Proteins used to treat epithelial barrier dysfunction

Abstract

The present disclosure relates to therapeutic proteins and pharmaceutical compositions comprising the proteins, which can be used to treat various human diseases. In particular aspects, the disclosed therapeutic proteins can be used to treat human gastrointestinal inflammatory diseases and gastrointestinal disorders associated with reduced epithelial cell barrier function or integrity. In addition, the disclosed therapeutic proteins can be used to treat human inflammatory bowel diseases, including Crohn's disease and ulcerative colitis.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 611,334, filed December 28, 2017, U.S. Provisional Application No. 62 / 607,706, filed December 19, 2017, and U.S. Provisional Application No. 62 / 482,963, filed April 7, 2017, the entire contents of which are incorporated herein by reference in their entirety.

[0003] Electronic Application Text Instructions

[0004] The contents of the text submitted in electronic form are incorporated herein in their entirety: Computer Readable Form Sequence Listing File Name: SEGE_001_03WO_SeqList_ST25.txt, Creation Date, April 4, 2018, File Size ≈ 45.3 kilobytes. Technical Field

[0005] The present disclosure relates to novel proteins and pharmaceutical compositions comprising the proteins, which are particularly useful for treating gastrointestinal inflammatory diseases and epithelial barrier dysfunction. In some embodiments, the proteins and pharmaceutical compositions described herein have specific applications in treating or preventing disease states associated with abnormally permeable epithelial barriers and inflammatory bowel diseases or disorders. Background Art

[0006] Inflammatory bowel disease (IBD) is a heterogeneous disease of unknown etiology that results in frequent and bloody bowel movements and histopathological destruction of the gastrointestinal mucosa (Zhang et al., 2017, Front Immunol, 8:942). Although the specific triggers of the disease are unclear, a research program on disease progression suggests that disruption of intestinal barrier function allows bacteria or bacterial components to translocate into mucosal tissues (Coskun, 2014, Front Med (Lausanne), 1:24; Martini et al., 2017, Cell Mol Gastroenterol Hepatol, 4:33-46). Bacterial translocation leads to activation of inflammatory signal transduction, which triggers additional barrier disruption, resulting in a cyclic amplification loop of barrier disruption, bacterial translocation, and inflammation. Although many current therapies target inflammation, the lack of therapies that promote mucosal healing provides an opportunity for new therapies that promote epithelial repair and intestinal barrier integrity.

[0007] Based on the hypothesis that bacterial translocation can trigger IBD, recent studies have shown that harmful changes or dysbiosis of the intestinal microbiota may promote the development of IBD.

[0008] Currently, many IBD therapies available on the market are designed only to target and inhibit the inflammatory response associated with IBD in question. Although helpful, this narrow therapeutic mode of action ignores the important role of epithelial barrier integrity in disease etiology.

[0009] Therefore, there is a great need in the art to develop a therapeutic agent that not only suppresses the inflammatory response of the immune system, but also acts to restore the epithelial barrier function of an individual. Likewise, there is a need to produce a protein therapeutic as described herein that is stable during manufacture and / or processing of the protein therapeutic and under long-term storage conditions. Summary of the invention

[0010] The disclosure satisfies the important demand of the medical profession for therapeutic agents that can effectively treat objects suffering from gastrointestinal diseases such as inflammatory bowel disease (IBD). On the one hand, novel protein therapeutic agents that can maintain epithelial barrier integrity and / or improve epithelial barrier repair are provided. In some embodiments, the epithelial barrier is an intestinal epithelial barrier. These protein therapeutic agents can also alleviate inflammation in the intestinal tract of the object and / or alleviate the symptoms relevant to intestinal inflammation.

[0011] The protein therapeutics provided herein can be used to treat a variety of diseases and / or symptoms that may be associated with decreased gastrointestinal epithelial barrier function or integrity.

[0012] In some embodiments, the present disclosure teaches novel protein therapeutics derived from microbiomes and methods of using the same. In a specific embodiment, a protein derived from a microbiome and comprising an amino acid sequence having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to SEQ ID NO: 19 is provided. In some embodiments, the therapeutic protein does not comprise an amino acid sequence identical to SEQ ID NO: 3. In other embodiments, the therapeutic protein comprises a non-naturally occurring amino acid sequence.

[0013] In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19. In other embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 3. In other embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 19.

[0014] In some embodiments, the protein comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to SEQ ID NO: 19, wherein the amino acid sequence has at least 1, 2, 3, or 4 amino acid substitutions relative to SEQ ID NO: 19 or relative to SEQ ID NO: 3. In other embodiments, the amino acid sequence has at least 2 and less than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acid substitutions relative to SEQ ID NO: 3. In other embodiments, the therapeutic protein comprises a non-naturally occurring amino acid sequence.

[0015] In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 3. In other embodiments of SEQ ID NO: 3, X53 is N, S, T, M, R, Q, and / or X83 is N, R or K, and / or X84 is G or A, and / or X147 is C, S, T, M, V, L, A or G, and / or X151 is C, S, T, M, V, L, A or G. In other embodiments, X53 is N, S or K, and / or X83 is N or R, and / or X84 is G or A, and / or X147 is C, V, L or A, and / or X151 is C, S, V, L or A.

[0016] In some embodiments, the protein is about 200 to 250 amino acids, 210 to 250 amino acids, 220 to 250 amino acids, 220 to 240 amino acids, 230 to 250 amino acids, 230 to 240 amino acids, or 230 to 235 amino acids, 220 to 275 amino acids, 220 to 260 amino acids, 230 to 260 amino acids, 240 to 250 amino acids, 250 to 260 amino acids, 230 to 256 amino acids, 240 amino acids to 256 amino acids, 245 amino acids to 256 amino acids in length. In other embodiments, the therapeutic protein is 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, or 260 amino acids in length.

[0017] In some embodiments, the present disclosure teaches antibodies or fragments thereof that specifically bind to a therapeutic protein comprising SEQ ID NO: 19 or a variant thereof. In other embodiments, the antibody or fragment thereof does not bind to a protein comprising the same amino acid sequence as SEQ ID NO: 3. In other embodiments, the antibody or fragment thereof binds to a protein comprising the same amino acid sequence as SEQ ID NO: 19, but does not bind to a protein comprising the same amino acid sequence as SEQ ID NO: 3.

[0018] In some embodiments, the protein increases the barrier function of the epithelial cell layer in an in vitro assay, wherein the increase is relative to the barrier function in the assay when the protein is not present. In other embodiments, the in vitro assay is a transepithelial electrical resistance (TEER) assay. In other embodiments, the increase in barrier function is an increase in resistance of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% greater than the resistance in the assay when the protein is not present. In some embodiments, the epithelial cell layer is an intestinal epithelial cell layer. In other embodiments, the intestinal epithelial cell layer is a cell layer comprising intestinal epithelial cells and goblet cells.

[0019] In some embodiments, the protein reduces the secretion of proinflammatory cytokines from cells in an in vitro assay. In other embodiments, the in vitro assay comprises incubating monocytes with heat-killed E. coli in the presence and absence of the protein. In other embodiments, at least one proinflammatory cytokine is selected from TNF-α, IL-17, IL-1β, IL-2, IFN-γ, IL-6, IL-12, IL-25, IL-33, IL-8, MCP-1, MIP-3α, CXCL1 and IL-23.

[0020] In some embodiments, the protein increases the secretion of anti-inflammatory cytokines from cells in an in vitro assay. In other embodiments, the in vitro assay comprises incubating monocytes with heat-killed E. coli in the presence and absence of the protein. In other embodiments, at least one anti-inflammatory cytokine is selected from IL-4, IL-10, IL-13, IFN-α and TGF-β.

[0021] In some embodiments, the protein reduces intestinal tissue damage in a subject to which the protein is administered. In some embodiments, the subject has intestinal tissue damage induced by treatment with a chemical. In other embodiments, the subject has intestinal tissue damage induced by treatment with the chemical dextran sulfate sodium (DSS). In other embodiments, the subject is a mammal. In other embodiments, the animal is a rodent. In other embodiments, the subject is a non-human primate.

[0022] In some embodiments, the therapeutic protein reduces gastrointestinal inflammation in a subject to which the protein is administered. In other embodiments, the protein reduces inflammation of the intestinal mucosa of a subject. In still other embodiments, the protein improves intestinal epithelial cell barrier function or integrity in a subject.

[0023] In some embodiments, the protein increases the amount of mucin in the intestinal tissue of a subject to which the protein is administered.

[0024] In some embodiments, the protein increases intestinal epithelial cell wound healing in a subject to which the protein is administered. In other embodiments, the protein increases intestinal epithelial cell wound healing in an in vitro assay.

[0025] In some embodiments, the protein prevents or reduces colon shortening in a subject to which the protein is administered.

[0026] In some embodiments, a therapeutic protein modulates (ie, increases or decreases) a cytokine in the blood, plasma, serum, tissues, and / or mucosa of a subject to which the protein is administered.

[0027] In some embodiments, the protein reduces the level of at least one proinflammatory cytokine in the blood, plasma, serum, tissues and / or mucosa of a subject. In other embodiments, the at least one proinflammatory cytokine is selected from TNF-α, IL-17, IL-1β, IL-2, IFN-γ, IL-6, IL-12, IL-25, IL-33, IL-8, MCP-1, MIP-3α, CXCL1 and IL-23.

[0028] In some embodiments, the protein increases the level of at least one anti-inflammatory cytokine in the blood, plasma, serum, tissues and / or mucosa of a subject. In other embodiments, the at least one anti-inflammatory cytokine is selected from IL-4, IL-10, IL-13, IFN-α and TGF-β.

[0029] In some embodiments, the protein reduces the level of at least one anti-inflammatory cytokine in the blood, plasma, serum, tissues and / or mucosa of a subject. In other embodiments, the at least one anti-inflammatory cytokine is selected from IL-4, IL-10, IL-13, IFN-α and TGF-β.

[0030] In some embodiments, the disclosure teaches polynucleotides encoding novel protein therapeutic agents and methods of expressing the nucleic acids in host cells. In a specific embodiment, the polynucleotide comprises a sequence encoding a protein that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to SEQ ID NO: 19. In other embodiments, the polynucleotide comprises a sequence encoding a protein that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to SEQ ID NO: 19 and less than 100% identical to SEQ ID NO: 3. In other embodiments, the polynucleotide encodes a protein that is a non-naturally occurring variant of SEQ ID NO: 1 or SEQ ID NO: 3. In other embodiments, the polynucleotide is codon optimized for expression in a recombinant host cell. In other embodiments, the polynucleotide is codon optimized for expression in E. coli.

[0031] In some embodiments, the disclosure teaches a nucleic acid comprising a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 20. In some embodiments, the nucleic acid comprises a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 20 and less than 100% identical to SEQ ID NO: 4. In other embodiments, the nucleic acid comprises a sequence that is a non-naturally occurring variant of SEQ ID NO:2 or SEQ ID NO:4.

[0032] In some embodiments, the protein is chemically modified at the N-terminus and / or C-terminus. In other embodiments, the N-terminus of the protein is chemically modified by acetylation. In other embodiments, the C-terminus is chemically modified by amidation.

[0033] In some embodiments, the protein is pegylated.

[0034] In some embodiments, the protein is substantially purified and is modified by glycosylation, ubiquitination, nitrosylation, methylation, acetylation, or lipidation.

[0035] In some embodiments, the protein is fused to a second protein. In other embodiments, the second protein is an immunoglobulin Fc domain or a human serum albumin protein domain.

[0036] In some aspects, the present disclosure provides a pharmaceutical composition for treating inflammatory bowel disease, comprising: a therapeutic protein comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 100% sequence identity to SEQ ID NO: 19, and a pharmaceutically acceptable carrier. In some embodiments, the therapeutic protein is purified or substantially purified. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 3. In another embodiment, the protein does not comprise a sequence identical to SEQ ID NO: 3, or the protein is a non-naturally occurring variant of SEQ ID NO: 3. In other embodiments, the protein comprises an amino acid sequence selected from SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19. In other embodiments, the protein comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:7 or SEQ ID NO:19.

[0037] In some embodiments, the pharmaceutical composition is formulated for rectal, parenteral, intravenous, topical, oral, dermal, transdermal or subcutaneous administration. In other embodiments, the pharmaceutical composition is a liquid, gel or cream. In other embodiments, the pharmaceutical composition is a solid composition comprising an enteric coating.

[0038] In some embodiments, the pharmaceutical composition is a cream, capsule, liquid, gel, or emulsion.

[0039] In some embodiments, the pharmaceutical composition is formulated to provide delayed release. In other embodiments, the delayed release is released into the gastrointestinal tract. In other embodiments, the delayed release enters the mouth, small intestine, large intestine and / or rectum.

[0040] In some embodiments, the pharmaceutical composition is formulated to provide sustained release. In other embodiments, the sustained release is released into the gastrointestinal tract. In other embodiments, the sustained release enters the oral cavity, small intestine, large intestine and / or rectum. In other embodiments, the sustained release composition releases the therapeutic agent over a period of about 1 to 20 hours, 1 to 10 hours, 1 to 8 hours, 4 to 12 hours, or 5 to 15 hours.

[0041] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In other embodiments, the second therapeutic agent is selected from antidiarrheal drugs, 5-aminosalicylic acid compounds, anti-inflammatory agents, antibiotics, anti-cytokine agents, anti-inflammatory cytokine agents, steroids, corticosteroids, immunosuppressants, JAK inhibitors, anti-integrin biologics, anti-IL12 / 23R biologics and vitamins.

[0042] In some embodiments, the pharmaceutical composition further comprises a protease inhibitor. In other embodiments, the protease inhibitor inhibits degradation of the therapeutic protein in the presence of feces and / or blood.

[0043] As previously described, these novel protein therapeutics are capable of promoting epithelial barrier function and integrity of a subject. In some embodiments, the epithelial barrier function is an intestinal epithelial barrier function. In addition, the therapeutic effects of the protein include inhibiting inflammatory immune responses in IBD individuals. Therefore, the present disclosure provides detailed guidance for methods of treating many gastrointestinal inflammatory conditions and disease states involving impaired gastrointestinal epithelial barrier integrity using the therapeutic proteins taught.

[0044] In some embodiments, a method for treating an inflammatory bowel disease or condition in a patient thereof is provided, comprising: administering to the patient a pharmaceutical composition comprising: i) a therapeutic protein comprising an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19; and ii) a pharmaceutically acceptable carrier. In other embodiments of the method, the protein comprises an amino acid sequence identical to SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19. In other embodiments, the protein is not identical to SEQ ID NO: 3 or is a non-naturally occurring variant of SEQ ID NO: 3.

[0045] In some embodiments, the patient has been diagnosed with intestinal inflammation. In other embodiments, the intestinal inflammation is in the small intestine and / or large intestine. In other embodiments, the intestinal inflammation is in the rectum. In other embodiments, the patient has been diagnosed with pouchitis.

[0046] In some embodiments, the patient has been diagnosed with intestinal ulcers. In other embodiments, the patient has been diagnosed with intestinal endothelial drainage and / or rectovaginal fistulas.

[0047] In some embodiments, the patient has been diagnosed with Crohn's disease (CD). In other embodiments, the CD is mildly active CD. In other embodiments, the CD is moderately to severely active CD. In other embodiments, the patient has been diagnosed with pediatric CD.

[0048] In some embodiments, the patient has been diagnosed with short bowel syndrome or irritable bowel syndrome.

[0049] In some embodiments, the patient has been diagnosed with mucositis. In other embodiments, the mucositis is oral mucositis. In other embodiments, the mucositis is chemotherapy-induced mucositis, radiation therapy-induced mucositis, chemotherapy-induced oral mucositis, or radiation therapy-induced oral mucositis. In other embodiments, the mucositis is gastrointestinal mucositis. In other embodiments, the gastrointestinal mucositis is mucositis of the small intestine, large intestine, or rectum.

[0050] In some embodiments, administration to a patient diagnosed with CD results in reduced intestinal endothelial drainage and / or the number of rectovaginal fistulas. In other embodiments, the administration maintains fistula closure in an adult patient with fistula disease.

[0051] In other embodiments, the patient has been diagnosed with ulcerative colitis (UC). In other embodiments, the UC is mildly active UC. In other embodiments, the UC is moderately to severely active UC. In other embodiments, the patient has been diagnosed with pediatric UC.

[0052] In some embodiments, the patient is in clinical remission from IBD. In other embodiments, the patient is in clinical remission from UC, pediatric UC, CD, or pediatric CD.

[0053] In some embodiments, the patient has an inflammatory bowel disease or condition other than Crohn's disease or ulcerative colitis. In other embodiments, the patient has at least one symptom associated with an inflammatory bowel disease.

[0054] In some embodiments, administration reduces gastrointestinal inflammation in the patient and / or reduces intestinal mucosal inflammation associated with inflammatory bowel disease. In other embodiments, administration improves intestinal epithelial cell barrier function or integrity in the patient.

[0055] In some embodiments, after administration, the patient experiences a reduction in at least one symptom associated with an inflammatory bowel disease or condition. In other embodiments, the at least one symptom is selected from abdominal pain, blood in the stool, pus in the stool, fever, weight loss, frequent diarrhea, fatigue, loss of appetite, nausea, cramps, anemia, tenesmus, and rectal bleeding. In other embodiments, after administration, the patient experiences a decrease in the frequency of diarrhea, a decrease in blood in the stool, and / or a decrease in rectal bleeding.

[0056] In some embodiments, the patient has an inadequate response to conventional therapy. In other embodiments, conventional therapy is treatment with aminosalicylates, corticosteroids, thiopurines, methotrexate, JAK inhibitors, sphingosine 1-phosphate (S1P) receptor inhibitors, anti-integrin biologics, anti-IL12 / 23R or anti-IL23 / p10 biologics, and / or anti-tumor necrosis factor agents or biologics.

[0057] In some embodiments, the administration modulates (ie, increases or decreases) cytokine levels in the patient's blood, plasma, serum, mucosa, or tissue.

[0058] In some embodiments, the administration suppresses the level of at least one proinflammatory cytokine in the patient. In other embodiments, the at least one proinflammatory cytokine is selected from TNF-α, IL-17, IL-1β, IL-2, IFN-γ, IL-6, IL-12, IL-25, IL-33, IL-8, MCP-1, MIP-3α, CXCL1 and IL-23.

[0059] In some embodiments, administration increases the level of at least one anti-inflammatory cytokine in the patient's blood, plasma, serum, mucosa or tissue. In other embodiments, the at least one anti-inflammatory cytokine is selected from IL-4, IL-10, IL-13, IFN-α and TGF-β.

[0060] In some embodiments, administration reduces the level of at least one anti-inflammatory cytokine in the patient's blood, plasma, serum, mucosa or tissue. In other embodiments, the at least one anti-inflammatory cytokine is selected from IL-4, IL-10, IL-13, IFN-α and TGF-β.

[0061] In some embodiments, the administering increases the amount of mucin in the intestinal lumen of the patient.

[0062] In some embodiments, the administering increases wound healing of intestinal epithelial cells in the patient.

[0063] In some embodiments, the administering prevents or reduces shortening of the patient's colon.

[0064] In some embodiments, administration comprises administering the pharmaceutical composition to a patient rectally, intravenously, parenterally, orally, topically, dermally, transdermally or subcutaneously. In other embodiments, administration is administration into the gastrointestinal lumen.

[0065] In some embodiments, at least one second therapeutic agent is also administered to the patient. In other embodiments, at least one second therapeutic agent is selected from an antidiarrheal, an anti-inflammatory, an antibody, an antibiotic, or an immunosuppressant. In other embodiments, at least one second therapeutic agent is an aminosalicylate, a steroid, or a corticosteroid. In other embodiments, at least one second therapeutic agent is selected from adalimumab, polyethylene glycol, golimumab, infliximab, vedolizumab, ustekinumab, tofacitinib, and certolizumab pegol or certolizumab pegol.

[0066] In some aspects, an expression vector is provided that comprises an exogenous polynucleotide encoding a protein comprising an amino acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:19.

[0067] In some embodiments, the polynucleotide encodes a protein comprising an amino acid sequence at least 99% or 100% identical to SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19. In other embodiments, the polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO: 3. In other embodiments, the polynucleotide encodes a protein comprising an amino acid sequence that is not identical to SEQ ID NO: 3.

[0068] In some aspects, an expression system is provided, which includes a host cell and an expression vector comprising the aforementioned exogenous polynucleotide.

[0069] In some embodiments, the host cell is prokaryotic or eukaryotic. In other embodiments, the host cell is a mammalian cell, a yeast cell or a bacterial cell. In other embodiments, the bacterial cell is Escherichia coli. In other embodiments, the mammalian cell is a CHO cell.

[0070] In some aspects, methods of producing proteins are provided.

[0071] In some embodiments, the method for producing a protein comprises transforming or transfecting the host cell with the expression vector, and culturing the transformed or transfected host cell under conditions sufficient to express the protein encoded by the exogenous polynucleotide. In other embodiments, the method further comprises purifying the protein from the transformed or transfected host cell and culture medium.

[0072] In some embodiments, a method for treating a disease, such as a disease related to intestinal epithelial barrier function, is provided, which utilizes any sequence disclosed in the present application and the sequence listing. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1A and Figure 1B Restoration of epithelial barrier integrity by SG-11 following inflammation-induced disruption as described in Example 2 is shown.

[0074] Figure 2A The effect of SG-11 administration on TNF-α production induced by heat-killed Escherichia coli (HK E. coli) as described in Example 3 is shown.

[0075] Figure 2B The effect of SG-11 administration on IL-23 production induced by HK E. coli as described in Example 3 is shown.

[0076] Figure 3 The effect of SG-11 administration on IL-10 production induced by HK E. coli as described in Example 4 is shown.

[0077] Figure 4 The effect of SG-11 administration on mucin expression after HK E. coli stimulation as described in Example 5 is shown.

[0078] Figure 5 The effect of SG-11 administration on epithelial cell wound healing as described in Example 6 is shown.

[0079] Figure 6 The effects of SG-11 administration on epithelial central barrier function readouts in the DSS model of inflammatory bowel disease as described in Example 7 are shown.

[0080] Figure 7 The effects of SG-11 administration on inflammatory readouts in response to impaired barrier function in the DSS model of inflammatory bowel disease as described in Example 7 are shown.

[0081] Figure 8The effect of SG-11 administration on body weight in the DSS model of inflammatory bowel disease as described in Example 7 is shown.

[0082] Fig. 9 The effect of SG-11 administration on gross pathology in the DSS model of inflammatory bowel disease as described in Example 7 is shown.

[0083] Fig. 10A , 10B and 10C show the proximal end of the DSS model of inflammatory bowel disease as described in Example 7 ( Fig. 10A ),remote( Fig. 10B ) and proximal and distal ( Fig. 10C ) tissue histopathological analysis results.

[0084] Fig.11A The effect of SG-11 administration on colon length in the DSS model of inflammatory bowel disease as described in Example 7 is shown.

[0085] Fig. 11B The effect of SG-11 administration on the ratio of colon weight to length in the DSS model of inflammatory bowel disease as described in Example 7 is shown.

[0086] Fig.12 The integrity of the epithelial barrier following SG-11 treatment of the DSS model of inflammatory bowel disease as described in Example 8 is shown.

[0087] Fig.13 An inflammation-centric readout of barrier function in the DSS model of inflammatory bowel disease as described in Example 8 is shown.

[0088] Fig.14 Prevention of weight loss in the DSS model of inflammatory bowel disease as described in Example 8 was demonstrated.

[0089] Fig.15A The effect of SG-11 administration on colon length in the DSS model of inflammatory bowel disease as described in Example 8 is shown.

[0090] Fig. 15B The effect of SG-11 administration on the ratio of colon weight to length in the DSS model of inflammatory bowel disease as described in Example 8 is shown.

[0091] Fig.16A , 16B and 16C show the proximal ( Fig.16A ),remote( Fig. 16B ) and proximal and distal ( Fig. 16C ) tissue histopathological analysis results.

[0092] Fig.17Shown are the results of a multiple sequence alignment analysis of SG-11 (SEQ ID NO: 7) with similar protein sequences from Roseburia species.

[0093] Figure 18 shows the Fig.18A , Fig.18B , Fig.18C , Fig.18D , Fig.18E , Fig.18F , Figure 18G , Fig.18H and Fig.18I The influence of conditions on the stability of SG-11. FIG. 18A to FIG. 18I For relevant conditions, please see Example 14.

[0094] Figure 19 shows the Fig.19A , Fig.19B , Fig.19C , Fig.19D , Fig.19E , Fig.19F , Figure 19G , Fig.19H and Fig.19I The influence of conditions on the stability of SG-11V5. FIG. 19A to FIG. 19I For relevant conditions, please see Example 14.

[0095] Fig. 20 Restoration of epithelial barrier integrity by SG-11 and SG-11 variants following inflammation-induced disruption as described in Example 15 is shown.

[0096] Fig.21A and Fig.21B Epithelial barrier integrity following treatment of the DSS model of inflammatory bowel disease with SG-11 and SG-11 variants as described in Example 16 is shown.

[0097] Fig.22A and Fig. 22B An inflammation-centric readout of barrier function in the DSS model of inflammatory bowel disease as described in Example 16 is shown.

[0098] Fig.23A and Fig. 23B The effect of treatment with SG-11 or SG-11 variants on weight loss in the DSS model of inflammatory bowel disease as described in Example 16 is shown.

[0099] Fig.24 The effect of administration of SG-11 or SG-11 variants on overall pathology in the DSS model of inflammatory bowel disease as described in Example 16 is shown.

[0100] Fig.25A and Fig.25BThe effect of treatment with SG-11 or SG-11 variants on colon length in the DSS model of inflammatory bowel disease as described in Example 16 is shown.

[0101] Fig.26A and Fig.26B The effect of treatment with SG-11 or SG-11 variants on the colon weight / length ratio in the DSS model of inflammatory bowel disease as described in Example 16 is shown.

[0102] Fig.27A The results of multiple sequence alignment analysis of SG-11 (SEQ ID NO: 7) and SG-11 variants (SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19) are shown, and Fig.27B Results of the percentage identity matrix based on multiple sequence alignment analysis are shown. The Clustal Omega program provided by EMBL-EBI was used for the multiple alignment analysis described herein. DETAILED DESCRIPTION OF THE INVENTION

[0104] The present disclosure provides novel protein therapeutic agents that can be used to treat subjects with symptoms associated with gastrointestinal diseases. For example, these proteins can promote or enhance epithelial barrier function and / or integrity. The protein can also inhibit the inflammatory immune response of IBD individuals. The protein therapeutic agents provided herein can be used to treat a variety of diseases associated with reduced gastrointestinal epithelial barrier function or intestinal integrity and inflammation.

[0105] Also provided in the present disclosure are protein variants having therapeutic activity comparable to or superior to that of the original protein, but having enhanced stability during manufacture and processing of protein therapeutic products and under long-term storage conditions.

[0106] definition

[0107] Unless otherwise defined, the scientific and technological terms used in this application have the same meanings as those generally understood by those of ordinary skill in the art to which the present invention belongs. Generally, the nomenclature and techniques used in chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry described herein are well known and commonly used in the art. Therefore, although the following terms are believed to be well known to those of ordinary skill in the art, the following definitions are given to aid in understanding the subject matter disclosed herein.

[0108] Throughout this document, the word "comprise" or its derivative forms such as "include" or "contain" should be understood to mean the inclusion of stated components or groups of components but not the exclusion of any other components or groups of components.

[0109] The term "a" or "an" means one or more entities, i.e., includes plural meanings. Therefore, "a" or "a kind", "one or more", and "at least one" can be used interchangeably. In addition, the use of "a" or "an" to indicate "an element" does not exclude the presence of more than one element, unless it is explicitly required that there is only or only one element.

[0110] The term "including" is used to mean "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0111] As used herein, the terms "gastrointestinal" or "gastrointestinal tract," "digestive tract," and "intestine" are used interchangeably to refer to a series of hollow organs extending from the mouth to the anus and including the mouth, esophagus, stomach, small intestine, large intestine, rectum, and anus. The terms "gastrointestinal" or "gastrointestinal tract," "digestive tract," and "intestine" are not always intended to be limited to a specific portion of the digestive tract.

[0112] As used herein, the term "SG-11" refers to a protein comprising the amino acid sequence of SEQ ID NO: 3, and also refers to variants thereof having the same or similar functional activity as described herein. Therefore, SG-11 herein may refer to a protein comprising or consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9, or a variant or fragment thereof. Examples of SG-11 variants include, but are not limited to, SEQ ID NO: 11 (SG-11V1), SEQ ID NO: 13 (SG-11V2), SEQ ID NO: 15 (SG-11V3), SEQ ID NO: 17 (SG-11V4) and SEQ ID NO: 19 (SG-11V5). In U.S. provisional patent applications (62 / 482,963, filed April 7, 2017; 62 / 607,706, filed December 19, 2017; 62 / 611,334, filed December 28, 2017, to which this specification claims priority and is incorporated herein by reference in its entirety), the term "experimental protein 1" and its variants are used and are synonymous with SG-11 or its variants as used herein.

[0113] "Signal sequence" (also called "prequel", "signal peptide", "leader sequence" or "leader peptide") refers to an amino acid sequence located at the N-terminus of a nascent protein and which promotes secretion of the protein from a cell. The resulting extracellular protein mature form lacks the signal sequence, which is cleaved off during the secretion process.

[0114] As used herein, the terms "sequence identity", "percentage of identity", "percentage of homology", or, for example, "a sequence that is 50% identical to ..." refer to the degree to which sequences are identical on the basis of nucleotides or amino acids in the comparison window. Therefore, the method for calculating the "percentage of sequence identity" can be to compare two optimally aligned sequences in the comparison window, determine the number of sites where the same nucleic acid bases (e.g., A, T, C, G, I) or amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) appear in the two sequences to obtain the number of matching sites, divide the number of matching sites by the total number of sites in the comparison window (i.e., the window size), and multiply the result by 100 to obtain the percentage of sequence identity.

[0115] The calculation of sequence similarity or sequence identity (these terms are used interchangeably herein) between sequences can be performed as follows. In order to determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences can be compared for the best comparison purpose (for example, a gap can be introduced in one or both of the first and second amino acid or nucleic acid sequences to achieve the best alignment, and non-homologous sequences can be ignored for the purpose of comparison). In some embodiments, the length of the reference sequence compared for the purpose of comparison is at least 30% of the length of the reference sequence, preferably at least 40%, more preferably at least 50%, 60%, even more preferably at least 70%, 80%, 90%, or 100%. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When the amino acid residue or nucleotide occupied at a position in the first sequence is the same as that at the corresponding position in the second sequence, then these molecules are the same at that position. The percentage identity between the two sequences is related to the number of common identical positions of the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for the best comparison of the two sequences.

[0116] In the context of at least two nucleic acids or polypeptides, the phrases "substantially similar" and "substantially identical" generally refer to polynucleotides or polypeptides comprising sequences having at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7% or even 99.8% sequence identity compared to a reference polynucleotide or polypeptide. In some embodiments, substantially identical polypeptides differ only by one or more conservative amino acid substitutions. In some embodiments, substantially identical polypeptides are immunologically cross-reactive. In some embodiments, substantially identical nucleic acid molecules hybridize to each other under stringent conditions (e.g., within a range of moderate to high stringency).

[0117] As is well known in the art, as used herein, the term "nucleotide changes" refers to, for example, nucleotide substitutions, deletions and / or insertions. For example, mutations include changes that produce silent substitutions, additions or deletions that do not alter the properties or activity of the encoded protein or the way the protein is made.

[0118] Related (and derived) proteins include "variant" proteins. Variant proteins can differ from another (i.e., parent) protein and / or from each other by a small number of amino acid residues. The variant can include one or more amino acid mutations (e.g., amino acid deletions, insertions or substitutions) compared to the parent protein from which the variant is derived.

[0119] "Conservatively modified variants" apply to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, a conservatively modified variant refers to a nucleic acid encoding the same amino acid sequence or an amino acid sequence having one or more "conservative substitutions". An example of a conservative substitution is the exchange of an amino acid from one of the following groups with another amino acid from the same group (see U.S. Pat. No. 5,767,063; Kyte and Doolittle (1982) J. Mol. Biol. 157: 105-132). (1) Hydrophobic: norleucine, Ile, Val, Leu, Phe, Cys, Met; (2) Neutral hydrophilic: Cys, Ser, Thr; (3) Acidic: Asp, Glu; (4) Basic: Asn, Gln, His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe; and (7) Small amino acids: Gly, Ala, Ser. Thus, the term "conservative substitution" with respect to amino acids refers to the substitution of one or more amino acids by another chemically similar residue, wherein the substitution generally does not affect the functional properties of the protein. Examples include substitutions of amino acid residues with similar characteristics, such as small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids, and aromatic amino acids. In some embodiments, the disclosure provides proteins having at least one non-naturally occurring conservative amino acid substitution relative to the amino acid sequence identified in SEQ ID NO: 3 or SEQ ID NO: 17. Some common illustrative examples of conservative amino acid substitutions are as follows.

