Proteins for the treatment of epithelial barrier dysfunction

Abstract

The present disclosure relates to therapeutic proteins and pharmaceutical compositions containing the proteins that are useful for treating various human diseases. In certain aspects, the disclosed therapeutic proteins are useful for treating human gastrointestinal inflammatory diseases and gastrointestinal conditions associated with impaired epithelial cell barrier function or integrity. Furthermore, the disclosed therapeutic proteins are useful for treating human inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, among others.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 611,334, filed December 28, 2017, U.S. Provisional Patent Application No. 62 / 607,706, filed December 19, 2017, and U.S. Provisional Patent Application No. 62 / 482,963, filed April 7, 2017, each of which is incorporated herein by reference in its entirety.

[0002] Explanation of the electronically submitted text file The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: Computer-Readable Copy of the Sequence Listing File name: SEGE_001_03WO_SeqList_ST25.txt; Creation date: April 4, 2018; File size: approximately 45.3 KB.

[0003] Field The present disclosure relates to novel proteins and pharmaceutical compositions comprising the proteins, which have application in the treatment of, inter alia, gastrointestinal inflammatory diseases and epithelial barrier dysfunction. In some aspects, the proteins and pharmaceutical compositions described herein have particular application in the treatment or prevention of disease states associated with abnormally permeable epithelial barriers and inflammatory bowel diseases or disorders. [Background technology]

[0004] background Inflammatory bowel disease (IBD) is a heterogeneous disorder of unknown etiology that results in frequent, bloody bowel movements accompanied by histopathological damage to the gastrointestinal mucosa (Zhang et al., 2017, Front Immunol, 8:942). Although the exact trigger of the disease has not been fully identified, one proposal for disease progression suggests that disruption of intestinal barrier function leads to the translocation of bacteria or bacterial components into mucosal tissue (Coskun, 2014, Front Med (Lausanne), 1:24; Martini et al., 2017, Cell Mol Gastroenterol Hepatol, 4:33-46). Bacterial translocation activates inflammatory signaling, which induces further barrier disruption, resulting in a cyclical amplification loop of barrier disruption, bacterial translocation, and inflammation. While many current treatments target inflammation, the lack of therapies that promote mucosal healing provides an opportunity for novel therapies that promote epithelial repair and intestinal barrier integrity.

[0005] Expanding on the hypothesis that bacterial translocation could be a trigger for IBD, more recent studies have demonstrated that adverse changes in the gut microbiota, or dysbiosis, can promote the development of IBD.

[0006] Currently, many commercially available IBD medications are solely aimed at targeting and suppressing the IBD-associated inflammatory responses discussed above. While beneficial, this narrow therapeutic mode of action ignores the significant contribution that epithelial barrier integrity plays in the pathogenesis of this disease.

[0007] Thus, there is a significant need in the art for the development of therapeutics that act in concert to not only suppress the inflammatory response of the immune system, but also restore epithelial barrier function in an individual. There is also a need for the production of protein therapeutics as described herein that are stable not only throughout the manufacture and / or processing of the protein therapeutic, but also under long-term storage conditions. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Zhang et al., 2017, Front Immunol, 8:942 [Non-patent document 2] Coskun, 2014, Front Med (Lausanne), 1:24 [Non-patent document 3] Martini et al., 2017, Cell Mol Gastroenterol Hepatol, 4:33-46 Summary of the Invention

[0009] Disclosure Overview The present disclosure addresses a significant need in the medical community for therapeutic agents that can effectively treat subjects suffering from gastrointestinal disorders, such as inflammatory bowel disease (IBD). In one aspect, novel protein therapeutic agents are provided that can maintain the integrity of the epithelial barrier and / or improve epithelial barrier repair. In some embodiments, the epithelial barrier is the intestinal epithelial barrier. Such protein therapeutic agents can also suppress intestinal inflammation and / or alleviate symptoms associated with intestinal inflammation in a subject.

[0010] The protein therapeutics provided herein are useful for treating a number of diseases and / or conditions that may be associated with a compromised gastrointestinal epithelial cell barrier function or integrity.

[0011] In some embodiments, the present disclosure teaches novel protein therapeutics derived from gut microbiota and methods of utilizing the protein therapeutics. In certain embodiments, proteins derived from gut microbiota are provided that comprise 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. In some embodiments, the therapeutic protein does not comprise an amino acid sequence identical to SEQ ID NO: 3. In yet other embodiments, the therapeutic protein comprises an amino acid sequence that is not naturally occurring.

[0012] 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 yet other embodiments, the protein comprises the amino acid sequence of SEQ ID NO:19.

[0013] 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 one, two, three, or four amino acid substitutions relative to SEQ ID NO:19 or SEQ ID NO:3. In other embodiments, the amino acid sequence has at least two and fewer 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 yet other embodiments, the therapeutic protein comprises a non-naturally occurring amino acid sequence.

[0014] In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:3. In other embodiments related to 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 still 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.

[0015] In some embodiments, the protein is about 200-250 amino acids, 210-250 amino acids, 220-250 amino acids, 220-240 amino acids, 230-250 amino acids, 230-240 amino acids, or 230-235 amino acids, 220-275 amino acids, 220-260 amino acids, 230-260 amino acids, 240-250 amino acids, 250-260 amino acids, 230-256 amino acids, 240-256 amino acids, or 245-256 amino acids. 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.

[0016] In some embodiments, the present disclosure teaches an antibody or fragment thereof that specifically binds 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 an amino acid sequence identical to SEQ ID NO:3. In yet other embodiments, the antibody or fragment thereof binds to a protein comprising an amino acid sequence identical to SEQ ID NO:19, but does not bind to a protein comprising an amino acid sequence identical to SEQ ID NO:3.

[0017] In some embodiments, the protein increases the barrier function of an epithelial cell layer in an in vitro assay; the increase is relative to the barrier function in the absence of the protein in the assay. In other embodiments, the in vitro assay is a transepithelial electrical resistance (TEER) assay. In still other embodiments, the increase in barrier function is an increase in electrical resistance in the assay that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% greater than the electrical resistance in the absence of the protein. In some embodiments, the epithelial cell layer is an intestinal epithelial cell layer. In still other embodiments, the intestinal epithelial cell layer is a cell layer comprising enterocytes and goblet cells.

[0018] In some embodiments, the protein reduces secretion of proinflammatory cytokines from cells in an in vitro assay. In other embodiments, the in vitro assay involves incubating monocyte-lineage cells with heat-killed E. coli in the presence and absence of the protein. In still other embodiments, the at least one proinflammatory cytokine is selected from the group consisting of 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.

[0019] In some embodiments, the protein increases secretion of an anti-inflammatory cytokine 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 still other embodiments, the at least one anti-inflammatory cytokine is selected from the group consisting of IL-4, IL-10, IL-13, IFN-α, and TGF-β.

[0020] In some embodiments, the protein reduces intestinal tissue pathology in a subject administered the protein. In some embodiments, the subject has been induced to have intestinal tissue damage by treatment with a chemical. In other embodiments, the subject has been treated with dextran sulfate sodium (DSS), a chemical that causes intestinal tissue damage. In still other embodiments, the subject is a mammal. In still other embodiments, the animal is a rodent. In other embodiments, the subject is a non-human primate.

[0021] In some embodiments, the therapeutic protein reduces gastrointestinal inflammation in a subject administered the protein. In other embodiments, the protein reduces inflammation of the intestinal mucosa in a subject. In yet other embodiments, the protein improves the barrier function or integrity of intestinal epithelial cells in a subject.

[0022] In some embodiments, the protein increases the amount of mucin in intestinal tissue in a subject receiving the protein.

[0023] In some embodiments, the protein increases wound healing of intestinal epithelial cells in a subject administered the protein, hi other embodiments, the protein increases wound healing of intestinal epithelial cells in an in vitro assay.

[0024] In some embodiments, the protein prevents or reduces colon shortening in a subject to which the protein is administered.

[0025] In some embodiments, a therapeutic protein modulates (ie, increases or decreases) cytokines in the blood, plasma, serum, tissues and / or mucosa of a subject to which the protein is administered.

[0026] In some embodiments, the protein reduces the level of at least one pro-inflammatory cytokine in the blood, plasma, serum, tissues, and / or mucosa of the subject. In other embodiments, the at least one pro-inflammatory cytokine is selected from the group consisting of 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.

[0027] 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 the subject, hi other embodiments, the at least one anti-inflammatory cytokine is selected from the group consisting of IL-4, IL-10, IL-13, IFN-α, and TGF-β.

[0028] 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 the subject, hi other embodiments, the at least one anti-inflammatory cytokine is selected from the group consisting of IL-4, IL-10, IL-13, IFN-α, and TGF-β.

[0029] In some embodiments, the present disclosure teaches polynucleotides encoding novel protein therapeutics and methods of expressing the nucleic acids in host cells. In certain embodiments, the polynucleotides comprise 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 that encodes 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 yet 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 yet other embodiments, the polynucleotide is codon-optimized for expression in a recombinant host cell. In yet other embodiments, the polynucleotide is codon-optimized for expression in E. coli.

[0030] In some embodiments, the present disclosure teaches nucleic acids 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 other 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%, or 99% identical to SEQ ID NO:20 and less than 100% identical to SEQ ID NO:4. In yet 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.

[0031] 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 yet other embodiments, the C-terminus is chemically modified by amidation.

[0032] In some embodiments, the protein is pegylated.

[0033] In some embodiments, the protein is substantially purified and modified by glycosylation, ubiquitination, nitrosylation, methylation, acetylation, or lipidation.

[0034] In some embodiments, the protein is fused to a second protein, hi other embodiments, the second protein is an immunoglobulin Fc domain or a human serum albumin protein domain.

[0035] 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 other embodiments, 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 the group consisting of 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 still other embodiments, the protein comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:7, or SEQ ID NO:19.

[0036] 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 yet other embodiments, the pharmaceutical composition is a solid composition comprising an enteric coating.

[0037] In some embodiments, the pharmaceutical composition is a cream, capsule, liquid, gel, or emulsion.

[0038] In some embodiments, the pharmaceutical composition is formulated to provide delayed release. In other embodiments, the delayed release is in the gastrointestinal tract. In still other embodiments, the delayed release is in the oral cavity, small intestine, large intestine, and / or rectum.

[0039] In some embodiments, the pharmaceutical composition is formulated to provide sustained release. In other embodiments, the sustained release is to the gastrointestinal tract. In still other embodiments, the sustained release is to the oral cavity, small intestine, large intestine, and / or rectum. In still other embodiments, the sustained release composition releases the therapeutic formulation over about 1-20 hours, 1-10 hours, 1-8 hours, 4-12 hours, or 5-15 hours.

[0040] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent, hi other embodiments, the second therapeutic agent is selected from the group consisting of an anti-diarrheal agent, a 5-aminosalicylate compound, an anti-inflammatory agent, an antibiotic, an anti-cytokine agent, an anti-inflammatory cytokine agent, a steroid, a corticosteroid, an immunosuppressant, a JAK inhibitor, an anti-integrin biologic, an anti-IL12 / 23R biologic, and a vitamin.

[0041] In some embodiments, the pharmaceutical composition further comprises a protease inhibitor, hi still other embodiments, the protease inhibitor inhibits degradation of the therapeutic protein in the presence of feces and / or blood.

[0042] As described above, these novel protein therapeutics can promote epithelial barrier function and integrity in a subject. In some embodiments, the epithelial barrier function is intestinal epithelial barrier function. Furthermore, the therapeutic effect of the proteins includes suppressing inflammatory immune responses in IBD individuals. Therefore, the present disclosure provides detailed guidance on how to utilize the taught therapeutic proteins to treat hosts with gastrointestinal inflammation and disease states in which the integrity of the gastrointestinal epithelial barrier is compromised.

[0043] In some embodiments, methods of treating an inflammatory bowel disease or disorder in a patient are provided, the methods comprising administering to the patient: i) a peptide sequence comprising 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: and ii) a pharmaceutical composition comprising: 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 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 yet other embodiments, the protein is not identical to SEQ ID NO:3 or is a non-naturally occurring variant of SEQ ID NO:3.

[0044] 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 yet other embodiments, the intestinal inflammation is in the rectum. In yet other embodiments, the patient has been diagnosed with pouchitis.

[0045] In some embodiments, the patient has been diagnosed with an intestinal ulcer, hi other embodiments, the patient has been diagnosed with a draining enterocutaneous and / or rectovaginal fistula.

[0046] In some embodiments, the patient has been diagnosed with Crohn's disease (CD). In other embodiments, the CD is mild CD. In yet other embodiments, the CD is moderate to severe CD. In yet other embodiments, the patient has been diagnosed with pediatric CD.

[0047] In some embodiments, the patient has been diagnosed with short bowel syndrome or irritable bowel syndrome.

[0048] In some embodiments, the patient has been diagnosed with mucositis. In other embodiments, the mucositis is oral mucositis. In still other embodiments, the mucositis is chemotherapy-induced mucositis, radiation therapy-induced mucositis, chemotherapy-induced oral mucositis, or radiation therapy-induced oral mucositis. In still other embodiments, the mucositis is gastrointestinal mucositis. In still other embodiments, the gastrointestinal mucositis is small intestinal, large intestinal, or rectal mucositis.

[0049] In some embodiments, the administration to patients diagnosed with CD results in a decrease in the number of draining enterocutaneous and / or rectovaginal fistulas, hi other embodiments, the administration maintains fistula closure in adult patients with fistulizing disease.

[0050] In other embodiments, the patient has been diagnosed with ulcerative colitis (UC). In other embodiments, the UC is mild UC. In yet other embodiments, the UC is moderate to severe UC. In yet other embodiments, the patient has been diagnosed with pediatric UC.

[0051] In some embodiments, the patient is in clinical remission from IBD, hi other embodiments, the patient is in clinical remission from UC, pediatric UC, CD, or pediatric CD.

[0052] In some embodiments, the patient has an inflammatory bowel disease or disorder other than Crohn's disease or ulcerative colitis, hi other embodiments, the patient has at least one symptom associated with inflammatory bowel disease.

[0053] In some embodiments, the administration reduces gastrointestinal inflammation and / or reduces inflammation of the intestinal mucosa associated with inflammatory bowel disease in the patient, hi other embodiments, the administration improves the barrier function or integrity of intestinal epithelial cells in the patient.

[0054] In some embodiments, after administration, the patient experiences a reduction in at least one symptom associated with inflammatory bowel disease or disorder. In other embodiments, the at least one symptom is selected from the group consisting of abdominal pain, bloody stool, pus-filled stool, fever, weight loss, frequent diarrhea, fatigue, loss of appetite, nausea, cramps, anemia, tenesmus, and rectal bleeding. In still 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.

[0055] In some embodiments, the patient has experienced an inadequate response to conventional therapy, hi other embodiments, the conventional therapy is treatment with an 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.

[0056] In some embodiments, the administration modulates (ie, increases or decreases) the level of a cytokine in the patient's blood, plasma, serum, mucosa, or tissue.

[0057] In some embodiments, the administration suppresses the level of at least one pro-inflammatory cytokine in the patient, hi other embodiments, the at least one pro-inflammatory cytokine is selected from the group consisting of 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.

[0058] In some embodiments, the administration increases the level of at least one anti-inflammatory cytokine in the patient's blood, plasma, serum, mucosa, or tissue, hi other embodiments, the at least one anti-inflammatory cytokine is selected from the group consisting of IL-4, IL-10, IL-13, IFN-α, and TGF-β.

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

[0060] In some embodiments, the administration increases the amount of mucin in the intestinal lumen of the patient.

[0061] In some embodiments, the administration increases wound healing of intestinal epithelial cells in the patient.

[0062] In some embodiments, the administration prevents or reduces colon shortening in the patient.

[0063] In some embodiments, the administering comprises rectal, intravenous, parenteral, oral, topical, dermal, transdermal, or subcutaneous administration of the pharmaceutical composition to the patient, while in other embodiments, the administering is to the gastrointestinal lumen.

[0064] In some embodiments, the patient is also administered at least one second therapeutic agent. In other embodiments, the at least one second therapeutic agent is selected from the group consisting of an antidiarrheal agent, an anti-inflammatory agent, an antibody, an antibiotic, or an immunosuppressant. In still other embodiments, the at least one second therapeutic agent is an aminosalicylate, a steroid, or a corticosteroid. In other embodiments, the at least one second therapeutic agent is selected from the group consisting of adalimumab, pegol, golimumab, infliximab, vedolizumab, ustekinumab, tofacitinib, and certolizumab or certolizumab pegol.

[0065] In some aspects, an expression vector is provided, the vector comprising an exogenous polynucleotide encoding a 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%, or 100% identity to SEQ ID NO:19.

[0066] 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 still other embodiments, the polynucleotide encodes a protein comprising an amino acid sequence that is not identical to SEQ ID NO:3.

[0067] In some aspects, an expression system is provided, which includes a host cell and an expression vector comprising the exogenous polynucleotide described above.

[0068] In some embodiments, the host cell is a prokaryotic or eukaryotic cell. In other embodiments, the host cell is a mammalian cell, a yeast cell, or a bacterial cell. In yet other embodiments, the bacterial cell is E. coli. In yet other embodiments, the mammalian cell is a CHO cell.

[0069] In some aspects, methods of producing the proteins are provided.

[0070] In some embodiments, the method of producing the protein comprises transforming or transfecting the host cell with the expression vector and culturing the transformed or transfected host cell under conditions sufficient for expression of the protein encoded by the exogenous polynucleotide, hi other embodiments, the method further comprises purifying the protein from the transformed or transfected host cell and culture medium.

