is an inflammatory disease mediated by the production of proinflammatory cytokines such as TNF, IL-1, IL-6, and/or other inflammatory mediators.
[0444] Embodiment 6F: The method of Embodiment IF, wherein administration of IL-33 is indicated for treatment or prevention of the condition, disease, or disorder.
[0445] Embodiment 7F: The method of Embodiment IF, wherein administration of IL-37 is indicated for treatment or prevention of the condition, disease, or disorder.
[0446] Embodiment 8F: The method of Embodiment IF, wherein the condition, disease, or disorder is characterized by pain.
[0447] Embodiment 9F: The method of Embodiment 8F, wherein the pain is selected from inflammatory pain and neuropathic pain.
[0448] Embodiment 10F: The method of Embodiment IF, wherein the condition is osteoarthritis or chronic pain.
[0449] Embodiment 1 IF: The method of Embodiment IF, wherein the condition is a neurodegenerative disease.
[0450] Embodiment 12F: The method of Embodiment IF, wherein the condition is characterized by local or systemic inflammation, immune activation, and/or lymphoproliferation.
[0451] In any one of the embodiments or sequences disclosed herein, the IL-33 and/or the IL-37 fragment, variant or homolog thereof may optionally lack an N-terminus Methionine. EXAMPLES
[0452] The materials and methods disclosed herein will now be illustrated with reference to the following examples, which set forth particularly advantageous embodiments. However, it should be noted that these embodiments are illustrative and are not to be construed as restricting the claimed subject-matter in any way.
Example 1
Animal Studies-ALS with IL-33 and IL-37 fragments
[0453] Eighty male, B6SJLTgN (SODlG93A)lGur (J AX Stock No: 002726; Jackson Laboratory, USA) were assigned to the study. Their average age, at the beginning of the study, was 42+1 days (range 40-46 days). Mice were individually housed and were provided laboratory food and water ad libitum. The mice were divided into four study groups, balanced for age and litter. Each group received different treatment regiments, as shown in the table below. Lyophilized IL-33 (Seri 12-Thr270 fragment) and IL-37 (Ser2-Aspl92 fragment) were purchased from R&D Systems (Bio-Techne) and were re-constituted in IX PBS. Within each group, there were two cohorts: 1) The “survival cohort” included 12 mice monitored for grip strength (Ix/week), body weights (5x/week), NeuroScores (5x/week), disease onset and survival. This cohort was sacrificed when mice reached the humane endpoint, tissues (brain, spinal cord, plasma) were collected, flash frozen with liquid nitrogen and stored at -80 C; 2) The “pharmacodynamic (PD) cohort” included 8 mice monitored for body weights (5x/week) and NeuroScores (5x/week) and were harvested at approximately age 105±l days (i.e. when 25% of the PD cohort presented with symptoms of the disease), were perfused with saline, brains and spinal cords were collected, submerged in RPMI buffer and were sent on ice for study.
Figure imgf000079_0001
Figure imgf000080_0001
Grip Strength Analysis
[0454] SOD1G93A mice underwent grip strength testing on a weekly basis for eleven consecutive weeks, using a Bioseb grip strength apparatus. Each grip strength test consisted of three trials, and the average of the three trials was used for the analysis. For each trial, the experimenter gently pulled the mouse away from a platform and the machine recorded the force that the mouse exerted on the platform sensors, with all its limbs simultaneously, to resist the force of the pull. In the event where a mouse was unable to complete the test, either because it was severely progressed or diseased, the last recorded grip strength value was progressed forward for purpose of the analyses. The data are presented as a ratio of the baseline grip strength for each group (shown in FIG. 8). Each point represents the average force for 12 mice per group ± SEM.
[0455] Beginning at two weeks post-treatment initiation, a clear' trend emerged where all of the treated groups (IL-33, IL-37, IL-33/IL-37) had increased grip strength compared to the control group (untreated mice). The control group was consistently the weakest of the four groups, as the disease progressed throughout the duration of the study. A 2-way ANOVA revealed a statistically significant overall treatment effect (p<0.0499), with all three treatment regimens performing better compared to the CONTROL group. Further, Sidak’s multiple comparisons test, revealed the following statistically significant differences at specific time points: WEEK 7_C0NTR0L vs. IL-33/IL-37_p=0.0187; WEEK 8_C0NTR0L vs. IL- 33_p=0.0420; WEEK 9_C0NTR0L vs. IL-33_p=0.0345. There were no significant differences between the three treated groups at any time point.