[0120] The term "amino acid" or "any amino acid" refers to any and all amino acids, including naturally occurring amino acids (e.g., α-amino acids), unnatural amino acids, modified amino acids, and unnatural or non-natural amino acids. It includes D- and L-amino acids. Natural amino acids include those found in nature, such as the 23 amino acids that are combined into peptide chains to form the structural modules of a large number of proteins. These are mainly L stereoisomers, although some D-amino acids are present in bacterial envelopes and some antibiotics, for example. The 20 "standard" natural amino acids are listed in the table above. "Non-standard" natural amino acids are pyrrolysine (present in methanogenic organisms and other eukaryotic organisms), selenocysteine ​​(present in many non-eukaryotic organisms and most eukaryotic organisms), and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts). "Unnatural" or "non-natural" amino acids are protein amino acids that are not naturally occurring or chemically synthesized (i.e., amino acids that are not naturally encoded or not found in the genetic code). There are more than 140 known unnatural amino acids, and there may be more than tens of thousands of combinations. "Modified" amino acids include amino acids (eg, natural amino acids) that have been chemically modified to include one or more groups or chemical moieties that are not naturally occurring on the amino acid.

[0121] As used herein, a "synthetic nucleotide sequence" or "synthetic polynucleotide sequence" is a nucleotide sequence that is not known to occur naturally or that does not occur naturally. Typically, such a synthetic nucleotide sequence will contain at least one nucleotide difference when compared to any other naturally occurring nucleotide sequence. As used herein, a "synthetic amino acid sequence" or "synthetic peptide sequence" or "synthetic polypeptide sequence" or "synthetic protein sequence" is an amino acid sequence that is not known to occur naturally in nature. Typically, such a synthetic amino acid sequence will contain at least one amino acid difference when compared to any other naturally occurring amino acid sequence.

[0122] As used herein, "synthetic protein" or "synthetic therapeutic protein" refers to a protein comprising an amino acid sequence comprising one or more amino acids substituted with a different amino acid relative to a naturally occurring amino acid sequence. That is, a "synthetic protein" comprises an amino acid sequence that has been altered to comprise at least one non-naturally occurring substitution modification at a given amino acid position relative to a naturally occurring amino acid sequence.

[0123] As used herein, with respect to % sequence identity or % sequence homology of a nucleic acid sequence or an amino acid sequence, the term "about" refers to up to and including ±1.0% in increments of 0.1%. For example, "about 90%" sequence identity includes 89.0%, 89.1%, 89.2%, 89.3%, 89.4%, 89.5%, 89.6%, 89.7%, 89.8%, 89.9%, 90%, 90.1%, 90.2%, 90.3%, 90.4%, 90.5%, 90.6%, 90.7%, 90.8%, 90.9% and 91%. If not used in the context of % sequence identity, "about" means ±1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, depending on the context of the relevant value.

[0124] In most cases, the names of natural and unnatural amino acid residues used herein follow the naming conventions recommended by the IUPAC Organic Nomenclature Commission and the IUPAC-IUB Biochemical Nomenclature Commission, as set forth in "Nomenclature of α-Amino Acids (Recommendations, 1974)" Biochemistry, 14(2), (1975). To the extent that the names and abbreviations of amino acids and aminoacyl residues used in this specification and the appended claims differ from those suggested, the reader will be made aware of them.

[0125] Throughout the specification, unless the full name of a natural amino acid is used (e.g., alanine, arginine, etc.), it is represented by a conventional three-letter or single-letter abbreviation (e.g., Ala or A for alanine, Arg or R for arginine, etc.). Unless otherwise indicated, the three-letter and single-letter abbreviations of amino acids refer to the L isomeric form of the amino acid. As used herein, the term "L-amino acid" refers to the "L" isomeric form of a peptide, and conversely, the term "D-amino acid" refers to the "D" isomeric form of a peptide (e.g., Dasp, (D)Asp or D-Asp; Dphe, (D)Phe or D-Phe). An amino acid residue in the D isomeric form can replace any L-amino acid residue as long as the peptide retains the desired function. When represented using single-letter abbreviations, D-amino acids are generally represented in lowercase.

[0126] In the case of less common or unnatural amino acids, unless referred to by their full names (e.g., sarcosine, ornithine, etc.), their residues are generally referred to by three or four character codes, including Sar or Sarc (sarcosine, i.e., N-methylglycine), Aib (α-aminoisobutyric acid), Dab (2,4-diaminobutyric acid), Dapa (2,3-diaminopropionic acid), γ-Glu (γ-glutamic acid), Gaba (γ-aminobutyric acid), β-Pro (pyrrolidine-3-carboxylic acid) and 8Ado (8-amino-3,6-dioxaoctanoic acid), Abu (2-aminobutyric acid), βhPro (β-homoproline), βhPhe (β-homophenylalanine) and Bip (β,β-diphenylalanine) and Ida (iminodiacetic acid).

[0127] The sequences disclosed herein are sequences in which a "Hy-" moiety is incorporated at the amino terminus (N-terminus) and a "-OH" moiety or a "-NH2" moiety is incorporated at the carboxyl terminus (C-terminus). In this case, unless otherwise indicated, the "Hy-" moiety at the N-terminus of the relevant sequence represents a hydrogen atom, corresponding to the presence of a free primary or secondary amino group at the N-terminus, and the "-OH" or "-NH2" moiety at the C-terminus of the sequence represents a hydroxyl or amino group, corresponding to the presence of an amide group (CONH2) at the C-terminus. In each sequence disclosed herein, the C-terminal "-OH" moiety can replace the C-terminal "-NH2" moiety, and vice versa.

[0128] As used herein, the term "Ac" refers to acetyl protection by acylation of the C- or N-terminus of a polypeptide. In certain peptides shown herein, the NH2 at the C-terminus of the peptide represents an amino group. As used herein, the term "carboxyl" refers to -CO2H.

[0129] The term "pharmaceutically acceptable salt" as used herein represents a salt or zwitterionic form of a peptide, protein or compound of the subject invention, which is water-soluble or oil-soluble or dispersible; which is suitable for treating disease without excessive toxicity, irritation and allergic response; which is commensurate with a reasonable benefit / risk ratio; and which is effective for its intended use. These salts can be prepared during the final isolation and purification of the compound, or they can be prepared separately by reacting an amino group with a suitable acid. Representative acid addition salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isethionate), lactate, maleate, m-benzenesulfonate, methanesulfonate, naphthalenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, p-toluenesulfonate and undecanoate. In addition, the amino groups in the compounds of the present disclosure can be quaternized with the following: methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dimethyl, diethyl, dibutyl and dimethyl sulfates; decyl, lauryl, myristyl and benzyl chlorides, bromides and iodides; and benzyl and phenethyl bromides. Examples of acids that can be used to form therapeutically acceptable addition salts include inorganic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid) and organic acids (such as oxalic acid, maleic acid, succinic acid and citric acid). Pharmaceutically acceptable salts can be suitably selected from, for example, acid addition salts and basic salts. Examples of acid addition salts include hydrochlorides, citrates and acetates. Examples of basic salts include salts in which cations are selected from alkali metal cations such as sodium or potassium ions, alkaline earth metal cations such as calcium or magnesium ions and substituted ammonium ions. Other examples of pharmaceutically acceptable salts are described in "Remington's Pharmaceutical Sciences", 17th edition, Alfonso R. Gennaro (ed.), Mark Publishing Company, Easton, Pennsylvania, USA, 1985 (and its latest edition), in "Encyclopaedia of Pharmaceutical Technology", 3rd edition, James Swarbrick (ed.), Informa Healthcare USA (Incorporated), New York, USA, 2007, and J. Pharm. Sci. 66: 2 (1977).Additionally, for a review of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use, eds. Stahl and Wermuth (Wiley-VCH, 2002).

[0130] As used herein, the terms "at least a portion" or "fragment" of a nucleic acid or polypeptide refer to a portion having a minimum size characteristic of such sequence, or any larger fragment of the full-length molecule, up to and including the full-length molecule.

[0131] As used herein, the term "primer" refers to an oligonucleotide capable of annealing to a target polynucleotide.

[0132] As used herein, the phrases "recombinant construct," "expression construct," "chimeric construct," "construct" and "recombinant DNA construct" are used interchangeably herein and are well known to the skilled artisan.

[0133] As used herein, the term "host cell" refers to a cell or cell line into which a recombinant expression vector for producing a polypeptide can be introduced so as to express the polypeptide.

[0134] As used herein, the terms "isolated," "purified," "separated," and "recovered" refer to a material (e.g., a protein, nucleic acid, or cell) that is removed from at least one component with which it is naturally associated, e.g., at a concentration of at least 90%, or at least 95%, or at least 98% by weight of the contained sample. For example, these terms can refer to a material that is substantially or essentially free of components that normally accompany it as found in its natural state, such as an intact biological system.

[0135] "Subject", "patient" and "individual" are used interchangeably and refer to humans or non-human animals. These terms include mammals, such as humans, non-human primates, livestock animals (such as cattle, pigs), animal companions (such as dogs, cats) and rodents (such as mice and rats). In certain embodiments, these terms refer to human patients. In exemplary embodiments, the term refers to a human patient suffering from a gastrointestinal inflammatory disease.

[0136] As used herein, "improvement" shall broadly include improvements in the characteristics of a determined disease state compared to a control, or compared to a known average quantity associated with the characteristic in question, which is considered by a person skilled in the art to be generally associated with or indicative of the disease in question. For example, "improved" epithelial barrier function associated with the use of the proteins of the present disclosure can be demonstrated by comparing the epithelial barrier integrity of a person treated with the protein of the present invention compared to the epithelial barrier integrity of an untreated person. Alternatively, the epithelial barrier integrity of a person treated with a protein of the present disclosure can be compared to the average epithelial barrier integrity of a person, as represented in a scientific or medical publication known to a person skilled in the art. In the present disclosure, "improvement" does not necessarily require that the data be statistically significant (i.e., p < 0.05); rather, any quantifiable difference indicating that one value (e.g., the average treatment value) is different from another value (e.g., the average control value) can be raised to the level of "improvement".

[0137] As used herein, "inhibit and suppress" and similar terms should not be interpreted as requiring complete inhibition or suppression, although this may be desirable in some embodiments. Therefore, "suppressed immune response" or "inhibition of inflammatory cytokines" does not require absolute suppression.

[0138] Thus, as used herein, the terms "increase," "inhibit," or "decrease," or grammatical equivalents thereof, refer to values ​​relative to a reference (e.g., baseline) measurement, such as a value under comparable conditions (e.g., a measurement in the same individual before starting a treatment described herein, or in a control individual (or multiple control individuals) without a treatment described herein). In some embodiments, a suitable control is a baseline measurement, such as a measurement in the same individual before starting a treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of a treatment described herein.

[0139] As used herein, the term "IBD" or "inflammatory bowel disease" refers to a condition in which an individual has a chronic or recurring immune response and inflammation of the gastrointestinal (GI) tract. The two most common inflammatory bowel diseases are ulcerative colitis (UC) and Crohn's disease (CD).

[0140] As used herein, the term "therapeutically effective amount" refers to the amount of a therapeutic agent (e.g., a peptide, polypeptide or protein of the present invention) that imparts a therapeutic effect to a subject being treated at a reasonable benefit / risk ratio applicable to any medical treatment. This therapeutic effect can be objective (i.e., measurable by a test or marker) or subjective (i.e., the subject of treatment shows a sign or feels the effect). In some embodiments, a "therapeutically effective amount" refers to an amount of a therapeutic agent or composition that effectively treats, alleviates or prevents (e.g., delays the onset) a related disease or condition and / or exhibits a detectable therapeutic or preventive effect, such as by alleviating symptoms associated with the disease, preventing or delaying the onset of the disease, and / or also alleviating the severity or frequency of the symptoms of the disease. The therapeutically effective amount is typically administered in a dosage regimen that may include multiple unit doses. For any specific therapeutic agent, the therapeutically effective amount (and / or a suitable unit dose within an effective dosage regimen) may vary, for example, according to the route of administration, in combination with other therapeutic agents. Alternatively or additionally, the specific therapeutically effective amount (and / or unit dose) for any particular patient can depend on a variety of factors, including the specific form of the disease being treated; the severity of the condition or pre-condition; the activity of the specific therapeutic agent used; the specific composition used; the patient's age, weight, general health, sex and diet; the administration time, administration route and / or excretion or metabolic rate of the specific therapeutic agent used; the duration of treatment; and similar factors well known in the medical field. The present disclosure utilizes a therapeutically effective amount of novel proteins and compositions thereof to treat a variety of diseases, such as: inflammatory diseases of the gastrointestinal tract or diseases involving gastrointestinal epithelial barrier dysfunction. In some embodiments, the administered protein or a therapeutically effective amount of a composition comprising the protein will reduce inflammation associated with IBD or repair gastrointestinal epithelial barrier integrity and / or function.

[0141] As used herein, the term "treatment" (also referred to as "treatment") refers to any administration of a therapeutic agent (e.g., a peptide, polypeptide or protein of the present invention) according to a treatment regimen that achieves the desired effect, such that it partially or completely alleviates, alleviates, attenuates, inhibits, delays the onset of one or more symptoms or features of a specific disease, disorder and / or condition (e.g., chronic or recurring gastrointestinal immune response and inflammation), reduces its severity and / or reduces its incidence; in some embodiments, the administration of a therapeutic agent according to a treatment regimen is associated with the achievement of a desired effect. Such treatment may be for subjects who do not show signs of the relevant disease, disorder and / or disorder and / or subjects who only show early signs of the disease, disorder and / or disorder. Alternatively or in addition, such treatment may be for subjects who show established signs of the relevant disease, disorder and / or disorder. In some embodiments, treatment may be performed on subjects who have been diagnosed with the relevant disease, disorder and / or condition. In some embodiments, treatment may be directed to subjects known to have one or more susceptibility factors that are statistically associated with an increased risk of developing the relevant disease, disorder and / or condition.

[0142] "Pharmaceutical" means that the composition, agent, method, etc. is capable of having a pharmaceutical effect and that the composition can also be safely administered to a subject. "Pharmaceutical effect" is not limited to and can mean that the composition, agent or method is capable of stimulating a desired biochemical, genetic, cellular, physiological or clinical effect in at least one individual, such as a mammalian subject, such as a human, in at least 5% of a subject population, at least 10%, at least 20%, at least 30%, at least 50% of the subjects, etc. "Pharmaceutically acceptable" means approved by a federal or state government regulatory agency, or listed in the U.S. Pharmacopeia or other generally accepted pharmacopeia for safety for use in animals, more specifically for safety for human use. "Pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a diluent, adjuvant, excipient or vehicle administered with a protein described herein.

[0143] "Prevention" refers to reducing the risk of developing a disease or condition (i.e., causing at least one of the clinical symptoms of the disease not to develop, or causing the symptoms to develop to a lesser severity than would occur in the absence of treatment, in a subject who may be exposed to or susceptible to the disease but does not yet experience or display symptoms of the disease). "Prevention" may refer to delaying the onset of a disease or condition.

[0144] The therapeutic pharmaceutical compositions taught herein may include one or more natural products. However, in certain embodiments, the therapeutic pharmaceutical compositions themselves are not naturally occurring. In addition, in certain embodiments, the therapeutic pharmaceutical compositions have significantly different characteristics compared to any individual naturally occurring counterparts or composition components that may exist in nature. That is, in certain embodiments, the pharmaceutical compositions taught herein (which contain a therapeutically effective amount of purified protein) have at least one structural and / or functional property that imparts significantly different characteristics to the composition as a whole compared to any individual component of the composition that may occur naturally. The court has determined that compositions comprising natural products having significantly different characteristics compared to any individual component that may occur naturally are statutory subjects. Therefore, the therapeutic pharmaceutical compositions taught generally have significantly different characteristics. These characteristics are illustrated in the data and examples taught herein.

[0145] The details of the present disclosure are set forth herein. Although methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of the present invention, exemplary methods and materials are described below. Other features, objects and advantages of the present invention will be apparent from the specification and claims.

[0146] Therapeutic proteins derived from the microbiome - open overview

[0147] A variety of diseases and disorders are associated with reduced barrier function or integrity of the gastrointestinal epithelium. These diseases and conditions are multifaceted and present in a variety of ways in diagnosis. One such disease is inflammatory bowel disease (IBD), the incidence and prevalence of which have increased over time and around the world, indicating that it has become a global disease. (Merchlinsky et al., Virology 142: 46-54, 2012). IBD is a general term used to describe conditions with chronic or recurrent gastrointestinal (GI) tract immune response and inflammation. The two most common inflammatory bowel diseases are ulcerative colitis (UC) and Crohn's disease (CD). Both are marked by abnormal responses of the gastrointestinal immune system. Normally, immune cells protect the body from infection. However, in people with IBD, this immune system mistakes food, bacteria, and other substances in the intestine for pathogens, and an inflammatory response enters the intestinal lining, causing chronic inflammation. When this happens, the patient experiences IBD symptoms.

[0148] IBD involves chronic inflammation of all or part of the digestive tract. Both UC and CD often involve symptoms such as severe diarrhea, abdominal pain, fatigue, and weight loss. IBD and related diseases can be debilitating and sometimes lead to life-threatening complications.

[0149] Regarding intestinal barrier integrity, loss of intestinal epithelial integrity plays a key pathogenic role in IBD. Maloy, Kevin J.; Powrie, Fiona, "Intestinal homeostasis and its breakdown in inflammatory bowel disease," (2011) Nature. 474(7351): 298-306. It is hypothesized that deleterious changes in the intestinal flora cause inappropriate or uncontrolled immune responses, leading to damage to intestinal epithelial cells. Disruptions in this critical intestinal epithelial barrier allow further infiltration of the microbiota, which in turn causes further immune responses. Therefore, IBD is a multifactorial disease driven in part by an excessive immune response to the intestinal microbiota, which can lead to defects in epithelial barrier function.

[0150] Microbiome profiling in IBD patients has shown different profiles, such as an increase in Proteobacteria, including adherent invasive Escherichia coli, often at the expense of potentially beneficial microorganisms such as Roseburia species (Machiels et al., 2014, Cut, 63: 1275-1283; Patterson et al., 2017, Front Immunol, 8: 1166; Shawki and McCole, 2017, Cell Mol Gastroenterol Hepatol, 3: 41-50). In addition, a decrease in Roseburia hominis has been associated with malnutrition in patients with ulcerative colitis. It has been found that individuals affected by IBD have a 30% to 50% reduction in the biodiversity of commensal bacteria, such as a decrease in Firmicutes (i.e., Lachnospiraceae) and Bacteroidetes. Further evidence for the role of the gut microbiota in inflammatory bowel disease is that individuals with IBD infection are more likely to have been prescribed antibiotics in the 2-5 years before diagnosis compared to unaffected individuals. See Aroniadis OC, Brandt LJ, "Fecal microbiota transplantation: past, present and future", (2013) Curr. Opin. Gastroenterol. 29(1) (2013): 79-84.

[0151] In various clinical and preclinical studies, protective bacterial communities, probiotics, and bacterial-derived metabolites have been shown to improve disease. For example, although fecal microbial transfer (FMT) remains challenging, FMT experiments have achieved some success in IBD patients (Moayyedi et al., 2015, Gastroenterology, 149: 102-109e106; Qazi et al., 2017, Gut Microbes, 8: 574-588; Narula et al., 2017, Inflamm Bowel Dis, 23: 1702-1709). In other studies, treatment with probiotics, including VSL#3, Lactobacillus spp., and Bifidobacterium spp., has also shown beneficial effects in humans and animal models (Srutkova et al., 2015, PLoS One, 10:e0134050; Pan et al., 2014, Benef Microbes, 5:315-322; Huynh et al., 2009, Inflamm Bowel Dis, 15:760-768; Bibiloni et al., 2005, Am J Gastroenterol, 100:1539-1546). Furthermore, bacterial products such as p40 from Lactobacillus rhamnosus GG and Amuc-1100 from Aspergillus mucilaginosus have been shown to promote barrier function and protect in animal models of IBD and metabolic diseases (Yan et al., 2011, J Clin Invest, 121: 2242-2253; Plovier et al., Nat Med, 23: 107-113).

[0152] Although the use of live microbial populations to treat diseases is becoming more and more common, this approach relies on the ability of the administered bacteria to survive in a host or patient and to interact with host tissues in a beneficial and therapeutic manner. Another approach provided herein is to identify proteins encoded by microorganisms and variants thereof that can affect host cell function and provide therapeutic benefits. Such proteins can be administered, for example, in the form of pharmaceutical compositions comprising substantially isolated or purified therapeutic bacteria-derived proteins or live biotherapeutic agents (bacteria) engineered to express therapeutic proteins as exogenous proteins. In addition, treatment methods including the administration of therapeutic proteins are not limited to the intestine (small intestine, large intestine, rectum), but can also include the treatment of other diseases in the digestive tract, such as oral mucositis.

[0153] In order to identify proteins of microbial origin that may have therapeutic applications in gastrointestinal inflammatory diseases, mucosal biopsies from healthy people or people diagnosed with IBD (UC) were analyzed to determine the microbial composition of these mucosal biopsies. By comparing the bacterial profiles of healthy and diseased subjects, bacteria that may be beneficial (larger in healthy subjects versus diseased patients) or harmful bacteria (smaller in healthy subjects versus diseased patients) can be determined. Consistent with the studies mentioned above, bacterial species considered to be beneficial include Roseburia hominis. Extensive bioinformatics analysis was then performed to predict bacterially encoded proteins and then identify proteins that may be secreted by bacteria. Proteins predicted to be secreted proteins were then characterized using a series of in vitro assays to study the effects of each protein on epithelial barrier integrity, cytokine production and / or release, and wound healing. Proteins identified as having the function of increasing epithelial barrier integrity were then evaluated in an in vivo mouse model of colitis. One such protein, identified herein as "SG-11," exhibits in vitro and in vivo activity suggesting its ability to provide therapeutic benefit for improving epithelial barrier integrity and treating diseases and disorders associated with epithelial barrier integrity and treating inflammatory gastrointestinal diseases, such as IBD, as described in more detail below.

[0154] SG-11 protein

[0155] The protein referred to herein as SG-11 is encoded within a 768 nucleotide sequence of the Roseburia hominis genome (SEQ ID NO: 2). The complete genomic sequence of the Roseburia hominis strain can be found in GenBank Accession No. CP003040 (which sequence is incorporated herein by reference in its entirety). The 16S rRNA gene sequence of the Roseburia hominis strain can be found in GenBank Accession No. AJ270482. The full-length protein encoded by the Roseburia hominis genomic sequence is 256 amino acids in length (SEQ ID NO: 1), of which residues 1-24 are predicted to be a signal peptide, which is cleaved in vivo to produce a mature protein of 232 amino acids (SEQ ID NO: 3; encoded by SEQ ID NO: 4). Recombinant SG-11 can be expressed with an N-terminal methionine (encoded by the codon ATG) to produce a mature protein of 233 amino acids (SEQ ID NO: 7).

[0156] As detailed in the Examples, e.g., Example 1, SG-11 is recombinantly expressed in different commercially available and commonly used expression vectors. For example, the following expression vectors were used to express SG-11 (a protein comprising SEQ ID NO: 3), a pGEX expression vector, which expresses a target protein having a GST tag and a protease site, which is cleaved after expression and purification, a pET-28 expression vector, a vector to which an N-terminal FLAG tag is added, and a pD451 expression vector, which is used to express an SG-11 protein consisting of SEQ ID NO: 7 and having no N-terminal tag. Experiments performed and repeated on these proteins showed that minor changes in the N-terminus and / or C-terminus due to the use of different protein expression systems and DNA constructs maintained the same functional activity in both in vivo and in vitro assays. It should be understood that, unless otherwise indicated, the term "SG-11" herein refers to such variants of the protein described herein as the amino acid sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 3 (including but not limited to SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 7). SG-11 variants may include variations (substitutions, insertions, deletions) of amino acid residues, as well as modifications such as fusion constructs and post-translational modifications (phosphorylation, glycosylation, etc.). Some exemplary embodiments of SG-11 proteins and encoding nucleic acids are provided in Table 11 below.

[0157] Table 1

[0158]

[0159]

[0160]

[0161]

[0162]

[0163] Epithelial barrier function in disease

[0164] Studies in recent years have determined that genetic and environmental factors play an important role in the pathogenesis of IBD. Markus Neurath, "Cytokines in Inflammatory BowelDisease", Nature Reviews Immunology, Vol. 14, 329-342 (2014). The combination of these IBD risk factors appears to cause deleterious changes in epithelial barrier function, thereby displacing luminal antigens (e.g., bacterial antigens from commensal flora) to the intestinal wall. Ibid. Subsequently, abnormal and excessive responses to such environmental triggers (e.g., increased release of proinflammatory cytokines) cause subclinical or acute mucosal inflammation in genetically susceptible hosts. Ibid. Therefore, the importance of proper epithelial barrier function in IBD is obvious, because in subjects who cannot resolve acute intestinal inflammation, chronic intestinal inflammation is caused by uncontrolled activation of the mucosal immune system. Specifically, mucosal immune cells, such as macrophages, T cells, and subsets of innate lymphoid cells (ILCs), appear to respond to microbial products or antigens of the commensal flora by, for example, producing cytokines that promote chronic inflammation in the gastrointestinal tract. Therefore, restoring proper epithelial barrier function in patients may be critical to resolving IBD.

[0165] The therapeutic activity of SG-11 is determined in part by its beneficial effects on epithelial barrier function in vivo and in vitro. As shown in Example 2, SG-11 is active in increasing epithelial barrier integrity as indicated by in vitro transepithelial electrical resistance (TEER) assays. TEER analysis is a well-known method for measuring the effects on the structural and functional integrity of epithelial cell layers (Srinivasan et al., 2015, J Lab Autom, 20: 107-126; Beduneau et al., 2014, Eur J Pharm Biopharm, 87: 290-298; Zolotarevsky et al., 2002, Gastroenterology, 123: 163-172; Dewi et al., (2004) J. Virol. Methods. 121: 171-180, and Mandic et al., (2004) Clin. Exp. Metast. 21: 699-704). The assay performed and described herein consists of an epithelial monolayer composed of enterocytes and goblet cells to more accurately model the structural and functional components of the intestinal epithelium. Cells are cultured until tight junctions are formed, and barrier function capacity is assessed by measuring transepithelial resistance. After adding damage such as heat-killed Escherichia coli, the resistance of the epithelial layer decreases. Control reagents for TEER assays include staurosporine and myosin light chain kinase inhibitors. Staurosporine is a broad-spectrum kinase inhibitor that originates from Streptomyces staurosporeus and can induce apoptosis. This reagent destroys about 98% of gap junctions, resulting in a decrease in resistance in the TEER assay. Myosin light chain kinase (MLCK) is the terminal effector of a signaling cascade induced by proinflammatory cytokines, which causes the perijunctional actomyosin ring to contract, resulting in gap junction separation. By inhibiting MLCK, the destruction of tight junctions can be prevented. MLCK inhibitors in TEER assays should reduce or prevent the decrease in resistance in TEER assays.

[0166] In some embodiments, the SG-11 protein or variant or fragment thereof as described herein can be characterized by its ability to increase the integrity of epithelial barrier function as assessed by an in vitro TEER assay. The SG-11 protein or variant or fragment thereof can increase the electrical resistance in a TEER assay by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% compared to a TEER assay performed in the absence of the protein.

[0167] In addition, Example 6 shows that the SG-11 protein can enhance or promote epithelial wound healing, and this activity can play a role in the maintenance or repair of epithelial barriers, such as intestinal or mucosal epithelial barriers.

[0168] Given the role of SG-11 in restoring barrier function integrity in vitro, the ability of SG-11 to reduce damage in rodent models of IBD was analyzed in vivo. Examples 7 and 8 (SG-11) and 16 (SG-11 variants) describe studies using the DSS (dextran sulfate sodium) animal model, which has been widely accepted for studying drugs for IBD (Chassaign et al., 2014, Curr Protoc Imunol, 104: Unit-15.25; Kieslerdeng, 2015, Cell Mol Gastroenterol Hepatol). DSS is a sulfated polysaccharide that is directly toxic to the colonic epithelium and causes epithelial cell damage, resulting in loss of barrier function due to the destruction of gap junctions. In these experiments, animals were treated with SG-11 before (Example 7) or after (Example 8) induction of colitis in mice. As a positive control, mice were also treated with Gly2-GLP2, a stable analog of glucagon-like peptide 2 (GLP2). In experimental mouse colitis models, Gly2-GLP2 is known to promote epithelial cell growth and reduce colonic damage. Results from the DSS study showed that the SG-11 protein was effective in reducing weight loss in the DSS model, an important indicator of clinical efficacy of IBD therapeutics. SG-11 treatment also reduced scores on gross pathology and intestinal histopathological analysis.

[0169] It should be noted that in Example 7, although SG-11 treatment improved 4Kda-FITC intestinal permeability readings and reduced serum levels of LPS binding protein (LBP, a marker of LPS exposure), no significant effects were observed with treatment with SG-11 or Gly2-GLP2 in Example 8. This is not surprising considering that the animals in Example 8 were treated with DSS for 7 days before normal drinking water was substituted for SG-11 or Gly2-GLP2 treatment. Prior exposure to DSS would result in damage to the intestinal epithelium, translocation of LPS across the disrupted epithelial barrier, and induction of LBP secretion. However, based on the measurements of 4KDa-FITC dextran, epithelial barrier repair appeared to occur rapidly within 3-4 days after replacing DSS with normal drinking water (data not shown, Fig.12). Therefore, at the time of measurement (6 days after treatment), it was difficult to detect an improvement in 4KDa-FITC permeability readings in treated and untreated animals. In addition, the level of LBP in serum can be independent of the repair of barrier function in animals exposed to DSS for an extended period of time before treatment with therapeutic agents (Example 8). For example, hepatocytes activated by LPS translocation during DSS exposure produce and secrete large amounts of LPB. Therefore, without being bound by theory, the short time period of this study may not allow sufficient time for hepatocytes to inactivate and clear LBP from the serum of DSS-treated animals. Therefore, it can be assumed that studies that continue to measure serum LBP will show a decrease in serum LBP levels at a later time point, but if barrier function is restored in treated and untreated animals before LBP is cleared from the serum, the serum LBP reduction in these two animals may be similar.

[0170] Variants of SG-11

[0171] Considering the therapeutic value of SG-11 and its use in treating diseases, the protein was further characterized and its sequence was modified to change its primary structure to optimize drug formulation and long-term storage of the protein.

[0172] As described in Example 9, a BLAST search of the GenBank non-redundant protein database was performed using SEQ ID NO: 7 to identify proteins that have similar amino acid sequences and that may be functional homologs of SG-11 or that have similar functions to SG-11. Three such proteins were identified, and the predicted mature sequence (without the N-terminal signal peptide) of each was aligned with SEQ ID NO: 7 to identify relatively conserved regions and individual positions in the protein. These three proteins are disclosed herein as SEQ ID NO: 21 (derived from GenBank Accession No. WP_006857001), SEQ ID NO: 22 (derived from GenBank Accession No. WP_075679733), and SEQ ID NO: 23 (derived from GenBank Accession No. WP_055301040) ( Fig.17 ), thus the present invention provides a pharmaceutical composition comprising one of the three proteins or a variant or fragment thereof, and a method for treating a disease associated with barrier dysfunction and / or a gastrointestinal disease or disorder, comprising administering to a subject in need thereof a pharmaceutical composition comprising any one of SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23 or a variant or fragment thereof.

[0173] Exemplary embodiments of SG-11 variants, SG-11V5 amino acid sequences and encoding nucleic acid sequences are provided in Table 2 below.

[0174] Table 2

[0175]

[0176]

[0177] In order to enhance the stability of SG-11 protein for pharmaceutical formulations and clinical applications, studies were conducted to identify and characterize post-translational modifications (PTMs) of purified SG-11 protein. These experiments are described in Examples 10-11. Such analyses indicate that SG-11 protein can undergo at least PTMs of methionine oxidation and asparagine deamidation. In addition, in the experiments described in Example 12, the cysteines in SG-11 are unlikely to form disulfide bonds in the purified functional conformation of the active protein, suggesting that free sulfhydryl groups in SG-11 may cause aggregation in solutions containing purified protein. Based on these stability studies, and regardless of the conservative nature of the residues in SG-11 as seen in the multiple sequence alignment ( Fig.17 ), it was decided to test whether the cysteine ​​at position 147 and / or 151 (reference SEQ ID NO: 7) could be substituted with other amino acids. In addition, substitutions of the conservative asparagine at positions 53 and 83 were considered. In an exemplary embodiment, the SG-11 sequence of SEQ ID NO: 7 was modified to introduce the substitutions of C147V and C151S to generate SEQ ID NO: 11 (SG-11V1). C147V and C151S substitutions are also present in the provided SG-11 variants SG-11V2 (SEQ ID NO: 13; comprising G84D, C147V, C151S), SG-11V3 (SEQ ID NO: 15; comprising N83S, C147V, C151S), SG-11V4 (SEQ ID NO: 17; comprising N53S, G84D, C147V, C151S) and SG-11V5 (SEQ ID NO: 19; comprising N53S, N83S, C147V, C151S).

[0178] Example 13 shows that PTMs (methionine oxidation and asparagine deamidation) are significantly reduced in SG-11V5 compared to SG-11 (SEQ ID NO: 7). The reduction was observed at different temperatures and in different storage buffers. Example 14 describes experiments conducted to determine whether a SG-11 variant (SG-11V5, SEQ ID NO: 19) containing a cysteine ​​substitution would affect aggregation of the protein in storage buffer. The results show that the SG-11V5 variant has reduced aggregation compared to SG-11 (SEQ ID NO: 7) when tested in different storage buffers.

[0179] It is noteworthy that although the amino acids substituted to generate SG-11V5 are present in relatively conserved regions of the SG-11 protein, it is possible to substitute these four residues without loss of functional activity (Examples 15 and 16, described in more detail below).