[0071] In some aspects, methods of treating disorders, such as disorders associated with intestinal epithelial barrier function, are provided, which utilize any of the sequences disclosed in this application and in the Sequence Listing. [Brief explanation of the drawings]

[0072] [Figure 1] 1A and 1B show the restoration of epithelial barrier integrity by SG-11 after inflammation-induced barrier disruption, which is described in Example 2. [Figure 2] Figure 2A shows the effect of SG-11 administration on TNF-α production induced by heat-killed E. coli (HK E. coli), which is described in Example 3. Figure 2B shows the effect of SG-11 administration on IL-23 production induced by HK E. coli, which is described in Example 3. [Figure 3] FIG. 3 shows the effect of SG-11 administration on IL-10 production induced by HK E. coli, which is described in Example 4. [Figure 4] FIG. 4 shows the effect of SG-11 administration on mucin expression after stimulation with HK E. coli, which is described in Example 5. [Figure 5] FIG. 5 shows the effect of SG-11 administration on epithelial cell wound healing, as described in Example 6. [Figure 6] FIG. 6 shows the effect of SG-11 administration on epithelial-centered barrier function readouts in the DSS model of inflammatory bowel disease, which is described in Example 7. [Figure 7]FIG. 7 shows the effect of SG-11 administration on inflammatory readouts in response to barrier dysfunction in the DSS model of inflammatory bowel disease, which is described in Example 7. [Figure 8] FIG. 8 shows the effect of SG-11 administration on body weight in the DSS model of inflammatory bowel disease, which is described in Example 7. [Figure 9] FIG. 9 shows the effect of SG-11 administration on macroscopic lesions in the DSS model of inflammatory bowel disease, which is described in Example 7. [Figure 10] Figures 10A, 10B, and 10C show the results of histopathological analysis of proximal (Figure 10A), distal (Figure 10B), and both proximal and distal (Figure 10C) tissues from a DSS model of inflammatory bowel disease, which is described in Example 7. [Figure 11] Figure 11A shows the effect of SG-11 administration on colon length in a DSS model of inflammatory bowel disease, which is described in Example 7. Figure 11B shows the effect of SG-11 administration on colon weight-to-length ratio in a DSS model of inflammatory bowel disease, which is described in Example 7. [Figure 12] FIG. 12 shows epithelial barrier integrity after SG-11 treatment in the DSS model of inflammatory bowel disease, which is described in Example 8. [Figure 13] FIG. 13 shows an inflammation-centric readout of barrier function in the DSS model of inflammatory bowel disease, which is described in Example 8. [Figure 14] FIG. 14 shows prevention of weight loss in the DSS model of inflammatory bowel disease, which is described in Example 8. [Figure 15] Figure 15A shows the effect of SG-11 administration on colon length in a DSS model of inflammatory bowel disease, which is described in Example 8. Figure 15B shows the effect of SG-11 administration on colon weight-to-length ratio in a DSS model of inflammatory bowel disease, which is described in Example 8. [Figure 16]Figures 16A, 16B, and 16C show the results of histopathological analysis of proximal (Figure 16A), distal (Figure 16B), and both proximal and distal (Figure 16C) tissues from a DSS model of inflammatory bowel disease, which is described in Example 8. [Figure 17] FIG. 17 shows the results of a multiple sequence alignment analysis of SG-11 (SEQ ID NO:7) with similar protein sequences from Roseburia species. [Figure 18] Figure 18 shows the effect of conditions from Figures 18A, 18B, 18C, 18D, 18E, 18F, 18G, 18H, and 18I on the stability of SG-11. See Example 14 for conditions related to Figures 18A-18I. [Figure 19] Figure 19 shows the effect of conditions from Figures 19A, 19B, 19C, 19D, 19E, 19F, 19G, 19H, and 19I on the stability of SG-11V5. See Example 14 for conditions related to Figures 19A-19I. [Figure 20] FIG. 20 shows restoration of epithelial barrier integrity by SG-11 and SG-11 mutants after inflammation-induced barrier disruption, as described in Example 15. [Figure 21] 21A and 21B show epithelial barrier integrity after treatment of the DSS model of inflammatory bowel disease with SG-11 and SG-11 mutants, which is described in Example 16. [Figure 22] 22A and 22B show inflammation-centric readouts of barrier function in the DSS model of inflammatory bowel disease, which is described in Example 16. [Figure 23] 23A and 23B show the effect of treatment with SG-11 or SG-11 mutants on weight loss in the DSS model of inflammatory bowel disease, which is described in Example 16. [Figure 24] FIG. 24 shows the effect of administration of SG-11 or SG-11 mutants on macroscopic lesions in the DSS model of inflammatory bowel disease, which is described in Example 16. [Figure 25] 25A and 25B show the effect of treatment with SG-11 or SG-11 mutants on colon length in the DSS model of inflammatory bowel disease, which is described in Example 16. [Figure 26] 26A and 26B show the effect of treatment with SG-11 or SG-11 mutants on colon weight-to-length ratio in the DSS model of inflammatory bowel disease, which is described in Example 16. [Figure 27] Figure 27A shows the results of a multiple sequence alignment analysis between 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), and Figure 27B shows the percent identity matrix results based on the multiple sequence alignment analysis. The multiple alignment analysis described herein used the Clustal Omega program provided by EMBL-EBI. DETAILED DESCRIPTION OF THE INVENTION

[0073] Detailed Description The present disclosure provides novel protein therapeutics useful for treating subjects suffering from symptoms associated with gastrointestinal disorders. For example, these proteins can promote or enhance epithelial barrier function and / or integrity. The proteins can also suppress inflammatory immune responses in IBD individuals. The protein therapeutics described herein are useful for treating many diseases associated with impaired barrier function or integrity of gastrointestinal epithelial cells and intestinal inflammation.

[0074] The present disclosure also provides protein variants that have therapeutic activity equal to or greater than that of the original protein, but that have improved stability throughout the manufacture and processing of protein therapeutic products, as well as under long-term storage conditions.

[0075] definition Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. Generally, the nomenclature used in connection with, and techniques related to, 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. Accordingly, while the following terms are believed to be well understood by those of ordinary skill in the art, the following definitions are provided to facilitate description of the subject matter disclosed herein.

[0076] Throughout this specification, the term "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of the stated component or group of components, but not the exclusion of other components or groups of components.

[0077] The term "a" or "an" can refer to one or more of that entity, i.e., it can refer to multiple referents. Thus, the terms "a" or "an," "one or more," "one or more," and "at least one" are used interchangeably herein. Also, reference to "an element" by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that one and only one element be present.

[0078] The term "including" is used to mean "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0079] As used herein, the terms "gastrointestinal" or "gastrointestinal tract," "digestive tract," and "intestine" can be used interchangeably to refer to the series of hollow organs extending from the mouth to the anus, including the mouth, esophagus, stomach, small intestine, large intestine, rectum, and anus. The terms "gastrointestinal" or "gastrointestinal tract," "digestive tract," and "intestine" are not necessarily intended to be limited to any particular portion of the digestive tract.

[0080] As used herein, the term "SG-11" refers to a protein comprising the amino acid sequence of SEQ ID NO:3, as well as variants thereof having the same or similar functional activity as described herein. Accordingly, SG-11 herein can 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 variants or fragments 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 (Nos. 62 / 482,963, filed April 7, 2017; 62 / 607,706, filed December 19, 2017; and 62 / 611,334, filed December 28, 2017; to which this application claims priority, each of which is incorporated herein by reference in its entirety), the term "experimental protein 1" and variants thereof was used, which term is synonymous with SG-11 or variants thereof as used herein.

[0081] A "signal sequence" (also called a "presequence," "signal peptide," "leader sequence," or "leader peptide") refers to a sequence of amino acids located at the N-terminus of a nascent protein that can facilitate secretion of the protein from a cell. The resulting mature form of the extracellular protein lacks the signal sequence, which is cleaved off during the secretion process.

[0082] As used herein, the terms "sequence identity," "percent identity," "percent homology," or statements including, for example, "a sequence 50% identical to" refer to the degree to which sequences are identical nucleotide-by-nucleotide or amino acid-by-amino acid over a comparison window. Thus, "percent sequence identity" can be calculated by: comparing two optimally aligned sequences over a comparison window; determining the number of positions in both sequences where the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) exists to obtain the number of matched positions; dividing the number of matched positions by the total number of positions within the comparison window (i.e., window size); multiplying the result by 100 to obtain the percentage of sequence identity.

[0083] Calculation of sequence similarity or sequence identity (these terms are used interchangeably herein) between sequences can be performed as follows. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). In certain embodiments, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the length of the reference sequence. Next, the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the two molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences.

[0084] The phrases "substantially similar" and "substantially identical" in the context of at least two nucleic acids or polypeptides typically mean that the polynucleotides or polypeptides contain sequences that have 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).

[0085] As used herein, the term "nucleotide change" refers to, for example, a nucleotide substitution, deletion, and / or insertion, as is well understood in the art. For example, mutations include modifications that result in silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded protein or the way the protein is made.

[0086] Related (and derivative) proteins include "mutant" proteins. Mutant proteins may differ from another protein (i.e., a parent protein) and / or from each other by a small number of amino acid residues. A variant can contain one or more amino acid mutations (e.g., amino acid deletions, insertions, or substitutions) compared to the parent protein from which it is derived.

[0087] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids that encode the same amino acid sequence or an amino acid sequence with one or more "conservative substitutions." Examples of conservative substitutions are the replacement of one 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 that affect chain direction: Gly, Pro; (6) aromatic: Trp, Tyr, Phe; and (7) small amino acids: Gly, Ala, Ser. Thus, with respect to amino acids, the term "conservative substitution" refers to the replacement of one or more amino acids with another, chemically similar residue, which substitution generally does not affect the functional properties of the protein. Examples include the substitution of amino acid residues with similar properties, such as small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids, aromatic amino acids, etc. In some aspects, the present disclosure provides proteins with 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 representative examples of conservative amino acid substitutions are provided below.

[0088] 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 non-natural or unnatural amino acids. It includes both D- and L-amino acids. Natural amino acids include those found in nature, such as the 23 amino acids that are linked into peptide chains that form the building blocks of countless proteins. These are primarily L-stereoisomers, although a few D-amino acids are present, for example, in bacterial envelopes and some antibiotics. The 20 "standard" natural amino acids are listed in the table above. "Non-standard" natural amino acids are pyrrolysine (found in methanogens and other eukaryotes), selenocysteine ​​(present in many non-eukaryotes and most eukaryotes), and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts). "Non-natural" or "unnatural" amino acids are non-proteinogenic amino acids (i.e., not naturally encoded or found in the genetic code) that occur in nature or are chemically synthesized. There are over 140 known non-natural amino acids, resulting in thousands of possible combinations. "Modified" amino acids include amino acids (e.g., naturally occurring amino acids) that have been chemically modified to include a group(s) or chemical moiety that does not naturally occur on the amino acid.

[0089] As used herein, a "synthetic nucleotide sequence" or a "synthetic polynucleotide sequence" refers to a nucleotide sequence that is not known to occur in nature or does not occur in nature. Generally, such a synthetic nucleotide sequence will contain at least one nucleotide difference compared to other naturally occurring nucleotide sequences. As used herein, a "synthetic amino acid sequence" or a "synthetic peptide sequence" or a "synthetic polypeptide sequence" or a "synthetic protein sequence" refers to an amino acid sequence that is not known to occur in nature or does not occur in nature. Generally, such a synthetic amino acid sequence will contain at least one amino acid difference compared to other naturally occurring amino acid sequences.

[0090] As used herein, "synthetic protein" or "synthetic therapeutic protein" refers to a protein that comprises an amino acid sequence in which one or more amino acids have been substituted with a different amino acid compared to a naturally occurring amino acid sequence. That is, a "synthetic protein" comprises an amino acid sequence that has been altered to include at least one non-naturally occurring substitution modification at a given amino acid position compared to a naturally occurring amino acid sequence.

[0091] As used herein, the term "about" with respect to percent sequence identity or percent sequence homology of a nucleic acid or amino acid sequence means up to ±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%. When not used in the context of percent sequence identity, "about" means ±1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, depending on the context of the value in question.

[0092] For the most part, the names of natural and unnatural aminoacyl residues used herein follow the naming conventions proposed by the IUPAC Commission on Organic Chemical Nomenclature and the IUPAC-IUB Commission on Biochemical Nomenclature as set forth in "Nomenclature of α-Amino Acids (Recommendations, 1974)" Biochemistry, 14(2), (1975). Where the names and abbreviations of amino acids and aminoacyl residues used in this specification and the appended claims differ from these proposals, this will be made clear to the reader.

[0093] Throughout this specification, unless natural amino acids are referred to by their full names (e.g., alanine, arginine, etc.), they are designated by their conventional three-letter or one-letter abbreviations (e.g., Ala or A for alanine, Arg or R for arginine, etc.). Unless otherwise specified, the three-letter and one-letter abbreviations of amino acids refer to the L-isomer of that amino acid. As used herein, the term "L-amino acid" refers to the "L" isomer of a peptide, and conversely, the term "D-amino acid" refers to the "D" isomer of a peptide (e.g., Dasp, (D)Asp, or D-Asp; Dphe, (D)Phe, or D-Phe). Any L-amino acid residue can be substituted for the D-isomer, so long as the desired function is retained by the peptide. D-amino acids, when referred to using one-letter abbreviations, can be conventionally indicated by lowercase letters.

[0094] For less common or unnatural amino acids, unless they are referred to by their full name (e.g., sarcosine, ornithine, etc.), three- or four-letter codes are frequently used for those residues; examples include Sar or Sarc (sarcosine, i.e., N-methylglycine), Aib (α-aminoisobutyric acid), Dab (2,4-diaminobutanoic acid), Dapa (2,3-diaminopropanoic acid), γ-Glu (γ-glutamic acid), Gaba (γ-aminobutanoic acid), β-Pro (pyrrolidine-3-carboxylic acid), 8Ado (8-amino-3,6-dioxaoctanoic acid), Abu (2-aminobutyric acid), βhPro (β-homoproline), βhPhe (β-homophenylalanine), and Bip (β,β-diphenylalanine), Ida (iminodiacetic acid), etc.

[0095] Some sequences disclosed herein incorporate a "Hy-" moiety at the amino-terminus (N-terminus) of the sequence and either an "-OH" or "-NH2" moiety at the carboxy-terminus (C-terminus) of the sequence. In such cases, unless otherwise specified, the "Hy-" moiety at the N-terminus of the sequence in question denotes a hydrogen atom, corresponding to the presence of a free primary or secondary amino group at the N-terminus, while the "-OH" or "-NH2" moiety at the C-terminus of the sequence denotes an amino group, corresponding to the presence of a hydroxy group or an amide (CONH2) group, respectively, at the C-terminus. In each sequence of the present disclosure, the C-terminal "-OH" moiety can be replaced by a C-terminal "-NH2" moiety, and vice versa.

[0096] As used herein, the term "Ac" refers to acetyl protection by acylation of the C-terminus or N-terminus of a polypeptide. In certain peptides shown herein, NH2 located at the C-terminus of the peptide represents an amino group. As used herein, the term "carboxy" refers to -CO2H.

[0097] As used herein, the term "pharmaceutically acceptable salts" refers to salts or zwitterionic forms of peptides, proteins, or compounds of the present disclosure, which salts are soluble or dispersible in water or oil, suitable for the treatment of diseases without undue toxicity, irritation, or allergic response, and are effective for their intended use at a reasonable benefit / risk ratio. Salts can be prepared during the final isolation and purification of the compounds or separately by reacting the 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, mesitylenesulfonate, methanesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, p-toluenesulfonate, and undecanoate salts. In addition, the amino group in the compounds of the present disclosure can be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfate; decyl, lauryl, myristyl, and steryl 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 base salts. Examples of acid addition salts include chloride salts, citrate salts, and acetate salts.Examples of basic salts include salts with cations 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 found in "Remington's Pharmaceutical Sciences", 17th edition, Alfonso R. Gennaro (ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and its latest edition); "Encyclopaedia of Pharmaceutical Technology", 3rd edition, James Swarbrick (ed.), Informa Healthcare USA (Inc.), NY, USA, 2007; and J. Pharm. Sci. 66: 2 (1977). Also, for a review of suitable salts, see "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, 2002).

[0098] As used herein, the term "at least a portion" or "fragment" of a nucleic acid or polypeptide means a portion having the minimum size characteristic of such sequence, or a larger fragment of the full-length molecule (up to and including the full-length molecule).

[0099] As used herein, the term "primer" refers to an oligonucleotide that is capable of annealing to a target polynucleotide.

[0100] As used herein, the terms "recombinant construct," "expression construct," "chimeric construct," "construct," and "recombinant DNA construct" are used interchangeably herein and are well known to those of skill in the art.

[0101] As used herein, the term "host cell" refers to a cell or cell line into which a recombinant expression vector can be introduced for expression of a polypeptide for the production of that polypeptide.

[0102] As used herein, the terms "isolated," "purified," "separated," and "recovered" refer to a material (e.g., a protein, nucleic acid, or cell) that has been 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 sample containing it. For example, these terms can refer to material that is substantially or essentially free from components that normally accompany it when found in its native state (e.g., in an intact biological system, etc.).

[0103] The terms "patient," "subject," and "individual" can be used interchangeably and refer to either a human or a non-human animal. These terms include mammals such as humans, non-human primates, livestock (e.g., cows, pigs), pets (e.g., dogs, cats), and rodents (e.g., mice, rats). In certain embodiments, these terms refer to a human patient. In exemplary embodiments, these terms refer to a human patient suffering from a gastrointestinal inflammatory disease.

[0104] As used herein, "improved" should be broadly interpreted to include an improvement in a confirmed characteristic of a disease state, which characteristic is generally correlated with or considered by those skilled in the art to be indicative of the disease in question, compared to a control or compared to a known average amount associated with the characteristic in question. For example, "improved" epithelial barrier function associated with the application of a protein of the present disclosure can be demonstrated by comparing the epithelial barrier integrity of a human treated with a protein of the present disclosure with that of an untreated human. Alternatively, the epithelial barrier integrity of a human treated with a protein of the present disclosure can be compared to the average epithelial barrier integrity of a human, as shown in scientific or medical publications known to those skilled in the art. In the present disclosure, "improved" does not necessarily require that the data be statistically significant (i.e., p<0.05); rather, a quantifiable difference indicating that one value (e.g., the average treatment value) is different from another value (e.g., the average control value) can reach the level of "improvement."

[0105] As used herein, "inhibit and suppress" and similar terms should not be construed as requiring complete inhibition or suppression, although complete inhibition or suppression may be desirable in some embodiments. Thus, an "inhibited immune response" or "inhibition of inflammatory cytokines" does not require absolute inhibition.

[0106] Thus, as used herein, the terms "increase," "inhibit," "decrease," or their grammatical equivalents refer to values ​​relative to a reference (e.g., baseline) measurement, e.g., a measurement obtained under comparable conditions (such as a measurement in the same individual before initiating a treatment described herein, or a measurement in a control individual(s) not receiving a treatment described herein). In some embodiments, a suitable control is a baseline measurement, e.g., a measurement in the same individual before initiating a treatment described herein, or a measurement in a control individual(s) not receiving a treatment described herein.