Disease Onset
[0456] The onset of the disease in the SOD1G93A model is defined as the age when the mice first exhibit signs of paresis in their hindlimbs. On phenotypic examination, paresis in the hindlimbs corresponds to a NeuroScore of 2. The Kaplan-Meier curve in FIG. 9 shows the proportion of mice that present with disease symptoms as a function of their ages (in days).
[0457] The control group mice (untreated) reached their disease onset earlier than all the treated groups. The median disease onset age for control mice was 102 days, for IL-33/IL-37 it was 106.5 days, for IL-37 it was 112 days, and for IL-33 it was 113 days. The 12-day delay in disease onset, compared to the control group, in the IL-33 group was very close to being statistically significant (p=0.0548; Log-rank (Mantel-Cox) test). The 11-day delay if the IL-37 group and the 4.5-day delay in the IL-33/IL-37 group were not statistically significant.
Survival Duration
[0458] SOD1G93A mice reach their endpoint when they are no longer able to right themselves up. On phenotypic examination this corresponds to a NeuroScore of 4. When mice reach their endpoint, they are humanely sacrificed. The Kaplan-Meier curve in FIG. 10 shows the proportion of mice that have died as a function of their age (in days). The control group mice (untreated) had a shorter survival duration than all the treated groups. The median survival age for control mice was 116.5 days, while all the treated groups had a median survival age that was 123+0.5 days or about 7-days longer than the controls. The survival extensions were not statistically significant for any of the treatments (Log-rank (Mantel-Cox) test).
Body Weight Maintenance
[0459] All groups gained and lost weight in the manner expected for the SOD1G93A model of ALS. The treated groups appeared to maintain their body weight better than the control mice during the latter stages of their disease (FIG. 11 dotted area), though these differences were not statistically significant (2-way ANOVA with repeated measures). Conclusion
[0460] All the treatment regimens tested performed better than the control group in several different metrics used to assess disease progression in the SOD1G93A model of ALS (i.e. by maintaining grip strength and body weight, delaying disease onset, and extending the survival duration).
Example 2
Fusion Protein for IL-33 and IL-37
[0461] A novel fusion protein comprising an IL-33 fragment fused to an IL-37 fragment via an intervening peptide linker is prepared. The IL-33 fragment is selected from the group consisting of amino acid sequence 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), and 112-270 (SEQ ID NO: 5). The IL-37 fragment is an amino acid sequence selected from SEQ ID NO: 6-10. The linker is SEQ ID NO: 11.
[0462] The recombinant coding sequence for this fusion protein is subcloned into a eukaryotic expression vector. The expression construct is stably transfected into mammalian cells. The fusion protein is detected in cell media. A western blot is used to detect the size and to confirm the identity of the fusion protein. The fusion protein may be purified by chromatography .
[0463] Binding of the fusion protein to IL-33 receptor is analyzed by flow cytometry.
[0464] Binding of the fusion protein to IL-37 receptor is analyzed by flow cytometry.
Example 3
Animal Studies-ALS
[0465] B6SJLTgN (SODlG93A)lGur (Jackson Laboratory, USA) mice are used as an ALS model.
[0466] Mice receive IL-33-IL-37 fusion protein twice per week i.p. at a dosage of 0.1 pg/mousc to 1 pg/mousc. Treatment is continued for 50 days or more, for example 100 days or more. Treatment may begin before or after onset of ALS symptoms. The onset of the disease in the SOD1G93A model is defined as the age when the mice first exhibit signs of paresis in their hindlimbs. A wire-hang test may be conducted, for example as described in Example 1 . Mice receiving PBS or untreated mice arc used as a control.
Animal Studies-Alzheimers
[0467] APP/Ps 1 mice are used as an Alzheimer’ s disease (AD) model because they exhibit amyloid plaque deposition and cognitive impairment.