[0180] Based on the experimental data and analysis described herein, it is contemplated that variants of SG-11 (e.g., SEQ ID NO: 3 or SEQ ID NO: 5) are designed to substitute any one or more of the amino acids N53, N83, G84, C147, and C151 of SEQ ID NO: 7 (wherein the substitutions indicated are relative to the residue positions of SEQ ID NO: 7). An embodiment of the variant is provided in Table 3 below in the form of SEQ ID NO: 33, wherein the residues at each of positions 53, 83, 84, 147, and 151 are indicated as Xs, indicating that one or more of these five residues can each be substituted with any of the 20 possible amino acids. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 33. In other embodiments, X53 is N, S, T, M, R, Q, and / or X83 is N, R or K, and / or X84 is G or A, and / or X147 is C, S, T, M, V, L, A or G, and / or X151 is C, S, T, M, V, L, A or G. In other embodiments, X53 is N, S or K, and / or X83 is N or R, and / or X84 is G or A, and / or X147 is C, V, L or A, and / or X151 is C, S, V, L or A.

[0181] In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 34, wherein X53 is any amino acid except N, X83 is any amino acid except N, X84 is any amino acid except G, X147 is any amino acid except C, and / or X151 is any amino acid except C.

[0182] Table 3

[0183]

[0184]

[0185] In another example, certain amino acids of the taught proteins can replace other amino acids in the protein structure without significantly losing the ability to interact with the structure, for example, the antigen binding region of an antibody, a receptor, a binding site on a substrate molecule, etc. Therefore, these proteins will be biological functional equivalents of the disclosed proteins (e.g., comprising SEQ ID NO: 3 or a variant thereof). So-called "conservative" changes will not destroy the biological activity of the protein because these structural changes will not affect the ability of the protein to perform its designed function. Therefore, the inventors believe that a variety of different changes can be made to the sequences of the genes and proteins disclosed herein while still achieving the purposes of the present disclosure.

[0186] Also provided herein are variants of SG-11: SEQ ID NO: 11 (C147V, C151S, "SG11-V1"), SEQ ID NO: 13 (G84D, C147V, C151S "SG11-V2"), SEQ ID NO: 15 (N83S, C147V, C151S "SG11-V3"), SEQ ID NO: 17 (N53S, G84D, C147V, C151S "SG11-V4") and SEQ ID NO: 19 (N53S, N83S, C147V, C151S "SG11-V5").

[0187] Importantly, the SG-11 variant protein comprising SEQ ID NO: 19 maintained its activity relative to the TEER assay (Example 15) and the in vivo DSS mouse model (Example 16), indicating that variants of SG-11 can maintain therapeutic functions equivalent to wild-type SG-11. Specifically, in vitro TEER and in vivo DSS model experiments were performed, in which SG-11 (SEQ ID NO: 7) and SG-11V5 (SEQ ID NO: 19) were used in parallel. Example 15 shows that SG-11 and SG-11V5 have essentially the same functional ability to reduce TEER in vitro. As described in Examples 7 and 8, in which DSS model mice were treated with SG-11 before or after DSS treatment, Example 16 was performed to compare the in vivo efficacy of SG-11 and SG-11 variants. Example 16 also compares the administration of protein to mice before DSS treatment (described as Example 16A) and after DSS treatment (described as Example 16B). SG-11 and SG-11 variants reduced weight loss ( Fig.23A and 23B ) and overall pathological clinical score ( Fig.24 ). Similarly, in Example 16A, SG-11 reduced intestinal permeability and serum LBP levels, while SG-11V5 showed a dose-dependent reduction in intestinal permeability and serum LBP levels ( Fig.21A and Fig.22A ). Similar to the results observed in Examples 7 and 8, SG-11 and SG-11 variant proteins did not reduce intestinal permeability or serum LBP levels in Example 16B, where the therapeutic protein was administered after chronic DSS challenge and the results were observed for a limited period of time. As described above, it is believed that continued studies will show a reduction in both permeability and serum LBP levels.

[0188] In view of the data provided herein, the therapeutic protein has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a protein comprising the amino acid sequence of SEQ ID NO: 3 or a fragment thereof. In alternative embodiments, the therapeutic protein has at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to SEQ ID NO: 19 or SEQ ID NO: 7 or a fragment thereof. In some embodiments, the therapeutic protein comprises an amino acid sequence identical to SEQ ID NO: 19 or SEQ ID NO: 5. Alternatively, the therapeutic protein can be one of the variants of SEQ ID NO: 3 or SEQ ID NO: 7, wherein the therapeutic protein has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions relative to SEQ ID NO: 7. In some embodiments, the variant therapeutic protein comprises a non-naturally occurring variant of SEQ ID NO: 3. In other words, the therapeutic protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-naturally occurring amino acid substitutions relative to SEQ ID NO: 3. In some embodiments, the therapeutic protein does not comprise an amino acid sequence identical to the sequence of residues 2 to 233 of SEQ ID NO: 7.

[0189] In some embodiments, the SG-11 protein can be modified or altered by one or more amino acid insertions or deletions. Insertion can be the addition of 1 or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 1 to 10, 1 to 20, 1 to 30, 1 to 40 or 1 to 50) amino acids to the N-terminus and / or C-terminus of the protein and / or can be the insertion of 1 or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 1 to 10, 1 to 20, 1 to 30, 1 to 40 or 1 to 50) amino acids at a position between the N-terminal and C-terminal amino acids. Similarly, deletion of 1 or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 1 to 10, 1 to 20, 1 to 30, 1 to 40 or 1 to 50) amino acids can occur at any one of the N-terminal and C-terminal ends as well as within.

[0190] In some embodiments, a modified or variant protein is provided that comprises at least one non-naturally occurring amino acid substitution relative to SEQ ID NO: 3. In other embodiments, the variant protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions relative to SEQ ID NO: 3 or SEQ ID NO: 7. In other embodiments, the modified protein comprises an amino acid sequence as set forth in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 (SG-11V1), SEQ ID NO: 13 (SG-11V2), SEQ ID NO: 15 (SG-11V3), SEQ ID NO: 17 (SG-11V4) or SEQ ID NO: 19 (SG-11V5).

[0191] In some embodiments, the therapeutic protein according to the present invention includes any variant protein (e.g., SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17; or SEQ ID NO: 19), which also retains one or more activities of the full-length mature protein shown in, for example, SEQ ID NO: 3 or SEQ ID NO: 7.

[0192] Polynucleotide sequences encoding these proteins are also contemplated. It is well known to those of ordinary skill that due to the degeneracy of the genetic code, two polynucleotide sequences encoding a single polypeptide sequence can share relatively low sequence identity. For example, if each codon in a polynucleotide encoding a 233 amino acid sequence contains at least 1 substituent at its third position, this would calculate a sequence identity of about 67% between the two polynucleotides. The polynucleotides of the present disclosure include sequences encoding proteins that are at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 19. Thus, in some embodiments, the polynucleotide comprises a sequence that is at least 67% identical to SEQ ID NO: 4 or SEQ ID NO: 8, or about 67% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 90% to 100% or 95% to 100% identical to SEQ ID NO: 20, or a fragment thereof. In some embodiments, the polynucleotide comprises a sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20, or a fragment thereof.

[0193] In some embodiments, the taught protein has significantly different structural and / or functional characteristics compared to a protein comprising or consisting of SEQ ID NO:3.

[0194] As used herein, the term "SG-11 variant" may include SG-11 proteins that are, for example, identical or different from a protein comprising the sequence of SEQ ID NO: 3 and may be further modified by, for example, PTM or linkage or fusion with a second substance (e.g., a protein or peptide).

[0195] Protein PTMs occur in vivo and can increase the functional diversity of the proteome by covalent addition of functional groups or proteins, proteolytic cleavage of regulatory subunits, or degradation of the entire protein. The isolated proteins prepared according to the present disclosure may undergo one or more PTMs in vivo or in vitro. The type of modification depends on the host cell in which the protein is expressed, including but not limited to phosphorylation, glycosylation, ubiquitination, nitrosylation (e.g., S-nitrosylation), methylation, acetylation (e.g., N-acetylation), lipidation (myristoylation, N-myristoylation, S-palmitoylation, farnesylation, S-pentenoylation, S-palmitoylation) and proteolysis may affect almost all aspects of normal cell biology and pathogenesis. The isolated and / or purified SG-11 protein or its variants or fragments as disclosed herein may contain one or more of the above-mentioned post-translational modifications.

[0196] The SG-11 protein or its variant or fragment may be a fusion protein in which the N- and / or C-terminal domain is fused to a second protein via a peptide bond. Common fusion partners known to those skilled in the art include, but are not limited to, human serum albumin and the crystallizable fragment or constant domain of IgG, Fc. In some embodiments, the SG-11 protein or its variant or fragment is linked to a second protein or peptide via a disulfide bond, wherein the second protein or peptide comprises a cysteine ​​residue.

[0197] As described above, the structures of the proteins disclosed herein can be modified and / or altered (e.g., substituted, inserted, deleted). Therefore, the present disclosure contemplates sequence changes of these proteins, and the nucleic acids encoding them, wherein they are still able to retain significant activity relative to the functional activity evaluated in various in vitro and in vivo assays and in the therapeutic aspects of the present disclosure. With respect to functional equivalents, those skilled in the art fully understand that inherent in the definition of "biologically functional equivalent" proteins and / or polynucleotides is the concept that there is a limited number of changes that can be made within a defined portion of the molecule while retaining an acceptable level of equivalent biological activity.

[0198] It is also contemplated that the SG-11 protein or a variant or fragment thereof is a protein that, when administered to a subject, can reduce disease-related weight loss, improve clinical pathology scores, and / or minimize shortening of the subject's colon. In some embodiments, the subject is a mammal with a genetic or clinically induced inflammatory disease or dysfunctional epithelial barrier function. Alternatively, the animal suffers from an idiopathic gastrointestinal disease, including reduced epithelial barrier function or an inflammatory bowel disease. In other embodiments, the mammal is a human, a non-human primate, or a rodent. The rodent can be a mouse or a rat.

[0199] In some embodiments, the SG-11 protein or its variant or fragment according to the present disclosure is a protein that can regulate the production and / or secretion of cytokines in an object to which the protein is administered in vitro or is administered. In some embodiments, the secretion of cytokines is reduced in vitro. The level of cytokines produced and / or secreted in an object to which the protein is administered in vitro is likely to be measured in the blood, serum and / or plasma of the object. Administration of the protein may result in a decrease in the serum level of one or more of proinflammatory cytokines, such as TNF-α, IL-17, IL-1, IL-2, IFN-γ, IL-6, IL-12, IL-25, IL-33, IL-8, MCP-1, MIP-3, CXCL1 and IL-23. Alternatively, the cytokine may be an anti-inflammatory cytokine, in which case the administration of the protein results in an increase in the serum level of anti-inflammatory cytokines such as IL-4, IL-10, IL-13, IFN-α, and TGF-β.

[0200] In some embodiments, when the SG-11 protein or its variant or fragment thereof is administered to a subject such as a mammal (e.g., a rodent, a non-human primate, or a human), it has the functional ability to reduce gastrointestinal inflammation. In other embodiments, when administered to a subject, the protein has the functional ability to reduce inflammatory (i.e., pro-inflammatory) cytokines. In other embodiments, when the protein is administered to a subject, the protein is capable of reducing TNF-α and / or IL-23. In other embodiments, when administered to a subject, the protein has the functional ability to increase anti-inflammatory cytokines. In some aspects, when administered to a subject, the protein of the present invention is capable of increasing IL-10.

[0201] The SG-11 protein or variant or fragment thereof according to the present disclosure is a protein that, when administered to a subject (e.g., a rodent, a non-human primate or a human), can improve gastrointestinal epithelial cell barrier function, reduce disease-associated weight loss, improve clinical scores, improve colon length and / or colon weight to length ratio readouts, induce or increase mucin gene expression (e.g., muc2 expression), increase the structural integrity and / or function of the gastrointestinal mucus barrier (e.g., small intestine, large intestine, oral cavity and / or esophagus) and / or reduce inflammation of the gastrointestinal tract.

[0202] In some embodiments, the SG-11 protein or its variant or fragment produced by such substitution, insertion and / or deletion of amino acids relative to SEQ ID NO: 3 or SEQ ID NO: 7 maintains a functional activity level substantially at the same level as the protein of SEQ ID NO: 7 or SEQ ID NO: 19 (e.g., capable of increasing electrical resistance in a TEER assay in which an epithelial cell layer is disrupted by, for example, heat-killed E. coli). The variant protein can be used as a therapeutic agent for treating or preventing a variety of conditions, including, but not limited to, inflammatory conditions and / or barrier dysfunction, including, but not limited to, inflammation of the mucosa of the gastrointestinal tract (including the oral cavity, esophagus and intestine), and impaired integrity of intestinal epithelial cell gap junctions. In some embodiments, when administered to an individual suffering from or susceptible to inflammatory diseases and / or barrier dysfunction, the modified protein has one or more of the following effects: improving the integrity of the epithelial barrier, for example, after inflammation-induced damage; inhibiting the production of at least one proinflammatory cytokine (e.g., TNF-α and / or IL-23) by one or more immune cells; inducing mucin production in epithelial cells; and / or improving epithelial wound healing. In addition, modified or variant SG-11 proteins can be used to treat or prevent diseases or disorders such as, but not limited to, inflammatory bowel disease, ulcerative colitis, Crohn's disease, short bowel syndrome, gastrointestinal mucositis, oral mucositis, chemotherapy-induced mucositis, radiation-induced mucositis, necrotizing enterocolitis, pouchitis, metabolic diseases, celiac disease, inflammatory bowel syndrome, or chemotherapy-associated steatohepatitis (CASH).

[0203] As demonstrated, for example, in Example 6, SG-11 protein can enhance epithelial wound healing. Thus, provided herein is a therapeutic protein comprising an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 7, or a variant or fragment thereof, wherein the protein can increase wound healing in an in vitro assay.

[0204] Treatment

[0205] The SG-11 proteins described herein, including their variants (e.g., amino acid substitutions, deletions, insertions), modified forms (e.g., glycosylation, acetylation), SG-11 fragments and fusions thereof, are contemplated for use in treating subjects diagnosed with or suffering from a disease associated with inflammation in the gastrointestinal tract and / or epithelial barrier dysfunction in the gastrointestinal tract.

[0206] Provided herein is a method for treating an object in need thereof, the method comprising administering to the object a pharmaceutical composition comprising an SG-11 protein or a fragment or variant thereof as described in the present disclosure. The object may be diagnosed with inflammatory bowel disease, ulcerative colitis, pediatric UC, Crohn's disease, pediatric Crohn's disease, short bowel syndrome, mucositis GI mucositis, oral mucositis, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (colon) and / or rectal mucositis, chemotherapy-induced mucositis, radiation-induced mucositis, necrotizing enterocolitis, pouchitis, metabolic disease, celiac disease, irritable bowel syndrome or chemotherapy-associated steatohepatitis (CASH). Administration of the SG-11 pharmaceutical composition described herein may also be used for wound healing applications.

[0207] Inflammatory bowel disease

[0208] Inflammatory bowel disease (IBD) generally includes ulcerative colitis (UC) and Crohn's disease (CD). The pathogenesis of inflammatory bowel disease is unclear. Genetic susceptibility has been proposed, and many environmental factors, including bacteria, viruses and antigens in the diet, may trigger the ongoing intestinal inflammatory cascade. Same as above. IBD may involve severe diarrhea, pain, fatigue and weight loss. IBD can be debilitating and sometimes lead to life-threatening complications. Therefore, in some embodiments, the treatment methods described herein can effectively alleviate, prevent or eliminate any one or more of the above symptoms, wherein the method comprises administering a therapeutically effective amount of a pharmaceutical composition to a patient in need thereof, the pharmaceutical composition comprising an SG-11 protein or a variant or fragment thereof. In some embodiments, the treatment method results in relief.

[0209] Ulcerative colitis

[0210] Ulcerative colitis is an inflammatory bowel disease that causes long-term inflammation and pain (ulcers) in the lining of the large intestine (colon) and rectum.

[0211] Ulcerative colitis typically presents as a superficial, continuous inflammation that extends proximally from the rectum to include the entire colon in many patients. There are no fistulas, fissures, abscesses, or small bowel involvement. Patients with limited disease (e.g., proctitis) typically have mild but frequently recurring symptoms, whereas patients with pancreatitis more commonly have severe symptoms that often require hospitalization. Botoman et al., "Management of Inflammatory Bowel Disease," Am. Fam. Physician, Vol. 57(1): 57-68 (Jan. 1, 1998) (internal citations omitted). Thus, ulcerative colitis is a type of IBD that causes long-term inflammation and sores (ulcers) in the innermost lining of your large intestine (colon) and rectum.

[0212] Crohn's disease

[0213] Unlike ulcerative colitis, Crohn's disease can affect the entire intestine, from mouth to anus, with discrete focal ulcers, fistula formation, and perianal involvement. The terminal ileum is most commonly affected, with varying degrees of colon involvement. A subset of patients suffer from perianal disease, with fissures and fistula formation. Among patients with Crohn's disease, only 2% to 3% have clinically significant involvement of the upper gastrointestinal tract. Botoman et al., "Management of Inflammatory Bowel Disease," Am. Fam. Physician, Vol. 57(1): 57-68 (January 1, 1998) (internal citations omitted). Therefore, Crohn's disease is an IBD that causes inflammation of the lining of the digestive tract. In Crohn's disease, the inflammation often extends deep into the affected tissues. Inflammation can involve different areas of the digestive tract - the large intestine, the small intestine, or both. Collagenous colitis and lymphocytic colitis are also considered inflammatory bowel diseases but are usually considered separately from classic inflammatory bowel disease.

[0214] Clinical parameters of inflammatory bowel disease

[0215] As previously mentioned, inflammatory bowel diseases include ulcerative colitis and Crohn's disease. A number of scores and clinical markers are known to those skilled in the art that can be used to assess the efficacy of the administered proteins described herein in treating these conditions.

[0216] There are two general approaches to evaluating patients with IBD. Given that IBD presents as a manifestation of inflammation and ulceration in the GI tract, the first involves a visual inspection of the mucosa and relies on observation for signs of mucosal damage. Any method that can evaluate the mucosa can be used. Examples include barium enema, X-rays, and endoscopy. Endoscopy can be an examination of the esophagus, stomach, and duodenum (esophagogastroduodenoscopy), the small intestine (enteroscopy), or the large intestine / colon (colonoscopy, sigmoidoscopy). These techniques are used to identify areas of inflammation, ulceration, and abnormal growths such as polyps.

[0217] Scoring systems based on this visual inspection of the gastrointestinal tract exist to determine the status and severity of IBD, and although these patients may be evaluated by different medical professionals, these scoring systems are intended to ensure uniform assessment of different patients in the diagnosis and monitoring of these diseases and in clinical research evaluations. Daperno M et al. (J Crohns Colitis. 20115: 484-98) discussed and compared examples of assessments based on visual inspection of UC.

[0218] Clinical scoring systems also exist that have the same purpose. The results of endoscopy or other mucosal examinations can be incorporated into these clinical scoring systems, but these scoring systems also incorporate symptom-based data such as stool frequency, rectal bleeding, and the physician's global assessment. IBD has a variety of symptoms that affect quality of life, so some scoring systems also consider quantitative assessments of the impact on quality of life as well as quantification of symptoms.

[0219] An example of a UC scoring system is the Mayo scoring system (Schroeder et al., N Eng J Med, 1987, 317:1625-1629), but there are other less commonly used methods, including the ulcerative colitis endoscopic index of severity (UCEIS) score (Travis et al., 2012, Gut, 61:535-542), the Baron score (Baron et al., 1964, BMJ, 1:89), the ulcerative colitis colonoscopy severity index (UCCIS) (Thia et al., 2011, Inflamm Bowel Disease Index, 2011, 535-542), and the ulcerative colitis colonoscopy severity index (UCCIS) score (Thia et al., 2011, Inflamm Bowel Disease Index, 2011, 535-542). Dis, 17: 1757-1764), Rachmilewitz endoscopic index (Rachmilewitz, 1989, BMJ, 298: 82-86), Sutherland index (also known as UC disease activity index (UCDAI) scoring system; Sutherland et al., 1987, Gastroenterology, 92: 1994-1998), Matts score (Matts, 1961, QJM, 30: 393-407), and Blackstone index (Blackstone, 1984, Inflammatory bowel disease. In: Blackstone MO (ed.) Endoscopic interpretation: normal and pathologic appearances of the gastrointestinal tract, 1984, pp. 464-494). For a review, see Paine, 2014, Gastroenterol Rep 2: 161-168. Thus, also contemplated herein are methods for treating subjects diagnosed with UC and suffering from UC, wherein the treatment comprises administering an SG-11 protein or variant or fragment thereof as described herein, and wherein the treatment results in a reduction in UC pathology, as determined by measuring the UCEIS score, Baron score, UCCIS score, Rachmilewitz endoscopic index, Sutherland index and / or Blackstone index.

[0220] An example of a CD scoring system is the Crohn's Disease Activity Index (CDAI) (Sands B et al., 2004, N Engl J Med 350(9):876-85); most major studies use the CDAI to define disease response or remission. The CDAI score is calculated by including the number of liquid stools over 7 days, the instances and severity of abdominal pain over 7 days, the overall health status over 7 days, extraintestinal complications (e.g., arthritis / arthralgia, iritis / uveitis, erythema nodosum, pyoderma gangrenosum, aphthous stomatitis, anal fissure / fistula / abscess, and / or fever>37.8°C), the use of antidiarrheal medications within 7 days, the presence of abdominal masses, hematocrit, and body weight, displayed as the ideal / observed ratio or the percentage deviation from the standard weight. According to the CDAI score, CD is divided into asymptomatic remission (0 to 149 points), mild to moderately active CD (150 to 220 points), moderate to severely active CD (221 to 450 points), or severely active fulminant disease (451 to 1000 points). In some embodiments, the method of treating a patient diagnosed with CD by administering a therapeutically effective amount of SG-11 protein or its variant or fragment results in a reduction in the diagnostic score of CD. For example, the score can change the diagnosis from severe activity to mild or moderate activity or asymptomatic remission.

[0221] The Harvey-Bradshaw Index is a simple version of the CDAI consisting only of clinical parameters (Harvey et al., 1980, Lancet 1(8178): 1134-1135). The Inflammatory Bowel Disease Questionnaire (IBDQ) also addresses the impact on quality of life (Irvine et al., 1994, Gastroenterology 106: 287-296). Alternative methods also include the CDEIS and SES CD (see, e.g., Levesque et al., (2015) Gastroentrol. 148: 3757).

[0222] In some embodiments, a method for treating IBD, such as UC, is provided, wherein the treatment is effective in reducing the Mayo score. The Mayo score is an endoscopic and clinical comprehensive scoring scale for assessing the severity of UC, with a score range of 1-12. The Mayo score is a combination of sub-scores for stool frequency, rectal bleeding, flexible sigmoidoscopy or colonoscopy results, and the physician's overall evaluation results (Paine, 2014, Gastroenterol Rep 2: 161-168). For rectal bleeding, blood spots appearing in the stool less than half of the time are assigned 1 point, blood in most stools is assigned 2 points, and pure blood passed is assigned 3 points. Regarding the number of stools, 0 points are assigned to a normal daily number of stools, 1 point is assigned to 1 or 2 more stools than normal, 2 points are assigned to 3 or 4 more stools than normal, and 3 points are assigned to 5 or more stools than normal. For the endoscopic component, a score of 0 indicates normal mucosa or inactive UC, a score of 1 for mild disease with mild friability, reduced vascular pattern, and signs of mucosal erythema, a score of 2 for moderate disease showing friability, erosions, complete loss of vascular morphology, and marked erythema, and a score of 3 for ulceration and spontaneous bleeding (Schroeder et al., 1987, N Engl J Med, 317: 1625-1629). The overall assessment by the physician is: 0 for normal findings, 1 for mild colitis, 2 for moderate colitis, and 3 for severe colitis. Thus, in some embodiments, a patient treated with SG-11 therapeutic protein, or a variant or fragment thereof, is successfully treated when the patient experiences a decrease in Mayo score of at least 1, 2, or 3 points in at least one of the following: rectal bleeding, visible blood spots in the stool, endoscopic score, and physician's overall assessment. In some embodiments, the method comprising administering a therapeutically effective amount of SG-11 protein or a variant or fragment thereof to a patient diagnosed with UC results in a decrease in the diagnostic score for UC. For example, the score can change a diagnostic score, such as a Mayo score, by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 points.

[0223] Pouchitis

[0224] Additionally or alternatively, compositions comprising SG-11 therapeutic proteins or variants and methods of administration described herein can be used to treat pouchitis. Pouchitis is an inflammation of the cyst wall produced surgically in the treatment of UC. Specifically, subjects with severe UC may have the diseased colon removed and the intestine reconnected by a procedure called interileoileal anastomosis (IPAA) or J-pouch surgery. Cases of pouchitis can recur in many patients, manifesting as acute recurrent pouchitis or chronic refractory pouchitis. Therefore, methods for treating pouchitis, acute pouchitis, or recurrent pouchitis are provided herein.

[0225] The activity of pouchitis can be classified as remission (no active pouchitis), mild to moderate activity (increased stool frequency, urinary urgency and / or infrequent urinary incontinence), or severe activity (frequent urinary incontinence and / or patient hospitalization due to dehydration). The duration of pouchitis can be defined as short-term (less than or equal to four weeks) or long-term (four weeks or longer), and its pattern can be classified as infrequent (1-2 acute attacks), recurrent (three or fewer attacks), or continuous attacks. The response to drug treatment can be labeled as responsive to treatment or refractory to treatment, and drugs are specified for both conditions. Thus, in some embodiments, a method for treating a subject diagnosed with pouchitis is provided, wherein treatment with a pharmaceutical composition comprising SG-11 or a variant or fragment thereof results in a reduction in the severity of the pouchitis and / or results in remission.

[0226] Mucositis and mucosal barriers

[0227] The mucosa of the gastrointestinal (GI) tract is a complex microenvironment involving epithelial barriers, immune cells, and microorganisms. A delicate balance is maintained in a healthy colon. Intraluminal microorganisms are physically isolated from the host immune system by a barrier composed of epithelium and mucus. Although the pathogenesis of IBD has not been fully elucidated, it may involve host maladaptive responses to altered commensal flora with abnormal mucosal barrier function. See, Boltin et al., "Mucin Function in Inflammatory Bowel Disease An Update", J. Clin. Gastroenterol., Vol. 47(2): 106-111 (February 2013).

[0228] Mucositis occurs when cancer treatments (especially chemotherapy and radiation therapy) break down the rapidly dividing epithelial cells of the intestinal wall (from the mouth to the anus), making the mucosal tissue susceptible to ulcers and infections. Mucosal tissue, also known as mucosa or mucosa, covers all body passages that communicate with the air, such as the respiratory and digestive tracts, and has cells and associated glands that secrete mucus. This lining portion that covers the mouth is called the oral mucosa, which is one of the most sensitive parts of the human body and is particularly susceptible to damage from chemotherapy and radiation. The mouth is the most common location for mucositis. Although the oral mucosa is the most common site of mucosal toxicity and the resulting mucositis, it should be understood that mucositis can also occur along the entire digestive tract, including the esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (colon) and rectum. In some embodiments, a pharmaceutical composition comprising SG-11 or a variant or fragment thereof is therapeutically effective in treating the mucosa of the mouth, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (colon) and / or rectum.

[0229] Oral mucositis can cause a variety of problems, including pain, nutritional problems due to the inability to eat, and an increased risk of infection due to open ulcers on the mucosa. It has a significant impact on the patient's quality of life and can be dose-limiting (i.e., requiring a reduction in subsequent chemotherapy doses). The World Health Organization has an oral toxicity scale for diagnosing oral mucositis: Grade 1: soreness ± erythema, Grade 2: erythema, ulcers; patients can swallow solid food; Grade 3: extensive erythema ulcers; patients cannot swallow solid food; Grade 4: mucositis to the point of being unable to digest. Grades 3 and 4 oral mucositis are considered severe mucositis. Therefore, a method for treating a subject diagnosed with oral mucositis is provided herein, wherein administration of a pharmaceutical composition comprising SG-11 or a variant or fragment thereof can reduce the grade of oral toxicity from 1 to 4 by at least 1 point.

[0230] Shortened colon

[0231] Ulcerative colitis is an idiopathic inflammatory bowel disease that affects the colonic mucosa and is clinically characterized by diarrhea, abdominal pain, and blood in the stool. The extent of the disease is variable and may involve only the rectum (ulcerative proctitis), the left side of the colon to splenic varices, or the entire colon (pancreatitis). The severity of the disease can also vary quite a bit histologically, ranging from minimal to small-scale ulcers and dysplasia. Carcinomas may develop. The typical histological (microscopic) lesion of ulcerative colitis is a crypt abscess, in which the crypt epithelium is disrupted and the lumen is filled with polymorphonuclear cells. The lamina propria is infiltrated with leukocytes. As the crypts are destroyed, the normal mucosal architecture disappears, scarring shortens and may narrow the colon. Therefore, shortening of the colon may be a result of colitis disease and is often used for diagnosis. For example, non-invasive abdominal planar X-rays can show a gaseous outline of the transverse colon in acute patients. Planar films as well as double contrast barium enema can also demonstrate shortening of the colon and disappearance of the gastric mucosa. Indications of ulcerative disease include loss of mucosal detail, cobblestone-filled defects, and areas of segmental involvement. See: “Ulcerative Colitis: Introduction—Johns Hopkins Medicine,” available at: www.hopkinsmedicine.org / gastroenterology_hepatology / _pdfs / small_large_intestine / ulcerative_colitis.pdf.

[0232] In addition, in vivo models of colitis recognized in the art will utilize the shortening of colon length when assessing the severity of colitis in the model. See Kim et al., "Investigating Intestinal Inflammation in DSS-induced Model of IBD," Journal of Visualized Experiments, Vol. 60, pp. 2-6 (February 2012).

[0233] Epithelial barrier function in non-IBD diseases

[0234] A dysfunctional epithelial barrier is increasingly implicated in conditions such as IBD and mucositis. In addition, studies have shown that a number of other diseases are also caused, linked, associated, and / or exacerbated by a dysfunctional epithelial barrier. These diseases include: (1) metabolic diseases, including: obesity, type 2 diabetes, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), liver disease, and alcoholic steatohepatitis (ASH); (2) celiac disease; (3) necrotizing enterocolitis; (4) irritable bowel syndrome (IBS); (5) intestinal infections (e.g., Clostridium difficile); (6) other general gastrointestinal diseases; (7) interstitial cystitis; (8) neurological diseases or cognitive disorders (e.g., Alzheimer's disease, Parkinson's disease, multiple sclerosis, and autism); (9) chemotherapy-associated steatohepatitis (CASH); and (10) pediatric forms of the above diseases.See, e.g., Everard et al., "Responses of Gut Microbiota and Glucose and Lipid Metabolism to Prebiotics in Genetic Obese and Diet-Induced Leptin-Resistant Mice," Diabetes, Vol. 60 (November 2011), pp. 2775-2786; Everard et al., "Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity," PNAS, Vol. 110, No. 22, (May 2013), pp. 9066-9071; Cani et al., "Changes in Gut Microbiota Control Metabolic Endotoxemia-Induced Inflammation in High-Fat Diet-Induced Obesity and Diabetes in Mice." "Metabolic Endotoxemia-Induced Inflammation in High-Fat Diet-Induced Obesity and Diabetes in Mice", Diabetes, Volume 57, (June 2008), pages 1470-1481; Delzenne et al., "Targeting gut microbiota in obesity: effects of prebiotics and probiotics", Nature Reviews, Volume 7, (November 2011), pages 639-646. Therefore, restoring proper epithelial barrier function for patients may be crucial to resolving the aforementioned disease states.

[0235] A properly functioning epithelial barrier in the lumen of the digestive tract (including the mouth, esophagus, stomach, small intestine, large intestine, and rectum) is essential for controlling and maintaining the microbiome within the gastrointestinal and digestive tracts. The ecosystem of the microbiome includes the environment, barriers, tissues, mucus, mucins, enzymes, nutrients, food, and microbial communities that reside in the gastrointestinal and digestive tracts. The integrity and permeability of the intestinal mucosal barrier influence health in many critical ways.

[0236] Loss of mucosal barrier integrity in gastrointestinal diseases due to altered mucin secretion may be related to host immune changes, luminal microbial factors, or directly acting genetic or environmental determinants. Therefore, an imbalance in the mucus barrier may be central to the pathogenesis of IBD. Boltin et al., "Mucin Function in Inflammatory Bowel Disease An Update", J. Clin. Gastroenterol., Vol. 47(2): 106-111 (February 2013).

[0237] Mucins are the major components of the mucus layer located in the gastrointestinal tract. At least 21 mucin (MUC) genes are known in the human genome, encoding secretory or membrane-bound mucins. The major mucins in the normal colorectum are MUC1, MUC2, MUC3A, MUC3B, MUC4, MUC13, and MUC17.1. MUC2 is the major secretory gel component of intestinal mucus produced in goblet cells. See, Boltin et al., "Mucin Function in Inflammatory Bowel Disease An Update," J. Clin. Gastroenterol., Vol. 47(2): 106-111 (February 2013). Goblet cell secretion of MUC2, together with other secreted mucins (e.g., MUC1, 3A, 3B, 4, 13, and 17.1), forms a protective barrier on colonic epithelial cells, thereby reducing exposure to intestinal contents that may damage epithelial cells or prime immune responses.