[0107] 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).

[0108] As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic agent (e.g., a peptide, polypeptide, or protein of the present disclosure) that confers a therapeutic effect on a treated subject at a reasonable benefit / risk ratio applicable to any medical treatment. Such a therapeutic effect can be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject indicates or feels an effect). In some embodiments, a "therapeutically effective amount" refers to an amount of a therapeutic agent or composition effective to treat, ameliorate, or prevent (e.g., delay the onset of) the relevant disease or condition and / or to exhibit a detectable therapeutic or prophylactic effect, for example, by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and / or reducing the severity or frequency of symptoms of the disease. A therapeutically effective amount is typically administered in a dosing regimen that may include multiple unit doses. The therapeutically effective amount of a particular therapeutic agent (and / or an appropriate unit dose within an effective dosing regimen) may vary, for example, depending on the route of administration or on the use in combination with other therapeutic agents. Alternatively, or in addition, the specific therapeutically effective amount (and / or unit dose) for a particular patient may depend on various factors, such as the specific form of the disease being treated; the severity of the disease or pre-disease; the activity of the specific therapeutic agent used; the specific composition used; the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and / or excretion rate or metabolic rate of the specific therapeutic agent used; duration of treatment; and similar factors well known in the medical field. The present disclosure utilizes therapeutically effective amounts of novel proteins and compositions comprising the same to treat various diseases, such as gastrointestinal inflammatory diseases or diseases involving dysfunction of the gastrointestinal epithelial barrier. The therapeutically effective amount of the administered protein or composition comprising the same will, in some embodiments, reduce inflammation associated with IBD or restore the integrity and / or function of the gastrointestinal epithelial barrier.

[0109] As used herein, the term "treatment" ("treat" or "treating") refers to administering a therapeutic agent (e.g., a peptide, polypeptide, or protein of the present disclosure) according to a therapeutic regimen that achieves a desired effect in that it partially or completely alleviates, improves, alleviates, suppresses, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition (e.g., chronic or recurrent immune responses and inflammation of the gastrointestinal (GI) tract); in some embodiments, administering a therapeutic agent according to a therapeutic regimen correlates with achieving a desired effect. Such treatment may be treatment for a subject who does not exhibit symptoms of the associated disease, disorder, and / or condition and / or who exhibits only early signs of the disease, disorder, and / or condition. Alternatively, or in addition, such treatment may be treatment for a subject who exhibits one or more established signs of the associated disease, disorder, and / or condition. In some embodiments, treatment may be treatment for a subject who has been diagnosed with the associated disease, disorder, and / or condition. In some embodiments, treatment may be for subjects known to have one or more susceptibility factors that statistically correlate with an increased risk of developing the relevant disease, disorder and / or condition.

[0110] "Pharmaceutical" means that a composition, reagent, method, etc., is capable of producing a pharmaceutical effect, and further that the composition can be safely administered to a subject. "Pharmaceutical effect" can mean, without limitation, that a composition, reagent, or method is capable of stimulating a desired biochemical, genetic, cellular, physiological, or clinical effect in at least one individual mammalian subject, e.g., human, in at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, or a similar percentage of the subject population. "Pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for safe use in animals, particularly in humans. "Pharmaceutically acceptable vehicle" or "pharmaceutically acceptable excipient" refers to a diluent, adjuvant, excipient, or carrier administered with a protein described herein.

[0111] "Preventing" or "prevention" refers to reducing the risk of acquiring a disease or disorder (i.e., preventing at least one clinical symptom of the disease from developing in a subject who may be exposed to or predisposed to the disease but who has not yet experienced or manifested symptoms of the disease, or causing symptoms to develop less severely than would occur in the absence of treatment). "Preventing" or "prevention" can also refer to delaying the onset of a disease or disorder.

[0112] The therapeutic pharmaceutical compositions taught herein can include one or more natural products. However, in certain embodiments, the therapeutic pharmaceutical compositions themselves do not exist in nature. Furthermore, in certain embodiments, the therapeutic pharmaceutical compositions have significantly different properties compared to individual natural counterparts or composition components that may exist in nature. That is, in certain embodiments, the pharmaceutical compositions taught herein (including a therapeutically effective amount of purified protein) have at least one structural and / or functional property that confers significantly different properties to the composition as a whole compared to a single individual component of the composition that may exist in nature. Courts have determined that compositions containing natural products that have significantly different properties compared to individual components that may exist in nature are statutory subject matter. Thus, the therapeutic pharmaceutical compositions taught herein have significantly different properties as a whole. These properties are illustrated in the data and examples taught herein.

[0113] The details of the present disclosure are now described. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, exemplary methods and materials are now described. Other features, objects, and advantages of the present disclosure will be apparent from the detailed description and claims.

[0114] Microbiota-derived therapeutic proteins - Overview of the disclosure Many diseases and disorders are associated with impaired gastrointestinal epithelial cell barrier function or integrity. These diseases and disorders are multifaceted and can be diagnosed in numerous ways. One such disease is inflammatory bowel disease (IBD), whose incidence and prevalence have been increasing over time in various regions around the world, indicating its emergence as a global disease (Molodecky et al., Gastroenterol 142:46-54, 2012). IBD is a collective term describing conditions involving chronic or recurrent immune responses and inflammation of the gastrointestinal (GI) tract. The two most common inflammatory bowel diseases are ulcerative colitis (UC) and Crohn's disease (CD). Both are characterized by abnormal responses of the GI 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, resulting in an inflammatory response in the intestinal lining and chronic inflammation. When this occurs, patients experience the symptoms of IBD.

[0115] IBD involves chronic inflammation of all or part of the digestive tract. Both UC and CD typically present with symptoms such as severe diarrhea, abdominal pain, fatigue, and weight loss. IBD and related disorders can be debilitating and sometimes lead to life-threatening complications.

[0116] Regarding the integrity of the intestinal barrier, loss of intestinal epithelial integrity plays an important pathogenetic 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 adverse changes in the intestinal microbiota induce inappropriate or uncontrolled immune responses that result in damage to the intestinal epithelium. Disruption of this crucial intestinal epithelial barrier allows further infiltration of the microbiota, which in turn induces further immune responses. Therefore, IBD is a multifactorial disease driven, in part, by an excessive immune response against the intestinal microbiota, which can cause defects in epithelial barrier function.

[0117] Microbiota profiling of IBD patients revealed distinct profiles, often characterized by an increase in Proteobacteria species, including adherent and invasive Escherichia coli, at the expense of potentially beneficial microorganisms such as Roseburia spp. (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). Furthermore, a decrease in Roseburia hominis has been linked to dysbiosis in patients with ulcerative colitis. Individuals with IBD have been found to have a 30-50% reduction in commensal bacterial biodiversity, including a reduction in Firmicutes (i.e., Lachnospiraceae) and Bacteroidetes. Further evidence of the role of the gut flora in the pathogenesis of inflammatory bowel disease is that individuals with IBD are more likely than non-IBD individuals to have been prescribed antibiotics 2-5 years before diagnosis. See Aroniadis OC, Brandt LJ, "Fecal microbiota transplantation: past, present, and future," (2013) Curr. Opin. Gastroenterol. 29 (1) (2013): 79-84.

[0118] Protective bacterial communities, probiotics, and bacterial-derived metabolites have been demonstrated to ameliorate disease in various clinical and preclinical studies. For example, fecal microbial transplantation (FMT) experiments have shown some success in IBD patients, but challenges remain (Moayyedi et al., 2015, Gastroenterology, 149:102-109 e106; Qazi et al., 2017, Gut Microbes, 8:574-588; Narula et al., 2017, Inflamm Bowel Dis, 23:1702-1709). Other studies have shown that treatment with probiotics such as VSL#3, Lactobacillus spp., and Bifidobacterium spp. also has 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 L. rhamnosus GG and Amuc-1100 from A. muciniphila, have been shown to promote barrier function and protect in animal models of IBD and metabolic disease, respectively (Yan et al., 2011, J Clin Invest, 121:2242-2253; Plovier et al., Nat Med, 23:107-113).

[0119] While the use of live microbial populations to treat disease is becoming increasingly common, such methods rely on the ability of the administered bacteria to survive within the host or patient's body and interact with host tissues in a beneficial, therapeutic manner. Another approach provided herein is to identify microbially encoded proteins and their variants that can affect cellular function within the host, resulting in a therapeutic effect. Such proteins can be administered, for example, as pharmaceutical compositions containing substantially isolated or purified therapeutic bacterial proteins, or as live biotherapeutics (bacteria) genetically engineered to express the therapeutic protein as an exogenous protein. Furthermore, therapeutic methods involving the administration of therapeutic proteins are not limited to the intestine (small intestine, large intestine, rectum) but can also include the treatment of other disorders within the gastrointestinal tract, such as oral mucositis.

[0120] To identify microbial proteins with therapeutic potential for gastrointestinal inflammatory diseases, we analyzed mucosal biopsies from healthy individuals or individuals diagnosed with IBD (UC) to examine their microbial composition. Comparison of bacterial profiles from healthy individuals and patients identified bacteria likely to be beneficial (higher abundance in healthy individuals than in patients) or harmful (lower abundance in healthy individuals than in patients). Among the bacterial species identified as beneficial was Roseburia hominis, consistent with the above-mentioned studies. Next, extensive bioinformatics analysis was performed to predict proteins encoded by the bacteria and subsequently identify proteins likely secreted by the bacteria. The predicted secreted proteins were subsequently characterized using a series of in vitro assays to study each protein's effects on epithelial barrier integrity, cytokine production and / or release, and wound healing. Proteins identified as functioning in enhancing epithelial barrier integrity were then evaluated in an in vivo mouse model of colitis. One such protein, identified herein as "SG-11," has demonstrated both in vitro and in vivo activity indicating its ability to provide therapeutic benefits for improving epithelial barrier integrity and for treating diseases and disorders associated with epithelial barrier integrity and for treating inflammatory gastrointestinal diseases such as IBD; this is described in more detail below.

[0121] SG-11 protein The protein referred to herein as SG-11 is encoded within a 768 nucleotide sequence (SEQ ID NO:2) present in the genome of Roseburia hominis. The complete genome sequence of a Roseburia hominis strain can be found in GenBank accession number CP003040, which is incorporated herein by reference in its entirety. The 16S rRNA gene sequence of a Roseburia hominis strain can be found in GenBank accession number AJ270482. The full-length protein encoded by the Roseburia hominis genome sequence is 256 amino acids in length (SEQ ID NO:1), with residues 1-24 predicted to be a signal peptide that is cleaved in vivo to generate a 232 amino acid mature protein (encoded by SEQ ID NO:3; SEQ ID NO:4). Recombinant SG-11, when expressed to include an N-terminal methionine (encoded by the codon ATG), produces a mature protein of 233 amino acids (SEQ ID NO:7).

[0122] As detailed in the Examples, e.g., Example 1, SG-11 was recombinantly expressed in a variety of commercially available and routinely used expression vectors. For example, SG-11 (a protein comprising SEQ ID NO:3) was expressed using the pGEX expression vector, which expresses the protein of interest containing a GST tag and a protease site that is cleaved after expression and purification; the pET-28 expression vector, which adds an N-terminal FLAG tag; and the pD451 expression vector, which was used to express the SG-11 protein consisting of SEQ ID NO:7 without an N-terminal tag. Repeated experiments with these proteins demonstrated that minor N- and / or C-terminal changes resulting from the use of different protein expression systems and DNA constructs retained equivalent functional activity in in vivo and in vitro assays. Unless otherwise specified, the term "SG-11" herein is understood to refer to the amino acid sequence set forth herein as SEQ ID NO:3, and such variants of the protein comprising 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 can include amino acid residue changes (substitutions, insertions, deletions), as well as modifications such as fusion constructs and post-translational modifications (phosphorylation, glycosylation, etc.). Some specific examples of SG-11 proteins and nucleic acids encoding them are shown in Table 1 below.

[0123] (Table 1) TIFF0007789303000001.tif229154TIFF0007789303000002.tif215154TIFF0007789303000003.tif222154TIFF0007789303000004.tif68154

[0124] Epithelial barrier function in disease Recent studies have identified the key roles of both genetic and environmental factors in the pathogenesis of IBD. Markus Neurath, “Cytokines in Inflammatory Bowel Disease,” Nature Reviews Immunology, Vol. 14, 329-342 (2014). The combination of these IBD risk factors appears to induce deleterious changes in epithelial barrier function, thereby enabling the translocation of luminal antigens (e.g., bacterial antigens from commensal microbiota) into the intestinal wall. Ibid. Subsequently, abnormal and exaggerated responses to such environmental triggers, such as increased release of inflammatory cytokines, lead to subclinical or acute mucosal inflammation in genetically susceptible hosts. Ibid. Therefore, the importance of proper epithelial barrier function in IBD is clear: subjects who are unable to resolve acute intestinal inflammation develop chronic intestinal inflammation induced by uncontrolled activation of the mucosal immune system. In particular, mucosal immune cells, such as macrophages, T cells, and subsets of innate lymphoid cells (ILCs), appear to respond to microbial products or antigens from the commensal microbiota by producing cytokines that can promote chronic inflammation in the gastrointestinal tract. Consequently, restoring proper epithelial barrier function to patients may be crucial in resolving IBD.

[0125] The therapeutic activity of SG-11 has been confirmed both in vitro and in vivo, in part, by its beneficial effects on epithelial barrier function. As shown in Example 2, SG-11 is active in enhancing epithelial barrier integrity, as demonstrated by an in vitro transepithelial electrical resistance (TEER) assay. The TEER assay 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. The cells are cultured until intercellular tight junctions form, and the functional capacity of the barrier is assessed by measuring transepithelial electrical resistance. Insults, such as heat-killed E. coli, decrease the electrical resistance across the epithelial layer. Useful control reagents for the TEER assay include staurosporine and myosin light chain kinase inhibitors. Staurosporine is a broad-spectrum kinase inhibitor derived from Streptomyces staurosporeus that induces apoptosis. This reagent disrupts approximately 98% of gap junctions, resulting in a decrease in electrical resistance in the TEER assay. Myosin light chain kinase (MLCK) is the terminal effector of a signaling cascade induced by inflammatory cytokines, causing contraction of the actomyosin ring around junctions, resulting in gap junction separation. MLCK inhibition prevents the disruption of tight junctions. MLCK inhibitors in the TEER assay should reduce or prevent the decrease in electrical resistance in the TEER assay.

[0126] In some embodiments, the SG-11 protein or variant or fragment thereof described herein can be characterized by its ability to enhance 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 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.

[0127] Additionally, Example 6 demonstrates that SG-11 protein can enhance or promote epithelial wound healing, a function that plays a role in maintaining or repairing epithelial barriers, such as the intestinal or mucosal epithelial barrier.

[0128] Given the efficacy of SG-11 in restoring barrier integrity in vitro, we analyzed its ability to reduce damage in rodent models of IBD in vivo. Examples 7 and 8 (SG-11) and Example 16 (SG-11 mutant) describe studies conducted using the DSS (dextran sodium sulfate) animal model, a widely accepted model for studying agents for IBD (Chassaign et al., 2014, Curr Protoc Imunol, 104:Unit-15.25; Kiesler et al., 2015, Cell Mol Gastroenterol Hepatol). DSS is a sulfated polysaccharide that is directly toxic to the colonic epithelium, causing epithelial cell damage that leads to impaired barrier function due to gap junction disruption. In these experiments, mice were treated with SG-11 either before (Example 7) or after (Example 8) induction of colitis. As a positive control, mice were also treated with Gly2-GLP2, a stable analog of glucagon-like peptide 2 (GLP2). Gly2-GLP2 is known to promote epithelial cell proliferation and reduce colonic damage in experimental mouse colitis models. Results from the DSS study showed that SG-11 protein was effective in reducing weight loss in the DSS model, an important indicator of the clinical efficacy of IBD medications. Treatment with SG-11 also reduced gross lesions and intestinal histopathological analysis scores.

[0129] Note that treatment with SG-11 improved the 4KDa-FITC intestinal permeability readout and reduced serum levels of LPS-binding protein (a marker of LBP exposure) in Example 7, whereas no significant effect was observed for treatment with SG-11 or Gly2-GLP2 in Example 8. This is not surprising, given that the animals in Example 8 were treated with DSS for 7 days prior to switching to regular drinking water and treatment with SG-11 or Gly2-GLP2. This prior exposure to DSS results in damage to the intestinal epithelium, translocation of LPS across the disrupted epithelial barrier, and induction of LBP secretion. However, based on 4KDa-FITC dextran measurements, repair of the epithelial barrier appears to occur rapidly within 3–4 days after switching to regular drinking water (data not shown; Figure 12). Therefore, at the time of measurement (after 6 days of treatment), it is difficult to detect an improvement in the 4KDa-FITC permeability readout in treated animals relative to untreated animals. Furthermore, serum LBP levels may be unrelated to the restoration of barrier function in animals exposed to DSS for extended periods prior to therapeutic treatment (Example 8). For example, hepatocytes activated by LPS transfusion during DSS exposure produce and secrete large amounts of LBP. Therefore, without being bound by theory, short-term studies may not allow enough time for hepatocyte inactivation and clearance of LBP from the serum of DSS-treated animals. Therefore, although measuring serum LBP at later time points in a continuing study would show a decrease in serum LBP levels, if barrier function is restored in both treated and untreated animals before LBP is cleared from the serum, the decrease in serum LBP would likely be similar in both animals.

[0130] SG-11 mutants Given the therapeutic value of SG-11 and its use in disease treatment, the protein was further characterized and sequence modified to alter its primary structure in a manner that would optimize its pharmaceutical formulation and long-term storage.

[0131] As described in Example 9, SEQ ID NO:7 was used to perform a BLAST search of the GenBank non-redundant protein database to identify proteins with similar amino acid sequences that are functional homologs of SG-11 or have similar function(s) to SG-11. Three such proteins were identified, and their predicted mature sequences (excluding the N-terminal signal peptide) were aligned with SEQ ID NO:7 to identify relatively conserved regions and individual positions within the proteins. These three proteins are disclosed herein as SEQ ID NO:21 (derived from GenBank accession number WP_006857001), SEQ ID NO:22 (derived from GenBank accession number WP_075679733), and SEQ ID NO:23 (derived from GenBank accession number WP_055301040) (FIG. 17). Accordingly, provided herein are pharmaceutical compositions comprising one of these three proteins or variants or fragments thereof, and methods of treating diseases associated with impaired barrier function and / or gastrointestinal diseases or disorders 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 variants or fragments thereof.