[0468] Mice receive IL-33-IL-37 fusion protein twice or more per week i.p. at a dosage of 0.1 p.g/mousc to 1 ug/mousc. Treatment is continued for 50 days or more, for example 100 days or more. Treatment may begin before or after onset of AD symptoms. Amyloid plaque pathology is studied in the mice. Contextual fear conditioning tests or open field tests are conducted. Mice receiving vehicle or PBS or untreated mice are used as a control.
Example 4
IL-33 Fragments
[0469] An IL-33 fragment is selected from the group consisting of amino acid sequence 95- 270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), and 112-270 (SEQ ID NO: 5).
[0470] The recombinant coding sequence for this IL-33 fragment is subcloned into a eukaryotic expression vector. The expression construct is stably transfected into mammalian cells. The fragment is detected in cell media. A western blot is used to detect the size and to confirm the identity of the fragment. The fragment may be purified by chromatography.
[0471] Binding of the fragment to IL-33 receptor is analyzed by flow cytometry.
IL-37
[0472] An IL-37 is selected from the group consisting of amino acid sequence SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.
[0473] The recombinant coding sequence for this IL-37 is subcloned into a eukaryotic expression vector. The expression construct is stably transfected into mammalian cells. The protein is detected in cell media. A western blot is used to detect the size and to confirm the identity of the IL-37. The IL-37 may be purified by chromatography.
[0474] Binding of the IL-37 to IL-37 receptor is analyzed by flow cytometry. Example 5
Animal Studies-ALS
[0475] B6SJLTgN (SODlG93A)lGur (Jackson Laboratory, USA) mice are used as an ALS model.
Mice receive:
(i) IL-33 fragment twice per week i.p. at a dosage of 0.1 pg/mousc to 1 pg/mousc; and/or
(ii) IL-37 twice per week i.p. at a dosage of 0.1 pg/mouse to 1 pg/mouse.
Treatment is continued for 50 days or more, for example 100 days or more. Treatment may begin before or after onset of ALS symptoms. The onset of the disease in the SOD1G93A model is defined as the age when the mice first exhibit signs of paresis in their hindlimbs. A wire-hang test may be conducted, for example as described in Example 1. Mice receiving PBS or untreated mice are used as a control.
Animal Studies-Alzheimers
[0476] APP/Ps 1 mice are used as an Alzheimer’ s disease model because they exhibit amyloid plaque deposition and cognitive impairment.
[0477] Mice receive TL-33 fragment and/or IL-37 twice or more per week i.p. at a dosage of 0.1 pg/mouse to 1 pg/mouse. Treatment is continued for 50 days or more, for example 100 days or more. Treatment may begin before or after onset of AD symptoms. Contextual fear conditioning tests or open field tests are conducted. Mice receiving vehicle or PBS or untreated mice are used as a control.
SEQUENCE LISTINGS OF IL-33 AND IL-37 AND THEIR RESPECTIVE FRAGMENTS OR ISOFORMS
[0478] SEQ ID NO: 1 represents amino acid sequence for full length human IL-33 (for 1- 270) (NCBI Accession 095760.1)
MKPKMKYSTNKISTAKWKNTASKALCFKLGKSQQKAKEVCPMYFMKLRSGLMIKKEA CYFRRETTKRPSLKTGRKHKRHLVLAACQQQSTVECFAFGISGVQKYTRALHDSSITGIS PITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGV DGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKT
DPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET
[0479] SEQ ID NO: 2 represents amino acid sequence for IL-33 (for 95-270 fragment)
AFGISGVQKYTRALHDSSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKK
DKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLP
DQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET
[0480] SEQ ID NO: 3 represents amino acid sequence for IL-33 fragment (for 107-270 fragment)
ALHDSSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQH
PSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHS
NCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET
[0481] SEQ ID NO: 4 represents amino acid sequence for IL-33 fragment (for 109-270 fragment)
HDSSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPS
NESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNC
VSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET
[0482] SEQ ID NO: 5 represents amino acid sequence for IL-33 fragment (for 112-270 fragment)
SITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNES
GDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSF
ECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET
[0483] SEQ ID NO: 6 represents amino acid sequence for full length human IL-37 (1-218) (NCBI Accession Q9NZH6.1).