[0238] Inflammatory mechanisms in IBD

[0239] There is a lot of evidence that certain cytokines are associated with IBD. Recent studies have shown that cytokines play a vital role in the pathogenesis of inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis, and they control multiple aspects of the inflammatory response. Markus Neurath, "Cytokines in Inflammatory Bowel Disease", Nature Reviews Immunology, Vol. 14, 329-342 (2014). In particular, the imbalance between pro-inflammatory and anti-inflammatory cytokines that occurs in IBD hinders the resolution of inflammation, but instead leads to disease perpetuation and tissue destruction. Ibid. Recent studies have shown that there is a regulatory cytokine network that is of great significance to the progression of the disease. Ibid. Therefore, experiments were conducted to study the effect of SG-11 on the production and / or secretion of pro-inflammatory and anti-inflammatory cytokines.

[0240] Pro-inflammatory cytokines

[0241] In short, proinflammatory cytokines are cytokines that are important in cell signaling and promote systemic inflammation. They are mainly produced by activated macrophages and participate in the upregulation of inflammatory responses. Proinflammatory cytokines come from genes that encode for translating small mediator molecules, which cause responses after upregulation. Interleukin-1 (IL-1), IL6, IL-12, IL-18, IL-23, CD40L, tumor necrosis factor (TNF) such as TNF-α, gamma-interferon (IFN-γ), granulocyte-macrophage colony stimulating factor and MCP-1 are well-characterized proinflammatory cytokines. Inflammation is characterized by the interaction between proinflammatory and anti-inflammatory cytokines.

[0242] Reducing the biological activity of proinflammatory cytokines can be used to treat certain diseases. For example, blocking IL-1 or TNF-α has successfully helped patients with rheumatoid arthritis, inflammatory bowel disease, or graft-versus-host disease. See Strober W, Fuss IJ (May 2011). "Proinflammatory cytokines in the pathogenesis of inflammatory bowel diseases", Gastroenterology, Vol. 140(6): 1756-67.

[0243] Anti-inflammatory cytokines

[0244] In short, anti-inflammatory cytokines are a series of immunomodulatory molecules that regulate the pro-inflammatory cytokine response. Therefore, these molecules regulate and help reduce the inflammatory response triggered by the pro-inflammatory cytokines. Anti-inflammatory cytokines include, for example, IL4, IL-10, IL-13, IFN-α, and transforming growth factor-β (TGF-β) are considered anti-inflammatory cytokines.

[0245] In some embodiments of the methods taught herein, administration of a pharmaceutical composition comprising an SG-11 protein or a variant or fragment thereof can result in a reduction in the production of at least one proinflammatory cytokine (e.g., TNF-α and / or IL-23) by immune cells of a patient to whom the composition is administered. In some embodiments, the administration can cause an increase in the production of at least one anti-inflammatory cytokine (e.g., IL-10) by immune cells of the patient. In some embodiments, the administration can cause a reduction in the production of at least one anti-inflammatory cytokine (e.g., IL-10) by immune cells of the patient. In some embodiments, the administration can improve the production of mucin in epithelial cells of the patient and / or the healing of epithelial wounds.

[0246] The dosage regimen for treatment depends on the desired therapeutic effect, the route of administration and the duration of treatment. The dosage varies from patient to patient, depending on the nature and severity of the disease, the patient's weight, the patient's subsequent special diet, concurrent medications and other factors that will be recognized by those skilled in the art.

[0247] Typically, a therapeutic protein dosage level of 0.0001 to 10 mg / kg body weight per day is administered to a patient, such as a patient with inflammatory bowel disease. The dosage range is typically about 0.5 mg to 100.0 g per patient per day, and can be administered in a single dose or multiple doses.

[0248] In some aspects, the dosage range will be about 0.5 mg to 10 g per patient per day, or 0.5 mg to 9 g per patient per day, or 0.5 mg to 8 g per patient per day, or 0.5 mg to 7 g per patient per day, or 0.5 mg to 6 g per patient per day, or 0.5 mg to 5 g per patient per day, or 0.5 mg to 4 g per patient per day, or 0.5 mg to 3 g per patient per day, or 0.5 mg to 2 g per patient per day, or 0.5 mg to 1 g per patient per day.

[0249] In some aspects, the dosage range will be about 0.5 mg to 900 mg per patient per day, or 0.5 mg to 800 mg per patient per day, or 0.5 mg to 700 mg per patient per day, or 0.5 mg to 600 mg per patient per day, or 0.5 mg to 500 mg per patient per day, or 0.5 mg to 400 mg per patient per day, or 0.5 mg to 300 mg per patient per day, or 0.5 mg to 200 mg per patient per day, or 0.5 mg to 100 mg per patient per day, or 0.5 mg to 50 mg per patient per day, or 0.5 mg to 40 mg per patient per day, or 0.5 mg to 30 mg per patient per day, or 0.5 mg to 20 mg per patient per day, or 0.5 mg to 10 mg per patient per day, or 0.5 mg to 1 mg per patient per day.

[0250] Combination therapies comprising therapeutic proteins

[0251] The pharmaceutical compositions comprising therapeutic proteins taught herein can be combined with other treatment methods and / or pharmaceutical compositions. For example, a patient with inflammatory bowel disease may already be taking a prescription drug prescribed by his or her doctor to treat the disease. In embodiments, the pharmaceutical compositions taught herein can be co-administered with a patient's current medication.

[0252] For example, the therapeutic proteins taught herein can be combined with one or more of the following: antidiarrheal drugs, 5-aminosalicylic acid compounds, anti-inflammatory agents, antibiotics, antibodies (e.g., antibodies targeting inflammatory cytokines, such as antibodies targeting anti-cytokine drugs, such as anti-TNF-α (e.g., adalimumab, pegylated certolizumab, golimumab, infliximab, V565) or anti-IL-12 / IL-23 (e.g., ustekinumab, risankizumab, brazikumab, ustekinumab), JAK inhibitors (e.g., tofacitinib, PF06700841, PF06651600, filgotinib, upadacitinib), anti-integrin agents (e.g., vedolizumab, etorizumab), S1P inhibitors (e.g., ertulimumab, ozanimod, amisimod), cell-based recombinant drugs (e.g., Cx601), steroids, corticosteroids, immunosuppressants (e.g., azathioprine and mercaptopurine), vitamins, and / or special diets.

[0253] A combination of a pharmaceutical composition according to the present invention and an agent for treating mucositis, such as oral mucositis, can be administered to a cancer patient who is undergoing chemotherapy or radiotherapy and who suffers from or is at risk of developing mucositis, such as oral mucositis. In some embodiments, a method of treatment comprises administering to a patient suffering from mucositis a pharmaceutical composition comprising SG-11 or a variant or fragment thereof and one or more selected from the group consisting of amifostine, benzocaine, phenylethylamine, ranitidine, omeprazole, capsaicin, glutamine, prostaglandin E2, vitamin E, sucralfate and a second therapeutic agent of allopurinol.

[0254] In some embodiments of the methods herein, a second therapeutic agent is administered simultaneously or sequentially with the SG-11 protein described herein. In some embodiments, the protein and the second agent act synergistically to treat or prevent the disease, disorder or symptom. In other embodiments, the protein and the second agent act additively to treat or prevent the disease, disorder or symptom.

[0255] Pharmaceutical compositions comprising SG-11 therapeutic protein

[0256] Provided herein are pharmaceutical compositions comprising an SG-11 protein, a variant or fragment thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient according to the present disclosure. In some embodiments, the pharmaceutical composition is formulated for administration to the lumen of the gastrointestinal tract, including the oral cavity, esophagus, small intestine, large intestine, rectum and / or anus.

[0257] In some embodiments, the composition comprises one or more other substances related to the source of the protein, for example, cellular components from the production host cell, or substances related to the chemical synthesis of the protein. In other embodiments, the pharmaceutical composition is formulated to include one or more second active agents as described herein. In addition, the composition may include components that maintain the structure and / or functional activity of the active agent or the composition itself. Such components include, but are not limited to, antioxidants and various antibacterial and antifungal agents, including, but not limited to, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or a combination thereof.

[0258] The term "pharmaceutical" or "pharmaceutically acceptable" refers to a composition that does not or preferably does not produce adverse, allergic or other untoward reactions when properly administered to an animal, such as a human. The preparation of pharmaceutical compositions or additional active ingredients will be known to those skilled in the art, for example, Remington's Pharmaceutical Sciences, 18th edition, Mark Printing Company, 1990, incorporated herein by reference. In addition, for administration to animals (such as humans), it is understood that the preparation should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA official biological standards.

[0259] The pharmaceutical composition of the present disclosure is formulated according to the intended route of administration and whether it is administered in, for example, solid, liquid or aerosol form. In a preferred embodiment, the composition can be administered rectally, but can also be administered directly to target cells bathed in a perfusion solution by injection, infusion, oral, intrathecal, intranasal, subcutaneous, mucosal, local administration, which is performed by catheter, by lavage or by other methods known to those of ordinary skill in the art or any combination of the foregoing methods. Liquid preparations containing therapeutically effective amounts of proteins can be administered rectally by enema, catheter, using a bulb syringe. Suppositories are examples of solid dosage forms formulated for rectal delivery. Typically, for suppositories, conventional carriers can include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories can be formed from a mixture, including, for example, an active ingredient range of about 0.5%-about 10%, and or about 1%-about 2%. Injectable liquid compositions are typically based on injectable sterile saline or phosphate buffered saline or other injectable carriers known in the art. Other liquid compositions include suspensions and emulsions. Solid compositions such as for oral administration can be in the form of tablets, pills, capsules (e.g., hard or soft shell gelatin capsules), oral compositions, lozenges, elixirs, suspensions, syrups, wafers, or combinations thereof. The active agent in such liquid and solid compositions, i.e., the protein described herein, is usually about 0.05% to 10% by weight of the component, with the remainder being injectable vehicles, etc.

[0260] The pharmaceutical composition can be formulated as a controlled release or sustained release composition, which provides an active agent including a therapeutic protein of the present disclosure released over an extended period of time, such as 30-60 minutes, or 1-10 hours, 2-8 hours, 8-24 hours, etc. Alternatively or additionally, the composition is formulated to be released to a specific site in the host. For example, the composition may have an enteric coating to prevent the active agent from being released in an acidic environment such as the stomach, so that it is released only in a more neutral or alkaline environment of the small intestine, colon or rectum. Alternatively or additionally, the composition can be formulated to provide delayed release in the oral cavity, small intestine or large intestine.

[0261] Depending on the intended route of administration, each of the above formulations may include at least one pharmaceutically acceptable excipient or carrier, e.g., a solid for rectal administration or a liquid for intravenous or parenteral administration or administration via a cannula. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, the like, and combinations thereof, which should be known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference).

[0262] Pharmaceutical compositions for administration may be present in unit dosage form to facilitate accurate dosing. For solid compositions, typical unit dosage forms include pre-filled, pre-measured ampoules or syringes of liquid compositions or suppositories, pills, tablets, capsules, etc. In some embodiments of such compositions, the active agent, i.e., a protein as described herein, may be a component (about 0.1 to 50 wt / wt%, 1 to 40 wt / wt%, 0.1 to 1 wt / wt%, or 1 to 10 wt / wt%), with the remainder being various carriers or carriers and processing aids to aid in forming the desired dosage form.

[0263] The actual dosage of the unit dosage form of the present disclosure administered to a patient may be determined by physical and physiological factors, such as body weight, severity of the condition, type of disease being treated, previous or concurrent therapeutic interventions, patient's idiopathic conditions and route of administration. In any event, the physician responsible for administration will determine the concentration of the active ingredient in the composition, as well as the appropriate dosage for the individual subject.

[0264] Protein expression systems and protein production

[0265] Provided herein are compositions and methods for producing the isolated proteins of the present disclosure, as well as expression vectors comprising polynucleotide sequences encoding the proteins and host cells harboring the expression vectors.

[0266] The proteins disclosed herein can be prepared by conventional recombinant methods, for example, culturing cells transformed or transfected with an expression vector comprising a nucleic acid encoding an SG-11 therapeutic protein, a variant or a fragment thereof. Host cells comprising any such vectors are also provided. The host cell may be prokaryotic or eukaryotic, and examples of host cells include Escherichia coli, yeast or mammalian cells. Further provided is a method for producing any protein described herein, comprising culturing the host cell under conditions suitable for expression of the desired protein, and recovering the desired protein from the cell culture. The recovered protein can then be separated and / or purified for use in in vitro and in vivo methods, as well as formulated into a pharmaceutically acceptable composition. In some embodiments, the protein is expressed in a prokaryotic cell such as Escherichia coli, and the separation and purification of the protein includes a step of reducing endotoxins to a level acceptable for therapeutic use in humans or other animals.

[0267] Expression vector

[0268] Expression vectors are provided herein, which contain polynucleotide sequences encoding proteins of the present disclosure or variants and / or fragments thereof. Polynucleotide sequences encoding proteins of the present disclosure can be obtained using standard recombinant techniques. The desired encoding polynucleotide sequence can be amplified from the genomic DNA of the source bacterium, i.e., R. hominis. Alternatively, a nucleotide synthesizer can be used to synthesize polynucleotides. Once obtained, the sequence encoding the polypeptide is inserted into a recombinant vector capable of replicating and expressing heterologous (exogenous) polynucleotides in a host cell. For the purposes of the present disclosure, many vectors known in the art can be used. The selection of a suitable vector will depend primarily on the size of the nucleic acid to be inserted into the vector and the specific host cell to be transformed with the vector. Each vector contains various components, depending on its function (amplification or expression of heterologous polynucleotides, or both) and its compatibility with the specific host cell in which it resides. Vector components generally include, but are not limited to, an origin of replication, a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, a heterologous nucleic acid insert, and a transcription termination sequence.

[0269] Typically, plasmid vectors containing control sequences and replicons derived from species compatible with host cells are used in combination with these hosts. The vector typically carries a replication site and a marker sequence that can provide phenotypic selection in the transformed cell. For example, pBR322, pUC, pET or pGEX vectors, a plasmid derived from an Escherichia coli species, are typically used to transform Escherichia coli. Such vectors include genes encoding ampicillin (Amp) and tetracycline (Tet) resistance, thereby providing a simple method for identifying transformed cells. These vectors and their derivatives or other microbial plasmids or phages may also include or be modified to include a promoter that can be used by microbial organisms to express endogenous proteins.

[0270] The expression vector of the present disclosure may include a promoter, which is an untranslated regulatory sequence located upstream (5′) and operably linked to the nucleotide sequence encoding the protein, so that the promoter regulates the transcription of the coding sequence. Prokaryotic promoters are generally divided into two categories, inducible and constitutive. An inducible promoter is a promoter that initiates an increase in the transcription level of the coding polynucleotide under its control in response to changes in culture conditions (e.g., the presence or absence of nutrients or changes in temperature). A large number of promoters recognized by a variety of potential host cells are well known, and technicians can select promoters based on the desired expression level. Promoters suitable for prokaryotic hosts include E. coli promoters, such as lac, trp, tac, trc and ara; viral promoters recognized by E. coli, such as λ and T5 promoters; and T7 and T7lac promoters derived from T7 phage. Host cells carrying vectors containing, for example, T7 promoters are engineered to express T7 polymerase. Such host cells include E. coli BL21 (DE3), Lemo21 (DE3) and NiCo21 (DE3) cells. In some embodiments, the promoter is an inducible promoter under the control of a chemical or environmental cue.

[0271] Other plasmid vectors include pIN vectors (Inouye et al., 1985) and pGEX vectors, which are used to generate soluble fusion proteins of glutathione S-transferase (GST) for subsequent purification and separation or cleavage. Other suitable fusion proteins are fusion proteins with β-galactosidase, ubiquitin, etc.

[0272] Suitable vectors for expression in prokaryotic and eukaryotic host cells are known in the art, and some are further described herein.

[0273] The vector of the present disclosure may further comprise a signal sequence that allows the translated recombinant protein to be recognized and processed by the host cell (i.e., cleaved by a signal peptidase). For prokaryotic host cells that do not recognize and process a signal sequence that is native to a heterologous polypeptide, the signal sequence is replaced by a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, Ipp or heat-stable enterotoxin II (STII) guides, LamB, PhoE, PeIB, OmpA and MBP. Well-known signal sequences for eukaryotic expression systems include, but are not limited to, interleukin-2, CD5, immunoglobulin kappa light chain, trypsinogen, serum albumin and prolactin.

[0274] As described herein, the SG-11 protein or its variant or fragment can be expressed as a fusion protein or polypeptide. Common fusion partners include, but are not limited to, human serum albumin and the crystallizable fragment or constant domain of IgG, Fc. Histidine tags or FLAG tags can also be used to simplify the purification of recombinant proteins from expression medium or recombinant cell lysates. The fusion partner can be fused to the N and / or C terminus of the target protein.

[0275] Host cells

[0276] Suitable host cells for cloning or expressing DNA in the vectors herein include prokaryotes, yeast or higher eukaryotic cells. A variety of cell lines and cultures can be used for host cells, which can be obtained, for example, through the American Type Culture Collection (ATCC), which is an archive of living cultures and genetic materials. Cell types that can be used for vector replication and / or expression include, but are not limited to, bacteria, such as Escherichia coli (e.g., Escherichia coli strains RR1, Escherichia coli LE392, Escherichia coli B, Escherichia coli X 1776 (ATCC No. 31537), Escherichia coli W3110 (F-, λ-, prototrophic, ATCC No. 273325), DH5α, JM109 and KC8, Bacillus (such as Bacillus subtilis); and other Enterobacteriaceae, such as Salmonella typhimurium, Serratia marcescens, various Pseudomonas species, and many commercially available bacterial hosts, such as Competent cells and SOLOPACK TM Gold Cell In certain embodiments, bacterial cells such as E. coli are specifically contemplated as host cells.

[0277] Examples of eukaryotic host cells for vector replication and / or expression include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, CHO, Saos, and PC12. Other eukaryotic host cells include yeast (e.g., Pichia pastoris and Saccharomyces cerevisiae) and cells derived from insects (e.g., Spodoptera frugiperda or Trichoplusia ni). Many host cells from various cell types and organisms are available and are well known to those skilled in the art. Similarly, viral vectors can be used in conjunction with eukaryotic or prokaryotic host cells, particularly in conjunction with host cells that allow vector replication or expression. The selection of suitable host cells is considered to be within the technical scope of the art.

[0278] It is a well-known method to introduce recombinant DNA, i.e., expression vector, into a host cell so that the DNA can be replicated, either as an extrachromosomal element or as a chromosomal integrant, thereby generating a host cell carrying the desired expression vector. Methods of transfection are known to the skilled artisan, such as by CaPO4 and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate for such cells. Calcium treatment with calcium chloride or electroporation, as described by Sambrook et al. above, is commonly used for prokaryotes or other cells containing a large cell wall barrier. General aspects of mammalian cell host system transformation have been described in U.S. Pat. No. 4,399,216. Transformation into yeast is generally performed according to the methods of Van Solingen et al., J. Bact, 130: 946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76: 3829. Other methods for introducing DNA into cells include nuclear microinjection, electroporation, fusion of bacterial protoplasts with intact cells, or introduction using polycations such as poly-p-phenylenediamine, poly-ornithine. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology. 185:527-537 (1990) and Mansour et al., Nature, 336:348-352 (1988).

[0279] Thus, provided herein is a recombinant vector or expression vector as described above, comprising a polynucleotide encoding an SG-11 therapeutic protein sequence of interest (e.g., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19 or variants and / or fragments thereof, as described herein). The polynucleotide may be, for example, any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20 or variants or fragments thereof. In addition, the present disclosure teaches a host cell carrying the vector. The host cell may be a eukaryotic or prokaryotic cell as described above. In a preferred embodiment, the host cell is a prokaryotic cell. In another preferred embodiment, the host cell is Escherichia coli.

[0280] In some embodiments, the polynucleotide encoding the target protein is codon optimized. A codon optimization algorithm is applied to the polynucleotide sequence encoding the protein so as to select the appropriate codon for a given amino acid based on the codon usage preference of the expression host. Many codon optimization algorithms also take into account other factors, such as mRNA structure, host GC content, ribosome entry site. Some examples of codon optimization algorithms and gene synthesis service providers are: AUTM: www.atum.bio / services / genegps; GenScript: www.genscript.com / codon-opt.html; ThermoFisher: www.thermofisher.com / us / en / home / life-science / cloning / gene-synthesis / geneart-gene-synthesis / geneoptimizer.html; and Integrated DNA Technologies: www.idtdna.com / CodonOpt. The nucleotide sequence is then synthesized and cloned into a suitable expression vector. In the present disclosure, the codon-optimized sequences in the present disclosure include SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 and SEQ ID NO:20.

[0281] Methods of producing proteins

[0282] Methods for producing the proteins described herein are provided, but they are well known to ordinary technicians. Host cells for protein production transformed or transfected with expression or cloning vectors described herein are cultured in conventional nutrient media, which are appropriately modified to induce promoters, select and / or maintain transformants, and / or express genes encoding the desired protein sequences. Culture conditions, such as culture media, temperature, pH, etc., can be selected by technicians without excessive experimentation. In general, principles, protocols and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: A Practical Approach (IRL Press, 1991), edited by M. Butler, and Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press).

[0283] Generally, "purified" refers to a particular protein composition that has been fractionated to remove non-protein components and various other proteins, polypeptides or peptides, and which composition substantially retains its activity, such as can be assessed by, for example, as described below or by protein assays known to those of ordinary skill in the art for the desired protein, polypeptide or peptide.

[0284] When the term "substantially purified" is used, this refers to a composition in which the particular protein, polypeptide or peptide forms the major component of the composition, for example, constituting about 50% or more of the protein in the composition. In preferred embodiments, the substantially purified protein will constitute 60%, 70%, 80%, 90%, 95%, 99% or more of the protein in the composition.

[0285] A peptide, polypeptide or protein "purified to homogeneity" as applied to the present disclosure means that the peptide, polypeptide or protein has a certain level of purity, wherein the peptide, polypeptide or protein is substantially free of other proteins and biological components. For example, a purified peptide, polypeptide or protein will generally be sufficiently free of other protein components so that degradation sequencing can be successfully performed.

[0286] Although preferred for use in certain embodiments, there is no general requirement to always provide a protein, polypeptide or peptide in its most purified state. In fact, it is expected that a substantially unpurified protein, polypeptide or peptide, although enriched in a desired protein composition relative to its native state, will have utility in certain embodiments.

[0287] Various methods of quantifying the degree of purification of proteins, polypeptides or peptides will be known to those skilled in the art in light of the present disclosure. These include, for example, determining the specific protein activity of a fraction by gel electrophoresis, or assessing the number of polypeptides within a fraction.

[0288] Another example is the use of a specific binding partner to purify a specific fusion protein. Such purification methods are routine in the art. Since the present disclosure provides the DNA sequence of a specific protein, any fusion protein purification method can now be practiced. This can be illustrated by generating a specific protein-glutathione S-transferase fusion protein, expressing it in E. coli and separating it to homogeneity using affinity chromatography on glutathione-agarose or generating a polyhistidine tag on the N- or C-terminus of the protein, followed by purification using Ni affinity chromatography. However, given that many DNAs and proteins are known or can be identified and amplified using the methods described herein, any purification method can now be used.

[0289] In other embodiments, a peptide-enriched preparation may be used instead of a purified preparation. In this article, whenever purified is used, enriched may also be used. The preparation may be enriched not only by purification methods but also by overexpression or overproduction of the peptide by bacteria compared to the wild-type. This may be accomplished using recombinant methods or by selecting conditions that induce expression of the peptide from wild-type cells.

[0290] The recombinantly expressed polypeptides of the invention can be recovered from the culture medium or host cell lysate. Suitable purification procedures include, for example, by fractionation on ion exchange (anion or cation) columns; ethanol precipitation; reverse phase HPLC; chromatographic separation on silica gel or cation exchange resins (such as DEAE); chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration or size exclusion chromatography (SEC), for example using Sephadex G-75; and metal chelate columns to bind epitope-tagged forms of the polypeptides of the invention. Various protein purification methods can be employed and are known in the art and described, for example, in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The purification step selected will depend, for example, on the nature of the production process used and the specific polypeptide produced.

[0291] Alternative methods well known in the art can be used to prepare the polypeptides of the present invention. For example, sequences encoding polypeptides or portions thereof can be produced by direct peptide synthesis using solid phase techniques (see, e.g., Stewart et al., 1969, Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco, CA; Merrifield. J. 1963, Am. Chem. Soc., 85: 2149-2154. In vitro protein synthesis can be performed using manual techniques or by automation. For example, automated synthesis can be performed using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using the manufacturer's instructions. Individual portions of the polypeptides of the present invention or portions thereof can be chemically synthesized separately and combined using chemical or enzymatic methods to produce full-length polypeptides or portions thereof.

[0292] In some embodiments, the present disclosure provides chimeric molecules comprising any polypeptide described herein fused to a heterologous polypeptide or amino acid sequence, and a polynucleotide encoding the chimeric molecule. Examples of such chimeric molecules include, but are not limited to, any polypeptide described herein fused to an antigenic determinant tag sequence, an immunoglobulin Fc region.

[0293] Recombinant bacterial delivery system

[0294] The present disclosure contemplates the use of delivery systems outside of traditional pharmaceutical formulations containing purified proteins. In some embodiments, the present disclosure utilizes a recombinant bacterial delivery system, a bacteriophage-mediated delivery system, a chitosan-DNA complex, or an AAV delivery system.

[0295] One particular recombinant bacterial delivery system is based on Lactococcus lactis. Essentially, a gene encoding a therapeutic protein (eg, SEQ ID NO: 3) can be cloned into an expression vector, which is then transformed into Lactococcus lactis. Subsequently, Lactococcus lactis can be administered to a patient. See, e.g., Bratt et al., "A phase 1 trial with transgenic bacteria expressing interleukin-10 in Crohn's disease," Clinical Gastroenterology and Hepatology, 2006, vol. 4, pp. 754-759 ("We treated Crohn's disease patients with a genetically modified Lactococcus lactis (LL-Thyl2) in which the thymidylate synthase gene was replaced with a synthetic sequence encoding mature human interleukin-10."); Shigemori et al., "Oral delivery of Lactococcus lactis that secretes bioactive heme oxygenase-1 alleviates development of acute colitis in mice," Microbial Cell Factories, 2015, vol. 14:189 (“Mucosal delivery of therapeutic proteins using genetically modified lactic acid bacteria strains (gmLAB) is being investigated as a novel therapeutic strategy.”); Steidler et al., “Treatment of murine colitis by Lactococcus lactis secreting interleukin-10”. "The cytokine interleukin-10 (IL-10) has been shown to treat inflammatory bowel disease (IBD) in clinical trials. Using two mouse models, we demonstrate that therapeutic doses of IL-10 can be reduced by local delivery of bacteria genetically engineered to secrete the cytokine. Intra-tail administration of IL-10-secreting Lactococcus lactis reduced colitis by 50% in mice treated with sodium dextran sulfate and prevented the onset of colitis in IL-102 / 2 mice. This approach may lead to better approaches for low-cost, long-term management of IBD in humans."”); Hanniffy et al., “Mucosal delivery of a pneumococcal vaccine using Lactococcus lactis affords protection against respiratory infection,” Journal of Infectious Diseases, 2007, volume 195, pages 185-193 (“Here, we evaluated intracellular production of pneumococcal surface protein A (PspA) by Lactococcus lactis as a mucosal vaccine to provide protection against pneumococcal disease.”); and Vandenbroucke et al., “Active delivery of trefoil factors by genetically modified Lactococcus lactis prevents and heals acute colitis in mice.” mice), Gastroenterology, 2004, vol. 127, pp. 502-513 ("We have proactively evaluated a novel treatment approach for acute and chronic colitis involving in situ secretion of murine TFFs by oral administration of Lactococcus lactis. This novel approach may lead to effective treatments for acute and chronic colitis and epithelial damage in humans.").

[0296] In another embodiment, "synthetic bacteria" can be used to deliver SG-11 protein or variants or fragments thereof, wherein the probiotic is engineered to express the SG-11 therapeutic protein (see, e.g., Durrer and Allen, 2017, PLoS One, 12: e0176286).

[0297] Bacteriophages have been genetically engineered to deliver specific DNA payloads or to alter host specificity. Transfer methods such as phages, plasmids, and transposons can be used to deliver and circulate engineered DNA sequences to microbial communities through processes such as transduction, transformation, and conjugation. For the purposes of this disclosure, it is fully understood that engineered phages can be a possible delivery system for the proteins of the invention by incorporating nucleic acids encoding the proteins into the phages and utilizing the phages to deliver the nucleic acids to the host microorganisms, which will then produce the proteins after the phages deliver the nucleic acids to their genomes.

[0298] Similar to the above-mentioned engineered phage method, a transposon delivery system can be used to incorporate nucleic acids encoding therapeutic proteins into host microorganisms that remain in the patient's microbiome. See, Sheth et al., "Manipulating bacterial communities by in situ microbiome engineering", Trends in Genetics, 2016, Volume 32, Issue 4, Pages 189-200.

[0299] The following examples are intended to illustrate and not to limit the scope of the disclosure herein. Example

[0300] The following experiments utilize a powerful mix of in vitro experiments combined with in vivo models of IBD to demonstrate the therapeutic capabilities of the proteins and methods taught.

[0301] Example 1

[0302] Expression of SG-11 and its variants

[0303] For the experiments described in the following examples, genomic DNA obtained from Roseburia hominis (A2-183; DSM type 16839 strain; see, e.g., Duncan, SH, Aminov, RI, Scott, KP, Louis, P., Stanton, TB, Flint, HJ (2006). Proposal of Roseburia faecis sp. nov., Roseburia hominis sp. nov., and Roseburia inulinivorans sp. nov., based on isolates from human feces) was amplified by PCR. faeces). Int. J. Syst. Evol. Microbiol. Volume 56, Pages 2437-2441) to obtain a polynucleotide encoding SG-11 (SEQ ID NO: 3). The encoding polynucleotide was then subcloned into an inducible expression vector and used to transform Escherichia coli BL21 (DE3) cells for expression and purification of SG-11 or its variants using conventional culture and purification methods in the art, as described below.

[0304] Expression of SG-11 (comprising SEQ ID NO: 3)

[0305] Expression and purification of proteins comprising the amino acid sequence of SG-11 (SEQ ID NO: 5) used in various experiments related to the present disclosure were achieved using the pGEX vector system, which is designed for inducible, high-level intracellular expression of genes or gene fragments. Expression in E. coli produces a tagged protein with a GST moiety at the amino terminus and the protein of interest at the carboxyl terminus. The vector has a tac promoter for chemically inducible high-level expression and an internal laq1 for use with any E. coli host. q Gene.

[0306] The polynucleotide containing the nucleotide sequence encoding SG-11 (SEQ ID NO: 3 from Roseburia hominis DSM 16839) was inserted into the multiple cloning site (BamHI and NotI sites) of pGEX-6P-1 (GE Healthcare Life Science, Pittsburgh, PA) to express SG-11 as a GST fusion protein, which was then cleaved at the Precision protease site to generate SG-11 having the amino acid sequence of SEQ ID NO: 5 (encoded by SEQ ID NO: 6) provided in Table 4 below. The protein was expressed and purified by two alternative methods. First, BL21 (DE3) transformants were grown in LB and 100 μg / ml carbenicillin and 1 μg / ml chloramphenicol at 30°C. When the culture density reached 0.6 OD 600 When 50 μL of 1% SG-11C was added, the expression was induced with 0.4 mM IPTG for 4 hours. The cells were collected by centrifugation, then lysed by sonication, and the soluble lysate was applied to a GST-resin column. The bound protein was washed with PBS, and the purified untagged SG-11C was eluted by adding PreScission protease to cleave the protein at the C-terminus of the GST tag.

[0307] Another expression and purification method using the same pGEX expression construct was performed by growing transformed BL21(DE3) cells in LB with 50 μg / ml carbenicillin at 37°C. When the culture reached 0.7 OD 600When the density reached , it was cooled to 16°C and expression was induced with 1 mM IPTG at 16°C for 15 hours. The cells were harvested and lysed by sonication, and the soluble lysate was then applied to the GSTrap column. The bound protein was washed with HEPES buffer, and the purified untagged SG-11 (SEQ ID NO: 5) was then eluted by adding HRV3C protease to cleave the protein at the C-terminus of the GST tag. The eluted fractions containing protein, as determined by SDS-PAGE and Coomassie Brilliant Blue staining, were identified and combined, and then applied to a HiTrap Q HP anion exchange column, and then to a Superdex 75 (26 / 60) preparative size exclusion column (SEC) to obtain the final preparation.

[0308] Table 4

[0309]

[0310] The expression and purification of mature SG-11 protein without a signal peptide was accomplished using the pD451-SR vector system (AUTM, Newark, CA). The expression vector utilizes an IPTG-inducible T7 promoter. The polynucleotide (SEQ ID NO: 4) encoding SG-11 (SEQ ID NO: 3) was codon-optimized at AUTM (Newark, CA) to produce the codon-optimized coding sequence of SEQ ID NO: 8 provided herein. The codon-optimized coding sequence was inserted into the pD451-SR vector, and the resulting construct provided expression of the 233 amino acid SG-11 protein of SEQ ID NO: 7 provided herein.