[0132] Specific examples of the amino acid sequence of SG-11V5, a mutant of SG-11, and the nucleic acid sequence encoding it are shown in Table 2 below.

[0133] (Table 2) TIFF0007789303000005.tif149154

[0134] To enhance the stability of SG-11 protein for use in pharmaceutical formulations and clinical applications, we conducted studies to identify and characterize post-translational modifications (PTMs) of purified SG-11 protein. These experiments are described in Examples 10-11. Such analyses revealed that SG-11 protein can undergo at least the PTMs of methionine oxidation and asparagine deamidation. Furthermore, experiments described in Example 12 suggest that the cysteines in SG-11 are unlikely to form disulfide bonds in the functional conformation of the purified, active protein, and that the free sulfhydryl groups in SG-11 may cause aggregation in solutions containing the purified protein. Based on these stability studies, despite the conserved nature of the residues in SG-11 observed in the multiple sequence alignment (Figure 17), we decided to test whether the cysteines at positions 147 and / or 151 (relative to SEQ ID NO:7) could be substituted with different amino acids. Substitution of the conserved asparagines at positions 53 and 83 was also explored. In a specific example, the SG-11 sequence of SEQ ID NO:7 is modified to introduce the C147V and C151S substitutions to generate SEQ ID NO:11 (SG-11V1). The C147V and C151S substitutions are also present in the provided SG-11 variants SG-11V2 (SEQ ID NO:13; containing G84D, C147V, C151S), SG-11V3 (SEQ ID NO:15; containing N83S, C147V, C151S), SG-11V4 (SEQ ID NO:17; containing N53S, G84D, C147V, C151S), and SG-11V5 (SEQ ID NO:19; containing N53S, N83S, C147V, C151S).

[0135] Example 13 shows that PTMs (methionine oxidation and asparagine deamidation) are significantly reduced in SG-11V5 compared to SG-11 (SEQ ID NO:7). This reduction was observed at different temperatures and in different storage buffers. Example 14 describes experiments performed to determine whether an SG-11 mutant (SG-11V5; SEQ ID NO:19) containing a cysteine ​​substitution affects aggregation of the protein in storage buffer. The results show that the SG-11V5 mutant exhibits less aggregation compared to SG-11 (SEQ ID NO:7) when tested in different storage buffers.

[0136] Notably, even though the amino acids substituted to generate SG-11V5 occur in relatively conserved regions of the SG-11 protein, these four residues could be substituted without loss of functional activity (Examples 15 and 16, described in more detail below).

[0137] Based on the experimental data and analysis described herein, a contemplated mutant of SG-11 (e.g., SEQ ID NO:3 or SEQ ID NO:5) was designed to substitute any one or more of amino acids N53, N83, G84, C147, and C151 of SEQ ID NO:7 (the indicated substitutions are residue positions relative to SEQ ID NO:7). A specific example of this mutant is provided in Table 3 below as SEQ ID NO:33, where each residue at positions 53, 83, 84, 147, and 151 is designated as X, indicating that one or more of these five residues may 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 yet 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.

[0138] In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:34, wherein X53 is any amino acid other than N, X83 is any amino acid other than N, X84 is any amino acid other than G, X147 is any amino acid other than C, and / or X151 is any amino acid other than C.

[0139] (Table 3) TIFF0007789303000006.tif42146

[0140] In another example, certain amino acids of the taught proteins can be substituted for other amino acids within the protein structure without significantly reducing the interactive binding ability with structures such as substrate molecules, receptors, or binding sites, e.g., antigen-binding regions of antibodies. Thus, these proteins are biologically functional equivalents of the disclosed proteins (e.g., including SEQ ID NO:3 or variants thereof). So-called "conservative" changes do not destroy the biological activity of the protein because the structural changes do not affect the protein's ability to perform its intended function. Thus, the inventors believe that various modifications can be made to the gene and protein sequences disclosed herein while still meeting the objectives of the present disclosure.

[0141] 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").

[0142] Importantly, the SG-11 mutant protein containing SEQ ID NO:19 maintained its activity in both TEER assays (Example 15) and in an in vivo DSS mouse model (Example 16), indicating that SG-11 mutants can maintain therapeutic function equivalent to that of wild-type SG-11. Specifically, in vitro TEER experiments and in vivo DSS model experiments were performed using SG-11 (SEQ ID NO:7) and SG-11V5 (SEQ ID NO:19) in parallel. Example 15 demonstrates that SG-11 and SG-11V5 have essentially the same functional ability to reduce TEER in vitro. Example 16 was performed to compare the in vivo efficacy of SG-11 and SG-11 mutants, as described in Examples 7 and 8 (DSS model mice were treated with SG-11 before or after DSS treatment). Example 16 also compared administering the protein to mice before (as described in Example 16A) and after (as described in Example 16B) DSS treatment. SG-11 and the SG-11 mutant reduced weight loss (Figures 23A and 23B) and clinical scores of gross lesions (Figure 24). Again, SG-11 reduced intestinal permeability and serum LBP levels, while SG-11V5 is shown to reduce intestinal permeability and serum LBP levels in Example 16A in a dose-dependent manner (Figures 21A and 22A). Similar to the results observed in Examples 7 and 8, SG-11 and SG-11 mutant proteins did not reduce intestinal permeability or serum LBP levels in Example 16B (where therapeutic proteins were administered after a prolonged challenge with DSS and results were observed over a limited period of time). As noted above, continued studies are expected to demonstrate reductions in both permeability and serum LBP levels.

[0143] In view of these data, provided herein are therapeutic proteins that are at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a protein comprising the amino acid sequence of SEQ ID NO:3, or a fragment thereof. In another embodiment, 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 a variant of SEQ ID NO:3 or SEQ ID NO:7, where 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 therapeutic variant protein comprises a non-naturally occurring variant of SEQ ID NO:3. Stated another way, the therapeutic protein contains 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 from residue 2 to residue 233 of SEQ ID NO:7.

[0144] In some embodiments, the SG-11 protein can be modified or altered by the insertion or deletion of one or more amino acids. Insertions can be the addition of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10, 1-20, 1-30, 1-40, or 1-50) amino acids to the N-terminus and / or C-terminus of the protein, and / or the insertion of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10, 1-20, 1-30, 1-40, or 1-50) amino acids at a position between the N-terminal and C-terminal amino acids. Similarly, deletions of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10, 1-20, 1-30, 1-40, or 1-50) amino acids can be present at either the N-terminus or C-terminus, as well as internally.

[0145] In some embodiments, modified or mutant proteins are provided that comprise at least one non-naturally occurring amino acid substitution relative to SEQ ID NO: 3. In other embodiments, the mutant 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 further embodiments, the modified protein comprises the amino acid sequence 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).

[0146] In some embodiments, therapeutic proteins according to the present disclosure include any one of variant proteins (e.g., SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19) that also retain one or more activities of the full-length mature protein, e.g., as shown in SEQ ID NO:3 or SEQ ID NO:7.

[0147] Polynucleotide sequences encoding these proteins are also contemplated. Those skilled in the art are aware that two polynucleotide sequences encoding a single polypeptide sequence can share relatively low sequence identity due to the degenerate nature of the genetic code. For example, if every codon in a polynucleotide encoding a 233-amino acid sequence contains at least one substitution at its third position, the sequence identity between the two polynucleotides is calculated to be approximately 67%. 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 the 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.

[0148] In some embodiments, the taught proteins have significantly different structural and / or functional properties compared to a protein comprising or consisting of SEQ ID NO:3.

[0149] As used herein, the term "SG-11 variant" can include, for example, an SG-11 protein that is identical or non-identical to a protein comprising the sequence of SEQ ID NO:3, and that is further modified, such as by a PTM or fusion or linkage to a second agent (e.g., a protein or peptide).

[0150] Protein PTMs occur in vivo and can increase the functional diversity of the proteome through the covalent addition of functional groups or proteins, proteolytic cleavage of regulatory subunits, or degradation of the entire protein. Proteins prepared and isolated according to the present disclosure can 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 and includes, but is 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-prenylation, S-palmitoylation), and proteolysis, which can affect nearly every aspect of normal cell biology and pathogenesis. The isolated and / or purified SG-11 protein or variants or fragments thereof disclosed herein can contain one or more of the above post-translational modifications.

[0151] The SG-11 protein or its variants or fragments can be a fusion protein in which the N-terminal and / or C-terminal domains are fused to a second protein via a peptide bond. Commonly used fusion partners well known to those skilled in the art include, but are not limited to, human serum albumin and its crystallizable fragments, or the constant domain Fc of IgG. In some embodiments, the SG-11 protein or its variants or fragments is linked to a second protein or peptide containing a cysteine ​​residue via a disulfide bond.

[0152] As noted above, modifications and / or changes (e.g., substitutions, insertions, deletions) can be made to the structure of the proteins described herein. Accordingly, the present disclosure contemplates variations in the sequences of these proteins and the nucleic acids encoding them, which may nonetheless retain comparable activity with respect to the functional activity assessed in various in vitro and in vivo assays and therapeutic aspects of the present disclosure. With respect to functional equivalents, as will be appreciated by those skilled in the art, inherent in the definition of "biologically functional equivalent" proteins and / or polynucleotides is the concept that there are limits to the number of changes that can be made to a particular portion of a molecule while still retaining an acceptable level of equivalent biological activity.

[0153] It is also believed that SG-11 protein or its variant or fragment can reduce disease-related weight loss, improve clinical pathology score, and / or minimize colon shortening in subjects when administered to them.In some embodiments, the subject is a mammal with genetically or clinically induced inflammatory disorder or epithelial barrier dysfunction.Alternatively, the animal has idiopathic gastrointestinal disorder accompanied by reduced epithelial barrier function or intestinal inflammatory disorder.In other embodiments, the mammal is a human, a non-human primate, or a rodent.The rodent can be a mouse or a rat.

[0154] In some embodiments, the SG-11 protein or variant or fragment thereof according to the present disclosure can regulate cytokine production and / or secretion in an in vitro assay or in a subject administered the protein. In some embodiments, cytokine secretion is reduced in vitro. The level of cytokines produced and / or secreted in an in vitro assay or in a subject administered the protein can be measured in the subject's blood, serum, and / or plasma. Administration of the protein can reduce serum levels of pro-inflammatory cytokines, such as one or more of 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. Alternatively, the cytokine is an anti-inflammatory cytokine, in which case administration of the protein increases serum levels of anti-inflammatory cytokines, such as IL-4, IL-10, IL-13, IFN-α, and TGF-β.

[0155] In some embodiments, the SG-11 protein or variant or fragment thereof has the functional ability to reduce gastrointestinal inflammation when administered to a subject, such as a mammal (e.g., a rodent, a non-human primate, or a human). In other embodiments, the protein has the functional ability to reduce inflammatory (i.e., pro-inflammatory) cytokines when administered to a subject. In still other embodiments, the protein can reduce TNF-α and / or IL-23 when administered to a subject. In still other embodiments, the protein has the functional ability to increase anti-inflammatory cytokines when administered to a subject. In some aspects, the proteins of the present disclosure can increase IL-10 when administered to a subject.

[0156] The SG-11 protein or variant or fragment thereof according to the present disclosure, 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 readout, induce or increase mucin gene expression (e.g., muc2 expression), enhance the structural integrity and / or functionality of the gastrointestinal (e.g., small intestine, large intestine, oral cavity, and / or esophagus) mucosal barrier, and / or reduce inflammation of the gastrointestinal tract.

[0157] In some embodiments, the SG-11 protein or variant or fragment thereof resulting from said amino acid substitutions, insertions, and / or deletions relative to SEQ ID NO:3 or SEQ ID NO:7 maintains substantially the same level of functional activity as the protein of SEQ ID NO:7 or SEQ ID NO:19 (e.g., is capable of increasing electrical resistance in a TEER assay in which an epithelial cell layer is disrupted, such as by heat-killed E. coli). The variant proteins may be useful as therapeutic agents for treating or preventing a variety of diseases, including, but not limited to, inflammatory diseases and / or barrier dysfunction, including inflammation of the gastrointestinal (including oral, esophageal, and intestinal) mucosa and impaired gap junction integrity of intestinal epithelial cells. In some embodiments, the modified protein, when administered to an individual suffering from or predisposed to an inflammatory disease and / or barrier dysfunction, has one or more of the following effects: improving epithelial barrier integrity, for example, after inflammation-induced barrier disruption; suppressing production of at least one inflammatory 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. Furthermore, modified or mutant SG-11 proteins can be used to treat or prevent disorders or diseases, such as, but not limited to, inflammatory bowel disease, ulcerative colitis, Crohn's disease, short bowel syndrome, GI mucositis, oral mucositis, chemotherapy-induced mucositis, radiation-induced mucositis, necrotizing enterocolitis, pouchitis, metabolic disease, celiac disease, inflammatory bowel syndrome, or chemotherapy-associated steatohepatitis (CASH).

[0158] SG-11 protein can promote epithelial wound healing, as demonstrated, for example, in Example 6. Accordingly, provided herein is a therapeutic protein comprising the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:7, or a variant or fragment thereof, which protein can enhance wound healing in an in vitro assay.

[0159] Treatment method The SG-11 proteins described herein, including their mutants (e.g., amino acid substitutions, deletions, insertions), modifications (e.g., glycosylation, acetylation), SG-11 fragments, and fusions thereof, are intended to be used to treat subjects diagnosed with or suffering from a disorder associated with inflammation in the gastrointestinal tract and / or epithelial barrier dysfunction in the gastrointestinal tract.

[0160] Provided herein is a method for treating a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising the SG-11 protein or a fragment or variant thereof described in the present disclosure.The subject may be diagnosed with inflammatory bowel disease, ulcerative colitis, pediatric UC, Crohn's disease, pediatric Crohn's disease, short bowel syndrome, GI mucositis, oral mucositis, mucositis of the esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (colon) and / or rectum, chemotherapy-induced mucositis, radiation-induced mucositis, necrotizing enterocolitis, pouchitis, metabolic disease, celiac disease, irritable bowel syndrome, or chemotherapy-associated steatohepatitis (CASH).Administering the SG-11 pharmaceutical composition described herein can also be useful for wound healing.

[0161] inflammatory bowel disease Inflammatory bowel disease (IBD) classically includes ulcerative colitis (UC) and Crohn's disease (CD). The etiology of IBD is unknown. Genetic predisposition has been suggested, and many environmental factors, including bacteria, viruses, and possibly dietary antigens, can trigger the ongoing intestinal inflammatory cascade. Ibid. IBD can cause severe diarrhea, pain, fatigue, and weight loss. IBD is debilitating and sometimes leads to life-threatening complications. Thus, in some embodiments, the therapeutic methods described herein are effective in reducing, preventing, or eliminating any one or more of the above symptoms, comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition containing an SG-11 protein or a variant or fragment thereof. In some embodiments, the therapeutic method results in remission.

[0162] ulcerative colitis Ulcerative colitis is an inflammatory bowel disease that causes long-term inflammation and pain (ulcers) in the innermost lining of the large intestine (colon) and rectum.

[0163] Ulcerative colitis typically presents as a shallow, continuous inflammation extending proximally from the rectum to involve the entire colon in many patients. Fistulas, fissures, abscesses, and small-bowel involvement are absent. Patients with localized disease (e.g., proctitis) usually have mild but frequently recurrent symptoms, whereas patients with global colitis generally have severe symptoms and often require hospitalization. Botoman et al., “Management of Inflammatory Bowel Disease,” Am. Fam. Physician, Vol. 57(1):57-68 (Jan 01, 1998) (internal citations omitted). Ulcerative colitis is therefore an IBD that causes long-term inflammation and pain (ulcers) in the innermost lining of the large intestine (colon) and rectum.

[0164] Crohn's disease Unlike ulcerative colitis, Crohn's disease can involve the entire intestinal tract, from the mouth to the anus, with discrete focal ulcerations, fistula formation, and perianal involvement. The terminal ileum is commonly affected most, usually with varying degrees of colonic involvement. Some patients have perianal disease with fissures and fistula formation. Only 2–3% of patients with Crohn's disease have clinically significant upper gastrointestinal involvement. Botoman et al., “Management of Inflammatory Bowel Disease,” Am. Fam. Physician, Vol. 57(1):57–68 (Jan 01, 1998) (internal citations omitted). Thus, Crohn's disease is an IBD that causes inflammation of the lining of the gastrointestinal tract. In Crohn's disease, inflammation often extends deep into the affected tissue. Such inflammation can involve various regions of the gastrointestinal tract, i.e., the large intestine, the small intestine, or both. Collagenous colitis and lymphocytic colitis are also considered inflammatory bowel diseases, but are usually considered separate entities from classical inflammatory bowel disease.

[0165] Clinical parameters of inflammatory bowel disease As previously mentioned, inflammatory bowel diseases include ulcerative colitis and Crohn's disease. There are numerous scores and clinical markers available and known to those skilled in the art that can be used to assess the effectiveness of the administered proteins described herein in treating these diseases.

[0166] There are two general approaches to evaluating patients with IBD. The first involves a visual examination of the mucosa, observing signs of mucosal damage, given the fact that IBD is evidenced by inflammation and the appearance of ulcers in the gastrointestinal tract. Any technique that allows for evaluation of the mucosa can be used. Examples include barium enema, X-ray, 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). Using these techniques, areas of inflammation, ulcers, and abnormal growths such as polyps are identified.

[0167] Scoring systems based on visual inspection of the gastrointestinal tract exist to determine the status and severity of IBD, and these scoring systems aim to ensure uniform evaluation of various patients in the diagnosis and monitoring of these diseases, as well as in clinical research evaluations, even though patients may be evaluated by different medical professionals. Examples of visual inspection-based evaluations of UC are discussed and compared in Daperno M et al. (J Crohns Colitis. 2011 5:484-98).