MSFVGENSGVKMGSEDWEKDEPQCCLEDPAGSPLEPGPSLPTMNFVHTSPKVKNLNPK
KFSIHDQDHKVLVLDSGNLIAVPDKNYIRPEIFFALASSLSSASAEKGSPILLGVSKGEFCL
YCDKDKGQSHPSLQLKKEKLMKLAAQKESARRPFIFYRAQVGSWNMLESAAHPGWFIC TSCNCNEPVGVTDKFENRKHIEFSFQPVCKAEMSPSEVSD
[0484] SEQ ID NO: 7 represents amino acid sequence for human IL-37 fragment having length of 192 residues (isoform 5; NCBI Accession NP_775297.1). MSGCDRRETETKGKNSFKKRLRGPKVKNLNPKKFSIHDQDHKVLVLDSGNLIAVPDKN YIRPEIFFALASSLSSASAEKGSPILLGVSKGEFCLYCDKDKGQSHPSLQLKKEKLMKLAA QKESARRPFIFYRAQVGSWNMLESAAHPGWFICTSCNCNEPVGVTDKFENRKHIEFSFQ PVCKAEMSPSEVSD
[0485] SEQ ID NO: 8 represents amino acid sequence human IL-37 fragment having length of 178 residues (Isoform 4; NCBI Accession NP_775296.1).
MSFVGENSGVKMGSEDWEKDEPQCCLEDPAGSPLEPGPSLPTMNFVHTK1FFALASSLSS ASAEKGSPILLGVSKGEFCLYCDKDKGQSHPSLQLKKEKLMKLAAQKESARRPFIFYRA QVGSWNMLESAAHPGWFICTSCNCNEPVGVTDKFENRKHIEFSFQPVCKAEMSPSEVSD
[0486] SEQ ID NO: 9 represents amino acid sequence for human IL-37 fragment having length of 197 residues (Isoform 2; NCBI Accession NP_775294.1).
MSFVGENSGVKMGSEDWEKDEPQCCLEGPKVKNLNPKKFSIHDQDHKVLVLDSGNLIA
VPDKNYIRPEIFFALASSLSSASAEKGSPILLGVSKGEFCLYCDKDKGQSHPSLQLKKEKL MKLAAQKESARRPFIFYRAQVGSWNMLESAAHPGWFICTSCNCNEPVGVTDKFENRKH IEFSFQPVCKAEMSPSEVSD
[0487] SEQ ID NO: 10 represents amino acid sequence for human IL-37 fragment having length of 157 residues (Isoform 3; NCBI Accession NP_775295.1).
MSFVGENSGVKMGSEDWEKDEPQCCLEEIFFALASSLSSASAEKGSPILLGVSKGEFCLY CDKDKGQSHPSLQLKKEKLMKLAAQKESARRPFIFYRAQVGSWNMLESAAHPGWFICT SCNCNEPVGVTDKFENRKHIEFSFQPVCKAEMSPSEVSD
[0488] SEQ ID NO: 11 represents the amino acid sequence of a peptide linker GGGGS. In some embodiments, the linker may be a multiple of SEQ ID NO: 11, for example a 2- or 3- or 4- time repeat.
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[0489] All publications and patents referred to herein are incorporated by reference. Various modifications and variations of the described subject matter will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the materials and methods disclosed herein have been described in connection with specific embodiments, it should be understood that the claimed subject-matter should not be unduly limited to these embodiments. Indeed, various modifications for carrying out the invention are obvious to those skilled in the ail and are intended to be within the scope of the following claims.

Claims

1. A composition comprising an intcrlcukin-33 (IL-33) (SEQ ID NO: 1), or a fragment, variant or homolog thereof, comprising at least one amino acid sequence selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), and 112-270 (SEQ ID NO: 5); an anti-inflammatory cytokine, or fragment, variant or homolog thereof; and an optional linker for creating linkage between the IL-33, or a fragment, variant or homolog thereof, and the anti-inflammatory cytokine or fragment, variant or homolog thereof.