[0311] BL21 (DE3) cells transformed with this construct were grown in the auto-induction medium MagicMedia (ThermoFisher). The culture was incubated at 25°C with shaking for 8 hours and then incubated at 16°C for up to 72 hours. The cells were centrifuged and precipitated, resuspended in 100mM Tris-HCl, pH 8.0 containing 50mM NaCl, 2mg / ml lysozyme and protease inhibitors, and then Triton X-100 was added to the suspension. The cells were then sonicated and a clarified lysate was prepared by centrifugation to purify the protein by standard column chromatography techniques.

[0312] SG-11 (SEQ ID NO: 7) was purified using two anion exchange columns, HiTrap Q HP and then Mono Q. Fractions containing partially purified protein, as determined by SDS-PAGE and Coomassie blue staining, were further purified using Mono Q. The purification method for Mono Q was the same as for HiTrap Q. Fractions containing SG-11 were combined and dialyzed in buffer (50 mM sodium phosphate, 150 mM NaCl, and 10% glycerol). Purity and homogeneity were analyzed using SDS-PAGE and analytical SEC, Superdex 200Increase 3.2 / 300, and the purity of the preparation was estimated to be approximately 92.7%.

[0313] The pD451-SR vector system was also used to express and purify the SG-11 variant SG-11V5 (SEQ ID NO: 19). To generate the expression construct, the codon optimized sequence of SG-11 (SEQ ID NO: 8) was modified to generate the polynucleotide of SEQ ID NO: 20 encoding SG-11V5 (SEQ ID NO: 19). The SG-11V5 coding sequence was cloned into the pD451-SR vector.

[0314] BL21(DE3) cells transformed with this construct were grown and processed to prepare clarified lysates as described above for expression of SG-11 (SEQ ID NO:7).

[0315] SG-11V5 protein was purified by HiTrap Q and then by hydrophobic interaction chromatography (HIC), HiTrapButyl HP from clarified lysates. Fractions containing SG-11V5 identified by SDS-PAGE and Coomassie blue staining were pooled and dialyzed in buffer (50 mM sodium phosphate, 150 mM NaCl and 10% glycerol). All preparative column chromatography described used Protein purification was performed using the GE Healthcare Life Sciences system (GE Healthcare Life Sciences, Pittsburgh, PA).

[0316] After SDS-PAGE and Coomassie blue staining, the purified proteins were quantified by densitometry using bovine serum albumin as a reference. nexgen-MCS TM Endotoxin levels were measured by ELISA (Charles River, Wilmington, MA). The endotoxin levels of the proteins used in the assays described herein were less than 1 EU / mg.

[0317] An expression construct was generated in which the pET-28 vector (Sigma Millipore) was used to contain and express a polynucleotide sequence (SEQ ID NO: 4) encoding SG-11 (SEQ ID NO: 3) with a FLAG tag (DYKDDDDK; SEQ ID NO: 32) at the N-terminus of SG-11. The complete FLAG-tagged SG-11 protein sequence is provided herein as SEQ ID NO: 9 (and encoded by SEQ ID NO: 10). Protein expression using this construct is under the control of the T7 promoter, which can be induced with isopropyl β-D-1-thiogalactopyranoside (IPTG). The N-terminal FLAG tag was incorporated into the construct using PCR and an oligonucleotide encoding DYKDDDDK. The transformed host cells were grown overnight in 2xYT medium at 37°C. The overnight culture was then inoculated into fresh 2xYT medium and incubated at 37°C for 4 hours. The 4-hour culture was then inoculated (1% inoculum) into MagicMedia TM E. coli expression medium (ThermoFisher). The cells were grown at 25°C for 8 hours and then at 16°C for up to 72 hours before being collected by centrifugation. The protein was expressed in a soluble form and could be recovered from the clarified lysate. The expressed protein was purified using a HiTrapQ anion exchange column followed by Superdex 200Increase 10 / 300GL SEC. Purity and homogeneity were analyzed using SDS-PAGE and analytical SEC, Superdex 200Increase 3.2 / 300, and the purity of the preparation was estimated to be approximately 93.3%.

[0318] Preparation of SG-11 protein for stability analysis

[0319] SG-11 (SEQ ID NO: 7) and its variant SG-11V5 (SEQ ID NO: 19) were purified using two anion exchange columns, HiTrap Q and then Mono Q. Fractions containing partially purified protein, as determined by SDS-PAGE and Coomassie blue staining, were further purified using Mono Q. The purification protocol for MonoQ was the same as for HiTrapQ. Fractions containing SG-11 were combined and dialyzed in buffer (50 mM sodium phosphate, 150 mM NaCl, and 10% glycerol).

[0320] For SG-11V5, after purification with HiTrap Q, the protein was further purified using hydrophobic interaction chromatography (HIC), HiTrap Butyl HP. Fractions containing SG-11V5 and identified by SDS-PAGE and Coomassie blue staining were pooled and dialyzed in buffer (50 mM sodium phosphate, 150 mM NaCl and 10% glycerol). All preparative column chromatography described used Protein purification was performed using the GE Healthcare Life Sciences (GE Healthcare Life Sciences, Pittsburgh, PA) system.

[0321] After SDS-PAGE and Coomassie blue staining, the purified proteins were quantified by densitometry using bovine serum albumin as a reference. nexgen-MCS TM Endotoxin levels were measured by ELISA (Charles River, Wilmington, MA). The endotoxin levels of the proteins used in the assays described herein were less than 1 EU / mg.

[0322] Example 2

[0323] Effect of SG-11 on restoration of epithelial barrier integrity after inflammation-induced disruption

[0324] The following experiment demonstrates the therapeutic ability of the SG-11 protein or variants thereof disclosed herein to restore gastrointestinal epithelial barrier integrity. Therefore, this experiment demonstrates the functional use of therapeutic proteins for treating gastrointestinal inflammatory diseases or diseases involving impaired epithelial barrier integrity / function.

[0325] The assay was performed in a Transwell plate as described below, where cells were separated by a permeable membrane for co-culture of multiple cell types. In the apical (top) chamber, human colonic epithelial cells consisting of a mixture of intestinal epithelial cells and goblet cells were cultured until the cells acquired tight junction formation and barrier function by measuring transepithelial electrical resistance (TEER). In the basal chamber, monocytes were cultured separately. Epithelial cells were primed with inflammatory cytokines. The assay measured the effects of therapeutic proteins, i.e., SG-11, on epithelial barrier function, muc2 gene expression, and cytokine production.

[0326] Cell culture. HCT8 human intestinal epithelial cells (ATCC catalog number CCL-244) were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 IU / ml penicillin, 100 μg / ml streptomycin, 10 μg / ml gentamicin, and 0.25 μg / ml amphotericin (cRPMI). HT29-MTX human goblet cells (Sigma-Aldrich (St. Louis, MO; catalog number 12040401)) were maintained in DMEM medium containing 10% fetal bovine serum, 100 IU / ml penicillin, 100 μg / ml streptomycin, 10 μg / ml gentamicin, and 0.25 μg / ml amphotericin (cDMEM). Epithelial cells were passaged after trypsinization and cells were used between 5 and 15 generations after thawing from liquid nitrogen stocks. U937 mononuclear cells (ATCC catalog number 700928) were maintained as suspension cultures in cRPMI medium and split as needed by dilution to maintain cells at 5 × 10 5 and 2×10 6 U937 cells were used up to passage 18 after thawing from liquid nitrogen.

[0327] Epithelial cell culture. A mixture of HCT8 intestinal epithelial cells and HT29-MTX goblet cells was plated in the top chamber of a Transwell plate at a ratio of 9:1 as described previously (Berget et al., 2017, Int J Mol Sci, 18:1573; Beduneau et al., 2014, Eur J Pharm Biopharm, 87:290-298). A total of 10 5 cells (9×10 4 HCT8 cells and 1×10 4 HT29-MTX cells). Epithelial cells were trypsinized from culture flasks and viable cells were determined by trypan blue counting. The correct volume of each cell type was combined in a single tube and centrifuged. The cell pellet was resuspended in cRPMI and added to the top chamber of the Transwell plate. Cells were cultured at 37°C + 5% CO2 for 8 to 10 days, with medium changes every 2 days.

[0328] Monocyte culture. On the 6th day of epithelial cell culture, 2×10 5 U937 monocytes were seeded into 96-well receiving plates at 10 cells / well. The cells were cultured at 37°C + 5% CO2, and the culture medium was changed every 24 hours for 4 days.

[0329] Co-culture assay. After 8-10 days of culture, the Transwell plates containing intestinal epithelial cells were treated with 10 ng / ml IFN-γ, which was added to the basolateral chamber of the transwell plate and treated for 24 hours at 37°C + 5% CO2. After 24 hours, fresh cRPMI was added to the epithelial cell culture plates. Transepithelial electrical resistance (TEER) readings were measured after IFN-γ treatment and used as pre-treatment TEER values. SG-11 was then added to the apical chamber of the Transwell plate at a final concentration of 1 μg / ml (40 nM). Myosin light chain kinase (MLCK) inhibitor peptide 18 (BioTechne, Minneapolis, MN) was used as a positive control at 50 nM, which prevents barrier disruption caused by inflammation (Zolotarevskky et al., 202, Gastroenterology, 123: 163-172). The bacterial-derived molecule staurosporine was used as a negative control at 100 nM, which induces apoptosis and exacerbates barrier disruption (Antonsson and Persson, 2009, Anticancer Res, 29: 2893-2898). The compounds were incubated on the intestinal epithelial cells for 1 hour or 6 hours. After pre-incubation with the test compound, the Transwell insert containing the intestinal cells was transferred to the top of the receiving plate containing U937 monocytes. Heat-killed Escherichia coli (HK E. coli) (bacteria heated to 80°C for 40 minutes) were then added to the top and basolateral chambers with a multiplicity of infection (MOI) of 10. The Transwell plates were incubated at 37°C + 5% CO2 for 24 hours, and then post-treatment TEER measurements were performed. TEER determinations were performed with mature SG-11 protein (SEQ ID NO: 5 or SEQ ID NO: 9).

[0330] Data analysis. Based on the surface area of ​​the Transwell insert (0.143 cm 2 ), convert the original resistance value in ohms (Ω) to ohms / square centimeter (Ωcm 2 ). In order to adjust the differential resistance generated after 10 days of culture, the single wells were treated with Ωcm 2 Readings were normalized to the pre-treatment Ωcm 2 Then the standardized Ωcm 2 Values ​​are expressed as the average Ωcm relative to the untreated samples. 2 The percentage change in value.

[0331] Both epithelial cells and monocytes were exposed to heat-killed Escherichia coli (HK E. coli) for 1 hour ( Figure 1A ) or 6 hours ( Figure 1B)Addition of SG-11 protein induces the production of inflammatory mediators by monocytes, leading to disruption of the epithelial monolayer, as indicated by a decrease in TEER. Myosin light chain kinase (MLCK) inhibitors were used as control compounds and have been shown to prevent barrier disruption and / or reverse barrier loss triggered by antimicrobial immune responses. Staurosporine was used as a control compound that causes apoptosis and / or death of epithelial cells, thus resulting in a dramatic decrease in TEER, indicating disruption and / or loss of epithelial cell barrier integrity / function. Figure 1A In the experiment, SG-11 increased the TEER destroyed by HK E. coli from 55.8% to 62%. Figure 1B In the experiment, SG-11 increased the TEER destroyed by HK E. coli from 53.5% to 60.6%. Figure 1A-1B The graphs in represent data collected from two separate experiments (n=6).

[0332] Example 3

[0333] Effect of SG-11 on TNF-α and IL-23 production induced by heat-killed Escherichia coli

[0334] The following experiment demonstrates the therapeutic ability of the SG-11 protein or variants thereof as disclosed herein to reduce immune activation as measured by cytokine production. Thus, this experiment demonstrates the potential functional utility of therapeutic proteins in gastrointestinal inflammatory diseases or diseases involving impaired epithelial barrier integrity / function, where modulation of cytokine levels will affect the host's disease state.

[0335] Proinflammatory cytokines TNF-α and IL-23 produced by monocytes were measured in tissue culture supernatants from the basal chamber of the co-culture TEER assay performed in Example 2. After the TEER reading, the supernatant was centrifuged at 10,000 g for 5 minutes at 4°C to remove cell debris. Luminex analysis was performed according to the manufacturer's instructions (Magpix instrument and xPonent software version 4.2; Luminex Group (Austin, TX)). Luminex analysis was performed to determine the pg / ml concentration of TNF-α and IL-23 produced by monocytes. Luminex analysis utilizes a microbead-based system to quantify multiple cytokines in a single sample. As with ELISA, Luminex magnetic beads are also coated with capture antibodies, and incubation with the sample allows the target to bind to the capture antibody. The beads are washed and incubated with fluorescently labeled detection antibodies to quantify the bound targets. Cytometry analysis is used to distinguish between bead populations loaded with different fluorescent dyes and quantify cytokine levels by measuring the detection antibody signal.

[0336] TNF-α and IL-23 production in untreated cells and cells preincubated with SG-11 for 6 h before HK E. coli treatment was normalized to the pg / ml concentration caused by HK E. coli, which was set as 1.0. Preincubation with SG-11 reduced TNF-α and IL-23 production. The results are shown in Figure 2A (TNF-α) and Figure 2B (IL-23). Figure 2A and Figure 2B The graphs in represent data collected from two separate experiments (n=6).

[0337] Example 4

[0338] Effect of SG-11 on IL-10 production induced by heat-killed Escherichia coli

[0339] The following experiments measured the effect of SG-11 administration on IL-10 production in a TEER assay.

[0340] IL-10 production was measured in tissue culture supernatant from the basolateral chamber of the co-culture TEER assay described in Example 2 containing monocytes. Luminex analysis was performed to determine the pg / ml concentration of IL-10 produced by monocytes. IL-10 production in untreated cells and cells pre-incubated with SG-11 for 6 hours prior to HK E. coli treatment was normalized to the pg / ml concentration caused by HK E. coli, which was set to 1.0. Pre-incubation with SG-11 reduced IL-10 production to 0.89. The results are shown in Figure 3 . Figure 3 The graphs in represent data collected from two separate experiments (n=6).

[0341] Example 5

[0342] Effect of SG-11 on mucin expression after stimulation with heat-killed Escherichia coli

[0343] The following experiment measures the effect of the SG-11 protein or variants thereof as disclosed herein to increase mucin expression in gastrointestinal tissue. Thus, this experiment demonstrates the functional use of the therapeutic protein SG-11 for the treatment of gastrointestinal inflammatory diseases or diseases involving impaired epithelial barrier integrity / function, where increased mucin expression would be beneficial.

[0344] Gene expression was measured in epithelial cell monolayers in the top chamber of the co-culture TEER assay described in Example 2. Total RNA was isolated from the epithelial cell monolayer and cDNA was synthesized. Before adding HK E. coli for 24 hours, cDNA produced by HCT8 and HT29-MTX cells treated with SG-11 (1 μg / ml; 40 nM) was subjected to qRT-PCR for 6 hours. Muc2 gene expression is expressed as mean fold change ± SEM. Statistical analysis was performed with HK E. coli by one-way ANOVA and multiple comparisons were performed using Fisher's LSD test.

[0345] Analysis of muc2 gene expression showed that HK E. coli alone increased expression by 13.6-fold compared to untreated cells (p = 0.0007). Cells stimulated with HK E. coli and treated with SG-11 increased expression by an additional 1.4-fold compared to HK E. coli (p = 0.03) ( Figure 4 ). Figure 4 The graph in represents data from a single experiment (n=3). A significant increase in muc2 production was observed in response to HK E. coli.

[0346] Example 6

[0347] Effect of SG-11 on wound healing of epithelial cells

[0348] The following experiment demonstrates the therapeutic ability of the proteins disclosed herein to increase wound healing of gastrointestinal epithelial cells. Thus, this experiment demonstrates the functional use of the therapeutic protein SG-11 for the treatment of gastrointestinal inflammatory diseases or diseases involving impaired epithelial barrier integrity / function, where increasing epithelial cell healing would be beneficial.

[0349] A 96-well Oris cell migration assay with a plug in the center of each well to prevent cell attachment was used according to the manufacturer's instructions (Platypus Technologies, Madison, WI).

[0350] Migration assay plates were warmed to room temperature before use and plugs were removed from 100% confluent wells before adding cells. HCT8 intestinal epithelial cells and HT29-MTX goblet cell lines were used in a 9:1 ratio, with a total of 5 × 10 cells added per well. 4 cells (4.5×10 4 HCT8 cells and 0.5×10 4HT29-MTX cells). The cells were incubated at 37°C + 5% CO2 for 24 hours. The plugs were then removed from all control and sample wells. The control wells included cells treated with diluted vehicle as a blank, 30 ng / ml epidermal growth factor (EGF) as a positive control, and 100 nM staurosporine as a negative control, all diluted in cRPMI. The sample wells contained SG-11 protein (SEQ ID NO: 5 and / or SEQ ID NO: 9) at a concentration of 1 μg / ml diluted in cRPMI. 100% and 0% wells were cultured in cRPMI. The treatments were added to the cells and incubated at 37°C + 5% CO2 for 48 hours. The plugs were removed from the 0% wells before staining the live cells. The treatment medium was removed and the cells were washed in PBS containing 0.9 mM CaCl2 and 0.5 mM MgCl2. Calcenin AM, a green fluorescent active dye, was added to all wells containing PBS with 0.9 mM CaCl2 and 0.5 mM MgCl2 at a concentration of 0.5 μg / ml, incubated at 37°C + 5% CO2 for 30 minutes, the dye was removed and washed in PBS containing 0.9 mM CaCl2 and 0.5 mM MgCl2, and the fluorescence was measured. The relative fluorescence value of 100% wells in which the plug was removed before cell plating was set to the maximum effect, and the relative fluorescence value of 0% wells in which the plug was retained until just before staining was used as the baseline. The samples were standardized between the 100% and 0% samples, and the values ​​were expressed as percentage growth.

[0351] like Figure 5 As shown, a significant increase in growth was observed after treatment with SG-11. The control compound modulated wound healing as expected, EGF increased proliferation, and staurosporine inhibited cell proliferation. Figure 5 The graph in represents data collected from 5 experiments (n=15). The data are representative of 5 independent replicates, where SG-11 SEQ ID NO: 5 was used in 1 experiment and SEQ ID NO: 9 was used in 4 experiments.

[0352] Example 7

[0353] SG-11 demonstrates therapeutic activity in a concurrent DSS model of inflammatory bowel disease

[0354] Examples 7 and 8 demonstrate the ability of the proteins disclosed herein to treat inflammatory bowel disease in an in vivo model. Therefore, this experiment demonstrates that the above-mentioned in vitro model (which describes important functions and possible mechanisms of action) will be translated into an in vivo model system of inflammatory bowel disease. Specifically, the mice in Examples 7 and 8 were treated with dextran sulfate sodium (DSS), a chemical known to induce intestinal epithelial damage and thereby reduce intestinal barrier integrity and function. DSS mice are a recognized model of colitis. In Example 7, mice were treated with SG-11 protein at approximately the same time as DSS administration (6 hours before). In Example 8, mice were treated with DSS for 6 days prior to treatment with SG-11 protein.

[0355] The graphs presented in Example 7 represent data from 3 independent experiments, each using 10 mice (n=30). The SG-11 protein used in these experiments was the mature protein (without signal peptide) without an N-terminal tag and comprising the amino acid sequence of SEQ ID NO: 3. For two experiments, the SG-11 protein consisted of SEQ ID NO: 5; for the third experiment, the SG-11 protein consisted of SEQ ID NO: 7.

[0356] Eight-week-old C57BL / 6 mice were housed, 5 animals were caged, and free access to food and water was provided for 7 days. After a 7-day adaptation period, treatment was initiated while 2.5% DSS was added to the drinking water. Preliminary tracking studies with fluorescently labeled bovine serum albumin following intraperitoneal (ip) injection of the protein showed that the protein reached the colon 6 hours after intraperitoneal delivery. Based on these results, mice were treated intraperitoneally with 50nmol / kg SG-11 (1.3mg / kg) or Gly2-GLP2 (0.2mg / kg) 6 hours before the addition of 2.5% DSS to the drinking water. Six hours after the initial treatment, the drinking water was changed to water containing 2.5% DSS. Mice were treated with 2.5% sodium dextran sulfate (DSS) in their drinking water for 6 days. Treatment was continued with intraperitoneal injections of SG-11 or Gly2-GLP2 at a dose of 50nmol / kg twice a day (bid) in the morning and evening (every 8 and 16 hours). Prepare fresh 2.5% DSS drinking water every 2 days.

[0357] On the sixth day, the mice were fasted for four hours and then orally fed 600 mg / kg of fluorescein isothiocyanate (FITC) labeled 4KDa dextran [4KDa-FITC]. One hour after the 4KDa-FITC-fed mice were sacrificed, blood was collected and the FITC signal was measured in the serum. A significant increase in the translocation of 4KDa-FITC dextran across the epithelial barrier was observed in untreated mice compared to DSS mice treated with vehicle. In addition, a significant decrease in 4KDa-FITC dextran was observed in mice that received DSS and treated with SG-11 compared to DSS mice treated with vehicle. The magnitude of 4KDa-FITC dextran translocation observed with SG-11 was similar to that of the positive control of Gly2-GLP2. The results are shown in Figure 6 , and are expressed as mean ± SEM. Figure 6 The graphs in represent data collected from 3 independent experiments (n=30).

[0358] SG-11 improves an inflammation-centric readout of barrier function in a concurrent DSS model of inflammatory bowel disease

[0359] The effect of SG-11 on the levels of LPS binding protein (LBP) in the blood of DSS animals administered with and without SG-11 was also evaluated. LPS binding protein (LBP), which is associated with clinical disease activity in inflammatory bowel disease subjects, was also measured by ELISA in the serum of mice tested in the DSS model described in Example 7. A significant increase in LBP concentrations in response to DSS was observed. In addition, a significant decrease in LBP was observed in SG-11 treated mice administered DSS compared to DSS mice treated with vehicle. In addition, SG-11 had a greater effect on LBP concentrations compared to the control peptide Gly2-GLP2, as significant differences were observed between DSS mice treated with Gly2-GLP2 and DSS mice treated with SG-11. The results are shown in Figure 7 , and are expressed as mean ± SEM. Figure 7 The graphs in represent data collected from 3 independent experiments (n=30).

[0360] SG-11 demonstrated to prevent weight loss in a concurrent DSS model of inflammatory bowel disease

[0361] The proteins disclosed herein are also evaluated for their therapeutic ability to improve weight loss in animals with inflammatory bowel disease. Weight loss is an important and potentially dangerous side effect of inflammatory bowel disease.

[0362] Body weight was measured daily from mice included in the DSS model described in this example. The percent change from day 0 weight was determined for each mouse. SG-11 administration resulted in significant improvements in body weight in DSS-treated mice compared to vehicle-treated DSS mice. The weight loss in mice treated with SG-11 on day 6 was similar to that observed with Gly2-GLP2. The results are shown in Figure 8 . Figure 8 The graphs in represent data collected from 2 independent experiments (n=20).

[0363] SG-11 significantly reduces overall pathology in the DSS model of inflammatory bowel disease

[0364] Gross pathology observations were made in the mice included in the concurrent DSS model performed in this example. Gross pathology in mice treated with SG-11 was significantly improved compared to vehicle-treated DSS mice. No differences in clinical scores were observed between mice given DSS and treated with Gly2-GLP2 or SG-11. The scoring system used was: (0) = no gross pathology, (1) = blood streaks visible in stool, (2) = completely bloody stool pellets, (3) bloody stool visible in the cecum, (4) bloody stool in the cecum and loose stools, (5) = rectal bleeding. The results are shown in Fig. 9 . Fig. 9 The graph in represents data collected from 3 independent experiments (n=30). These data indicate that SG-11 has a therapeutic effect in improving symptoms of IBD such as blood in stool.

[0365] In addition, histopathological analysis of proximal and distal colon tissues from DSS model animals was performed. Fig. 10A ) and remote ( Fig. 10B ) colon score (range 0-4) and the total colon score ( Fig. 10C ), which represents the sum of the proximal and distal colon scores (scored on a scale of 0-8). LMA = loss of mucosal architecture, Edema = edema, INF = inflammation, TMI = transmural inflammation, MH = mucosal hyperplasia, DYS = dysplasia. Figures represent data from two independent experiments and are plotted as mean ± SEM. Statistical analysis was performed by one-way ANOVA compared to DSS + vehicle followed by Fisher's LSD multiple comparison test.

[0366] SG-11 minimizes colon shortening in response to DSS treatment

[0367] The following experiments demonstrate the therapeutic ability of the proteins disclosed herein to treat inflammatory bowel disease in an in vivo model by showing the ability to prevent or minimize colon shortening.

[0368] Colon length was measured in mice included in the DSS model described in Example 7. SG-11 administration to DSS-treated mice prevented the shortening of the colon caused by DSS. Significant improvement in colon length was observed with Gly2-GLP2, and Gly2-GLP2 treatment was significantly better than SG-11 treatment. The results are shown in Fig.11AIn addition, treatment of DSS-exposed mice with Gly-2-GLP2 or SG-11 resulted in a significant improvement in the ratio of colon weight to length ( Fig. 11B ). Fig.11A and 11B The graphs in represent data pooled from 3 independent experiments (n=30). Data are plotted as mean±SEM and pooled from three independent experiments (n=30). Statistical analysis was performed by one-way ANOVA followed by Fisher's LSD multiple comparison test.

[0369] Example 8

[0370] SG-11 demonstrates therapeutic activity in the DSS model of inflammatory disease

[0371] In this example, experiments were performed to investigate the effects of SG-11 in a DSS mouse model when the SG-11 protein was administered to mice 7 days after DSS treatment. This is different from the treatment protocol of Example 7 above, in which the SG-11 protein was administered to mice shortly before treatment with DSS. This example further demonstrates the therapeutic ability of the proteins disclosed herein to treat inflammatory bowel disease in an in vivo model, thus demonstrating that the above in vitro model will translate into an in vivo model system for inflammatory bowel disease, which describes important functions and possible mechanisms of action.

[0372] Eight-week-old male C57BL / 6 mice were housed, 5 animals were caged, and free access to food and water was provided for 7 days. After acclimation for 7 days, mice were provided with drinking water containing 2.5% DSS for 7 days. During the 7-day DSS administration period, fresh 2.5% DSS water was prepared every 2 days. For this therapeutic DSS study, SG-11 used to treat animals was fused to a FLAG tag (DYKDDDDK; SEQ ID NO: 31) at its N-terminus.

[0373] On day 7, normal drinking water was restored and treatment with 50 nmol / kg SG-11 (1.3 mg / kg) or Gly2-GLP2 (0.2 mg / kg) was started. Treatment was given twice a day (bid), once in the morning and evening (once every 8 and 16 hours), for 6 days.

[0374] As described below, treatment outcomes were analyzed with respect to animal health, including body weight and gross pathology, histopathology of colonic tissue, assessment of barrier disruption, and levels of LPS-binding proteins.

[0375] Body weight was measured every day during the morning treatment period. Colon tissue was then harvested, and the length was measured in centimeters, and the tissue was weighed. The colon tissue was gently moved by a pair of tweezers to flush feces from the colon and remove residual PBS. The colon tissue was then weighed, and the ratio of colon weight to length in mg / mm was determined. After body weight measurement, proximal and distal colon tissues were stored for RNA and protein analysis, and the remaining tissues were fixed in 10% neutral buffered formalin for histopathological examination. Statistical analysis was performed by one-way ANOVA compared with DSS+ vehicle on serum 4KDa-FITC transport, serum LBP concentration, colon length, and colon weight to length ratio, and two-way ANOVA was performed to analyze body weight. In all analyses, Fisher's LSD multiple comparison test was used. The figure represents data from two experiments and is plotted as mean ± SEM.

[0376] This treatment model measures recovery from an established DSS insult. Because untreated mice also recovered after DSS was removed from their drinking water, as evidenced by the lack of an increase in 4KDa-FITC signal observed after 6 days of DSS treatment ( Fig.12 In addition, no decrease in LBP was observed after Gly2-GLP2 or SG-11 treatment ( Fig.13 ). Therefore, no changes in barrier function readouts were observed in the DSS-treated model.

[0377] Although no changes in barrier function readouts were observed in the therapeutic DSS model, significant improvements in clinical parameters such as body weight ( Fig.14 ), colon length ( Fig.15A ) and the ratio of colon weight to length ( Fig. 15B ). Similar to the barrier readings, the gross pathology scoring system based on bloody stools was no longer applicable, as even DSS mice recovered after 6 days of treatment. However, despite the absence of visible blood in the colon, colon thickening was still observed. As can be seen from the gross pathology observations, a decrease in the frequency of colon thickening was observed with SG-11 treatment (88% in DSS+vehicle, 25% in DSS+SG-11, Fisher's exact test p<0.0001, data not shown).

[0378] Histopathological analysis of proximal and distal colon tissues from the therapeutic DSS model described above was performed. Proximal ( Fig.16A ) and remote ( Fig. 16B ) colon score (range 0-4) and the total colon score, which represents the sum of the proximal and distal colon scores (range 0-8) ( Fig. 16C). LMA = loss of mucosal architecture, Edema = edema, INF = inflammation, TMI = transmural inflammation, MH = mucosal hyperplasia, DYS = dysplasia. Graphs represent data from two independent experiments and are plotted as mean ± SEM. Statistical analysis was performed by one-way ANOVA compared to DSS + vehicle followed by Fisher's LSD multiple comparison test.

[0379] SG-11 and Gly2-GLP2 treatment resulted in a modest but significant reduction in the loss of mucosal structure scores, while inflammation and transmural inflammation scores were unchanged. Similar to the results presented in Example 7, similar patterns of histopathological changes were observed with SG-11 and Gly2-GLP2, providing additional evidence that SG-11 can target epithelial cells.

[0380] Example 9

[0381] Design of stable and therapeutically active SG-11 variants

[0382] SG-11 is a therapeutic protein derived from the commensal bacterium Roseburia hominis. Administration of Roseburia hominis as a probiotic in the DSS model demonstrated efficacy in improving intestinal barrier function (4KDa-FITC and LBP), body weight, and clinical scores (data not shown).

[0383] Post-translational modifications (PTMs) may affect the recombinant production of therapeutic proteins, and post-translational modifications (PTMs) may occur during large-scale expression and purification and long-term storage. Such PTMs include, but are not limited to, oxidation of methionine, deamidation of asparagine, and intermolecular and / or intramolecular disulfide bonds between two cysteines. To reduce the risk of PTMs, SG-11 was modified by replacing residues that may affect protein stability. Examples 9-14 describe studies describing improved protein stability and maintenance of in vitro and in vivo activity.

[0384] In the first step, the SG-11 amino acid sequence (SEQ ID NO: 7) was compared with similar prokaryotic proteins. Residues identified based on the search results can be used for amino acid substitutions to enhance the stability of the therapeutic protein.

[0385] First, a blast search was performed against the GenBank non-redundant protein database (NCBI BLAST / default parameters / BLOSUM62 matrix) to identify other prokaryotic proteins that may be homologous to SG-11. The identified protein sequences are shown in Fig.17SEQ ID NO: 21 is a hypothetical protein from Roseburia intestinalis (GenBank: WP_006857001.1; BLAST E-value: 3e-90); SEQ ID NO: 22 is a hypothetical protein from Roseburia sp. 831b (GenBank: WP_075679733.1; BLAST E-value: 4e-58); SEQ ID NO: 23 is a hypothetical protein from Roseburia inulinivorans (GenBank: WP_055301040.1; BLAST E-value: 1e-83).

[0386] Each of SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23 is the predicted mature form of the indicated protein (lacking the signal peptide) and contains an N-terminal methionine. A multiple sequence alignment of these sequences with SG-11 (SEQ ID NO:7) was performed to identify regions conserved between the proteins. Fig.17 . This alignment is used to identify the most conserved residues in different proteins in order to assess the potential impact of replacing specific amino acids. Portions of SG-11 are conserved or highly conserved to some extent, where the amino acid at a specific position in the protein is the same in all 4 aligned proteins or in at least 2 (positions) or 3 (positions) of the 4 proteins. The high sequence conservation among these homologs of SG-11 suggests that SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23 may also have important functions in maintaining a healthy epithelial barrier. Therefore, the present invention provides methods for treating the diseases of the present invention using SEQ ID NOs: 21-23 and methods for preparing the same.

[0387] Example 10

[0388] Post-translational modification (PTM) analysis of SG-11

[0389] Studies were conducted using LC / MS / MS to identify SG-11 residues that are particularly susceptible to PTMs. Analysis was performed by LakePharma (Belmont, CA) to: 1) confirm the amino acid sequence of SG11 (SEQ ID NO: 9), and 2) determine any post-translational modifications, particularly deamidation and oxidation, that may result in reduced biological activity and immunogenicity.

[0390] For peptide mapping and PTM analysis, samples were treated with DTT and IAA followed by trypsin digestion. The digested samples were then analyzed by Waters ACQUITY UPLC coupled to a Xevo G2-XS QTOF mass spectrometer using a protein BEH C18 column.

[0391] Peptide mapping and sequencing confirmed the predicted amino acid sequence and also indicated multiple deamidation sites and one oxidation site. Among them, 7.84% of N53 and 3.77% of N83 were deamidated. These results given in Table 6 indicate that N53 and N83 are the main sites of deamidation under non-stress conditions. N53 represents the asparagine (Asn; N) located at position 53 in mature SG-11, with methionine at position 1 (SEQ ID NO: 7).