[0168] Clinical scoring systems also exist for the same purpose. Findings on endoscopic or other examinations of the mucosa are incorporated into these clinical scoring systems, but they also incorporate symptom-based data such as stool frequency, rectal bleeding, and the physician's global assessment. Because IBD has a variety of symptoms that affect quality of life, some of these scoring systems allow for not only the quantification of symptoms but also a quantitative assessment of the impact on quality of life.

[0169] One example of a scoring system for UC is the Mayo scoring system (Schroeder et al., N Eng J Med, 1987, 317:1625-1629), although other less commonly used scoring systems exist, 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 Colonoscopic Index of Severity (UCCIS) (Thia et al., 2011, Inflamm Bowel Dis, 17:1757-1764); and the Rachmilewitz Endoscopic Index (Rachmilewitz, 1989, BMJ, 298:82-86); Sutherland index (also known as the 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.Accordingly, also contemplated herein are methods for treating a subject diagnosed with and suffering from UC, wherein the treatment comprises administering an SG-11 protein or a variant or fragment thereof described herein, and wherein the treatment results in a reduction in UC symptoms as determined by measurement of the UCEIS score, Baron score, UCCIS score, Rachmilewitz endoscopic index, Sutherland index, and / or Blackstone index.

[0170] One example of a scoring system for CD is the Crohn's Disease Activity Index (CDAI) (Sands B et al 2004, N Engl J Med 350(9):876-85); most major studies have adopted the CDAI to define response or disease remission. Calculation of the CDAI score includes assessment of the following: number of liquid stools over a 7-day period, incidence and severity of abdominal pain over a 7-day period, general health status over a 7-day period, extraintestinal complications (e.g., arthritis / arthralgia, iritis / uveitis, erythema nodosum, pyoderma gangrenosum, aphthous stomatitis, anal fissures / fistulas / abscesses, and / or fever >37.8°C), use of antidiarrheal medications over a 7-day period, presence of abdominal masses, hematocrit, and weight as the ideal / observed weight ratio or percent deviation from standard weight. Based on the CDAI score, CD is classified as either asymptomatic remission (0-149 points), mild to moderately active CD (150-220 points), moderate to severely active CD (221-450 points), or severely active fulminant disease (451-1000 points). In some embodiments, a treatment method comprising administering a therapeutically effective amount of an SG-11 protein or a variant or fragment thereof to a patient diagnosed with CD results in a reduction in the CD diagnostic score. For example, the score can change the diagnosis from severely active to mild or moderately active, or to asymptomatic remission.

[0171] The Harvey-Bradshaw Index is a simpler version of the CDAI that consists of only clinical parameters (Harvey et al., 1980, Lancet 1(8178):1134-1135). The Inflammatory Bowel Disease Questionnaire (IBDQ) also addresses impact on quality of life (Irvine et al., 1994, Gastroenterology 106: 287-296). Alternative methods include the CDEIS and SES CD (see, e.g., Levesque, et al. (2015) Gastroenterol. 148:37 57).

[0172] In some embodiments, a method for treating IBD, e.g., UC, is provided, wherein the treatment is effective in reducing the Mayo score. The Mayo score is a combination of an endoscopic scale and a clinical scale used to assess the severity of UC, ranging from 1 to 12. The Mayo score is composed of subscores for stool frequency, rectal bleeding, flexible rectosigmoidoscopy or colonoscopy findings, and a physician's global assessment (Paine, 2014, Gastroenterol Rep 2:161-168). Regarding rectal bleeding, 1 point is assigned if blood streaks are present in half or less of the stool, 2 points are assigned if blood is present in most of the stool, and 3 points are assigned if pure blood is passed. Regarding stool frequency, 0 points are assigned for one stool per day, 1 or 2 more stools than usual, 2 points are assigned for 3 or 4 more stools than usual, and 3 points are assigned for 5 or more stools than usual. For the endoscopic component, a score of 0 indicates normal mucosa or inactive UC, a score of 1 is given for mild disease with findings of mild friability, reduced vascular pattern, and mucosal erythema, a score of 2 is given for moderate disease with friability, erosion, complete loss of vascular pattern, and marked erythema, and a score of 3 is given for ulceration and spontaneous bleeding (Schroeder et al., 1987, N Engl J Med, 317:1625-1629). The physician's global assessment assigns 0 points for normal findings, 1 point for mild colitis, 2 points for moderate colitis, and 3 points for severe colitis. Thus, in some embodiments, a patient treated with an SG-11 therapeutic protein or a variant or fragment thereof is successfully treated if the patient experiences a decrease in Mayo score of at least 1, 2, or 3 points in at least one of rectal bleeding, blood streaks in stool, endoscopy subscore, and physician's global assessment.In some embodiments, a treatment method comprising administering a therapeutically effective amount of an SG-11 protein or a variant or fragment thereof to a patient diagnosed with UC results in a reduction in the diagnostic score for UC, for example, the score can change the diagnostic score, such as the Mayo score, by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 points.

[0173] Pouchitis Additionally or alternatively, compositions and administration methods containing the SG-11 therapeutic protein or variant described herein can be used to treat pouchitis. Pouchitis is inflammation of the lining of the ileal pouch, which is surgically created to treat UC. Specifically, patients with severe UC have their affected colon removed and their intestines reconnected through a procedure called ileo-anal anastomosis (IPAA) or J-pouch surgery. Cases of pouchitis can recur in many patients, manifesting as either recurrent acute pouchitis or persistent chronic pouchitis. Accordingly, provided herein are methods for treating pouchitis, acute pouchitis, or recurrent pouchitis.

[0174] Pouchitis activity can be classified as remission (no active pouchitis), mild to moderate activity (increased bowel movements, urgency, and / or infrequent incontinence), or severe activity (frequent incontinence and / or patient hospitalization due to dehydration). The duration of pouchitis is defined as acute (four weeks or less) or chronic (four weeks or more), and the pattern can be classified as transient (one to two acute episodes), recurrent (three or fewer episodes), or persistent. Response to medical treatment can be classified as treatment-responsive or treatment-resistant, with medication prescribed in either case. Thus, in some embodiments, methods are provided for treating a subject diagnosed with pouchitis, in which treatment with a pharmaceutical composition comprising SG-11 or a variant or fragment thereof results in a decrease in the severity of pouchitis and / or causes remission.

[0175] Mucositis and the mucosal barrier The gastrointestinal (GI) tract mucosa is a complex microenvironment involving an epithelial barrier, immune cells, and microorganisms. A delicate balance is maintained in a healthy colon. Luminal microorganisms are physically separated from the host immune system by a barrier composed of epithelium and mucus. The pathogenesis of IBD is not fully understood but may involve an inappropriate host response to altered resident microbiota due to mucosal barrier dysfunction. See Boltin et al., “Mucin Function in Inflammatory Bowel Disease: An Update,” J. Clin. Gastroenterol., Vol. 47(2):106-111 (Feb. 2013).

[0176] Mucositis occurs when cancer treatments (especially chemotherapy and radiation) destroy the rapidly dividing epithelial cells lining the intestinal tract (from the mouth to the anus), leaving the mucosal tissue vulnerable to ulceration and infection. Mucosal tissue, also known as mucosa, lines all body passages that communicate with air, such as the respiratory and digestive tracts, and contains mucus-secreting cells and associated glands. The portion of this lining that covers the mouth, called the oral mucosa, is one of the most sensitive parts of the body and is particularly vulnerable to chemotherapy and radiation. The oral cavity is the most common site of mucositis. While the oral mucosa is the most common site of mucosal toxicity and resulting mucositis, it is understood that mucositis can occur along the entire digestive tract, including the esophagus, stomach, small intestine (duodenum, jejunum, and ileum), large intestine (colon), and rectum. In some embodiments, pharmaceutical compositions comprising SG-11 or a variant or fragment thereof are effective in treating mucositis of the mouth, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (colon), and / or rectum.

[0177] Oral mucositis can lead to several problems, including pain, nutritional issues resulting from the inability to eat, and an increased risk of infection due to open sores in the mucosa. It can significantly impact a patient's quality of life and can be dose-limiting (i.e., requiring a reduction in subsequent chemotherapy doses). The World Health Organization has established an oral toxicity scale for diagnosing oral mucositis: Grade 1: pain ± erythema; Grade 2: erythema, ulceration, patient able to eat solid foods; Grade 3: ulceration with widespread erythema, patient unable to eat solid foods; Grade 4: mucositis severe enough to prevent nutritional intake. Grades 3 and 4 oral mucositis are considered severe. Accordingly, provided herein are methods for treating a subject diagnosed with oral mucositis, in which administration of a pharmaceutical composition containing SG-11 or a variant or fragment thereof reduces the grade of oral toxicity by at least one point on a grade scale of 1 to 4.

[0178] Shortened colon Ulcerative colitis is an idiopathic inflammatory bowel disease affecting the colonic mucosa and is clinically characterized by diarrhea, abdominal pain, and bloody stools. The extent of the disease varies, involving only the rectum (ulcerative proctitis), the left side of the colon up to the splenic flexure, or the entire colon (pancolitis). Disease severity also varies considerably histologically, ranging from minimal to erythematous ulceration and dysplasia. Cancer may develop. The typical histological (microscopic) lesion of ulcerative colitis is a crypt abscess, in which the crypt epithelium is destroyed and the lumen is filled with polymorphonuclear cells. The lamina propria is infiltrated with leukocytes. Crypt destruction results in loss of normal mucosal architecture, and the resulting scarring can shorten and narrow the colon. Thus, colonic shortening can be a consequence of colitis and is often used for diagnosis. For example, noninvasive plain abdominal x-rays can demonstrate a gaseous outline of the transverse colon in patients with acute disease. Colonic shortening and loss of haustral markings can be demonstrated not only by plain film but also by double-contrast barium enema. Signs of ulcerative disease include loss of mucosal detail, cobblestone filling defects, and segmental areas of disease. See "Ulcerative Colitis: Introduction - Johns Hopkins Medicine," found at www.hopkinsmedicine.org / gastroenterology_hepatology / _pdfs / small_large_intestine / ulcerative_colitis.pdf.

[0179] Furthermore, art-recognized in vivo models of colitis utilize shortening of colon length to assess 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, pages 2-6 (February 2012).

[0180] Epithelial barrier function in non-IBD diseases An improperly functioning epithelial barrier is increasingly implicated in, for example, IBD and mucositis. Furthermore, there are many other diseases that studies have shown to be caused, associated, correlated, and / or exacerbated by an improperly functioning epithelial barrier. These diseases include: (1) metabolic diseases, such as obesity, type 2 diabetes, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), liver damage, 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 disorders; (7) interstitial cystitis; (8) neurological or cognitive disorders (e.g., Alzheimer's disease, Parkinson's disease, multiple sclerosis, autism); (9) chemotherapy-associated steatohepatitis (CASH); and (10) pediatric versions of the above diseases.For example, 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), pgs. 2775-2786; Everard et al., “Cross-talk between Akkermansia muciniphila and intestinal 9066-9071; Cani et al., “Changes in Gut Microbiota Control Metabolic Endotoxemia-Induced Inflammation in High-Fat Diet-Induced Obesity and Diabetes in Mice,” Diabetes, Vol. 57, (June 2008), pgs. See Delzenne et al., "Targeting gut microbiota in obesity: effects of prebiotics and probiotics," Nature Reviews, Vol. 7, (November 2011), pp. 639-646. Consequently, restoring proper epithelial barrier function to patients may be crucial in resolving the aforementioned disease states.

[0181] A properly functioning epithelial barrier in the lumen of the gastrointestinal tract, including the mouth, esophagus, stomach, small intestine, large intestine, and rectum, is important for controlling and maintaining the microbiota within the gastrointestinal and digestive tract. The microbiota ecosystem includes the environment, barriers, tissues, mucus, mucins, enzymes, nutrients, food, and the microbial communities that reside in the gastrointestinal and digestive tract. The integrity and permeability of the intestinal mucosal barrier affect health in many important ways.

[0182] Loss of mucosal barrier integrity in gastrointestinal disorders due to altered mucin secretion may be related to altered host immunity, luminal microbial factors, or directly acting genetic or environmental determinants. Thus, mucus barrier imbalance 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 (Feb. 2013).

[0183] Mucins are major components of the mucus layer lining the gastrointestinal tract. The human genome contains at least 21 known mucin (MUC) genes encoding secreted or membrane-bound mucins. The major mucins in the normal colon are MUC1, MUC2, MUC3A, MUC3B, MUC4, MUC13, and MUC17.1. MUC2 is the major secreted, gel-forming component of intestinal mucus produced by goblet cells. See Boltin et al., “Mucin Function in Inflammatory Bowel Disease An Update,” J. Clin. Gastroenterol., Vol. 47(2):106-111 (Feb. 2013). Together with additional secreted mucins, such as MUC1, 3A, 3B, 4, 13, and 17.1, goblet cell MUC2 secretion forms a protective barrier over colonic epithelial cells, reducing their exposure to intestinal contents that could potentially damage them or prime an immune response.

[0184] Inflammatory mechanisms in IBD There is significant evidence implicating specific cytokines in IBD. Recent studies have demonstrated that cytokines play a critical role in the pathogenesis of inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis, regulating many 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- and anti-inflammatory cytokines that occurs in IBD prevents inflammation resolution and instead leads to disease perpetuation and tissue destruction. Ibid. Recent studies suggest the existence of a regulatory cytokine network that is important for disease progression. Ibid. Therefore, we conducted experiments to study the effects of SG-11 on the production and / or secretion of pro- and anti-inflammatory cytokines.

[0185] Inflammatory cytokines Briefly, inflammatory cytokines are cytokines that play a key role in cell signaling and promote systemic inflammation. They are primarily produced by activated macrophages and are involved in the upregulation of inflammatory responses. Proinflammatory cytokines arise from genes encoding the translation of small mediator molecules that elicit responses after upregulation. Well-characterized proinflammatory cytokines include interleukin-1 (IL-1), IL-6, IL-12, IL-18, IL-23, CD40L, tumor necrosis factor (TNF), such as TNF-α, gamma interferon (IFN-γ), granulocyte-macrophage colony-stimulating factor (MCP-1), and MCP-1. Inflammation is characterized by the interplay between proinflammatory and anti-inflammatory cytokines.

[0186] Reducing the biological activity of proinflammatory cytokines may be useful in treating some diseases. For example, blocking IL-1 or TNF-α has been successful in helping 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.

[0187] Anti-inflammatory cytokines Simply put, anti-inflammatory cytokines are a group of immunoregulatory molecules that regulate the response of inflammatory cytokines. Thus, these molecules help regulate and reduce the inflammatory response caused by inflammatory cytokines. Anti-inflammatory cytokines include, for example, IL4, IL-10, IL-13, IFN-α, and transforming growth factor-β (TGF-β), which are recognized as anti-inflammatory cytokines.

[0188] 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 reduce the production of at least one pro-inflammatory cytokine (e.g., TNF-α and / or IL-23) by immune cells in a patient receiving the composition. In some embodiments, the administration can increase the production of at least one anti-inflammatory cytokine (e.g., IL-10) by immune cells in the patient. In some embodiments, the administration can reduce the production of at least one anti-inflammatory cytokine (e.g., IL-10) by immune cells in the patient. In some embodiments, the administration can result in improved mucin production in epithelial cells and / or epithelial wound healing in the patient.

[0189] The dosage regimen used for treatment depends on the desired therapeutic effect, the route of administration, and the duration of treatment. The dosage will vary from patient to patient depending on the nature and severity of the disease, the patient's weight, any special diet the patient follows, any concomitant medications, and other factors recognized by those skilled in the art.

[0190] Typically, dosage levels of 0.0001 to 10 mg / kg body weight / day of a therapeutic protein are administered to a patient, e.g., a patient suffering from inflammatory bowel disease. Dosage ranges generally from about 0.5 mg to 100.0 g per patient per day, and can be administered in single or multiple doses.

[0191] In some aspects, the dosage range is about 0.5 mg to 10 g per patient per day, 0.5 mg to 9 g per patient per day, 0.5 mg to 8 g per patient per day, 0.5 mg to 7 g per patient per day, 0.5 mg to 6 g per patient per day, 0.5 mg to 5 g per patient per day, 0.5 mg to 4 g per patient per day, 0.5 mg to 3 g per patient per day, 0.5 mg to 2 g per patient per day, or 0.5 mg to 1 g per patient per day.

[0192] In some aspects, the dosage range is about 0.5 mg to 900 mg per patient per day, about 0.5 mg to 800 mg per patient per day, about 0.5 mg to 700 mg per patient per day, about 0.5 mg to 600 mg per patient per day, about 0.5 mg to 500 mg per patient per day, about 0.5 mg to 400 mg per patient per day, about 0.5 mg to 300 mg per patient per day, about 0.5 mg to 500 mg per patient per day, about 0.5 mg to 400 mg per patient per day, about 0.5 mg to 500 mg per patient per day, about 0.5 mg to 500 mg per patient per day, about 0.5 mg to 600 mg per patient per day, about 0.5 mg to 700 mg per patient per day, about 0.5 mg to 700 mg per patient per day, about 0.5 mg to 800 mg per patient per day, about 0.5 mg to 800 mg per patient per day, about 0.5 mg to 900 mg per patient per day, about 0.5 mg to 1000 mg per patient per day, about 0.5 mg to 1200 mg per patient per day, about 0.5 mg to 1400 mg per patient per day, about 0.5 mg to 1600 mg per patient per day, about 0.5 mg to 18 ... about 0.5 mg to 200 mg per patient per day, about 0.5 mg to 100 mg per patient per day, about 0.5 mg to 50 mg per patient per day, about 0.5 mg to 40 mg per patient per day, about 0.5 mg to 30 mg per patient per day, about 0.5 mg to 20 mg per patient per day, about 0.5 mg to 10 mg per patient per day, or about 0.5 mg to 1 mg per patient per day.

[0193] Combination Therapies Containing Therapeutic Proteins The pharmaceutical compositions taught herein, including therapeutic proteins, can be combined with other therapies and / or pharmaceutical compositions. For example, a patient with inflammatory bowel disease may already be taking a medicine prescribed by a doctor to treat the disease. In some embodiments, the pharmaceutical compositions taught herein can be administered in conjunction with the patient's existing medicine.