2. The composition of claim 1, wherein the linkage is at least one selected from the group comprising: a) the IL-33 or a fragment, variant or homolog thereof, is fused with the antiinflammatory cytokine, or a fragment, variant or homolog thereof; b) the IL-33 or a fragment, variant or homolog thereof, is conjugated with the antiinflammatory cytokine, or a fragment, variant or homolog thereof; c) the IL-33 or a fragment, variant or homolog thereof, is co-administered with the anti-inflammatory cytokine, or a fragment, variant or homolog thereof; and d) a recombinant protein comprising: a first domain comprising IL-33, or a fragment, variant or homolog thereof, comprising any one sequence from SEQ ID NO: 1-5, a second domain comprising the anti-inflammatory cytokine, or fragment, variant or homolog thereof.
3. The composition of claim 2, wherein the recombinant protein further comprises a fourth domain having a cytokine receptor targeting moiety or cellular internalization moiety.
4. The composition of claim 1, wherein the fragment of IL-33 is derived from mast cells.
5. The composition of claim 1 , wherein the linker is a peptide or a non-peptide linker selected from the group consisting of direct linkage of two proteins, covalent linkers preferably non hydrolysable, PEGylated, Polyethylene glycol (PEG), organic linkers, water-soluble organic linkers, water-soluble polymers, Fc fusion proteins, Fc albumin fusion proteins, a peptide sequence, a peptidase with a cleaving site, a bifunctional linker, a polyglycine linker, carbohydrates, bridging factors, biostable and biodegradable polymers, diamino and or diacid units, natural or unnatural amino acids or derivatives thereof, as well as aliphatic moieties, including alkyl, aryl, heteroalkyl, heteroaryl, alkoxy and combinations thereof.
6. The composition of claim 1 or 5, wherein the linker comprises a cleavage site recognized by human serum or human cellular' enzymes.
7. The composition of claim 1, further comprising two or more anti-inflammatory therapeutic agents.
8. The composition of claim 1 , further comprising two or more anti-inflammatory proteins.
9. The composition of claim 1, further comprising one or more pharmaceutically acceptable carriers selected from the group of diluents, excipients, solvents, adjuvants, or formulating agents.
10. The composition of claim 1, wherein the IL-33, or fragment, variant or homolog thereof, is PEGylated at one or more specific sites to improve stability and half-life of the molecule.
11. The composition of any one of the preceding claims, wherein the anti-inflammatory cytokine is interleukin-37 (IL-37), or a fragment, variant or homolog thereof, and comprising an amino acid sequence selected from SEQ ID NO: 6-10.
12. A composition comprising an interleukin-33 (IL-33) (SEQ ID NO: 1), or a fragment, variant or homolog thereof comprising at least one amino acid sequence selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), and 112-270 (SEQ ID NO: 5).
13. The composition of claim 1, wherein the IL-33 fragment, variant or homolog thereof is modified at one or more sites.
14. The composition of claim 2, wherein the IL-33 fragment, variant or homolog thereof is modified at one or more sites by PEGylation.
15. A method for producing a protein comprising the steps of: a) introducing a target isolated nucleic acid sequence encoding IL-33 into one or more vectors along with a promoter expressed in one or more recombinant host cells to express full-length IL-33 proteins, wherein the IL-33 may optionally be cleaved into one or more fragments thereof by mast cell enzymes; b) introducing a target sequence of an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, into one or more vectors along with a promoter expressed in one or more recombinant host cells to express the antiinflammatory cytokine, or a fragment, variant or homolog thereof, and optionally linking the IL-33, or fragment, variant or homolog thereof, with the anti-inflammatory cytokine, or a fragment, variant or homolog thereof, using an optional linker; c) culturing the host cell under suitable conditions to allow the production of a desired recombinant protein; d) recovering the protein from the culture of the host cell; and e) optionally purifying the recovered protein.
16. The method of claim 15, wherein the protein comprises: a first domain comprising IL-33, or a fragment, variant or homolog thereof, a second domain comprising an anti-inflammatory cytokine, or a fragment, variant or homolog thereof, and a third domain comprising a linker.