[0392] Table 6: Post-translational modifications of SG-11

[0393]

[0394] 1 Amino acid position in SG-11 (SEQ ID NO: 7)

[0395] 2 Normalized to total peptide ion intensity

[0396] 3 Normalized to the total intensity of the corresponding precursor with or without modification

[0397] Embodiment 11

[0398] Forced Degradation of SG-11

[0399] SG-11 (SEQ ID NO: 9) was also tested under a range of stress conditions as shown in Table 7 below to further characterize the stability of recombinant purified SG-11. The stressed samples were analyzed by SEC-HPLC for the presence of aggregates and / or degradants. LC / MS / MS was performed to determine the level of deamidation and oxidation.

[0400] Table 7

[0401]

[0402]

[0403] For this analysis, SG-11 (SEQ ID NO: 9) was prepared at a concentration of 1 mg / ml in PBS + 10% glycerol (50 mM sodium phosphate, 150 mM NaCl, 10% glycerol, pH 8.0), except for testing at pH 4 and pH 9. For pH 4, SG-11 (SEQ ID NO: 9) was prepared at a concentration of 1 mg / ml in sodium acetate buffer (50 mM sodium acetate, 150 mM NaCl, pH 4). For pH 9, SG-11 (SEQ ID NO: 9) was prepared at a concentration of 1 mg / ml in CAPSO (3-cyclohexylamino-2-hydroxy-1-propanesulfonic acid) buffer (50 mM CAPSO, 150 mM NaCl, 10 mg / ml).

[0404] The analysis showed that the SG-11 (SEQ ID NO: 9) samples treated at 4°C had low levels of aggregates. Aggregation increased with increasing temperature. At 37°C, most aggregation occurred. In contrast, mechanical stress and repeated freeze-thaw cycles did not result in protein aggregation or degradation.

[0405] Three samples were treated by incubation at 40°C for two weeks, oxidation (H2O2 treatment) or high pH 9, and PTMs were analyzed by LC / MS / MS as described in Example 10. As shown in Table 8 below, N83 underwent significant deamidation after almost 100% deamidation at 40°C sample treatment. Significant deamidation of N83 (37%) and oxidation of M200 (63.9%) were observed in the hydrogen peroxide treated samples. 7.84% of N53 was deamidated without any treatment.

[0406] Table 8

[0407]

[0408]

[0409] 1 Amino acid position in SG-11 (SEQ ID NO: 7)

[0410] After reduction, free cysteines were artificially carbamoylmethylated with iodoacetamide to prevent cysteine ​​residues from being oxidized in the assay.

[0411] Example 12

[0412] Stability and cysteine ​​residues of SG-11

[0413] The stability of SG-11 (SEQ ID NO: 9) was evaluated after incubation in buffer C (100 mM sodium phosphate, pH 7.0, 0.5 M sorbitol) for 1 week at 37°C and 3 weeks at 4°C. The stability was evaluated by monitoring aggregate formation by analytical size exclusion chromatography (SEC) equilibrated with buffer D (100 mM sodium phosphate, pH 7.0, 10% glycerol). No significant changes were observed after storage at 4°C for 3 weeks compared to the freshly thawed protein, as both samples showed a single peak at 1.57 mL. However, after incubation at 37°C for 1 week, in addition to the monomer peak (the smallest peak) at 1.57 mL, the samples also clearly showed aggregation peaks at 1.29 and 1.41 mL. The cause of the aggregation was investigated as follows. There are two cysteine ​​residues at positions 147 and 151 (relative to SEQ ID NO: 7) in SG-11. Ellman's reagent assay revealed the presence of free thiol groups in SG-11 (SEQ ID NO: 9), indicating that Cys 147 and / or Cys 151 No stable disulfide bonds were formed. Since free sulfhydryl groups may cause aggregation by forming undesirable intermolecular disulfide bonds, it was investigated whether the presence of a reducing agent such as β-mercaptoethanol could prevent aggregation. After incubation at 37°C for 4 days, aggregation was greatly inhibited in the presence of 2.5% (v / v) β-mercaptoethanol in the buffer (50 mM sodium phosphate, 150 mM NaCl, and 10% glycerol), in contrast to the aggregates formed in the absence of β-mercaptoethanol. The results showed that Cys 147 and / or Cys 151 Provides free thiol groups which lead to aggregation.

[0414] Embodiment 13

[0415] Post-translational modifications of SG-11 variants

[0416] Although the SG-11 protein is stable at high temperatures, the formation of aggregates at 37°C within a week may be problematic during downstream processing. Deamidation of asparagine residues found by LC / MS / MS is also a risk factor. In order to improve the manufacturability of the protein comprising SEQ ID NO: 3 or its variants, the results of Examples 10-12 were taken into account in the design of SG-11 variants (e.g., SG-11V1 (SEQ ID NO: 11), SG-11V2 (SEQ ID NO: 13), SG-11V3 (SEQ ID NO: 15), SG-11V4 (SEQ ID NO: 17) and SG-11V5 (SEQ ID NO: 19)) to reduce the occurrence of deleterious PTMs.

[0417] Examples 13-16 describe experiments performed to characterize the effects of amino acid substitutions on the stability and function of SG-11 variant SG-11V5 (SEQ ID NO: 19, including N53S, N83S, C147V, C151S relative to SEQ ID NO: 7). SG-11V5 (expressed and purified as described in Example 1).

[0418] Based on the PTMs observed when SG-11 (SEQ ID NO: 9) was placed under stress conditions (Example 11), SG-11V5 (SEQ ID NO: 19) was subjected to LC-MS / MS analysis of post-translational modifications using the method described in Example 11 and compared with the PTMs of SG-11 (SEQ ID NO: 7).

[0419] For this analysis, the PTMs of wild-type SG-11 (SEQ ID NO: 7) and SG-11V5 (SEQ ID NO: 19) were compared. In the first analysis (results are provided in Table 9 below), the proteins were stored at a concentration of 1 mg / ml in Buffer 1 (50 mM NaPO4 - , pH 8, 150 mM NaCl, 10% glycerol) and stored at 4°C or 40°C for 2 weeks. The proteins were then treated with DTT and IAA, followed by trypsin digestion. The digested samples were then analyzed by Waters ACQUITY UPLC coupled to a Xevo G2-XS QTOF mass spectrometer using a protein BEH C18 column. Analysis of the proteins by LC-MS / MS showed that the percentages of both initial methionine oxidation and N137 deamidation of the SG11V5 protein were significantly reduced compared to SG-11 at 4°C and 40°C.

[0420] Table 9

[0421]

[0422]

[0423] In the second analysis, SG-11 (SEQ ID NO: 7) and SG-11V5 (SEQ ID NO: 19) proteins were stored in different buffers at 40°C. The results are listed in Table 10 below. The storage buffer used in this experiment was 100 mM NaPO4 -, pH 7, with 10% sorbitol (+Sor) or without 10% sorbitol (-Sor) and with 10% glycerol (+Gly) or without 10% glycerol (-Gly), as shown in Table 10. As shown in the data in Table 10, the SG-11V5 (SEQ ID NO: 19) protein has greatly reduced oxidation of the first methionine compared to the SG-11 (SEQ ID NO: 7) protein under all buffer conditions. There is also a difference in the level of N137 deamidation of the two proteins in the presence of at least glycerol and in the presence of sorbitol and glycerol, resulting in greatly reduced N137 deamidation. These data indicate that amino acid substitutions in the SG-11 protein can have a significant beneficial effect on the PTMs of this protein in solution.

[0424] Table 10

[0425]

[0426]

[0427] Embodiment 14

[0428] SG-11 variant construction and stability analysis

[0429] Although the SG-11 protein is very stable at high temperatures, aggregation formation within a week at 37°C can be a problem during downstream processing. Deamidation of asparagine residues found by LC / MS / MS is also a risk factor. In order to improve the manufacturability of proteins comprising SEQ ID NO: 3 or its variants, the protein represented as SG-11 (SEQ ID NO: 7) was mutated to contain the following 4 substitutions: N53S, N83S, C147V and C151S. This variant with 4 substitutions is referred to as SG-11V5 and is provided herein as SEQ ID NO: 19. The stability of purified SG-11 and SG-11V5 was tested in different storage buffer formulations. SG-11V5 (SEQ ID NO: 19) has approximately 98.3% sequence identity with SEQ ID NO: 7.

[0430] Stability Analysis of SG-11

[0431] Figure 18 shows the effect of various conditions on the stability of SG-11 (SEQ ID NO: 7). Specifically, purified SG-11 (SEQ ID NO: 7) was incubated at pH 5.2 ( Fig.18A , 18B and 18C), pH 7.0 ( Fig.18D , 18E and 18F) and pH 8.0 ( Figure 18GThe effect of the additives was also tested at 3 different pH conditions: 150 mM NaCl ( Fig.18A , 18D and 18G); 150 mM NaCl and 100 mM arginine ( Fig.18B , 18E and 18H); and 150 mM NaCl and 0.5 M sorbitol ( Fig.18C , 18F and 18I). Stability was analyzed by analytical SEC. Arrows indicate retention times of monomeric forms.

[0432] Stability Analysis of SG-11V5

[0433] Figure 19 shows the effects of various conditions on the stability of SG-11V5 (SEQ ID NO: 19). SG-11V5 (SEQ ID NO: 19) was incubated at pH 5.2 ( Fig.19A , 19B and 19C), pH 7.0 ( Fig.19D , 19E and 19F) and pH 8.0 ( Figure 19G The effect of the additives was also tested at 3 different pH conditions: 150 mM NaCl ( Fig.19A , 19D and 19G); 150 mM NaCl and 100 mM arginine ( Fig.19B , 19E and 19H); and 150 mM NaCl and 0.5 M sorbitol ( Fig.19C , 19F and 19I). Stability was analyzed by analytical SEC. Arrows indicate retention times of monomeric forms.

[0434] Aggregate formation of purified SG-11 (SEQ ID NO: 7) protein was greatly inhibited in the presence of 100 mM arginine at pH 7.0. However, some small peaks were observed at earlier retention times, indicating that there were other forms besides the monomeric form. SG-11V5 (SEQ ID NO: 19) did not show significant aggregation under all conditions tested in this example. Discrete monomeric peaks were observed even in the absence of any additives. 100 mM arginine or 0.5 M sorbitol inhibited the small aggregation peak at 1.34 mL. Purified SG-11 (SEQ ID NO: 7) and SG-11V5 (SEQ ID NO: 19) were precipitated at pH 5.2.

[0435] High temperatures increase the sensitivity of proteins to degradation and aggregation due to deamidation. To minimize the potential burden associated with deamidation and aggregation, mutations N53S, N83S C147V, and C151S were introduced into SG-11. As a result, SG-11V5 showed improved stability at pH 7.0 and pH 8.0.

[0436] Embodiment 15

[0437] In vitro functional analysis of SG-11V5

[0438] In vitro TEER assays were performed to demonstrate that SG-11 variants, such as SG-11V5, retain functions associated with maintaining epithelial barrier function as demonstrated for the SG-11 protein (see, e.g., Example 2).

[0439] Cell culture was performed as described in Example 2. Briefly, after 8-10 days of culture, the Transwell plates containing enterocytes were treated with 10 ng / ml IFN-γ added to the basolateral chamber of the transwell plates for 24 hours at 37°C + 5% CO2. After 24 hours, fresh cRPMI was added to the epithelial cell culture plates. Transepithelial electrical resistance (TEER) readings were measured after IFN-γ treatment and used as pre-treatment TEER values. SG-11 (SEQ ID NO: 9) or SG-11V5 (SEQ ID NO: 19) was then added to the apical chamber of the Transwell plates at a final concentration of 1 μg / ml (40 nM). Myosin light chain kinase (MLCK) inhibitor peptide 18 (BioTechne, Minneapolis, MN) was used at 50 nM as a positive control for preventing barrier disruption caused by inflammation (Zolotarevskky et al., 202, Gastroenterology, 123: 163-172). Compounds were incubated on intestinal epithelial cells for 6 hours. After pre-incubation with the test compounds, the Transwell inserts containing the intestinal cells were transferred to the top of the receiving plate containing U937 monocytes. Heat-killed Escherichia coli (HK E. coli) (bacteria heated to 80°C for 40 minutes) were then added to the apical and basolateral chambers with a multiplicity of infection (MOI) of 10. The Transwell plates were incubated at 37°C + 5% CO2 for 24 hours, followed by post-treatment TEER measurements. SG-11 (SEQ ID NO: 9) increased the TEER disrupted by HK E. coli from 78.6% to 89.5% (p < 0.0001), while SG-11V5 (SEQ ID NO: 19) increased it to 89.1% (p < 0.0001) ( Fig. 20 ). Statistical analysis was performed using one-way ANOVA compared with HK E. coli followed by Fisher's LSD multiple comparison test. Fig. 20 The graphs in represent data collected from four plates in two separate experiments (n=12).

[0440] Example 16

[0441] In vivo functional analysis of SG-11V5

[0442] Next, the DSS animal model experiments performed as described in Examples 7 and 8 above were repeated to test SG-11 or SG-11V5 (SEQ ID NO: 19) in parallel. In these experiments, SG-11 or SG-11V5 was administered to mice at the same time as the start of treatment with DSS (as in Example 7) or after the previous DSS administration. The only difference was that the mice in Example 8 were treated with SG-11 or SG-11V5 (SEQ ID NO: 19) for 4 days instead of 6 days.

[0443] In the first DSS mouse model (Example 16A; same method as Example 7), mice were treated with test compounds intraperitoneally (ip) on day 0, and DSS treatment was initiated 6 hours later. Doses included 50 mol / kg of SG-11 (SEQ ID NO: 9) (1.3 mg / m1) and Gly2-GLP2 (0.2 mg / kg), and dose responses of SG-11V5 (SEQ ID NO: 19), including 16 nmol / kg (0.4 mg / m1), 50 nmol / kg (1.3 mg / m1), and 158 nmol / kg (4.0 mg / kg). Mice were treated with 2.5% DSS in their drinking water for 6 days (from day 0 to day 6). During DSS exposure, therapeutic protein treatments were given twice a day.

[0444] In the second experiment (Example 16B; same method as Example 8), mice were provided with drinking water containing 2.5% DSS for 7 days. On the 7th day, normal drinking water was restored and intraperitoneal treatment with 50 nmol / kg SG-11 (SEQ ID NO: 9) (1.3 mg / kg), SG-11V5 (SEQ ID NO: 19) (1.3 mg / kg) or Gly2-GLP2 (0.2 mg / kg) was started. Treatment was performed twice a day (bid), once in the morning and evening (once every 8 and 16 hours) for 4 days. For both DSS models (Examples 16A and 16B), fresh 2.5% DSS water was prepared every 2 days during DSS administration.

[0445] At the end of each DSS experiment, mice were fasted for 4 hours and then orally administered 600 mg / kg of 4KDa dextran labeled with fluorescein isothiocyanate (FITC) [4KDa-FITC]. One hour after the 4KDa-FITC-fed mice were sacrificed, blood was collected and the FITC signal was measured in serum. For the first model, a significant increase in the translocation of 4KDa-FITC dextran across the epithelial barrier was observed in DSS mice treated with vehicle compared to untreated mice. The results are shown in Fig.21A Middle: SG-11 (SEQ ID NO: 9) significantly reduced the 4KDa-FITC signal (p = 0.04). SG-11V5 (SEQ ID NO: 19) also reduced the 4KDa-FITC signal, although the difference did not reach statistical significance (p = 0.21). Fig.21B In the Figures 1 and 2, similar to the previous results in Example 8, no increase in 4KDa-FITC was observed, and thus no effect of SG-11 or SG-11V5 treatment was observed. The data in both figures are plotted as mean ± SEM, and each figure represents data from a single experiment (n = 10 per group).

[0446] Effects of SG-11V5 on an inflammation-centric readout of barrier function in the DSS model of inflammatory bowel disease

[0447] After completion of the above DSS models, LBP levels were measured as an inflammation-centric readout of barrier function following the protocol detailed in Example 7. After completion of both DSS models (Examples 16A and 16B), blood was collected and serum was separated. LPS binding protein (LBP) levels were measured in serum using a commercially available ELISA kit (Enzo Life Sciences, Farmingdale, NY). Results are presented in Table 1. Fig.22A (Example 16A) and Fig. 22B (Example 16B). In response to DSS exposure, a significant increase in LBP was observed in the Example 16A DSS model. Similar reductions in LBP were observed with SG-11 (SEQ ID NO: 9) and SG-11V5 (SEQ ID NO: 19) at a dose of 50 nmol / kg, although neither was statistically significant. However, treatment with SG-11V5 (SEQ ID NO: 19) at a higher dose of 158 nmol / kg resulted in a significant reduction in LBP production (p=0.003) ( Fig.22A In Example 16B DSS model, exposure to DSS resulted in a significant increase in LBP production ( Fig. 22B). However, no reduction in LBP was observed with any treatment, and similar effects were observed for both SG-11 (SEQ ID NO: 9) and SG-11V5 (SEQ ID NO: 19). Without being bound by theory, it is believed that the long half-life of LBP in the circulation (reported to be 12-24 hours) may make it difficult to observe a reduction in systemic LBP levels in models where an LBP response (administration of DSS) is elicited prior to the start of treatment (Behrendt, D., J. Dembinski, A. Heep, and P. Bartmann. 2004. Lipopolysaccharide binding protein in preterm infants. Arch Dis Child Fetal Neonatal Ed 89: F551-554).

[0448] Effects of SG-11 and SG-11V5 on body weight in the DSS model of inflammatory bowel disease

[0449] Body weight was measured throughout the experimental models in Example 16A and Example 16B. In Example 16A DSS model ( Fig.23A ), similar changes in body weight were observed for 50 nmol / kg of SG-11 (SEQ ID NO: 9) and SG-11V5 (SEQ ID NO: 19) treatment, and a significant improvement in body weight was observed on day 6 for 158 nmol / kg of SG-11V5 (SEQ ID NO: 19). A similar pattern was observed in the therapeutic DSS model, with similar changes in body weight for 50 nmol / kg doses of SG-11 (SEQ ID NO: 9) and SG-11V5 (SEQ ID NO: 19), and both had statistically improved changes in body weight on day 11 (p < 0.05). Fig.23A and Fig. 23B , Data are plotted as mean ± SEM, and each graph represents data from a single experiment. Statistical analysis was performed using two-way ANOVA compared to the DSS+vehicle group with Fisher's LSD multiple comparison test.

[0450] Effects of SG-11 and SG-11V5 on overall pathology in the DSS model of inflammatory bowel disease

[0451] As described above in Example 7, gross pathological observations of colonic tissue were performed for Example 16A. Briefly, a scoring system based on visible blood levels and stool pellet consistency was used. The scoring system used was: (0) = no gross pathology, (1) = visible blood in stool, (2) = completely bloody stool pellets, (3) visible bloody stool in cecum, (4) bloody stool in cecum and loose stools, (5) = rectal bleeding. Similar results were obtained for SG-11 (SEQ ID NO: 9) and SG-11V5 (SEQ ID NO: 19) at a dose of 50 nmol / kg, and a dose-dependent effect was observed for SG-11V5 (SEQ ID NO: 19), with a significant improvement at a dose of 160 nmol / kg (p < 0.002). Fig.24 Data shown are expressed as mean ± SEM and include data from a single experiment. Statistical analysis was performed by one-way ANOVA followed by Fisher's LSD multiple comparison test.

[0452] Effects of SG-11 and SG-11V5 on colon length in the DSS model of inflammatory bowel disease

[0453] The effects of SG-11 and SG-11 variant proteins on colon length from the DSS model of Example 16 were also analyzed. Fig.25A ) or Example 16B ( Fig.25B )DSS model for colon length measurement. Similar results were obtained with SG-11 (SEQ ID NO: 9) and SG-11V5 (SEQ ID NO: 19) in both DSS models, with both treatment regimens resulting in a significant increase in colon length. However, in the DSS model of Example 16A, no dose-dependent effect of SG-11V5 (SEQ ID NO: 19) on colon length was observed. Data in both figures are presented as mean ± SEM and represent data from a single experiment. Statistical analysis was performed using a one-way ANOVA compared to DSS + vehicle followed by Fisher's LSD multiple comparison test.

[0454] Effects of SG-11 and SG-11V5 on the ratio of colon weight to length in the DSS model of inflammatory bowel disease

[0455] The effects of SG-11 and SG-11 variant proteins on the ratio of colon weight to length from the DSS model of Example 16 were also analyzed. Fig.26A ) and Example 16B ( Fig.26B)DSS model treatment regimen, the colon weight to length ratio was similar between SG-11 (SEQ ID NO: 9) and SG-11V5 (SEQ ID NO: 19). In the treatment of Example 16A, all treatments and doses significantly improved the colon weight to length ratio (p < 0.05). In the treatment regimen of Example 16B, both SG-11 (SEQ ID NO: 9) and SG-11V5 (SEQ ID NO: 19) significantly improved the colon weight to length ratio (p < 0.01), while the positive control Gly2-GLP2 did not. Statistical analysis was performed using a one-way ANOVA compared to DSS + vehicle, followed by Fisher's LSD multiple comparison test. Data are plotted as mean ± SEM, and each graph represents data from one experiment.

[0456] Although the foregoing disclosure provides some details by way of illustration and example for the purpose of clear understanding, it will be appreciated by those skilled in the art that various changes and modifications may be implemented without departing from the spirit or scope of the present invention, the scope of the present disclosure described in the appended claims. Therefore, this description should not be interpreted as limiting the scope of the present disclosure.

[0457] Table 11 shows the SEQ ID NOs of the present disclosure with detailed information.

[0458] Table 11

[0459]

[0460]

[0461] Numbered embodiments of the present disclosure

[0462] Notwithstanding the appended claims, the present disclosure shows the following numbered embodiments:

[0463] Treatment

[0464] 1. A method for treating gastrointestinal epithelial cell barrier dysfunction, comprising:

[0465] a. administering to a patient in need thereof a pharmaceutical composition comprising:

[0466] i. A therapeutic protein comprising an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and / or SEQ ID NO: 19; and

[0467] ii. a pharmaceutically acceptable carrier.

[0468] 2. The method of claim 1, wherein the gastrointestinal epithelial cell barrier dysfunction is a disease associated with reduced gastrointestinal wall integrity.

[0469] 3. The method according to any one of claims 1 to 2, wherein the gastrointestinal epithelial cell barrier dysfunction is a disease associated with reduced integrity of the gastrointestinal mucosal epithelium.

[0470] 4. The method according to any one of claims 1 to 3, wherein the gastrointestinal epithelial cell barrier dysfunction is a disease associated with reduced intestinal epithelial integrity.

[0471] 5. The method of any one of claims 1-4, wherein the gastrointestinal epithelial barrier dysfunction is selected from at least one of inflammatory bowel disease, Crohn's disease, ulcerative colitis, pouchitis, irritable bowel syndrome, intestinal infection, Clostridium difficile infection, metabolic disease, obesity, type 2 diabetes, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, liver disease, alcoholic steatohepatitis, celiac disease, necrotizing enterocolitis, gastrointestinal disease, short bowel syndrome, gastrointestinal mucositis, chemotherapy-induced mucositis, radiation-induced mucositis, oral mucositis, interstitial cystitis, neurological disease, cognitive impairment, Alzheimer's disease, Parkinson's disease, multiple sclerosis, autism, chemotherapy-associated steatohepatitis (CASH), and pediatric forms of the above diseases.

[0472] 6. The method according to any one of claims 1 to 5, wherein the gastrointestinal epithelial cell barrier dysfunction is inflammatory bowel disease.

[0473] 7. The method of any one of claims 1-6, wherein the gastrointestinal epithelial barrier dysfunction is Crohn's disease.

[0474] 8. The method according to any one of claims 1 to 6, wherein the gastrointestinal epithelial cell barrier dysfunction is ulcerative colitis.

[0475] 9. The method of any one of claims 1-8, wherein the therapeutic protein comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 and / or SEQ ID NO:19.

[0476] 10. The method of any one of claims 1-9, wherein the therapeutic protein comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 and / or SEQ ID NO:19.

[0477] 11. The method of any one of claims 1-10, wherein the therapeutic protein comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 and / or SEQ ID NO:19.

[0478] 12. The method of any one of claims 1-11, wherein the therapeutic protein comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 and / or SEQ ID NO:19.

[0479] 13. The method of any one of claims 1-12, wherein the therapeutic protein comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 and / or SEQ ID NO:19.

[0480] 14. The method of any one of claims 1-13, wherein the therapeutic protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19.

[0481] 15. The method of any one of claims 1-14, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO: 3.

[0482] 16. The method of any one of claims 1-14, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO: 7.

[0483] 17. The method of any one of claims 1-14, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO: 11.

[0484] 18. The method of any one of claims 1-14, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO: 13.

[0485] 19. The method of any one of claims 1-14, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO: 15.

[0486] 20. The method of any one of claims 1-14, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO: 17.

[0487] 21. The method of any one of claims 1-14, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO: 19.

[0488] 22. The method of any one of claims 1-21, wherein administration comprises rectal, parenteral, intravenous, topical, oral, cutaneous, transdermal or subcutaneous administration.

[0489] 23. The method of any one of claims 1-22, wherein the administration is to the oral cavity, gastrointestinal cavity and / or intestine of the patient.

[0490] 24. The method of any one of claims 1-23, wherein the patient experiences a reduction in at least one symptom associated with gastrointestinal epithelial barrier dysfunction.

[0491] 25. The method of any one of claims 1-24, wherein the patient experiences a reduction in at least one symptom associated with gastrointestinal epithelial barrier dysfunction selected from the group consisting of abdominal pain, blood in the stool, pus in the stool, fever, weight loss, frequent diarrhea, fatigue, decreased appetite, tenesmus, and rectal bleeding.

[0492] 26. The method of any one of claims 1-25, wherein administration reduces gastrointestinal inflammation in the patient.

[0493] 27. The method of any one of claims 1-25, wherein administration reduces intestinal mucosal inflammation in the patient.

[0494] 28. The method of any one of claims 1-25, wherein administration increases mucin production in the patient's intestinal tissue.

[0495] 29. The method of any one of claims 1-25, wherein administration increases intestinal epithelial wound healing in the patient.

[0496] 30. The method of any one of claims 1-25, wherein administration increases intestinal epithelial cell proliferation in the patient.

[0497] 31. The method of any one of claims 1-30, further comprising administering to the patient at least one second therapeutic agent.

[0498] 32. The method of any one of claims 1-31, further comprising administering to the patient at least one second therapeutic agent selected from the group consisting of an antidiarrheal agent, a 5-aminosalicylic acid compound, an anti-inflammatory agent, an antibiotic, an antibody, an anti-cytokine agent, an anti-inflammatory cytokine agent, a steroid, a corticosteroid, and an immunosuppressant.

[0499] Pharmaceutical composition

[0500] 1. A pharmaceutical composition comprising:

[0501] a. A therapeutic protein comprising an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and / or SEQ ID NO: 19; and

[0502] b. Pharmaceutically acceptable carrier.

[0503] 2. The pharmaceutical composition of claim 1, wherein the therapeutic protein comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 and / or SEQ ID NO:19.

[0504] 3. The pharmaceutical composition of any one of claims 1-2, wherein the therapeutic protein comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and / or SEQ ID NO: 19.

[0505] 4. The pharmaceutical composition of any one of claims 1-3, wherein the therapeutic protein comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and / or SEQ ID NO: 19.

[0506] 5. The pharmaceutical composition of any one of claims 1-4, wherein the therapeutic protein comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and / or SEQ ID NO: 19.

[0507] 6. The pharmaceutical composition of any one of claims 1-5, wherein the therapeutic protein comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and / or SEQ ID NO: 19.

[0508] 7. The pharmaceutical composition of any one of claims 1-6, wherein the therapeutic protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19.

[0509] 8. The pharmaceutical composition of any one of claims 1-7, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO: 3.

[0510] 9. The pharmaceutical composition of any one of claims 1-7, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO: 7.

[0511] 10. The pharmaceutical composition of any one of claims 1-7, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO: 11.

[0512] 11. The pharmaceutical composition of any one of claims 1-7, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO: 13.

[0513] 12. The pharmaceutical composition of any one of claims 1-7, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO: 15.

[0514] 13. The pharmaceutical composition of any one of claims 1-7, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO: 17.

[0515] 14. The pharmaceutical composition of any one of claims 1-7, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO: 19.

[0516] 15. The pharmaceutical composition of any one of claims 1-14, formulated for rectal, parenteral, intravenous, topical, oral, cutaneous, transdermal or subcutaneous administration.

[0517] 16. The pharmaceutical composition of any one of claims 1-15, formulated such that the therapeutic protein is active in the gastrointestinal lumen and / or intestine of a patient.

[0518] Expression vector

[0519] 1. An expression vector comprising: a polynucleotide encoding a protein comprising an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and / or SEQ ID NO: 19.

[0520] 2. The expression vector of claim 1, wherein the encoded protein comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 and / or SEQ ID NO:19.

[0521] 3. The expression vector of any one of claims 1-2, wherein the encoded protein comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 and / or SEQ ID NO:19.

[0522] 4. The expression vector of any one of claims 1-3, wherein the encoded protein comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 and / or SEQ ID NO:19.

[0523] 5. The expression vector of any one of claims 1-4, wherein the encoded protein comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 and / or SEQ ID NO:19.

[0524] 6. The expression vector of any one of claims 1-5, wherein the encoded protein comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 and / or SEQ ID NO:19.

[0525] 7. The expression vector of any one of claims 1 to 6, wherein the encoded protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19.

[0526] 8. The expression vector of any one of claims 1 to 7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO: 3.

[0527] 9. The expression vector of any one of claims 1 to 7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO: 5.

[0528] 10. The expression vector of any one of claims 1 to 7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO: 7.

[0529] 11. The expression vector of any one of claims 1 to 7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO: 9.

[0530] 12. The expression vector of any one of claims 1 to 7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO: 11.

[0531] 13. The expression vector of any one of claims 1 to 7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO: 13.

[0532] 14. The expression vector of any one of claims 1 to 7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO: 15.

[0533] 15. The expression vector of any one of claims 1 to 7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO: 17.

[0534] 16. The expression vector of any one of claims 1 to 7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO: 19.

[0535] Host cells

[0536] 1. A host cell comprising: an exogenous polynucleotide encoding a protein comprising an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and / or SEQ ID NO: 19.

[0537] 2. The host cell of claim 1, wherein the encoded protein comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 and / or SEQ ID NO:19.

[0538] 3. The host cell of any one of claims 1-2, wherein the encoded protein comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and / or SEQ ID NO: 19.

[0539] 4. The host cell of any one of claims 1-3, wherein the encoded protein comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and / or SEQ ID NO: 19.

[0540] 5. The host cell of any one of claims 1-4, wherein the encoded protein comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and / or SEQ ID NO: 19.

[0541] 6. The host cell of any one of claims 1-5, wherein the encoded protein comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and / or SEQ ID NO: 19.

[0542] 7. The host cell of any one of claims 1-6, wherein the encoded protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19.

[0543] 8. The host cell of any one of claims 1-7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO: 3.

[0544] 9. The host cell of any one of claims 1-7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO: 5.

[0545] 10. The host cell of any one of claims 1-7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO: 7.

[0546] 11. The host cell of any one of claims 1-7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO: 9.

[0547] 12. The host cell of any one of claims 1-7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO: 11.

[0548] 13. The host cell of any one of claims 1-7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO: 13.

[0549] 14. The host cell of any one of claims 1-7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO: 15.

[0550] 15. The host cell of any one of claims 1-7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO: 17.

[0551] 16. The host cell of any one of claims 1-7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO: 19.

[0552] 17. The host cell of any one of claims 1-16, wherein the exogenous polynucleotide further encodes a host cell-specific signal sequence.

[0553] 18. The host cell of any one of claims 1-17, wherein the exogenous polynucleotide comprises a nucleic acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:20.

[0554] 19. The host cell of any one of claims 1-18, wherein the host cell is a prokaryotic cell.

[0555] 20. The host cell of any one of claims 1-19, wherein the host cell is an E. coli cell.

[0556] 21. The host cell of any one of claims 1-18, wherein the host cell is a eukaryotic cell.

[0557] 22. The host cell of any one of claims 1-18 and 21, wherein the host cell is a Chinese hamster ovary cell.

[0558] 23. A method for producing a protein, comprising: culturing the host cell of any one of claims 1-22 under conditions sufficient to express the encoded protein.

[0559] Isolated protein

[0560] 1. An isolated therapeutic protein comprising: an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and / or SEQ ID NO:19.

[0561] 2. The isolated therapeutic protein of claim 1, comprising: an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and / or SEQ ID NO:19.

[0562] 3. The isolated therapeutic protein of any one of claims 1-2, comprising: an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and / or SEQ ID NO:19.

[0563] 4. The isolated therapeutic protein of any one of claims 1-3, comprising: an amino acid sequence having at least about 97% sequence identity to SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and / or SEQ ID NO:19.

[0564] 5. The isolated therapeutic protein of any one of claims 1-4, comprising: an amino acid sequence having at least about 98% sequence identity to SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and / or SEQ ID NO:19.

[0565] 6. The isolated therapeutic protein of any one of claims 1-5, comprising: an amino acid sequence having at least about 99% sequence identity to SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and / or SEQ ID NO:19.

[0566] 7. The isolated therapeutic protein of any one of claims 1-6, comprising: an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19.

[0567] 8. The isolated therapeutic protein of any one of claims 1-7, comprising: the amino acid sequence of SEQ ID NO: 3.

[0568] 9. The isolated therapeutic protein of any one of claims 1-7, comprising: the amino acid sequence of SEQ ID NO: 5.