[0194] For example, a therapeutic protein taught herein can be combined with one or more of the following: an anti-diarrheal agent, a 5-aminosalicylate compound, an anti-inflammatory agent, an antibiotic, an antibody, e.g., an antibody targeting an inflammatory cytokine, e.g., an antibody targeting an anti-cytokine agent, such as anti-TNF-α (e.g., adalimumab, certolizumab pegol, golimumab, infliximab, V565) or anti-IL-12 / IL-23 (e.g., ustekinumab, risankizumab, bradykinumab, ribozyme, ribozyme, ribozyme) zikumab, ustekinumab), JAK inhibitors (e.g., tofacitinib, PF06700841, PF06651600, filgotinib, upadacitinib), anti-integrin agents (e.g., vedolizumab, etrolizumab), S1P inhibitors (e.g., etrasimod, ozanimod, amiselimod), recombinant cell-based agents (e.g., Cx601), steroids, corticosteroids, immunosuppressants (e.g., azathioprine, mercaptopurine), vitamins, and / or special diets.

[0195] Cancer patients undergoing chemotherapy or radiation therapy and suffering from or at risk of developing mucositis, e.g., oral mucositis, can be administered a pharmaceutical composition according to the present disclosure in combination with an agent used to treat mucositis, such as oral mucositis. In some embodiments, a method of treatment comprises administering to a patient with mucositis a pharmaceutical composition comprising SG-11 or a variant or fragment thereof in combination with one or more second therapeutic agents selected from the group consisting of amifostine, benzocaine, benzydamine, ranitidine, omeprazole, capsaicin, glutamine, prostaglandin E2, vitamin E, sucralfate, and allopurinol.

[0196] In some embodiments of the methods described herein, a second therapeutic agent is administered in conjunction with the SG-11 protein described herein, either simultaneously or sequentially. In some embodiments, the protein and the second therapeutic agent act synergistically to treat or prevent a disease, condition, or symptom. In other embodiments, the protein and the second therapeutic agent act additively to treat or prevent a disease, condition, or symptom.

[0197] Pharmaceutical compositions containing SG-11 therapeutic protein Pharmaceutical compositions are provided herein, which contain an SG-11 protein, a variant or fragment thereof, or a pharmaceutically acceptable salt thereof according to the present disclosure, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated for administration to the gastrointestinal lumen, such as the mouth, esophagus, small intestine, large intestine, rectum, and / or anus.

[0198] In some embodiments, the composition includes one or more other substances associated with the source of the protein, such as cellular components from the producing host cell or substances associated with the chemical synthesis of the protein. In other embodiments, the pharmaceutical composition is formulated to include one or more second active agents described herein. In addition, the composition may include ingredients that preserve the structural and / or functional activity of the active agent(s) or the composition itself. Such ingredients include, but are not limited to, antioxidants, various antibacterial and antifungal agents, such as parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0199] The terms "pharmaceutical" or "pharmaceutically acceptable" refer to a composition that does not, or preferably does not, produce adverse, allergic, or other untoward reactions, as appropriate, when administered to an animal, e.g., a human. The preparation of pharmaceutical compositions or additional active ingredients will be known to those of skill in the art in light of the present disclosure, and are exemplified in Remington's Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990, incorporated herein by reference. Furthermore, it will be understood that for administration to animals (e.g., humans), preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biological Standards.

[0200] The pharmaceutical compositions of the present disclosure are formulated according to the intended route of administration and whether they are administered in solid, liquid, or aerosol form. In a preferred embodiment, the compositions are administered rectally, but they can also be administered topically, by injection, by infusion, orally, intrathecally, intranasally, subcutaneously, mucosally, by local irrigation to directly bathe target cells, via a catheter, via lavage, or by other methods or any combination of the foregoing, as would be known to those skilled in the art. Liquid formulations containing a therapeutically effective amount of the protein can be administered rectally via enema, catheter, or bulb syringe. Suppositories are an example of solid dosage forms formulated for rectal delivery. In general, for suppositories, conventional carriers include, for example, polyalkylene glycols, triglycerides, or combinations thereof. In certain embodiments, suppositories can be formed from mixtures containing, for example, about 0.5% to about 10% and / or about 1% to about 2% of the active ingredient. Liquid compositions for injection are typically based on sterile saline or phosphate-buffered saline for injection, or other injectable carriers known in the art. Other liquid compositions include suspensions and emulsions. Solid compositions, such as those for oral administration, can be in the form of tablets, pills, capsules (e.g., hard-shell or soft-shell gelatin capsules), buccal compositions, troches, elixirs, suspensions, syrups, wafers, or combinations thereof. The active agent, i.e., the protein described herein, in such liquid and solid compositions typically comprises about 0.05% to 10% by weight, with the remainder being the injectable carrier, etc.

[0201] Pharmaceutical compositions can be formulated as controlled- or sustained-release compositions that provide release of the active agent, including a therapeutic protein of the present disclosure, over an extended period of time, e.g., over 30-60 minutes, or over 1-10 hours, 2-8 hours, 8-24 hours, etc. Alternatively, or in addition, the compositions are formulated for release at a specific site within the host body. For example, the compositions may have an enteric coating that prevents release of the active agent in acidic environments such as the stomach, allowing release only in the more neutral or basic environments of the small intestine, colon, or rectum. Alternatively, or in addition, the compositions may be formulated to provide delayed release in the oral cavity, small intestine, or large intestine.

[0202] Each of the above formulations can contain at least one pharmaceutically acceptable excipient or carrier depending on the intended route of administration, 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, coating agents, 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, such similar materials, and combinations thereof, known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference).

[0203] Pharmaceutical compositions for administration can be presented in unit dosage forms to facilitate accurate dosing. Typical unit dosage forms include pre-filled ampoules or syringes for liquid compositions, or suppositories, pills, tablets, capsules, etc. for solid compositions. In some embodiments of such compositions, the active agent, i.e., a protein described herein, can be a component (about 0.1-50 wt / wt%, 1-40 wt / wt%, 0.1-1 wt / wt%, or 1-10 wt / wt%), with the remainder being various vehicles or carriers and processing aids useful for forming the desired dosage form.

[0204] The actual dosage in a 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 disease, the type of illness being treated, previous or concurrent therapeutic interventions, the patient's idiopathic disease (idiopathy), and the route of administration. In any event, the physician responsible for administration will determine the concentration of active ingredient(s) in the composition and the appropriate dose for the individual subject.

[0205] Protein Expression Systems and Protein Production Provided herein are compositions and methods for producing the isolated proteins of the disclosure, as well as expression vectors comprising polynucleotide sequences encoding the proteins and host cells harboring the expression vectors.

[0206] The proteins of the present disclosure can be prepared by conventional recombinant methods, for example, by culturing cells transformed or transfected with an expression vector containing a nucleic acid encoding the SG-11 therapeutic protein, a variant, or a fragment thereof. Host cells containing such vectors are also provided. Host cells can be prokaryotic or eukaryotic, and examples of host cells include E. coli, yeast, or mammalian cells. Methods for producing any of the proteins described herein are further provided, comprising culturing host cells under conditions suitable for expression of the protein of interest and recovering the protein of interest from the cell culture. The recovered protein can then be isolated and / or purified for use in in vitro and in vivo methods, as well as for formulation into pharmaceutically acceptable compositions. In some embodiments, the protein is expressed in prokaryotic cells such as E. coli, and isolation and purification of the protein includes reducing endotoxin to levels acceptable for therapeutic use in humans or other animals.

[0207] Expression vector Provided herein are expression vectors containing 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 coding polynucleotide sequence can be amplified from genomic DNA of the source bacterium, i.e., Roseburia hominis. Alternatively, the polynucleotide can be synthesized using a nucleotide synthesizer. Once obtained, the polypeptide-encoding sequence is inserted into a recombinant vector capable of replicating and expressing heterologous (exogenous) polynucleotides in host cells. Many vectors known and available in the art can be used for the purposes of the present disclosure. Selection of an appropriate vector depends 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 the heterologous polynucleotide, 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, the heterologous nucleic acid insert, and a transcription termination sequence.

[0208] Plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are generally used with these hosts. The vectors usually contain a replication site and marking sequences capable of providing phenotypic selection of transformed cells. For example, Escherichia coli is typically transformed using pBR322, pUC, pET, or pGEX vectors, which are plasmids derived from E. coli species. Such vectors contain genes encoding ampicillin (Amp) and tetracycline (Tet) resistance, providing a convenient means for identifying transformed cells. These vectors and their derivatives, or other microbial plasmids or bacteriophages, may contain or be modified to contain promoters that can be used by the microorganism for expression of endogenous proteins.

[0209] The expression vectors of the present disclosure can include a promoter located upstream (5') of the untranslated regulatory sequence operably linked to a protein-encoding nucleotide sequence, which regulates transcription of the coding sequence. Prokaryotic promoters are generally divided into two classes: inducible promoters and constitutive promoters. Inducible promoters are promoters that initiate increased levels of transcription of a coding polynucleotide under their control in response to changes in culture conditions, such as the presence or absence of a nutrient or a change in temperature. Numerous promoters recognized by a variety of potential host cells are well known, and those skilled in the art can select a promoter based on the desired expression level. Suitable promoters for use with prokaryotic hosts include E. coli promoters such as lac, trp, tac, trc, and ara; viral promoters recognized by E. coli, such as the lambda and T5 promoters; and the T7 and T7lac promoters from the T7 bacteriophage. Host cells harboring vectors containing the T7 promoter can be engineered to express, for example, 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 chemical or environmental factors.

[0210] Further useful plasmid vectors include the pIN vectors (Inouye et al., 1985); and the pGEX vectors, which are used to generate soluble glutathione S-transferase (GST) fusion proteins for subsequent purification and isolation or cleavage. Other suitable fusion proteins are those with β-galactosidase, ubiquitin, etc.

[0211] Suitable vectors for expression in both prokaryotic and eukaryotic host cells are known in the art, and some are further described herein.

[0212] The vectors of the present disclosure can further include a signal sequence that enables the translated recombinant protein to be recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In the case of prokaryotic host cells that do not recognize and process the signal sequence native to the heterologous polypeptide, the signal sequence is replaced with a prokaryotic signal sequence selected from the group consisting of, for example, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, PeIB, OmpA, and MBP. Well-known signal sequences used in eukaryotic expression systems include, but are not limited to, interleukin-2, CD5, immunoglobulin kappa light chain, trypsinogen, serum albumin, and prolactin.

[0213] The SG-11 protein or its variants or fragments described herein can be expressed as a fusion protein or polypeptide. Commonly used fusion partners include, but are not limited to, human serum albumin and its crystallizable fragments, or the constant domain Fc of IgG. Histidine tags or FLAG tags can also be used to simplify purification of recombinant proteins from expression media or recombinant cell lysates. Fusion partners can be fused to the N-terminus and / or C-terminus of the protein of interest.

[0214] host cell Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells. Numerous cell lines and cultures are available for use as host cells and can be obtained, for example, through the American Type Culture Collection (ATCC), an organization that serves as an archive of living cultures and genetic material. Cell types available for replication and / or expression of vectors include, but are not limited to, bacteria such as E. coli (e.g., E. coli strain RR1, E. coli LE392, E. coli B, E. coli X1776 (ATCC No. 31537), and E. coli W3110 (F-, λ-, prototrophic, ATCC No. 273325), DH5α, JM109, and KC8), bacilli such as Bacillus subtilis; and other Enterobacteriaceae such as Salmonella typhimurium, Serratia marcescens, various Pseudomonas species, as well as many commercially available bacterial hosts, such as SURE® Competent Cells and SOLOPACK™ Gold Cells (STRATAGENE®, La In certain embodiments, bacterial cells such as E. coli are specifically contemplated as host cells.

[0215] Examples of eukaryotic host cells for replication and / or expression of vectors include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Additional eukaryotic host cells include yeast (e.g., Pichia pastoris, Saccharomyces cerevisiae) and insect-derived cells (e.g., Spodoptera frugiperda, Trichoplusia ni). Many host cells from a variety of cell types and organisms are available and would be known to those skilled in the art. Similarly, viral vectors can be used with eukaryotic or prokaryotic host cells, particularly those permissive for replication or expression of the vector. The selection of an appropriate host cell is deemed to be within the skill of one of ordinary skill in the art.

[0216] Methods for introducing recombinant DNA, i.e., expression vectors, into host cells so that the recombinant DNA is replicable either as an extrachromosomal element or as a chromosomal integrant, thereby generating host cells carrying an expression vector of interest, are well known. Transfection methods, such as those using CaPO4 and electroporation, are known to those skilled in the art. Depending on the host cell used, transformation is carried out using standard techniques appropriate for such cells. Calcium treatment using calcium chloride, as described in Sambrook et al., supra, or electroporation, is generally used for prokaryotic or other cells containing a strong cell wall barrier. General aspects of transformation of mammalian cell host systems are described in U.S. Pat. No. 4,399,216. Transformation into yeast is usually carried out 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 (1979). Other methods for introducing DNA into cells include nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or introduction using polycations such as polybrene and polyornithine.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).

[0217] Thus, the present disclosure provides a recombinant vector or expression vector 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, SEQ ID NO:19, or a variant and / or fragment thereof described herein). The polynucleotide may be, for example, 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, SEQ ID NO:20, or any one of variants or fragments thereof. Furthermore, the present disclosure teaches a host cell carrying the vector. The host cell may be a eukaryotic or prokaryotic cell, as detailed above. In a preferred embodiment, the host cell is a prokaryotic cell. In a more preferred embodiment, the host cell is E. coli.

[0218] In some embodiments, the polynucleotide encoding the protein of interest is codon-optimized. A codon optimization algorithm is applied to the polynucleotide sequence encoding the protein to select appropriate codons for specific amino acids based on the codon usage bias of the expression host. Many codon optimization algorithms also take into account other factors such as mRNA structure, the GC content of the host, and ribosome entry sites. Examples of codon optimization algorithms and gene synthesis service providers are listed below: 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 can then be synthesized and cloned into an appropriate expression vector. Codon-optimized sequences in this 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.

[0219] Protein production methods Methods for producing the proteins described herein are provided, but these methods are well known to those skilled in the art. Host cells transformed or transfected with the expression or cloning vectors described herein for protein production are cultured in conventional nutrient media; the media is modified as needed to induce promoters, select and / or maintain transformants, and / or express genes encoding the desired protein sequences. Culture conditions, such as media, temperature, and pH, can be selected by those skilled in the art without undue experimentation. In general, principles, protocols, and practical techniques for maximizing cell culture productivity can be found in Mammalian Cell Biotechnology: A Practical Approach, edited by M. Butler (IRL Press, 1991) and Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press).

[0220] Generally, "purified" refers to a particular protein composition that has been subjected to fractionation to remove non-proteinaceous components and various other proteins, polypeptides, or peptides, and the composition substantially retains its activity, which can be assessed, for example, by protein assays such as those described herein below or known to those of skill in the art for the desired protein, polypeptide, or peptide.

[0221] When the term "substantially purified" is used, it refers to a composition in which a particular protein, polypeptide, or peptide forms a major component of the composition, such as making up about 50% or more of the protein in the composition. In preferred embodiments, a substantially purified protein makes up more than 60%, 70%, 80%, 90%, 95%, 99%, or even more of the protein in the composition.

[0222] A "purified to homogeneity" peptide, polypeptide, or protein as applied to this disclosure means that the peptide, polypeptide, or protein has a purity such that it is substantially free from other proteins and biological components. For example, a purified peptide, polypeptide, or protein will often be sufficiently free of other protein components to successfully perform degradative sequencing.

[0223] Although preferred for use in certain embodiments, there is no general requirement that proteins, polypeptides, or peptides always be provided in the most purified state, and indeed, less fully purified proteins, polypeptides, or peptides that are nonetheless enriched in the desired protein composition compared to the native state are considered useful in certain embodiments.

[0224] Various methods for quantifying the degree of purification of a protein, polypeptide, or peptide will be known to those of skill in the art in light of the present disclosure, including, for example, measuring the specific protein activity of a fraction or assessing the number of polypeptides within a fraction by gel electrophoresis.

[0225] Another example is the purification of a specific fusion protein using a specific binding partner. Such purification methods are routinely used in the art. Because the present disclosure provides the DNA sequences of the specific proteins, any fusion protein purification method is currently feasible. This is exemplified by: creating a specific protein-glutathione S-transferase fusion protein, expressing it in E. coli, and isolating it to homogeneity using affinity chromatography on glutathione-agarose, or adding a polyhistidine tag to 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 is currently available.

[0226] In other aspects, a peptide-enriched preparation can be used instead of a purified preparation. Wherever purified is used in this document, enriched preparations can also be used. In addition to being enriched by purification methods, preparations can also be enriched by bacterial overexpression or overproduction of the peptide compared to the wild-type. This can be achieved using recombinant methods or by selecting conditions that induce expression of the peptide from wild-type cells.

[0227] Recombinantly expressed polypeptides of the present disclosure can be recovered from culture medium or host cell lysates. Suitable purification methods include, for example, fractionation on an ion exchange (anion or cation) column; ethanol precipitation; reverse-phase HPLC; chromatography on a cation exchange resin such as silica or DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration or size exclusion chromatography (SEC), for example, using Sephadex G-75; and metal chelate columns that bind epitope-tagged forms of the polypeptides of the present disclosure. Various protein purification methods can be used, and such methods are known in the art and are described, for example, in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The purification step(s) selected will depend, for example, on the production process used and the properties of the specific polypeptide produced.

[0228] Alternative methods known in the art can be used to prepare the polypeptides of the present invention. For example, sequences encoding the polypeptides or portions thereof can be generated by direct peptide synthesis using solid-phase methods (see, e.g., Stewart et al., 1969, Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, Calif.; Merrifield, J. 1963, Am. Chem. Soc., 85:2149-2154). In vitro protein synthesis can be performed manually or by automation. Automated synthesis can be performed, for example, using an Applied Biosystems peptide synthesizer (Foster City, Calif.) according to the manufacturer's instructions. Various portions of the polypeptides or portions thereof of the present invention can be chemically synthesized separately and combined using chemical or enzymatic methods to generate the full-length polypeptide or portions thereof.

[0229] In some aspects, the present disclosure provides chimeric molecules comprising any of the polypeptides described herein fused to a heterologous polypeptide or amino acid sequence, and polynucleotides encoding the chimeric molecules. Examples of such chimeric molecules include, but are not limited to, any of the polypeptides described herein fused to an epitope tag sequence of an immunoglobulin Fc region.