17. The method of claim 16, wherein the recombinant protein further comprises a fourth domain having a cytokine receptor targeting moiety or a cellular’ internalization moiety.
18. The method of claim 15, wherein the linker is a non-hydrolysable covalent linker used to produce IL-33, or a fragment, variant or homolog thereof, comprising at least one amino acid residue fragment of IL-33 selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4) or 112-270 (SEQ ID NO: 5) conjugated with the anti-inflammatory cytokine, or a fragment, variant or homolog thereof.
19. The method of claim 15, wherein any one of the sequences encoding IL-33 or a fragment, variant or homolog thereof is directly fused with the target sequence of the anti- inflammatory cytokine, or a fragment, variant or homolog thereof, to produce a fusion protein.
20. The method of claim 15, wherein the linker is a peptide linker or a non-peptide linker selected from the group consisting of direct linkage of two proteins, covalent linkers preferably non hydrolysable, PEGylated, polyethylene glycol (PEG), organic linkers, water-soluble organic linkers, water-soluble polymers, Fc fusion proteins, Fc albumin fusion proteins, a peptide sequence, a peptidase with a cleaving site, a bifunctional linker, a polyglycine linker, carbohydrates, bridging factors, biostable and biodegradable polymers, diamino and or diacid units, natural or unnatural amino acids or derivatives thereof, as well as aliphatic moieties, including alkyl, aryl, heteroalkyl, heteroaryl, alkoxy and combinations thereof.
21. The method of claim 15 or 20, wherein the linker comprises a cleavage site recognized by human serum or human cellular enzymes.
22. The method of claim 15, wherein the fragment of IL-33 is derived from mast cells comprising the step of expressing the full-length IL-33 polypeptide and processing the full-length IL-33 polypeptide using mast cell enzymes to cleave it into one or more amino acid residue fragments of IL-33 selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), or 112-270 (SEQ ID NO: 5) of IL-33.
23. The method of claim 15, wherein the IL-33, or fragment, variant or homolog thereof, is PEGylated at one or more specific sites to enhance stability and half-life of the molecule.
24. The method as of claim 15, wherein the anti-inflammatory cytokine, or a fragment, valiant or homolog thereof, is an interleukin-37 (IL-37), or a fragment, valiant or homolog thereof.
25. Use of a pharmaceutical composition comprising IL-33, or a fragment, variant or homolog thereof, comprising at least one amino acid residue fragment selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4) or 112-270 (SEQ ID NO: 5), an optional linker and an anti-inflammatory cytokine, or a fragment, valiant or homolog thereof, for treating, preventing, or alleviating neurodegenerative and auto-immune disorders.
26. The use of claim 25, wherein the linker is a peptide linker or a non-peptide linker selected from the group consisting of direct linkage of two proteins, covalent linkers preferably non hydrolysable, PEGylated, polyethylene glycol (PEG), organic linkers, water-soluble organic linkers, water-soluble polymers, Fc fusion proteins, Fc albumin fusion proteins, a peptide sequence, a peptidase with a cleaving site, a bifunctional linker, a polyglycine linker, carbohydrates, bridging factors, biostable and biodegradable polymers, diamino and or diacid units, natural or unnatural amino acids or derivatives thereof, as well as aliphatic moieties, including alkyl, aryl, heteroalkyl, heteroaryl, alkoxy and combinations thereof.
27. A compound for treating, preventing, or alleviating neurodegenerative and auto-immune disorders, comprising interleukin-33 (IL-33), or a fragment thereof, comprising at least one amino acid sequence selected from 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), or 112-270 (SEQ ID NO: 5) wherein the IL-33, or fragment thereof, is PEGylated at one or more specific sites.
28. A method for the treatment of a condition, disease, or disorder, the method comprising administering an effective amount of a composition comprising:
IL-33, or a variant, fragment, or homolog thereof,
IL-37, or a variant, fragment, or homolog thereof, and an optional linker for creating linkage between the IL-33, or a fragment, variant or homolog thereof, and IL-37, or fragment, variant or homolog thereof, to a patient in need thereof.
29. The method of claim 28, wherein the IL-33, or variant, fragment, or homolog thereof, comprises at least one amino acid sequence selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), and 112-270 (SEQ ID NO: 5).