[0569] 10. The isolated therapeutic protein of any one of claims 1-7, comprising: the amino acid sequence of SEQ ID NO: 7.

[0570] 11. The isolated therapeutic protein of any one of claims 1-7, comprising: the amino acid sequence of SEQ ID NO: 9.

[0571] 12. The isolated therapeutic protein of any one of claims 1-7, comprising: the amino acid sequence of SEQ ID NO: 11.

[0572] 13. The isolated therapeutic protein of any one of claims 1-7, comprising: the amino acid sequence of SEQ ID NO: 13.

[0573] 14. The isolated therapeutic protein of any one of claims 1-7, comprising: the amino acid sequence of SEQ ID NO: 15.

[0574] 15. The isolated therapeutic protein of any one of claims 1-7, comprising: the amino acid sequence of SEQ ID NO: 17.

[0575] 16. The isolated therapeutic protein of any one of claims 1-7, comprising: the amino acid sequence of SEQ ID NO: 19.

[0576] 17. The isolated therapeutic protein of any one of claims 1-16, wherein the protein increases electrical resistance in an in vitro transepithelial electrical resistance assay.

[0577] 18. The isolated therapeutic protein of any one of claims 1-17, wherein the protein increases electrical resistance in an in vitro transepithelial electrical resistance assay by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% compared to an assay performed in the absence of the protein.

[0578] 19. The isolated therapeutic protein of any one of claims 1-18, wherein the protein increases electrical resistance in an in vitro transepithelial electrical resistance assay compared to a kinase inhibitor control.

[0579] 20. The isolated therapeutic protein of any one of claims 1-19, wherein the protein increases electrical resistance in an in vitro transepithelial electrical resistance assay compared to a staurosporine or myosin light chain kinase control.

[0580] Synthetic therapeutic proteins

[0581] 1. A synthetic therapeutic protein comprising: an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 19.

[0582] 2. The protein of claim 1, comprising: an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 19.

[0583] 3. The protein of any one of claims 1-2, comprising: an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 19.

[0584] 4. The protein of any one of claims 1-3, comprising: an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 19.

[0585] 5. The protein of any one of claims 1-4, comprising: an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 19.

[0586] 6. The protein according to any one of claims 1 to 5, comprising the amino acid sequence of SEQ ID NO: 19.

[0587] 7. The protein of any one of claims 1 to 6, wherein the amino acid at position 147 is valine.

[0588] 8. The protein of any one of claims 1 to 6, wherein the amino acid at position 151 is serine.

[0589] 9. The protein of any one of claims 1-6, wherein the amino acid at position 147 is valine, and the amino acid at position 151 is serine.

[0590] 10. The protein of any one of claims 1 to 5, wherein the amino acid at position 84 is aspartic acid.

[0591] 11. The protein of any one of claims 1-5, wherein the amino acid at position 84 is aspartic acid, the amino acid at position 147 is valine, and the amino acid at position 151 is serine.

[0592] 12. The protein of any one of claims 1-6, wherein the amino acid at position 83 is serine.

[0593] 13. The protein of any one of claims 1-6, wherein the amino acid at position 83 is serine, the amino acid at position 147 is valine, and the amino acid at position 151 is serine.

[0594] 14. The protein of any one of claims 1-6, wherein the amino acid at position 53 is serine.

[0595] 15. The protein of any one of claims 1-5, wherein the amino acid at position 53 is serine, and the amino acid at position 84 is aspartic acid, and the amino acid at position 147 is valine, and the amino acid at position 151 is serine.

[0596] 16. The protein of any one of claims 1-6, wherein the amino acid at position 53 is serine, and the amino acid at position 83 is serine, and the amino acid at position 147 is valine, and the amino acid at position 151 is serine.

[0597] 17. The protein of any one of claims 1-6, wherein the amino acid at position 147 is not cysteine, the amino acid at position 151 is not cysteine, the amino acid at position 83 is not asparagine, and / or the amino acid at position 53 is not asparagine.

[0598] 18. The protein of any one of claims 1-17, wherein the protein increases electrical resistance in an in vitro transepithelial electrical resistance assay.

[0599] 19. A pharmaceutical composition comprising: the protein according to any one of claims 1 to 17 and a pharmaceutically acceptable carrier.

[0600] 20. A method for treating gastrointestinal epithelial cell barrier dysfunction, comprising:

[0601] a. administering to a patient in need thereof a pharmaceutical composition comprising:

[0602] i. A therapeutic protein comprising an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and / or SEQ ID NO: 19; and

[0603] ii. a pharmaceutically acceptable carrier.

[0604] Incorporation by Reference

[0605] All publications, patents, and patent applications cited herein are incorporated by reference in their entirety for all purposes.

[0606] However, the citation of any reference, article, publication, patent, patent publication and patent application herein is not and should not be taken as an admission or any form of suggestion that they form part of the valid prior art or common general knowledge in any country in the world.

[0607] References

[0608] Molodecky et al. 2012. Increasing incidence and prevalence of the inflammatory bowel diseases withtime, based on systematic review. Gastroenterology 142(1): 46-54.

[0609] Aroniadis et al., 2013. Fecal microbiota transplantation: past, present and future. Curr. Opin. Gastroenterol. 29(1): 79-84.

[0610] Maloy et al., 2011, Intestinal homeostasis and its breakdown in inflammatory bowel disease, Nature 474(7351): 298-306.

[0611] Needleman et al., 1970. A general method applicable to the search for similarities in the amino acid sequences of two proteins. J. Mol. Biol., 48(3): 444-453.

[0612] Myers et al., 1988, Optimal alignments in linear space, CABIOS 4: 11-17.

[0613] Altschul, et al., 1990, Basic local alignment search tool, J. Mol. Biol. 215: 403-410.

[0614] Altschul et al., 1997, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25(17): 3389-3402.

[0615] Henikoff et al., 1992, Amino acid substitution matrices from protein blocks, Proc. Natl. Acad. Sci. 89(22): 10915-10919.

[0616] Karlin et al., 1993, Applications and statistics for multiple high-scoring segments in molecular sequences, Proc. Natl. Acad. Sci. 90: 5873-5877.

[0617] Higgins et al., 1988, CLUSTAL: a package for performing multiple sequence alignment on a microcomputer, Gene 73: 237-244.

[0618] Person et al., 1988, Improved tools for biological sequence comparison, Proc. Natl. Acad. Sci. 85: 2444-2448.

[0619] Bowie et al., 1990, Deciphering the message in protein sequences: tolerance to amino acid substitutions, Science 247: 1306-1310.

[0620] IUPAC Commission on Organic Nomenclature (CNOC) and IUPAC-IUB Commission on Biochemical Nomenclature (CBN), 1975, Nomenclature of α-Amino Acids (Recommendations, 1974), Biochemistry 14: 449-462.

[0621] Remington's Pharmaceutical Sciences, 17th edition, Alfonso R. Gennaro (ed.), Mark Publishing Company, Easton, Pennsylvania, USA, 1985.

[0622] Encyclopaedia of Pharmaceutical Technology, 3rd edition, James Swarbrick (ed.), Informa Healthcare USA (Inc.), New York, USA, 2007.

[0623] Berge et al., 1977, Pharmaceutical salts, J. Pharm. Sci. 66: 1-19.

[0624] Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Stahl and Wermuth, Wiley-VCH, 2002.

[0625] In re Bergstrom, 427 F.2d 1394, (CCPA 1970)

[0626] In re Bergy, 596 F.2d 952 (CCPA 1979)

[0627] Parke-Davis & Co. v. H.K. Mulford & Co., 189 F.95 (SDNY 1911) affirmed in part, modified in part, 196 F.496 (2d Cir. 1912)

[0628] Merck & Co., Inc. v. Olin Mathieson Chemical Corporation, 253 F.2d 156 (4th Cir. 1958)

[0629] Horhota et al., 2006, Glycerolnucleoside triphosphates: Synthesis and polymerase substrate activities, Organic Letters 8: 5345-5347.

[0630] Botoman et al., 1998, Management of Inflammatory Bowel Disease, Am. Fam. Physician, 57(1): 57-68.

[0631] Neurath, 2014, Cytokines in Inflammatory Bowel Disease, Nature Reviews Immunology 14: 329-342.

[0632] Zhang et al., 2007, Cytokines, Inflammation and Pain, Int. Anesthesiol. Clin. 45(2): 27-37.

[0633] Strober et al., Proinflammatory cytokines in the pathogenesis of inflammatory bowel diseases, Gastroenterology 140(6): 1756-1767.

[0634] Boltin et al., 2013, Mucin Function in Inflammatory Bowel Disease: An Update, J. Clin. Gastroenterol. 47(2): 106-111.

[0635] Kim et al., 2012, Investigating Intestinal Inflammation in DSS-induced Model of IBD. Journal of Visualized Experiments, 60: 2-6.

[0636] Levesque et al., 2015. Converging goals of treatment of inflammatory bowel disease from clinical trials and practice. Gastroenterology 148: 37-51.

[0637] Best et al., 1976. Development of a Crohn's disease activity index. National Cooperative Crohn's Disease Study. Gastroenterol. 70: 439-444.

[0638] Johansson, et al., 2014. Bacteria penetrate the normally impenetrable inner colon mucus layer in both murine colitis models and patients with ulcerative colitis. Gut 63: 281-291.

[0639] Simmonds et al., 2014. Paneth cell metaplasia in newly diagnosed inflammatory bowel disease in children. BMC Gastroenterol. 14: 93.

[0640] Gassler et al., 2001. Inflammatory bowel disease is associated with changes of enterocytic junctions. Am. J. Physiol. Gastrointest. Liver Physiol. 281: G216-G228.

[0641] Dewi, et al., 2004. In vitro assessment of human endothelial cell permeability: effects of inflammatory cytokines and dengue virus infection. J. Virol. Methods. 121: 171-180.

[0642] Mandic, et al., 2004, Evaluation of head and neck squamous cell carcinoma invasiveness by the electrical resistance breakdown assay, Clin. Exp. Metast. 21: 699-704.

[0643] Dewi, et al., 2008. Peripheral blood mononuclear cells increase the permeability of dengue virus-infected endothelial cells in association with down-regulation of vascular endothelial cadherin. J. Gen. Virol. 89: 642-652.

[0644] Sands, 2004. From symptom to diagnosis: clinical distinctions among various forms of intestinal inflammation. Gastroenterology 126: 1518-1532.

[0645] Danese et al., 2006, Etiology of inflammatory bowel diseases, World J. Gastroenterol. 12: 4807-4812.

[0646] Duncan, SH, Aminov, RI, Scott, KP, Louis, P., Stanton, TB, Flint, HJ (2006). Proposal of Roseburia faecis sp. nov., Roseburia hominis sp. nov. and Roseburiainulinivorans sp. nov., based on isolates from human faeces. Int. J. Syst. Evol. Microbiol. Vol. 56, pp. 2437-2441.

[0647] International Human Genome Sequencing Consortium (2004) Finishing the euchromatic sequence of the human genome. Nature. 431, 931-45.

[0648] Jensen ON (2004) Modification-specific proteomics: Characterization of post-translational modifications by mass spectrometry. Curr Opin Chem Biol. 8, 33-41.

[0649] Ayoubi TA and Van De Ven WJ (1996) Regulation of gene expression by alternative promoters. FASEB J. 10, 453-60.

[0650] Walsh C. (2006) Posttranslational modification of proteins: Expanding nature's inventory. Englewood, Colorado: Roberts and Co. Publishers. xxi, p. 490.

[0651] Gaston BM et al. (2003) S-nitrosylation signaling in cell biology. Mol Interv. 3, 253-63.

[0652] Jaffrey SR and Snyder SH (2001) The biotin switch method for the detection of S-nitrosylated proteins. SciSTKE. 2001, p11.

[0653] Han P. and Chen C. (2008) Detergent-free biotin switch combined with liquid chromatography / tandem mass spectrometry in the analysis of S-nitrosylated proteins. Rapid Commun Mass Spectrom. 22, 1137-45.

[0654] Imai S. et al. (2000) Transcriptional silencing and longevity protein SIR2 is an NAD-dependent histone deacetylase. Nature. 403, 795-800.

[0655] Glozak MA et al. (2005) Acetylation and deacetylation of non-histone proteins. Gene. 363, 15-23.

[0656] Yang XJ and Seto E. (2008) Lysine acetylation: Codified crosstalk with other posttranslational modifications. Mol Cell. 31, 449-61.

[0657] Bratt, et al., "A phase 1 trial with transgenic bacteria expressing interleukin-10 in Crohn's disease," Clinical Gastroenterology and Hepatology, 2006, volume 4, pages 754-759.

[0658] Shigemori, et al., “Oral delivery of Lactococcus lactis that secretes bioactive heme oxygenase-1 alleviates development of acute colitis in mice.” Microbial Cell Factories, 2015, Vol. 14: 189.

[0659] Steidler, et al., "Treatment ofmurine colitis by Lactococcus lactis secreting interleukin-10," Science, 2000, Vol. 289, pp. 1352-1355.

[0660] Hanniffy, et al., "Mucosal delivery of a pneumococcal vaccine using Lactococcus lactis affords protection against respiratory infection," Journal of Infectious Diseases, 2007, Vol. 195, pp. 185-193.

[0661] Vandenbroucke, et al., "Active delivery of trefoil factors by genetically modified Lactococcus lactis prevents and heals acute colitis in mice," Gastroenterology, 2004, Vol. 127, pp. 502-513.

[0662] Sheth, et al., “Manipulating bacterial communities by in situ microbiome engineering,” Trends in Genetics, 2016, Vol. 32, No. 4, pp. 189-200.

[0663] Lerner et al., “Dysbiosis may trigger autoimmune diseases via inappropriate post-translational modification of host proteins.” Frontiers in Microbiology, 2016, Vol. 7, Article 84.

[0664] Machiels, K., M. Joossens, J. Sabino, V. De Preter, I. Arijs, V. Eeckhaut, V. Ballet, K. Claes, F. Van Immerseel, K. Verbeke, M. Ferrante, J. Verhaegen, P. Rutgeerts, and S. Vermeire. 2014. A decrease of the butyrate-producing species Roseburia hominis and Faecalibacterium prausnitzii defines dysbiosis in patients with ulcerative colitis. Gut 63: 1275-1283.

[0665] Patterson, AM, IE Mulder, AJ Travis, A. Lan, N. Cerf-Bensussan, V. Gaboriau-Routhiau, K. Garden, E. Logan, MIDelday, AGP Coutts, E. Monnais, VC Ferraria, R. Inoue, G. Grant, and RIA Minov. 2017. Human Gut Symbiont Roseburia hominis Promotes and Regulates Innate Immunity. Front Immunol 8: 1166.