[0230] Recombinant bacterial delivery systems The present disclosure contemplates utilizing delivery systems other than traditional pharmaceutical formulations containing purified proteins. In some embodiments, the present disclosure utilizes recombinant bacterial delivery systems, phage-mediated delivery systems, chitosan-DNA complexes, or AAV delivery systems.

[0231] One particular recombinant bacterial delivery system is based on Lactococcus lactis. Essentially, a gene encoding a therapeutic protein (e.g., SEQ ID NO:3) can be cloned into an expression vector, which can then be transformed into L. lactis, which is then administered to a patient. See, for example, 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 patients with Crohn's disease using a recombinant Lactococcus lactis (LL-Thy12) 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 a genetically engineered strain of lactic acid bacteria (gmLAB) is being investigated as a novel therapeutic strategy."); Steidler, et al., "Treatment of murine colitis by "Lactococcus lactis secreting interleukin-10," Science, 2000, Vol. 289, pgs. 1352-1355 ("The cytokine interleukin-10 (IL-10) has shown promise in clinical trials for the treatment of inflammatory bowel disease (IBD). Using two mouse models, we show that the therapeutic dose of IL-10 can be reduced by local delivery of bacteria genetically engineered to secrete the cytokine."Intragastric administration of IL-10-secreting Lactococcus lactis caused a 50% reduction in colitis in dextran sulfate sodium-treated mice and prevented the development of colitis in IL-102 / 2 mice. This approach may lead to cost-effective 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, Vol. 195, pp. 185-193 ("Here, we evaluated Lactococcus lactis, which produces pneumococcal surface protein A (PspA) intracellularly, as a mucosal vaccine to protect against pneumococcal disease."); and 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 ("We demonstrated the in vivo effects of orally administered L. lactis on murine TFF." "We positively evaluated a new therapeutic approach for acute and chronic colitis with in situ secretion. This novel approach may lead to effective management of acute and chronic colitis and epithelial damage in humans."

[0232] In another embodiment, "synthetic bacteria" can be used to deliver the SG-11 protein or variants or fragments thereof, in which probiotic bacteria are engineered to express the SG-11 therapeutic protein (see, e.g., Durrer and Allen, 2017, PLoS One, 12:e0176286).

[0233] Phages have been genetically engineered to deliver specific DNA payloads or alter host specificity. Phage, plasmid, transposon, and other transfer methods can be used to deliver and circulate engineered DNA sequences into microbial communities through processes such as transduction, transformation, and conjugal transfer. For the purposes of this disclosure, it is sufficient to understand that engineered phage can be one possible delivery system for the proteins of this disclosure. The delivery system involves incorporating nucleic acid encoding the protein into a phage and using the phage to deliver the nucleic acid to a host microorganism. The microorganism will then produce the protein after allowing the phage to deliver the nucleic acid to its genome.

[0234] Similar to the engineered phage approach described above, transposon delivery systems can be used to integrate nucleic acids encoding therapeutic proteins into host microorganisms present in a patient's microbiome. See Sheth, et al., "Manipulating bacterial communities by in situ microbiome engineering," Trends in Genetics, 2016, Vol. 32, Issue 4, pp. 189-200.

[0235] The following examples are intended to illustrate, but not limit, the present disclosure. [Example]

[0236] The following experiments utilize a robust mixed model of in vitro experiments combined with an in vivo model of IBD to demonstrate the therapeutic potential of the taught proteins and methods.

[0237] Example 1 Expression of SG-11 and its mutants In the experiments described in the following Examples, a polynucleotide encoding SG-11 (SEQ ID NO:3) was obtained by PCR amplification of genomic DNA from Roseburia hominis (A2-183; DSM 16839 type strain; see, e.g., Duncan, SH, Aminov, RI, Scott, KP, Louis, P., Stanton, TB, Flint, HJ (2006)). The proposed Roseburia faecis sp. nov., Roseburia hominis sp. nov., and Roseburia inulinivorans sp. nov. were based on isolates from human feces. Int. J. Syst. Evol. Microbiol. Vol. 56, pp. 2437-2441. The encoding polynucleotide was then subcloned into an inducible expression vector and used to transform E. coli BL21(DE3) cells for expression and purification of SG-11 or its variants, as detailed below, using culture and purification methods routine in the art.

[0238] Expression of SG-11 (including SEQ ID NO:3) Expression and purification of a protein containing the amino acid sequence of SG-11 (SEQ ID NO:5) for use in various experiments related to this disclosure described below was achieved using the pGEX vector system, which is designed for inducible, high-level, intracellular expression of genes or gene fragments. Expression in E. coli results in a tagged protein with a GST moiety at the amino terminus and the protein of interest at the carboxyl terminus. This vector contains the tac promoter for chemically inducible, high-level expression and an internal laq1 promoter for use in E. coli hosts. q I have the gene.

[0239] A polynucleotide containing a nucleotide sequence encoding SG-11 (SEQ ID NO:3 from R. hominis DSM 16839) was inserted into the multiple cloning site (BamHI and NotI sites) of pGEX-6P-1 (GE Healthcare Life Sciences, 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) shown in Table 4 below. This protein was expressed and purified by two alternative methods. In the first method, BL21(DE3) transformants were grown at 30°C in LB containing 100 μg / ml carbenicillin and 1 μg / ml chloramphenicol. OD 600 When the culture reached a confluency of 0.6, expression was induced with 0.4 mM IPTG for 4 hours. Cells were harvested by centrifugation, lysed by sonication, and the soluble lysate was applied to a GST resin column. Bound proteins were washed with PBS, and then purified tag-free SG-11C was eluted by adding PreScission protease to cleave the protein at the C-terminus of the GST tag.

[0240] An alternative expression and purification method using the same pGEX expression construct was performed by growing transformed BL21(DE3) cells in LB containing 50 μg / ml carbenicillin at 37°C. 600Once the cells reached a density of 0.7, they were cooled to 16°C, and expression was induced with 1 mM IPTG at 16°C for 15 hours. Cells were harvested, lysed by sonication, and the soluble lysate was applied to a GSTrap column. Bound proteins were washed with HEPES buffer, and purified tag-free 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. Elution fractions containing the protein, as determined by SDS-PAGE and Coomassie Brilliant Blue staining, were identified, pooled, and applied to a HiTrap Q HP anion exchange column followed by a Superdex 75 (26 / 60) preparative size exclusion column (SEC) to obtain the final preparation.

[0241] (Table 4) TIFF0007789303000007.tif148154

[0242] Expression and purification of the mature SG-11 protein without a signal peptide was performed using the pD451-SR vector system (AU™, Newark, CA). This expression vector utilizes an IPTG-inducible T7 promoter. The polynucleotide (SEQ ID NO:4) encoding SG-11 (SEQ ID NO:3) was codon-optimized by AU™ (Newark, CA) to obtain the codon-optimized coding sequence provided herein as SEQ ID NO:8. This codon-optimized coding sequence was inserted into the pD451-SR vector. The resulting construct results in expression of the 233-amino acid SG-11 protein provided herein as SEQ ID NO:7.

[0243] BL21(DE3) cells transformed with the construct were grown in autoinduction medium MagicMedia (ThermoFisher). The culture was incubated with shaking at 25°C for 8 hours, followed by incubation at 16°C for up to 72 hours. Cells were pelleted by centrifugation and resuspended in 100 mM Tris-HCl, pH 8.0, containing 50 mM NaCl, 2 mg / ml lysozyme, and protease inhibitors. Triton X-100 was then added to the suspension. Cells were then sonicated, and a cleared lysate was prepared by centrifugation for protein purification by standard column chromatography techniques.

[0244] SG-11 (SEQ ID NO:7) was purified on two anion exchange columns: HiTrap Q HP followed by Mono Q. Fractions containing partially purified protein, as confirmed by SDS-PAGE and Coomassie blue staining, were further purified on Mono Q. The purification protocol for Mono Q was the same as that for HiTrap Q. SG-11-containing fractions were pooled and dialyzed against buffer (50 mM sodium phosphate, 150 mM NaCl, and 10% glycerol). Purity and homogeneity were analyzed by SDS-PAGE and analytical SEC on a Superdex 200 Increase 3.2 / 300 column, and the preparation was estimated to be approximately 92.7% pure.

[0245] The pD451-SR vector system was also used to express and purify the SG-11 mutant, SG-11V5 (SEQ ID NO:19). To create 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, which encodes SG-11V5 (SEQ ID NO:19). The SG-11V5 coding sequence was cloned into the pD451-SR vector.

[0246] BL21(DE3) cells transformed with the construct were grown and processed for preparation of cleared lysates as described above for expression of SG-11 (SEQ ID NO:7).

[0247] SG-11V5 protein was purified from the cleared lysate by HiTrap Q purification followed by hydrophobic interaction chromatography (HIC) on HiTrap Butyl HP. SG-11V5-containing fractions, as determined by SDS-PAGE and Coomassie blue staining, were pooled and dialyzed against buffer (50 mM sodium phosphate, 150 mM NaCl, and 10% glycerol). All column chromatography described for the preparation was performed using the AKTA Protein Purification System (GE Healthcare Life Sciences, Pittsburgh, PA).

[0248] Purified proteins were quantified by densitometry after SDS-PAGE and Coomassie Brilliant Blue staining using bovine serum albumin as a reference. Endotoxin levels were measured using Endosafe® nexgen-MCS™ (Charles River, Wilmington, MA) according to the manufacturer's instructions. The endotoxin levels of the proteins used in the assays described herein were less than 1 EU / mg.

[0249] An expression construct was created using the pET-28 vector (Sigma Millipore) to carry 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 its N-terminus. The complete FLAG-tagged SG-11 protein sequence is provided herein as SEQ ID NO:9 (encoded by SEQ ID NO:10). Protein expression using this construct is under the control of a T7 promoter inducible by isopropyl β-D-1-thiogalactopyranoside (IPTG). The N-terminal FLAG tag was incorporated into the construct using PCR and an oligonucleotide encoding DYKDDDDK. Transformed host cells were grown overnight at 37°C in 2xYT medium. 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% inoculation) into MagicMedia™ E. coli Expression Medium (ThermoFisher). Cells were grown at 25°C for 8 hours, then at 16°C for up to 72 hours, before being harvested by centrifugation. The protein was expressed in a soluble form, allowing for recovery from a clear lysate. The expressed protein was purified using a HiTrapQ anion exchange column followed by Superdex 200 Increase 10 / 300 GL SEC. Purity and homogeneity were analyzed by SDS-PAGE and analytical SEC on Superdex 200 Increase 3.2 / 300, and the preparation was estimated to be approximately 93.3% pure.

[0250] Preparation of SG-11 protein for stability analysis SG-11 (SEQ ID NO:7) and its mutant SG-11V5 (SEQ ID NO:19) were purified on two anion exchange columns: HiTrap Q HP followed by Mono Q. Fractions containing partially purified protein, as confirmed by SDS-PAGE and Coomassie blue staining, were further purified on Mono Q. The purification protocol for Mono Q was the same as that for HiTrap Q. SG-11-containing fractions were pooled and dialyzed against buffer (50 mM sodium phosphate, 150 mM NaCl, and 10% glycerol).

[0251] In the case of SG-11V5, after purification on HiTrap Q, the protein was further purified by hydrophobic interaction chromatography (HIC) on HiTrap Butyl HP. SG-11V5-containing fractions, as determined by SDS-PAGE and Coomassie blue staining, were pooled and dialyzed against buffer (50 mM sodium phosphate, 150 mM NaCl, and 10% glycerol). All column chromatography described for the preparation was performed using the AKTA Protein Purification System (GE Healthcare Life Sciences, Pittsburgh, PA).

[0252] Purified proteins were quantified by densitometry after SDS-PAGE and Coomassie blue staining using bovine serum albumin as a reference. Endotoxin levels were measured using Endosafe® nexgen-MCS™ (Charles River, Wilmington, MA) according to the manufacturer's instructions. The endotoxin levels of the proteins used in the assays described herein were less than 1 EU / mg.

[0253] Example 2 Effect of SG-11 on the restoration of epithelial barrier integrity after inflammation-induced barrier disruption The following experiment demonstrates the therapeutic ability of the SG-11 protein or its variants described herein to restore gastrointestinal epithelial barrier integrity. Thus, this experiment demonstrates the functional utility of the therapeutic protein for treating gastrointestinal inflammatory diseases or diseases associated with impaired epithelial barrier integrity / function.

[0254] The assay was performed in transwell plates as described below, where multiple cell types were co-cultured using a permeable membrane to separate the cells. Human colonic epithelial cells, consisting of a mixture of enterocytes and goblet cells, were cultured in the apical (upper) chamber until the cells acquired tight junction formation and functional barrier capacity, as assessed by measuring transepithelial electrical resistance (TEER). Monocytes were cultured separately in the basolateral chamber. Epithelial cells were stimulated with inflammatory cytokines. The assay measured the effect of a therapeutic protein, i.e., SG-11, on epithelial barrier function, muc2 gene expression, and cytokine production.

[0255] Cell Culture: HCT8 human intestinal cell line (ATCC catalog no. CCL-244) was maintained in RPMI-1640 medium (cRPMI) supplemented with 10% fetal bovine serum, 100 IU / ml penicillin, 100 μg / ml streptomycin, 10 μg / ml gentamicin, and 0.25 μg / ml amphotericin. HT29-MTX human goblet cells (Sigma-Aldrich, St. Louis, MO; catalog no. 12040401) were maintained in DMEM medium (cDMEM) containing 10% fetal bovine serum, 100 IU / ml penicillin, 100 μg / ml streptomycin, 10 μg / ml gentamicin, and 0.25 μg / ml amphotericin. Epithelial cells were passaged by trypsinization and used at passages 5–15 after thawing from liquid nitrogen stocks. U937 monocytes (ATCC catalog no. 700928) were maintained in cRPMI medium as suspension cultures at 5 × 10 5 ~2×10 6 Cells were split by dilution as needed to maintain a cell density of 1 / ml. U937 cells were used up to passage 18 after thawing from liquid nitrogen stock.

[0256] Epithelial cell culture: A mixture of HCT8 intestinal cells and HT29-MTX goblet cells was seeded in the apical chamber of a transwell plate at a ratio of 9:1, respectively, as previously described (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 cells were seeded in each well. 5 Cells were seeded (9 x 10 per well) 4 1 x 10 HCT8 cells and 1 x 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 into a single tube and centrifuged. The cell pellet was resuspended in cRPMI and added to the apical chamber of a transwell plate. Cells were cultured at 37°C + 5% CO2 for 8-10 days, with medium changes every 2 days.

[0257] Monocyte culture: On day 6 of epithelial cell culture, 2 × 10 5 U937 monocytes were seeded at 0.1 cells / well in a 96-well receiver plate. Cells were cultured at 37°C + 5% CO2 with medium changes every 24 hours for 4 days.

[0258] Co-culture assay: After 8–10 days of culture, transwell plates containing intestinal cells were treated with 10 ng / ml IFN-γ added to the basolateral chamber of the transwell plate for 24 hours at 37°C + 5% CO2. After 24 hours, fresh cRPMI was added to the epithelial cell culture plate. Transepithelial electrical resistance (TEER) readouts were measured after IFN-γ treatment and used as the pretreatment TEER value. SG-11 was then added to the apical chamber of the transwell plate at a final concentration of 1 μg / ml (40 nM). As a positive control to prevent inflammation-induced barrier disruption, myosin light chain kinase (MLCK) inhibitor peptide 18 (BioTechne, Minneapolis, MN) was used at 50 nM (Zolotarevskky et al., 202, Gastroenterology, 123:163–172). Staurosporine, a bacterial molecule, was used at 100 nM as a negative control to induce apoptosis and exacerbate barrier disruption (Antonsson and Persson, 2009, Anticancer Res, 29:2893-2898). Compounds were incubated on enterocytes for 1 or 6 hours. After preincubation with test compounds, transwell inserts containing enterocytes were transferred to the top of receiver plates containing U937 monocytes. Heat-killed Escherichia coli (HK E. coli) (bacteria heated to 80°C for 40 minutes) were then added to both the apical and basolateral chambers at a multiplicity of infection (MOI) of 10. Transwell plates were incubated at 37°C + 5% CO2 for 24 hours, and post-treatment TEER measurements were performed. TEER assays were performed using mature SG-11 protein (SEQ ID NO:5 or SEQ ID NO:9).

[0259] Data Analysis: Raw electrical resistance values ​​in ohms (Ω) were calculated based on the surface area of ​​the transwell insert (0.143 cm 2 ) based on ohms per square centimeter (Ωcm 2 ) after treatment in individual wells to adjust for the differential resistance that developed over the 10-day culture. 2 Readings were taken as Ωcm before treatment. 2Normalized to the reading. Then, the normalized Ωcm 2 Values ​​are the average Ωcm of the untreated samples 2 The values ​​were expressed as a percentage change from the original values.

[0260] SG-11 protein was added 1 hour (Figure 1A) or 6 hours (Figure 1B) before exposing both epithelial cells and monocytes to heat-killed E. coli (HK E. coli). Heat-killed E. coli induces monocytes to produce inflammatory mediators, resulting in the destruction of the epithelial monolayer, as indicated by a decrease in TEER. A myosin light chain kinase (MLCK) inhibitor was used as a control compound; it has been shown to prevent and / or reverse barrier destruction caused by antimicrobial immune responses. Staurosporine was used as a control compound, as it induces apoptosis and / or death of epithelial cells, thus resulting in a dramatic decrease in TEER, indicating the destruction and / or loss of epithelial cell barrier integrity / function. In Figure 1A, SG-11 increased TEER from 55.8% destruction by HK E. coli to 62%. In Figure 1B, SG-11 increased TEER from 53.5% destruction by HK E. coli to 60.6%. The graphs in Figures 1A and 1B represent pooled data from two separate experiments (n=6).

[0261] Example 3 Effect of SG-11 on TNF-α and IL-23 production induced by heat-killed Escherichia coli The following experiments demonstrate the therapeutic ability of the SG-11 protein or variants thereof described herein to reduce immune activation as measured by cytokine production, thereby demonstrating the potential functional utility of therapeutic proteins for treating gastrointestinal inflammatory diseases or diseases involving impaired epithelial barrier integrity / function, where modulation of cytokine levels impacts the host disease state.