30. The method of claim 28, wherein the interleukin-37 (IL-37), or a fragment, variant or homolog thereof, comprises an amino acid sequence selected from SEQ ID NO: 6-10.
31. The method of claim 28, wherein the composition comprises an optional linker for creating linkage between the IL-33, or fragment, variant or homolog thereof, and IL-37, or fragment, variant or homolog thereof.
32. The method of claim 31, wherein the linkage is at least one selected from: a) the IL-33 or at least one fragment of IL-33 selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4) or 112-270 (SEQ ID NO: 5) is fused with the anti-inflammatory cytokine, or a fragment, variant or homolog thereof; b) the IL-33 or at least one fragment of IL-33 selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), or 112-270 (SEQ ID NO: 5) is conjugated with the anti-inflammatory cytokine, or a fragment, variant or homolog thereof; c) the IL-33 or at least one amino acid residue fragment of IL-33 selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), or 112-270 (SEQ ID NO: 5) is co-administered with the antiinflammatory cytokine, or a fragment, variant or homolog thereof; and d) a recombinant protein comprising: a first domain comprising IL-33, or a fragment, variant or homolog thereof, comprising any one sequence from SEQ ID NO: 1-5, a second domain comprising the anti-inflammatory cytokine, or fragment, variant or homolog thereof.
33. The method of claim 28, wherein the condition, disease, or disorder is an inflammatory disease mediated by the production of proinflammatory cytokines such as TNF, IL- 1 , IL- 6, and/or other inflammatory mediators.
34. The method of claim 28, wherein administration of IL-33 is indicated for treatment or prevention of the condition, disease, or disorder.
35. The method of claim 28, wherein administration of IL-37 is indicated for treatment or prevention of the condition, disease, or disorder.
36. The method of claim 28, wherein the condition, disease, or disorder is characterized by pain.
37. The method of claim 36, wherein the pain is selected from inflammatory pain and neuropathic pain.
38. The method of claim 28, wherein the condition is osteoarthritis or chronic pain.
39. The method of claim 28, wherein the condition is a neurodegenerative disease.
40. The method of claim 28, wherein the condition is characterized by local or systemic inflammation, immune activation, and/or lymphoproliferation.
41 . A method for the treatment of a condition, disease, or disorder, the method comprising administering an effective amount of: a composition comprising IL-33, or a variant, fragment, or homolog thereof, and a composition comprising IL-37, or a variant, fragment, or homolog thereof, to a patient in need thereof.
42. The method of claim 41, wherein the composition comprising IL-33, or a variant, fragment, or homolog thereof and the composition comprising IL-37, or a variant, fragment, or homolog thereof are administered simultaneously or sequentially.
43. The method of claim 41, wherein the IL-33, or variant, fragment, or homolog thereof, comprises at least one amino acid sequence selected from the group consisting of 95-270 (SEQ ID NO: 2), 107-270 (SEQ ID NO: 3), 109-270 (SEQ ID NO: 4), and 112-270 (SEQ ID NO: 5).
44. The method of claim 41, wherein the interleukin-37 (IL-37), or a fragment, variant or homolog thereof, comprises an amino acid sequence selected from SEQ ID NO: 6-10.
45. The method of claim 41, wherein the condition, disease, or disorder is an inflammatory disease mediated by the production of proinflammatory cytokines such as TNF, IL- 1 , IL-6 and/or other inflammatory mediators.
46. The method of claim 41, wherein administration of IL-33 is indicated for treatment or prevention of the condition, disease, or disorder.
47. The method of claim 41, wherein administration of IL-37 is indicated for treatment or prevention of the condition, disease, or disorder.
48. The method of claim 41 , wherein the condition, disease, or disorder is characterized by pain.
49. The method of claim 48, wherein the pain is selected from inflammatory pain and neuropathic pain.
50. The method of claim 41, wherein the condition is osteoarthritis or chronic pain.
51. The method of claim 41, wherein the condition is a neurodegenerative disease.
52. The method of claim 41, wherein the condition is characterized by local or systemic inflammation, immune activation, and/or lymphoproliferation.
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