[0666] Shawki, A., and DF McCole. 2017. Mechanisms of Intestinal Epithelial Barrier Dysfunction by Adherent-Invasive Escherichia coli. Cell Mol Gastroenterol Hepatol 3: 41-50. Sequence Listing <110> Second Genome, Inc. A.W. Han (Han, Andrew Wonhee) A. W. Goodyear (Andrew Whitman) T.Gujral (Gujral, Tarunmeet) T.Z. DeSantis (Todd Zachary) Dabbagh, Karim T·Takeuchi (Takeuchi, Toshihiko) Y. Jin (Jin, Ye) Willcoxon, Michi Izumi Banas, Stefanie <120> Proteins for the treatment of epithelial barrier dysfunction <130> SEGE-001 / 03WO 321077-2031 <150> US 62 / 611,334 <151> December 28, 2017 <150> US 62 / 607,706 <151> December 19, 2017 <150> US 62 / 482,963 <151> April 7, 2017 <160> 34 <170> PatentIn version 3.5 <210> 1 <211> 256 <212> PRT <213> Roseburia hominis <400> 1 Met Lys Arg Leu Val Cys Thr Val Cys Ser Val Leu Leu Cys Ala Gly 1 5 10 15 Leu Leu Ser Gly Cys Gly Thr Ser Leu Glu Gly Glu Glu Ser Val Val 20 25 30 Tyr Val Gly Lys Lys Gly Val Ile Ala Ser Leu Asp Val Glu Thr Leu 35 40 45 Asp Gln Ser Tyr Tyr Asp Glu Thr Glu Leu Lys Ser Tyr Val Asp Ala 50 55 60 Glu Val Glu Asp Tyr Thr Ala Glu His Gly Lys Asn Ala Val Lys Val 65 70 75 80 Glu Ser Leu Lys Val Glu Asp Gly Val Ala Lys Leu Lys Met Lys Tyr 85 90 95 Lys Thr Pro Glu Asp Tyr Thr Ala Phe Asn Gly Ile Glu Leu Tyr Gln 100 105 110 Gly Lys Val Val Ala Ser Leu Ala Ala Gly Tyr Val Tyr Asp Gly Glu 115 120 125 Phe Ala Arg Val Glu Glu Gly Lys Val Val Gly Ala Ala Thr Lys Gln 130 135 140 Asp Ile Tyr Ser Glu Asp Asp Leu Lys Val Ala Ile Ile Arg Ala Asn 145 150 155 160 Thr Asp Val Lys Val Asp Gly Glu Ile Cys Tyr Val Ser Cys Gln Asn 165 170 175 Val Lys Leu Thr Gly Lys Asp Ser Val Ser Ile Arg Asp Gly Tyr Tyr 180 185 190 Leu Glu Thr Gly Ser Val Thr Ala Ser Val Asp Val Thr Gly Gln Glu 195 200 205 Ser Val Gly Thr Glu Gln Leu Ser Gly Thr Glu Gln Met Glu Met Thr 210 215 220 Gly Glu Pro Val Asn Ala Asp Asp Thr Glu Gln Thr Glu Ala Ala Ala 225 230 235 240 Gly Asp Gly Ser Phe Glu Thr Asp Val Tyr Thr Phe Ile Val Tyr Lys 245 250 255 <210> 2 <211> 768 <212> DNA <213> Roseburia hominis <400> 2 atgaagagat tagtgcac ggtctgcagt gtactgttgt gtgcgggact tctctccgga 60 tgcggtacct cgctggaggg agaggaaagt gtcgtgtacg tgggaaagaa aggcgtgata 120 gcgtcgctgg atgtggagac gctcgatcag tcctactacg atgagacgga actgaagtcc 180 tatgtggatg cagaggtgga agattacacc gcggagcatg gtaaaaatgc agtcaaggtg 240 gagagcctta aggtggaaga cggtgtggcg aagcttaaga tgaagtacaa gacaccggag 300 gattataccg catttaatgg aattgaactc tatcagggga aagtcgttgc ttccctggcg 360 gcaggatacg tctacgacgg ggagttcgcc cgcgtggagg aaggcaaggt tgtgggagct 420 gccacaaaac aggatattta ctctgaggat gatttgaaag ttgccatcat ccgtgccaat 480 acggatgtga aggtggacgg tgagatctgc tatgtctcct gtcagaatgt gaagctgacc 540 ggaaaagaca gtgtgtcgat ccgtgacgga tattatcttg agacgggaag cgtgacggca 600 tccgtggatg tgaccggaca ggagagcgtc gggaccgagc agcttcggg aaccgaacag 660 atggagatga ccggggagcc ggtgaatgcg gatgataccg agcagacaga ggcggcggcc 720 ggtgacggtt cgttcgagac agacgtatat acttcattg tctacaa 768 <210> 3 <211> 232 <212> PRT <213> Roseburia hominis (Roseburia hominis) <400> 3 Leu Glu Gly Glu Ser Val Val Tyr Val Gly Lys Lys Gly Val Ile 1 5 10 15 Only Ser Leu Asp Val Glu Thr Leu Asp Gln Ser Tyr Tyr Asp Glu Thr 20 25 30 Glu Leu Lys Ser Tyr Val Asp Ala Glu Val Glu Asp Tyr Thr Ala Glu 35 40 45 His Gly Lys Asn Ala Val Lys Val Glu Ser Leu Lys Val Glu Asp Gly 50 55 60 Val Ala Lys Leu Lys Met Lys Tyr Lys Thr Pro Glu Asp Tyr Thr Ala 65 70 75 80 Phe Asn Gly Ile Glu Leu Tyr Gln Gly Lys Val Val Ala Ser Leu Ala 85 90 95 Ala Gly Tyr Val Tyr Asp Gly Glu Phe Ala Arg Val Glu Glu Gly Lys 100 105 110 Val Val Gly Ala Ala Thr Lys Gln Asp Ile Tyr Ser Glu Asp Asp Leu 115 120 125 Lys Val Ala Ile Ile Arg Ala Asn Thr Asp Val Lys Val Asp Gly Glu 130 135 140 Ile Cys Tyr Val Ser Cys Gln Asn Val Lys Leu Thr Gly Lys Asp Ser 145 150 155 160 Val Ser Ile Arg Asp Gly Tyr Tyr Leu Glu Thr Gly Ser Val Thr Ala 165 170 175 Ser Val Asp Val Thr Gly Gln Glu Ser Val Gly Thr Glu Gln Leu Ser 180 185 190 Gly Thr Glu Gln Met Glu Met Thr Gly Glu Pro Val Asn Ala Asp Asp 195 200 205 Thr Glu Gln Thr Glu Ala Ala Ala Gly Asp Gly Ser Phe Glu Thr Asp 210 215 220 Val Tyr Thr Phe Ile Val Tyr Lys 225 230 <210> 4 <211> 696 <212> DNA <213> Roseburia hominis <400> 4 ctggagggag aggaaagtgt cgtgtacgtg ggaaagaaag gcgtgatagc gtcgctggat gtggagacgc tcgatcagtc ctactacgat gagacggac tgaagtccta tgtggatgca gaggtggaag attacaccgc gaggcatggt aaaaatgcag tcaaggtgga gagccttaag gtggaagacg gtgtggcga gcttaagatg aagtacaaga caccggagga ttataccgca tttaatgga ttgaactcta tcaggggaa gtcgttgctt ccctggcggc aggatacgtc 360. tacgacgggg agttcgcccg cgtggagga ggcaaggttg tgggagctgc cacaaaacag gatatttact ctgaggatga tttgaaagtt gccatcatcc gtgccaatac ggatgtgaag 420 gtggacggtg agatctgcta tgtctcctgt cagaatgtga agctgaccgg aaaagacagt 480 gtgtcgatcc gtgacggata ttatcttgag acgggaagcg tgacggcatc cgtggatgtg 540 accggacagg agagcgtcgg accggagcag ctttcggga ccgaacagat ggagatgacc ggggagccgg tgaatgcgga tgataccgag cagacagagg cggcggccgg tgacggttcg 660 ttcgagacag acgtatac tttcattgtc tacaaa <210> 5 <211> 241 <212> PRT <213> artificial sequence <220> <223> SG11 expressed in the pGEX6 vector and cut by Precision protein <400> 5 Gly Pro Leu Gly Ser Leu Glu Gly Glu Glu Ser Val Val Tyr Val Gly 1 5 10 15 Lys Lys Gly Val Ile Ala Ser Leu Asp Val Glu Thr Leu Asp Gln Ser 20 25 30 Tyr Tyr Asp Glu Thr Glu Leu Lys Ser Tyr Val Asp Ala Glu Val Glu 35 40 45 Asp Tyr Thr Ala Glu His Gly Lys Asn Ala Val Lys Val Glu Ser Leu 50 55 60 Lys Val Glu Asp Gly Val Ala Lys Leu Lys Met Lys Tyr Lys Thr Pro 65 70 75 80 Glu Asp Tyr Thr Ala Phe Asn Gly Ile Glu Leu Tyr Gln Gly Lys Val 85 90 95 Val Ala Ser Leu Ala Ala Gly Tyr Val Tyr Asp Gly Glu Phe Ala Arg 100 105 110 Val Glu Glu Gly Lys Val Val Gly Ala Ala Thr Lys Gln Asp Ile Tyr 115 120 125 Ser Glu Asp Asp Leu Lys Val Ala Ile Ile Arg Ala Asn Thr Asp Val 130 135 140 Lys Val Asp Gly Glu Ile Cys Tyr Val Ser Cys Gln Asn Val Lys Leu 145 150 155 160 Thr Gly Lys Asp Ser Val Ser Ile Arg Asp Gly Tyr Tyr Leu Glu Thr 165 170 175 Gly Ser Val Thr Ala Ser Val Asp Val Thr Gly Gln Glu Ser Val Gly 180 185 190 Thr Glu Gln Leu Ser Gly Thr Glu Gln Met Glu Met Thr Gly Glu Pro 195 200 205 Val Asn Ala Asp Asp Thr Glu Gln Thr Glu Ala Ala Ala Gly Asp Gly 210 215 220 Ser Phe Glu Thr Asp Val Tyr Thr Phe Ile Val Tyr Lys Ala Ala Ala 225 230 235 240 Ser <210> 6 <211> 723 <212> DNA <213> Artificial sequence <220> <223> SG11 expressed in pGEX6 vector and cleaved by Precision protease <400> 6 gggcccctgg gatccctgga gggagaggaa agtgtcgtgt acgtggggaaa gaaaggcgtg 60 atagcgtcgc tggatgtgga gacgctcgat cagtcctact acgatgagac ggaactgaag 120 tcctatgtgg atgcagaggt ggaagattac accgcggagc atggtaaaaa tgcagtcaag 180 gtggagagcc ttaaggtgga agacggtgtg gcgaagctta agatgaagta caagacaccg 240 gaggattata ccgcatttaa tggaattgaa ctctatcagg ggaaagtcgt tgcttccctg 300 gcggcaggat acgtctacga cggggagttc gcccgcgtgg aggaaggcaa ggttgtggga 360 gctgccacaa aacaggatat ttactctgag gatgatttga aagttgccat catccgtgcc 420 aatacggatg tgaaggtgga cggtgagatc tgctatgtct cctgtcagaa tgtgaagctg 480 accggaaaag acagtgtgtc gatccgtgac ggatattatc ttgagacggg aagcgtgacg 540 gcatccgtgg atgtgaccgg acaggagagc gtcgggaccg agcagctttc gggaaccgaa 600 cagatggaga tgaccgggga gccggtgaat gcggatgata ccgagcagac agaggcggcg 660 gccggtgacg gttcgttcga gacagacgta tatactttca ttgtctacaa agcggccgca 720 tcg 723 <210> 7 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> SG11 with Initiating Methionine <400> 7 Met Leu Glu Gly Glu Glu Ser Val Val Tyr Val Gly Lys Lys Gly Val 1 5 10 15 Ile Ala Ser Leu Asp Val Glu Thr Leu Asp Gln Ser Tyr Tyr Asp Glu 20 25 30 Thr Glu Leu Lys Ser Tyr Val Asp Ala Glu Val Glu Asp Tyr Thr Ala 35 40 45 Glu His Gly Lys Asn Ala Val Lys Val Glu Ser Leu Lys Val Glu Asp 50 55 60 Gly Val Ala Lys Leu Lys Met Lys Tyr Lys Thr Pro Glu Asp Tyr Thr 65 70 75 80 Ala Phe Asn Gly Ile Glu Leu Tyr Gln Gly Lys Val Val Ala Ser Leu 85 90 95 Ala Ala Gly Tyr Val Tyr Asp Gly Glu Phe Ala Arg Val Glu Glu Gly 100 105 110 Lys Val Val Gly Ala Ala Thr Lys Gln Asp Ile Tyr Ser Glu Asp Asp 115 120 125 Leu Lys Val Ala Ile Ile Arg Ala Asn Thr Asp Val Lys Val Asp Gly 130 135 140 Glu Ile Cys Tyr Val Ser Cys Gln Asn Val Lys Leu Thr Gly Lys Asp 145 150 155 160 Ser Val Ser Ile Arg Asp Gly Tyr Tyr Leu Glu Thr Gly Ser Val Thr 165 170 175 Ala Ser Val Asp Val Thr Gly Gln Glu Ser Val Gly Thr Glu Gln Leu 180 185 190 Ser Gly Thr Glu Gln Met Glu Met Thr Gly Glu Pro Val Asn Ala Asp 195 200 205 Asp Thr Glu Gln Thr Glu Ala Ala Ala Gly Asp Gly Ser Phe Glu Thr 210 215 220 Asp Val Tyr Thr Phe Ile Val Tyr Lys 225 230 <210> 8 <211> 699 <212> DNA <213> Artificial sequence <220> <223> SG11 with a starting methionine <400> 8 atgttggagg gtgaagagtc tgttgtctat gtgggtaaga aaggtgtgat cgcgtccctg 60 gacgtcgaga ctctggacca gtcttactat gatgaaaccg agctgaagtc gtatgtggac 120 gccgaagttg aggattacac ggccgagcac ggcaaaaatg ccgtcaaagt tgagagcttg 180 aaagttgagg acggcgtggc aaagctgaag atgaaataca agaccccaga ggactacacg 240 gcgttcaatg gtatcgagct gtatcagggc aaagtcgtcg catccctggc agcgggctat 300 gtgtacgacg gtgagtttgc gcgcgtcgaa gaaggcaaag ttgtgggtgc ggctacgaaa 360 caagatatct acagcgaaga tgacctgaaa gtcgcgatta ttcgtgctaa caccgatgtt 420 aaagttgatg gcgagatttg ctacgttagc tgtcaaaacg taaagctgac gggtaaagat 480 agcgtgagca ttcgtgatgg ctattatctg gaaaccggta gcgttacggc gagcgtcgat 540 gttaccggtc aagagagcgt gggtaccgaa cagctgagcg gcaccgaaca gatggaaatg 600 accggtgaac cggttaacgc agacgacacg gaacaaaccg aagccgcggc aggcgacggt 660 agcttcgaga ctgacgtgta cacctttatc gtgtacaag 699 <210> 9 <211> 246 <212> PRT <213> Artificial Sequence <220> <223> SG11 with N - terminal FLAG tag <400> 9 Met Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Ser His Met Leu Glu 1 5 10 15 Gly Glu Glu Ser Val Val Tyr Val Gly Lys Lys Gly Val Ile Ala Ser 20 25 30 Leu Asp Val Glu Thr Leu Asp Gln Ser Tyr Tyr Asp Glu Thr Glu Leu 35 40 45 Lys Ser Tyr Val Asp Ala Glu Val Glu Asp Tyr Thr Ala Glu His Gly 50 55 60 Lys Asn Ala Val Lys Val Glu Ser Leu Lys Val Glu Asp Gly Val Ala 65 70 75 80 Lys Leu Lys Met Lys Tyr Lys Thr Pro Glu Asp Tyr Thr Ala Phe Asn 85 90 95 Gly Ile Glu Leu Tyr Gln Gly Lys Val Val Ala Ser Leu Ala Ala Gly 100 105 110 Tyr Val Tyr Asp Gly Glu Phe Ala Arg Val Glu Glu Gly Lys Val Val 115 120 125 Gly Ala Ala Thr Lys Gln Asp Ile Tyr Ser Glu Asp Asp Leu Lys Val 130 135 140 Ala Ile Ile Arg Ala Asn Thr Asp Val Lys Val Asp Gly Glu Ile Cys 145 150 155 160 Tyr Val Ser Cys Gln Asn Val Lys Leu Thr Gly Lys Asp Ser Val Ser 165 170 175 Ile Arg Asp Gly Tyr Tyr Leu Glu Thr Gly Ser Val Thr Ala Ser Val 180 185 190 Asp Val Thr Gly Gln Glu Ser Val Gly Thr Glu Gln Leu Ser Gly Thr 195 200 205 Glu Gln Met Glu Met Thr Gly Glu Pro Val Asn Ala Asp Asp Thr Glu 210 215 220 Gln Thr Glu Ala Ala Ala Gly Asp Gly Ser Phe Glu Thr Asp Val Tyr 225 230 235 240 Thr Phe Ile Val Tyr Lys 245 <210> 10 <211> 738 <212> DNA <213> Artificial sequence <220> <223> SG11 with N-terminal FLAG tag (not codon-optimized) <400> 10 atggactaca aagacgatga cgacaagggc agcagccata tgctggaggg agaggaaagt 60 gtcgtgtacg tgggaaagaa aggcgtgata gcgtcgctgg atgtggagac gctcgatcag 120 tcctactacg atgagacgga actgaagtcc tatgtggatg cagaggtgga agattacacc 180 gcggagcatg gtaaaaatgc agtcaaggtg gagagcctta aggtggaaga cggtgtggcg 240 aagcttaaga tgaagtacaa gacaccggag gattataccg catttaatgg aattgaactc 300 tatcagggga aagtcgttgc ttccctggcg gcaggatacg tctacgacgg ggagttcgcc 360 cgcgtggagg aaggcaaggt tgtgggagct gccacaaaac aggatttta ctctgaggat 420 gatttgaaag ttgccatcat ccgtgccaat acggatgtga aggtggacgg tgagatctgc 480 tatgtctcct gtcagaatgt gaagctgacc ggaaaagaca gtgtgtcgat ccgtgacgga 540 tattatcttg agcgggag cgtgacggca tccgtggatg tgaccggaca ggagagcgtc gggaccgagc agctttcggg aaccgaacag atggagatga ccggggagcc ggtgaatgcg gatgataccg agcagacaga ggcggcggcc ggtgacggtt cgttcgagac agcgtatat 720 actttcattg tctacaaa 738 <210> 11 <211> 233 <212> PRT <213> The snowstorm <220> <223> SG11 Small Screw (C147V, C151S) <400> 11 Met Leu Glu Gly Glu Glu Ser Val Val Tyr Val Gly Lys Lys Gly Val 1 5 10 15 Ile Ala Ser Leu Asp Val Glu Thr Leu Asp Gln Ser Tyr Tyr Asp Glu 20 25 30 Thr Glu Leu Lys Ser Tyr Val Asp Ala Glu Val Glu Asp Tyr Thr Ala 35 40 45 Glu His Gly Lys Asn Ala Val Lys Val Glu Ser Leu Lys Val Glu Asp 50 55 60 Gly Val Ala Lys Leu Lys Met Lys Tyr Lys Thr Pro Glu Asp Tyr Thr 65 70 75 80 Ala Phe Asn Gly Ile Glu Leu Tyr Gln Gly Lys Val Val Ala Ser Leu 85 90 95 Ala Ala Gly Tyr Val Tyr Asp Gly Glu Phe Ala Arg Val Glu Glu Gly 100 105 110 Lys Val Val Gly Ala Ala Thr Lys Gln Asp Ile Tyr Ser Glu Asp Asp 115 120 125 Leu Lys Val Ala Ile Ile Arg Ala Asn Thr Asp Val Lys Val Asp Gly 130 135 140 Glu Ile Val Tyr Val Ser Ser Gln Asn Val Lys Leu Thr Gly Lys Asp 145 150 155 160 Ser Val Ser Ile Arg Asp Gly Tyr Tyr Leu Glu Thr Gly Ser Val Thr 165 170 175 Ala Ser Val Asp Val Thr Gly Gln Glu Ser Val Gly Thr Glu Gln Leu 180 185 190 Ser Gly Thr Glu Gln Met Glu Met Thr Gly Glu Pro Val Asn Ala Asp 195 200 205 Asp Thr Glu Gln Thr Glu Ala Ala Ala Gly Asp Gly Ser Phe Glu Thr 210 215 220 Asp Val Tyr Thr Phe Ile Val Tyr Lys 225 230 <210> 12 <211> 699 <212> DNA <213> Artificial sequence <220> <223> Artificial variant of SG11 (C147V, C151S) (codon optimized) <400> 12 atgttggagg gtgaagagtc tgttgtctat gtgggtaaga aaggtgtgat cgcgtccctg 60 gacgtcgaga ctctggacca gtcttactat gatgaaaccg agctgaagtc gtatgtggac 120 gccgaagttg aggattacac ggccgagcac ggcaaaaatg ccgtcaaagt tgagagcttg 180 aaagttgagg acggcgtggc aaagctgaag atgaaataca agaccccaga ggactacacg 240 gcgttcaatg gtatcgagct gtatcagggc aaagtcgtcg catccctggc agcgggctat 300 gtgtacgacg gtgagtttgc gcgcgtcgaa gaaggcaaag ttgtgggtgc ggctacgaaa 360 caagatatct acagcgaaga tgacctgaaa gtcgcgatta ttcgtgctaa caccgatgtt aaagttgatg gcgagattgt gtacgttagc agccaaaacg taaagctgac gggtaaagat agcgtgagca ttcgtgatgg ctattatctg gaaaccggta gcgttacggc gagcgtcgat 540 gttaccggtc aagagagcgt gggtaccgaa cagctgagcg gcaccgaaca gatggaaatg accggtgaac cggttaacgc agacgacacg gaacaaaccg aagccgcggc aggcgacggt agcttcgaga ctgacgtgta cacctttatc gtgtacaag 699 <210> 13 <211> 233 <212> PRT <213> The snowstorm <220> <223> SG11 Small Scale (G84D, C147V, C151S) <400> 13 Met Leu Glu Gly Glu Glu Ser Val Val Tyr Val Gly Lys Lys Gly Val 1 5 10 15 Ile Ala Ser Leu Asp Val Glu Thr Leu Asp Gln Ser Tyr Tyr Asp Glu 20 25 30 Thr Glu Leu Lys Ser Tyr Val Asp Ala Glu Val Glu Asp Tyr Thr Ala 35 40 45 Glu His Gly Lys Asn Ala Val Lys Val Glu Ser Leu Lys Val Glu Asp 50 55 60 Gly Val Ala Lys Leu Lys Met Lys Tyr Lys Thr Pro Glu Asp Tyr Thr 65 70 75 80 Ala Phe Asn Asp Ile Glu Leu Tyr Gln Gly Lys Val Val Ala Ser Leu 85 90 95 Ala Ala Gly Tyr Val Tyr Asp Gly Glu Phe Ala Arg Val Glu Glu Gly 100 105 110 Lys Val Val Gly Ala Ala Thr Lys Gln Asp Ile Tyr Ser Glu Asp Asp 115 120 125 Leu Lys Val Ala Ile Ile Arg Ala Asn Thr Asp Val Lys Val Asp Gly 130 135 140 Glu Ile Val Tyr Val Ser Ser Gln Asn Val Lys Leu Thr Gly Lys Asp 145 150 155 160 Ser Val Ser Ile Arg Asp Gly Tyr Tyr Leu Glu Thr Gly Ser Val Thr 165 170 175 Ala Ser Val Asp Val Thr Gly Gln Glu Ser Val Gly Thr Glu Gln Leu 180 185 190 Ser Gly Thr Glu Gln Met Glu Met Thr Gly Glu Pro Val Asn Ala Asp 195 200 205 Asp Thr Glu Gln Thr Glu Ala Ala Ala Gly Asp Gly Ser Phe Glu Thr 210 215 220 Asp Val Tyr Thr Phe Ile Val Tyr Lys 225 230 <210> 14 <211> 699 <212> DNA <213> Artificial sequence <220> <223> Artificial variant of SG11 (G84D, C147V, C151S) (codon-optimized) <400> 14 atgttggagg gtgaagagtc tgttgtctat gtgggtaaga aaggtgtgat cgcgtccctg 60 gacgtcgaga ctctggacca gtcttactat gatgaaaccg agctgaagtc gtatgtggac 120 gccgaagttg aggattacac ggccgagcac ggcaaaaatg ccgtcaaagt tgagagcttg 180 aaagttgagg acggcgtggc aaagctgaag atgaaataca agaccccaga ggactacacg 240 gcgttcaatg acatcgagct gtatcagggc aaagtcgtcg catccctggc agcgggctat 300 gtgtacgacg gtgagtttgc gcgcgtcgaa gaaggcaaag ttgtgggtgc ggctacgaaa 360 caagatatct acagcgaaga tgacctgaaa gtcgcgatta ttcgtgctaa caccgatgtt 420 aaagttgatg gcgagattgt gtacgttagc agccaaaacg taaagctgac gggtaaagat 480 agcgtgagca ttcgtgatgg ctattatctg gaaaccggta gcgttacggc gagcgtcgat 540 gttaccggtc aagagagcgt gggtaccgaa cagctgagcg gcaccgaaca gatggaaatg 600 accggtgaac cggttaacgc agacgacacg gaacaaaccg aagccgcggc aggcgacggt 660 agcttcgaga ctgacgtgta cacctttatc gtgtacaag 699 <210> 15 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> Artificial Variant of SG11 (N83S, C147V, C151S) <400> 15 Met Leu Glu Gly Glu Glu Ser Val Val Tyr Val Gly Lys Lys Gly Val 1 5 10 15 Ile Ala Ser Leu Asp Val Glu Thr Leu Asp Gln Ser Tyr Tyr Asp Glu 20 25 30 Thr Glu Leu Lys Ser Tyr Val Asp Ala Glu Val Glu Asp Tyr Thr Ala 35 40 45 Glu His Gly Lys Asn Ala Val Lys Val Glu Ser Leu Lys Val Glu Asp 50 55 60 Gly Val Ala Lys Leu Lys Met Lys Tyr Lys Thr Pro Glu Asp Tyr Thr 65 70 75 80 Ala Phe Ser Gly Ile Glu Leu Tyr Gln Gly Lys Val Val Ala Ser Leu 85 90 95 Ala Ala Gly Tyr Val Tyr Asp Gly Glu Phe Ala Arg Val Glu Glu Gly 100 105 110 Lys Val Val Gly Ala Ala Thr Lys Gln Asp Ile Tyr Ser Glu Asp Asp 115 120 125 Leu Lys Val Ala Ile Ile Arg Ala Asn Thr Asp Val Lys Val Asp Gly 130 135 140 Glu Ile Val Tyr Val Ser Ser Gln Asn Val Lys Leu Thr Gly Lys Asp 145 150 155 160 Ser Val Ser Ile Arg Asp Gly Tyr Tyr Leu Glu Thr Gly Ser Val Thr 165 170 175 Ala Ser Val Asp Val Thr Gly Gln Glu Ser Val Gly Thr Glu Gln Leu 180 185 190 Ser Gly Thr Glu Gln Met Glu Met Thr Gly Glu Pro Val Asn Ala Asp 195 200 205 Asp Thr Glu Gln Thr Glu Ala Ala Ala Gly Asp Gly Ser Phe Glu Thr 210 215 220 Asp Val Tyr Thr Phe Ile Val Tyr Lys 225 230 <210> 16 <211> 699 <212> DNA <213> Artificial Sequence <220> <223> Artificial Variant of SG11 (N83S, C147V, C151S) (Codon Optimized) <400> 16 atgttggagg gtgaagagtc tgttgtctat gtgggtaaga aaggtgtgat cgcgtccctg 60 gacgtcgaga ctctggacca gtcttactat gatgaaaccg agctgaagtc gtatgtggac 120 gccgaagttg aggattacac ggccgagcac ggcaaaaatg ccgtcaaagt tgagagcttg 180 aaagttgagg acggcgtggc aaagctgaag atgaaataca agaccccaga ggactacacg 240 gcgttcagcg gtatcgagct gtatcagggc aaagtcgtcg catccctggc agcgggctat 300 gtgtacgacg gtgagtttgc gcgcgtcgaa gaaggcaaag ttgtgggtgc ggctacgaaa 360 caagatatct acagcgaaga tgacctgaaa gtcgcgatta ttcgtgctaa caccgatgtt 420 aaagttgatg gcgagattgt gtacgttagc agccaaaacg taaagctgac gggtaaagat 480 agcgtgagca ttcgtgatgg ctattatctg gaaaccggta gcgttacggc gagcgtcgat 540 gttaccggtc aagagagcgt gggtaccgaa cagctgagcg gcaccgaaca gatggaaatg 600 accggtgaac cggttaacgc agacgacacg gaacaaaccg aagccgcggc aggcgacggt 660 agcttcgaga ctgacgtgta cacctttatc gtgtacaag 699 <210> 17 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> Artificial Variant of SG11 (N53S, G84D, C147V, C151S) <400> 17 Met Leu Glu Gly Glu Glu Ser Val Val Tyr Val Gly Lys Lys Gly Val 1 5 10 15 Ile Ala Ser Leu Asp Val Glu Thr Leu Asp Gln Ser Tyr Tyr Asp Glu 20 25 30 Thr Glu Leu Lys Ser Tyr Val Asp Ala Glu Val Glu Asp Tyr Thr Ala 35 40 45 Glu His Gly Lys Ser Ala Val Lys Val Glu Ser Leu Lys Val Glu Asp 50 55 60 Gly Val Ala Lys Leu Lys Met Lys Tyr Lys Thr Pro Glu Asp Tyr Thr 65 70 75 80 Ala Phe Asn Asp Ile Glu Leu Tyr Gln Gly Lys Val Val Ala Ser Leu 85 90 95 Ala Ala Gly Tyr Val Tyr Asp Gly Glu Phe Ala Arg Val Glu Glu Gly 100 105 110 Lys Val Val Gly Ala Ala Thr Lys Gln Asp Ile Tyr Ser Glu Asp Asp 115 120 125 Leu Lys Val Ala Ile Ile Arg Ala Asn Thr Asp Val Lys Val Asp Gly 130 135 140 Glu Ile Val Tyr Val Ser Ser Gln Asn Val Lys Leu Thr Gly Lys Asp 145 150 155 160 Ser Val Ser Ile Arg Asp Gly Tyr Tyr Leu Glu Thr Gly Ser Val Thr 165 170 175 Ala Ser Val Asp Val Thr Gly Gln Glu Ser Val Gly Thr Glu Gln Leu 180 185 190 Ser Gly Thr Glu Gln Met Glu Met Thr Gly Glu Pro Val Asn Ala Asp 195 200 205 Asp Thr Glu Gln Thr Glu Ala Ala Ala Gly Asp Gly Ser Phe Glu Thr 210 215 220 Asp Val Tyr Thr Phe Ile Val Tyr Lys 225 230 <210> 18 <211> 699 <212> DNA <213> Artificial sequence <220> <223> Artificial variants of SG11 (N53S, G84D, C147V, C151S) (codon optimized) <400> 18 atgttggagg gtgaagagtc tgttgtctat gtgggtaaga aaggtgtgat cgcgtccctg 60 gacgtcgaga ctctggacca gtcttactat gatgaaaccg agctgaagtc gtatgtggac 120 gccgaagttg aggattacac ggccgagcac ggcaaatccg ccgtcaaagt tgagagcttg 180 aaagttgagg acggcgtggc aaagctgaag atgaaataca agaccccaga ggactacacg 240 gcgttcaatg acatcgagct gtatcagggc aaagtcgtcg catccctggc agcgggctat 300 gtgtacgacg gtgagtttgc gcgcgtcgaa gaaggcaaag ttgtgggtgc ggctacgaaa 360 420 aaagttgatg gcgagattgt gtacgttagc agccaaaacg taaagctgac gggtaaagat 480 agcgtgagca ttcgtgatgg ctattatctg gaaaccggta gcgttacggc gagcgtcgat 540 gttaccggtc aagagagcgt gggtaccgaa cagctgagcg gcaccgaaca gatggaaatg 600 accggtgaac cggttaacgc agacgacacg gaacaaccg aagccgcgg aggcgacggt 660 699 <210> 19 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> Artificial variant of SG11 (N53S, N83S, C147V, C151S) <400> 19 Met Leu Glu Gly Glu Glu Ser Val Val Tyr Val Gly Lys Lys Gly Val 1 5 10 15 Ile Ala Ser Leu Asp Val Glu Thr Leu Asp Gln Ser Tyr Tyr Asp Glu 20 25 30 Thr Glu Leu Lys Ser Tyr Val Asp Ala Glu Val Glu Asp Tyr Thr Ala 35 40 45 Glu His Gly Lys Ser Ala Val Lys Val Glu Ser Leu Lys Val Glu Asp 50 55 60 Gly Val Ala Lys Leu Lys Met Lys Tyr Lys Thr Pro Glu Asp Tyr Thr 65 70 75 80 Ala Phe Ser Gly Ile Glu Leu Tyr Gln Gly Lys Val Val Ala Ser Leu 85 90 95 Ala Ala Gly Tyr Val Tyr Asp Gly Glu Phe Ala Arg Val Glu Glu Gly 100 105 110 Lys Val Val Gly Ala Ala Thr Lys Gln Asp Ile Tyr Ser Glu Asp Asp 115 120 125 Leu Lys Val Ala Ile Ile Arg Ala Asn Thr Asp Val Lys Val Asp Gly 130 135 140 Glu Ile Val Tyr Val Ser Ser Gln Asn Val Lys Leu Thr Gly Lys Asp 145 150 155 160 Ser Val Ser Ile Arg Asp Gly Tyr Tyr Leu Glu Thr Gly Ser Val Thr 165 170 175 Ala Ser Val Asp Val Thr Gly Gln Glu Ser Val Gly Thr Glu Gln Leu 180 185 190 Ser Gly Thr Glu Gln Met Glu Met Thr Gly Glu Pro Val Asn Ala Asp 195 200 205 Asp Thr Glu Gln Thr Glu Ala Ala Ala Gly Asp Gly Ser Phe Glu Thr 210 215 220 Asp Val Tyr Thr Phe Ile Val Tyr Lys 225 230 <210> 20 <211> 699 <212> DNA <213> Artificial sequence <220> <223> Artificial variants of SG11 (N53S, N83S, C147V, C151S) (codon optimized) <400> 20 atgttggagg gtgaagagtc tgttgtctat gtgggtaaga aaggtgtgat cgcgtccctg 60 gacgtcgaga ctctggacca gtcttactat gatgaaaccg agctgaagtc gtatgtggac 120 gccgaagttg aggattacac ggccgagcac ggcaaatccg ccgtcaaagt tgagagcttg 180 aaagttgagg acggcgtggc aaagctgaag atgaaataca agaccccaga ggactacacg 240 gcgttcagcg gtatcgagct gtatcagggc aaagtcgtcg catccctggc agcgggctat 300 gtgtacgacg gtgagtttgc gcgcgtcgaa gaaggcaaag ttgtgggtgc ggctacgaaa 360 caagatatct acagcgaaga tgacctgaaa gtcgcgatta ttcgtgctaa caccgatgtt 420 aaagttgatg gcgagattgt gtacgttagc agccaaaacg taaagctgac gggtaaagat 480 agcgtgagca ttcgtgatgg ctattatctg gaaaccggta gcgttacggc gagcgtcgat 540 gttaccggtc aagagagcgt gggtaccgaa cagctgagcg gcaccgaaca gatggaaatg 600 accggtgaac cggttaacgc agacgacacg gaacaaaccg aagccgcggc aggcgacggt 660 agcttcgaga ctgacgtgta cacctttatc gtgtacaag 699 <210> 21 <211> 227 <212> PRT <213> Roseburia intestinalis <400> 21 Met Leu Asp Ala Asp Thr Asp Thr Val Tyr Val Gln Lys Asn Gly Thr 1 5 10 15 Val Leu Ser Val Asp Val Glu Thr Leu Asp Lys Asp Tyr Tyr Asp Glu 20 25 30 Thr Glu Leu Lys Asp Tyr Val Thr Asp Ala Val Ser Thr Tyr Thr Gly 35 40 45 Glu His Gly Lys Ser Ala Val Lys Leu Glu Asn Leu Ser Val Lys Asp 50 55 60 Gly Thr Ala Thr Leu Lys Met Lys Tyr Lys Thr Pro Glu Asp Tyr Thr 65 70 75 80 Gly Phe Asn Gly Ile Glu Leu Tyr Glu Gly Lys Val Val Lys Ala Leu 85 90 95 Ala Ala Gly Tyr Asp Phe Lys Thr Asp Phe Val Ser Val Glu Asp Gly 100 105 110 Lys Val Thr Gly Thr Ala Thr Lys Glu Glu Ile Tyr Ser Gly Glu Asp 115 120 125 Leu Lys Val Val Ile Ile Lys Ala Asn Arg Asp Val Lys Val Asp Gly 130 135 140 Thr Ile Cys Tyr Val Ser Ser Glu Asn Val Lys Leu Thr Gly Thr Asp 145 150 155 160 Ser Val Ser Ile Arg Asp Gly Tyr Ser Leu Asn Ser Gly Ser Thr Ala 165 170 175 Asp Glu Ser Asp Ser Asp Glu Asn Ile Ala Asp Gly Thr Glu Ser Ile 180 185 190 Gly Gly Ser Thr Glu Val Ser Asp Thr Asp Val Asn Asp Asp Thr Thr 195 200 205 Tyr Val Lys Asp Asp Gly Ala Phe Glu Thr Asp Val Tyr Thr Tyr Ile 210 215 220 Ile Tyr Lys 225 <210> 22 <211> 215 <212> PRT <213> Rothia sp. 831b <400> 22 Met Leu Asp Val Glu Glu Ser Thr Val Tyr Val Gln Lys Asn Gly Ser 1 5 10 15 Val Ile Ser Thr Asp Ile Glu Asp Phe Ser Ala Asp Tyr Tyr Asp Glu 20 25 30 Asp Glu Leu Lys Asp Tyr Ile Gly Asp Glu Ile Ser Ser Tyr Thr Ser 35 40 45 Glu Asn Gly Lys Lys Ser Val Ser Leu Glu Ser Val Ser Val Lys Asp 50 55 60 Ser Val Ala Lys Leu Thr Met Lys Tyr Lys Thr Ala Glu Asp Tyr Thr 65 70 75 80 Asn Phe Asn Gly Val Glu Leu Tyr Thr Gly Thr Ile Val Lys Ala Met 85 90 95 Ala Ala Gly Tyr Asp Phe Gly Val Asp Phe Val Ser Val Lys Asp Gly 100 105 110 Ala Val Thr Gly Thr Ala Thr Lys Asp Glu Ile Val Asp His Asp Asp 115 120 125 Tyr Lys Val Ala Val Ile Lys Ala Asn Thr Asp Val Lys Val Asp Gly 130 135 140 Thr Ile Val Tyr Val Ser Ser Gln Asn Val Lys Val Thr Gly Lys Asn 145 150 155 160 Thr Val Ser Ile Arg Glu Gly Tyr Leu Ala Ala Asp Thr Thr Asn Val 165 170 175 Val Gly Ser Thr Glu Thr Val Ala Glu Thr Glu Ala Glu Glu Ala Asn 180 185 190 Gln Thr Glu Ala Val Leu Glu Asp Glu Phe Ala Ser Glu Ser Asp Val 195 200 205 Tyr Thr Tyr Val Ile Phe Lys 210 215 <210> 23 <211> 236 <212> PRT <213> Roseburia inulinivorans <400> 23 Met Leu Glu Ala Asp Thr Asn Thr Val Tyr Val Ser Lys His Gly Lys 1 5 10 15 Val Val Ser Met Asp Val Glu Gln Leu Asp Gln Ser Tyr Tyr Asp Glu 20 25 30 Thr Glu Leu Lys Glu Phe Val Asp Ser Ala Val Asp Glu Tyr Asn Thr 35 40 45 Glu Asn Gly Lys Asn Ser Val Lys Val Asp Asp Leu Thr Val Glu Asp 50 55 60 Gly Thr Ala Lys Leu Arg Met Asp Tyr Glu Thr Val Asp Asp Tyr Thr 65 70 75 80 Ala Phe Asn Gly Val Glu Leu Tyr Glu Gly Lys Ile Val Gln Ala Leu 85 90 95 Ala Ala Gly Tyr Asp Phe Asp Thr Asp Phe Ala Gly Val Asp Lys Asp 100 105 110 Gly Cys Val Thr Gly Val Thr Arg Gly Asp Ile Leu Ala Gln Glu Asp 115 120 125 Leu Lys Val Val Ile Ile Lys Ala Asn Thr Asp Val Lys Ile Asp Gly 130 135 140 Lys Ile Leu Tyr Val Ser Cys Asp Asn Val Thr Val Thr Gly Lys Asp 145 150 155 160 Ser Val Ser Ile Lys Glu Gly Thr Gly Ile Glu Lys Thr Trp Ile Thr 165 170 175 Glu Ala Glu Glu Val Pro Ser Thr Glu Ala Val Leu Glu Thr Glu Ser 180 185 190 Thr Glu Asp Ala Gly Asp Val Ile Glu Gly Glu Val Ile Ile Gly Thr 195 200 205 Glu Glu Ala Ser Gly Asn Asp Val Val Thr Asn Leu Ser Gly Gly Ser 210 215 220 Ser Gly Thr Asp Val Tyr Thr Tyr Tyr Ile Ile Tyr Lys 225 230 235 <210> twenty four <211> 13 <212> PRT <213> Artificial sequence <220> <223> Fragment of SG11 with initiator methionine <400> twenty four Met Leu Glu Gly Glu Glu Ser Val Val Tyr Val Gly Lys 1 5 10 <210> 25 <211> twenty two <212> PRT <213> Roseburia hominis <400> 25 Gly Val Ile Ala Ser Leu Asp Val Glu Thr Leu Asp Gln Ser Tyr Tyr 1 5 10 15 Asp Glu Thr Glu Leu Lys 20 <210> 26 <211> 17 <212> PRT <213> Roseburia hominis <400> 26 Thr Pro Glu Asp Tyr Thr Ala Phe Asn Gly Ile Glu Leu Tyr Gln Gly 1 5 10 15 Lys <210> 27 <211> 6 <212> PRT <213> Roseburia hominis <400> 27 Ala Asn Thr Asp Val Lys 1 5 <210> 28 <211> 14 <212> PRT <213> Roseburia hominis <400> 28 Val Asp Gly Glu Ile Cys Tyr Val Ser Cys Gln Asn Val Lys 1 5 10 <210> 29 <211> 67 <212> PRT <213> Roseburia hominis <400> 29 Gly Tyr Tyr Leu Glu Thr Gly Ser Val Thr Ala Ser Val Asp Val Thr 1 5 10 15 Gly Gln Glu Ser Val Gly Thr Glu Gln Leu Ser Gly Thr Glu Gln Met 20 25 30 Glu Met Thr Gly Glu Pro Val Asn Ala Asp Asp Thr Glu Gln Thr Glu 35 40 45 Ala Ala Ala Gly Asp Gly Ser Phe Glu Thr Asp Val Tyr Thr Phe Ile 50 55 60 Val Tyr Lys 65 <210> 30 <211> 4 <212> PRT <213> Roseburia hominis <400> 30 Asn Ala Val Lys 1 <210> 31 <211> 17 <212> PRT <213> Roseburia hominis <400> 31 Thr Pro Glu Asp Tyr Thr Ala Phe Ser Gly Ile Glu Leu Tyr Gln Gly 1 5 10 15 Lys <210> 32 <211> 8 <212> PRT <213> Artificial sequence <220> <223> FLAG tag <400> 32 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 33 <211> 233 <212> PRT <213> Artificial sequence <220> <223> Artificial variant of SG11 <220> <221> misc_feature <222> (53)..(53) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (83)..(84) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (147)..(147) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (151)..(151) <223> Xaa can be any naturally occurring amino acid <400> 33 Met Leu Glu Gly Glu Glu Ser Val Val Tyr Val Gly Lys Lys Gly Val 1 5 10 15 Ile Ala Ser Leu Asp Val Glu Thr Leu Asp Gln Ser Tyr Tyr Asp Glu 20 25 30 Thr Glu Leu Lys Ser Tyr Val Asp Ala Glu Val Glu Asp Tyr Thr Ala 35 40 45 Glu His Gly Lys Xaa Ala Val Lys Val Glu Ser Leu Lys Val Glu Asp 50 55 60 Gly Val Ala Lys Leu Lys Met Lys Tyr Lys Thr Pro Glu Asp Tyr Thr 65 70 75 80 Ala Phe Xaa Xaa Ile Glu Leu Tyr Gln Gly Lys Val Val Ala Ser Leu 85 90 95 Ala Ala Gly Tyr Val Tyr Asp Gly Glu Phe Ala Arg Val Glu Glu Gly 100 105 110 Lys Val Val Gly Ala Ala Thr Lys Gln Asp Ile Tyr Ser Glu Asp Asp 115 120 125 Leu Lys Val Ala Ile Ile Arg Ala Asn Thr Asp Val Lys Val Asp Gly 130 135 140 Glu Ile Xaa Tyr Val Ser Xaa Gln Asn Val Lys Leu Thr Gly Lys Asp 145 150 155 160 Ser Val Ser Ile Arg Asp Gly Tyr Tyr Leu Glu Thr Gly Ser Val Thr 165 170 175 Ala Ser Val Asp Val Thr Gly Gln Glu Ser Val Gly Thr Glu Gln Leu 180 185 190 Ser Gly Thr Glu Gln Met Glu Met Thr Gly Glu Pro Val Asn Ala Asp 195 200 205 Asp Thr Glu Gln Thr Glu Ala Ala Ala Gly Asp Gly Ser Phe Glu Thr 210 215 220 Asp Val Tyr Thr Phe Ile Val Tyr Lys 225 230 <210> 34 <211> 233 <212> PRT <213> Artificial sequence <220> <223> Artificial variant of SG11 <220> <221> misc_feature <222> (53)..(53) <223> Xaa is any amino acid except Asn <220> <221> misc_feature <222> (83)..(83) <223> Xaa is any amino acid except Asn <220> <221> misc_feature <222> (84)..(84) <223> Xaa is any amino acid except Gly <220> <221> misc_feature <222> (147)..(147) <223> Xaa is any amino acid except Cys <220> <221> misc_feature <222> (151)..(151) <223> Xaa is any amino acid except Cys <400> 34 Met Leu Glu Gly Glu Glu Ser Val Val Tyr Val Gly Lys Lys Gly Val 1 5 10 15 Ile Ala Ser Leu Asp Val Glu Thr Leu Asp Gln Ser Tyr Tyr Asp Glu 20 25 30 Thr Glu Leu Lys Ser Tyr Val Asp Ala Glu Val Glu Asp Tyr Thr Ala 35 40 45 Glu His Gly Lys Xaa Ala Val Lys Val Glu Ser Leu Lys Val Glu Asp 50 55 60 Gly Val Ala Lys Leu Lys Met Lys Tyr Lys Thr Pro Glu Asp Tyr Thr 65 70 75 80 Ala Phe Xaa Xaa Ile Glu Leu Tyr Gln Gly Lys Val Val Ala Ser Leu 85 90 95 Ala Ala Gly Tyr Val Tyr Asp Gly Glu Phe Ala Arg Val Glu Glu Gly 100 105 110 Lys Val Val Gly Ala Ala Thr Lys Gln Asp Ile Tyr Ser Glu Asp Asp 115 120 125 Leu Lys Val Ala Ile Ile Arg Ala Asn Thr Asp Val Lys Val Asp Gly 130 135 140 Glu Ile Xaa Tyr Val Ser Xaa Gln Asn Val Lys Leu Thr Gly Lys Asp 145 150 155 160 Ser Val Ser Ile Arg Asp Gly Tyr Tyr Leu Glu Thr Gly Ser Val Thr 165 170 175 Ala Ser Val Asp Val Thr Gly Gln Glu Ser Val Gly Thr Glu Gln Leu 180 185 190 Ser Gly Thr Glu Gln Met Glu Met Thr Gly Glu Pro Val Asn Ala Asp 195 200 205 Asp Thr Glu Gln Thr Glu Ala Ala Ala Gly Asp Gly Ser Phe Glu Thr 210 215 220 Asp Val Tyr Thr Phe Ile Val Tyr Lys 225 230

Claims

1. Use of a therapeutic protein in the preparation of a medicament for treating inflammatory bowel disease, wherein the therapeutic protein is SEQ ID NO: 7 or SEQ ID NO:

19.

2. The use according to claim 1, wherein the medicament is a rectal preparation, a parenteral preparation, an intravenous preparation, a topical preparation, an oral preparation, a dermal preparation, a transdermal preparation or a subcutaneous preparation.

3. The use of claim 1, wherein the medicament alleviates at least one symptom associated with inflammatory bowel disease.

4. The use of claim 1, wherein the medicament alleviates at least one symptom associated with inflammatory bowel disease, the symptom selected from the group consisting of abdominal pain, blood in the stool, pus in the stool, fever, weight loss, frequent diarrhea, fatigue, decreased appetite, tenesmus and rectal bleeding.

5. The use of claim 1, wherein the medicament reduces gastrointestinal inflammation.

6. The use of claim 1, wherein the drug reduces inflammation of the intestinal mucosa.

7. The use of claim 1, wherein the drug increases the production of mucin in intestinal tissue.

8. The use of claim 1, wherein the medicament increases intestinal epithelial wound healing.

9. The use of claim 1, wherein the drug increases intestinal epithelial cell proliferation.

10. The use of claim 1, wherein the medicament further comprises a second therapeutic agent.

11. The use of claim 10, wherein the second therapeutic agent is selected from the group consisting of antidiarrheal drugs, 5-aminosalicylic acid compounds, anti-inflammatory agents, antibiotics, antibodies, anti-cytokine agents, anti-inflammatory cytokine agents, steroids, corticosteroids and immunosuppressants.

12. A pharmaceutical composition comprising: a. A therapeutic protein, which is SEQ ID NO: 7 or SEQ ID NO: 19; and b. Pharmaceutically acceptable carrier.

13. The pharmaceutical composition of claim 12, formulated for rectal, parenteral, intravenous, topical, oral, cutaneous, transdermal or subcutaneous administration.

14. The pharmaceutical composition of claim 12, formulated such that the therapeutic protein is active in the gastrointestinal lumen and / or intestine of a patient.

15. An expression vector comprising: a polynucleotide encoding the protein of SEQ ID NO: 7 or SEQ ID NO:

19.

16. A host cell comprising: an exogenous polynucleotide encoding the protein of SEQ ID NO: 7 or SEQ ID NO:

19.

17. The host cell of claim 16, wherein the exogenous polynucleotide further encodes a host cell-specific signal sequence.

18. The host cell of claim 16, wherein the exogenous polynucleotide comprises the nucleic acid sequence SEQ ID NO: 8 or SEQ ID NO:

20.

19. The host cell of claim 16, wherein the host cell is a prokaryotic cell.

20. The host cell of claim 16, wherein the host cell is an E. coli cell.

21. The host cell of claim 16, wherein the host cell is a eukaryotic cell.

22. The host cell of claim 16, wherein the host cell is a Chinese hamster ovary cell.

23. A method for producing a protein, comprising: The host cell of claim 16 is cultured under conditions sufficient to express the encoded protein.

24. An isolated therapeutic protein which is SEQ ID NO: 7 or SEQ ID NO:

19.

25. The isolated therapeutic protein of claim 24, wherein the protein increases electrical resistance in an in vitro transepithelial electrical resistance assay.

26. The isolated therapeutic protein of claim 24, wherein the protein increases electrical resistance in an in vitro transepithelial electrical resistance assay by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% compared to an assay performed in the absence of the protein.

27. The isolated therapeutic protein of claim 24, wherein the protein increases electrical resistance in an in vitro transepithelial electrical resistance assay compared to a kinase inhibitor control.

28. The isolated therapeutic protein of claim 24, wherein the protein increases electrical resistance in an in vitro transepithelial electrical resistance assay compared to a staurosporine or myosin light chain kinase control.

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