[0262] Production of the inflammatory cytokines TNF-α and IL-23 by monocytes was measured in tissue culture supernatants from the basolateral chambers of the co-culture TEER assay performed in Example 2. After TEER readings, the supernatants were centrifuged at 10,000 g for 5 minutes at 4°C to remove cellular debris. Luminex analysis was performed according to the manufacturer's instructions (Magpix instrument and xPonent software version 4.2; Luminex Corporation, Austin, TX). Luminex analysis was performed to measure the pg / ml concentrations of TNF-α and IL-23 produced by monocytes. Luminex analysis utilizes a bead-based system for quantification of multiple cytokines from a single sample. Similar to ELISA, Luminex beads are coated with capture antibodies, and incubation with the sample allows targets to bind to the capture antibodies. The beads are washed and incubated with fluorescently labeled detection antibodies for quantification of bound targets. Cytometric analysis is used to distinguish between different fluorescent dye-loaded bead populations and quantify cytokine levels by measuring the detection antibody signal.

[0263] TNF-α and IL-23 production in untreated cells and in cells preincubated with SG-11 for 6 hours before HK E. coli treatment was normalized to the pg / ml concentration induced by HK E. coli, which was set at 1.0. Preincubation with SG-11 reduced the production of both TNF-α and IL-23. The results are shown in Figure 2A (TNF-α) and Figure 2B (IL-23). ​​The graphs in Figure 2A and Figure 2B represent pooled data from two separate experiments (n = 6).

[0264] Example 4 Effect of SG-11 on IL-10 production induced by heat-killed Escherichia coli In the following experiments, the effect of SG-11 administration on IL-10 production is measured by TEER assay.

[0265] IL-10 production was measured in tissue culture supernatants from the basolateral chamber of the co-culture TEER assay described in Example 2 containing monocytes. Luminex analysis was performed to measure the pg / ml concentration of IL-10 produced by monocytes. IL-10 production in untreated cells and in cells preincubated with SG-11 for 6 hours before HK E. coli treatment was normalized to the pg / ml concentration induced by HK E. coli, which was set at 1.0. Preincubation with SG-11 reduced IL-10 production to 0.89. The results are shown in Figure 3. The graph in Figure 3 represents pooled data from two separate experiments (n=6).

[0266] Example 5 Effect of SG-11 on mucin expression after stimulation with heat-killed Escherichia coli The following experiment measures the effect of the SG-11 protein described herein on increasing mucin expression in gastrointestinal tissue.Therefore, this experiment demonstrates the functional utility of the therapeutic protein SG-11 for treating inflammatory diseases of the gastrointestinal tract or diseases associated with impaired epithelial barrier integrity / function, where increased mucin expression is beneficial.

[0267] Gene expression was measured in epithelial cell monolayers from the apical chamber of the co-culture TEER assay described in Example 2. Total RNA was isolated from the epithelial cell monolayer, and cDNA was synthesized. qRT-PCR was performed on cDNA generated from HCT8 and HT29-MTX cells treated with SG-11 (1 μg / ml; 40 nM) for 6 hours before adding HK E. coli for 24 hours. Expression of the muc2 gene is graphed as the mean fold change ± SEM. Statistical analysis was performed by one-way ANOVA compared to HK E. coli, with Fisher's LSD test used for multiple comparisons.

[0268] Analysis of muc2 gene expression revealed that HK E. coli treatment alone resulted in a 13.6-fold increase compared to untreated cells (p=0.0007). Cells stimulated with HK E. coli and treated with SG-11 showed an additional 1.4-fold increase over HK E. coli (p=0.03) (Figure 4). The graph in Figure 4 represents data from a single experiment (n=3). A significant increase in muc2 production was observed in response to HK E. coli.

[0269] Example 6 Effect of SG-11 on wound healing in epithelial cells The following experiment demonstrates the therapeutic potential of the proteins described herein to increase wound healing in gastrointestinal epithelial cells. Thus, this experiment demonstrates the functional utility of the therapeutic protein SG-11 for treating gastrointestinal inflammatory diseases or diseases associated with impaired epithelial barrier integrity / function, where increased wound healing in epithelial cells is beneficial.

[0270] A 96-well Oris Cell Migration Assay containing a plug in the center of each well to prevent cell attachment was used according to the manufacturer's instructions (Platypus Technologies, Madison, WI).

[0271] Migration assay plates were warmed to room temperature before use, and plugs were removed from 100% confluent wells prior to cell addition. HCT8 intestinal cell line and HT29-MTX goblet cell line were used at a 9:1 ratio, for a total of 5 × 10 cells per well. 4 cells were added (4.5 × 10 4 0.5 x 10 HCT8 cells and 0.5 x 10 4HT29-MTX cells). Cells were incubated at 37°C + 5% CO2 for 24 hours. Plugs were then removed from all control and sample wells. Control wells included cells treated with diluted vehicle as a blank, cells treated with 30 ng / ml epidermal growth factor (EGF) as a positive control, and cells treated with 100 nM staurosporine as a negative control (all diluted in cRPMI). Sample wells contained SG-11 protein (SEQ ID NO:5 and / or SEQ ID NO:9) diluted in cRPMI at a concentration of 1 μg / ml. 100% and 0% wells were cultured in cRPMI. Cells were treated and incubated at 37°C + 5% CO2 for 48 hours. Plugs were removed from 0% wells before staining for live cells. Treatment medium was removed, and cells were washed with PBS containing 0.9 mM CaCl2 and 0.5 mM MgCl2. The green fluorescent viability dye, Calcenin AM, was added to all wells at a concentration of 0.5 μg / ml in PBS containing 0.9 mM CaCl2 and 0.5 mM MgCl2 and incubated at 37°C + 5% CO2 for 30 minutes. The dye was removed, and cells were washed with PBS containing 0.9 mM CaCl2 and 0.5 mM MgCl2, and fluorescence was measured. The relative fluorescence value of the 100% well, in which the plugs were removed prior to cell seeding, was set as the maximum effect, and the 0% well, in which the plugs remained in place until immediately before staining, was used as the baseline. Samples were normalized between the 100% and 0% samples, and values ​​were expressed as percent proliferation.

[0272] As shown in Figure 5, a significant increase in proliferation was observed upon treatment with SG-11. The control compound modulated wound healing as expected, EGF increased proliferation, and staurosporine inhibited cell proliferation. The graph in Figure 5 represents pooled data from five experiments (n=15). Data represent five independent replicate experiments, in which SG-11 of SEQ ID NO:5 was used in one experiment and SEQ ID NO:9 was used in four experiments.

[0273] Example 7 SG-11 exhibits therapeutic activity in a simultaneous DSS model of inflammatory bowel disease Examples 7 and 8 demonstrate the ability of the proteins described herein to treat inflammatory bowel disease in an in vivo model. Thus, this experiment demonstrates that the aforementioned in vitro model, which described key functions and potential modes of action, can be translated into an in vivo model system for inflammatory bowel disease. Specifically, the mice in Examples 7 and 8 were treated with dextran sulfate sodium (DSS), a chemical known to induce damage to the intestinal epithelium, thereby reducing the integrity and function of the intestinal barrier. DSS mice are a widely accepted model of colitis. In Example 7, mice were treated with SG-11 protein approximately simultaneously with (6 hours before) administration of DSS. In Example 8, mice were treated with DSS for 6 days before treatment with SG-11 protein.

[0274] The graph presented in Example 7 represents pooled data from three independent experiments (n=30) using 10 mice each. 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. In two experiments, the SG-11 protein consisted of SEQ ID NO:5; in the third experiment, the SG-11 protein consisted of SEQ ID NO:7.

[0275] Eight-week-old C57BL / 6 mice were housed five per cage and provided with food and water ad libitum for 7 days. After a 7-day acclimation period, treatment was initiated simultaneously with the addition of 2.5% DSS to the drinking water. Preliminary follow-up studies with fluorescently labeled bovine serum albumin after intraperitoneal (ip) injection demonstrated that the protein reached the colon 6 hours after ip delivery. Based on these results, mice were treated ip with 50 nmol / kg of SG-11 (1.3 mg / kg) or Gly2-GLP2 (0.2 mg / kg) 6 hours before the addition of 2.5% DSS to the drinking water. Six hours after this initial treatment, the drinking water was changed to water containing 2.5% DSS. Mice were treated with 2.5% dextran sulfate sodium (DSS) in drinking water for 6 days. Treatment continued with SG-11 or Gly2-GLP2 at 50 nmol / kg twice daily (bid) in the morning and evening (every 8 and 16 hours). Fresh 2.5% DSS drinking water was provided every 2 days.

[0276] On day 6, mice were fasted for 4 hours and then gavaged with 600 mg / kg of fluorescein isothiocyanate (FITC)-labeled 4KDa dextran [4KDa-FITC]. One hour after gavage, mice were euthanized, blood was collected, and serum FITC signal was measured. A significant increase in 4KDa-FITC dextran translocation across the epithelial barrier was observed in untreated mice compared with vehicle-treated DSS mice. Furthermore, a significant decrease in 4KDa-FITC dextran was observed in mice treated with DSS and SG-11 compared with vehicle-treated DSS mice. The extent of 4KDa-FITC dextran translocation observed with SG-11 was similar to that observed with the Gly2-GLP2 positive control. Results are shown in Figure 6 and are expressed as mean ± SEM. The graph in Figure 6 represents pooled data from three independent experiments (n=30).

[0277] SG-11 improves inflammation-centric readout of barrier function in a simultaneous DSS model of inflammatory bowel disease SG-11 was also evaluated for its effect on LPS-binding protein (LBP) levels in the blood of DSS animals treated with or without SG-11. LPS-binding protein (LBP), which is associated with clinical disease activity in patients with inflammatory bowel disease, was measured by ELISA in the serum of mice tested in the DSS model described in Example 7. A significant increase in LBP levels was observed in response to DSS. Furthermore, a significant decrease in LBP was observed in mice treated with DSS and SG-11 compared to vehicle-treated DSS mice. Furthermore, a significant difference was observed between DSS mice treated with Gly2-GLP2 and SG-11, indicating that SG-11 had a greater effect on LBP levels than the control peptide Gly2-GLP2. The results are shown in Figure 7 and are expressed as mean ± SEM. The graph in Figure 7 represents pooled data from three independent experiments (n = 30).

[0278] SG-11 prevents weight loss in a concurrent DSS model of inflammatory bowel disease The therapeutic potential of the proteins described herein to ameliorate weight loss in animals suffering from inflammatory bowel disease was also evaluated, which is a serious and potentially dangerous side effect of inflammatory bowel disease.

[0279] The body weight of mice included in the DSS model described in this example was measured daily. The percent change from the starting weight on day 0 was determined for each mouse. Administration of SG-11 to DSS-treated mice significantly improved body weight compared to vehicle-treated DSS mice. The weight loss on day 6 in SG-11-treated mice was similar to that observed with Gly2-GLP2. The results are shown in Figure 8. The graph in Figure 8 represents pooled data from two independent experiments (n=20).

[0280] SG-11 significantly reduces macroscopic lesions in the DSS model of inflammatory bowel disease Macroscopic lesions were observed in mice included in the simultaneous DSS model performed in this example. Administration of SG-11 to DSS-treated mice significantly improved macroscopic lesions compared to vehicle-treated DSS mice. No differences in clinical scores were observed between mice administered DSS and treated with Gly2-GLP2 or SG-11. The scoring system used was as follows: (0) = no macroscopic lesions, (1) = blood streaks in the feces, (2) = completely bloody fecal masses, (3) bloody fecal material in the cecum, (4) bloody fecal material in the cecum and loose stool, and (5) = rectal bleeding. The results are shown in Figure 9. The graph in Figure 9 represents pooled data from three independent experiments (n = 30). These data demonstrate that SG-11 is therapeutically effective in improving IBD symptoms, such as blood in the feces.

[0281] Furthermore, histopathological analysis was performed on proximal and distal colon tissues from DSS model animals. Proximal (Figure 10A) and distal (Figure 10B) colon scores (range 0-4) and a total colon score (Figure 10C) representing the sum of the proximal and distal colon scores (rated on a scale of 0-8) are shown. LMA = loss of mucosal structure, Edema = edema, INF = inflammation, TMI = transmural inflammation, MH = mucosal hyperplasia, and DYS = dysplasia. Graphs represent pooled 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 test for multiple comparisons.

[0282] SG-11 minimizes colon shortening effects in response to DSS treatment The following experiments demonstrate the therapeutic potential of the proteins described herein to treat inflammatory bowel disease in an in vivo model by showing their ability to prevent or minimize colon shortening.

[0283] Colon length was measured in mice included in the DSS model described in Example 7. Administration of SG-11 to DSS-treated mice prevented DSS-induced colon shortening. Significant improvements in colon length were observed with Gly2-GLP2, with Gly2-GLP2 treatment showing significant improvements compared to SG-11 treatment. The results are shown in Figure 11A. Furthermore, treatment of DSS-exposed mice with either Gly2-GLP2 or SG-11 resulted in significant improvements in colon weight-to-length ratio (Figure 11B). The graphs in Figures 11A and 11B represent pooled data from three independent experiments (n = 30). Data are graphed as mean ± SEM and are pooled from three independent experiments (n = 30). Statistical analysis was performed by one-way ANOVA followed by Fisher's LSD multiple comparison test.

[0284] Example 8 SG-11 exhibits therapeutic activity in DSS models of inflammatory disease In this example, an experiment was conducted to examine the effects of SG-11 in a mouse model of DSS when the SG-11 protein was administered to mice after 7 days of DSS treatment. This differs from the treatment regimen in Example 7 above, in which the SG-11 protein was administered to mice immediately before DSS treatment. This example further demonstrates the therapeutic potential of the proteins described herein to treat inflammatory bowel disease in an in vivo model, thereby demonstrating that the aforementioned in vitro model, which described important functions and potential modes of action, can be translated into an in vivo model system for inflammatory bowel disease.

[0285] Eight-week-old male C57BL / 6 mice were housed five per cage and given free access to food and water for 7 days. After a 7-day acclimation period, mice were given drinking water containing 2.5% DSS for 7 days. Fresh 2.5% DSS water was prepared every 2 days during the 7-day DSS administration. In 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.

[0286] On day 7, mice were returned to normal drinking water and treated with 50 nmol / kg of SG-11 (1.3 mg / kg) or Gly2-GLP2 (0.2 mg / kg) i.p. twice daily (bid) in the morning and evening (every 8 and 16 hours) for 6 days.

[0287] Treatment outcomes were analyzed for animal health, including body weight and gross lesions, colonic tissue pathology, assessment of barrier disruption, and levels of LPS-binding proteins, as detailed below.

[0288] Body weight was measured daily during the morning treatment. Colonic tissue was then harvested, its length measured in centimeters, and the tissue was weighed. Fecal material was flushed from the colon, and residual PBS was removed by gently handling the colonic tissue with a single forceps. Colonic tissue was then weighed, and the colon weight-to-length ratio, expressed in mg / mm, was determined. Following weight measurement, proximal and distal colonic tissue was saved for RNA and protein analysis, while the remaining tissue was fixed in 10% neutral-buffered formalin for histopathology. Statistical analysis was performed using one-way ANOVA compared to DSS + vehicle for serum 4KDa-FITC translocation, serum LBP concentration, colon length, and colon weight-to-length ratio, while two-way ANOVA was used for body weight analysis. Fisher's LSD test for multiple comparisons was used for all analyses. Graphs represent pooled data from two experiments and are plotted as mean ± SEM.

[0289] In this treatment model, recovery from established DSS injury was measured. No increase in 4KDa-FITC signal was observed after 6 days of DSS treatment, as untreated mice also showed recovery after DSS was removed from their drinking water (Figure 12). Furthermore, no decrease in LBP was observed after treatment with Gly2-GLP2 or SG-11 (Figure 13). Therefore, no changes in barrier function readouts were observed in this DSS treatment model.

[0290] In the therapeutic DSS model, no changes in barrier function readouts were observed, but significant improvements in clinical parameters, such as body weight (Figure 14), colon length (Figure 15A), and colon weight-to-length ratio (Figure 15B), were observed. Similar to the barrier function readouts, a macroscopic lesion scoring system based on bloody stool was no longer appropriate, as even DSS mice recovered after 6 days of treatment. However, although no visible blood remained in the colon, thickened colons were still observed. Based on macroscopic lesion findings, a reduction in the incidence of thickened colons was observed with SG-11 treatment (88% for DSS + vehicle and 25% for DSS + SG-11, p < 0.0001 by Fisher's exact test, data not shown).

[0291] Histopathological analysis was performed on proximal and distal colon tissues from the therapeutic DSS model described above. Proximal (Figure 16A) and distal (Figure 16B) colon scores (range 0-4) are shown, as well as a total colon score (range 0-8) (Figure 16C), representing the sum of the proximal and distal colon scores. LMA = loss of mucosal architecture, Edema = edema, INF = inflammation, TMI = transmural inflammation, MH = mucosal hyperplasia, and DYS = dysplasia. Graphs represent pooled 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 test for multiple comparisons.

[0292] Treatment with SG-11 and Gly2-GLP2 resulted in a slight but significant decrease in mucosal structure score; there was no change in inflammation score and transmural inflammation score. Similar to the results obtained in Example 7, a similar pattern of histopathological changes was observed with SG-11 and Gly2-GLP2, providing further evidence that SG-11 can target epithelial cells.

[0293] Example 9 Design of stable and therapeutically active SG-11 mutants SG-11 is a therapeutic protein derived from the commensal bacterium Roseburia hominis. Administration of R. hominis as a probiotic in the DSS model demonstrated efficacy with improvements in intestinal barrier function (4KDa-FITC and LBP), body weight, and clinical scores (data not shown).

[0294] Post-translational modifications (PTMs) can affect the recombinant production of therapeutic proteins, and therapeutic proteins can be affected by PTMs that can occur during large-scale expression and purification, as well as during long-term storage. Such PTMs include, but are not limited to, methionine oxidation, asparagine deamidation, and inter- and / or intramolecular disulfide bonds between two cysteines. In an attempt to mitigate the risk of PTMs, we modified SG-11 by replacing residues that could affect protein stability. Studies on improving protein stability and maintaining in vitro and in vivo activity are described in Examples 9-14.

[0295] As a first step, the SG-11 amino acid sequence (SEQ ID NO:7) was aligned to similar prokaryotic proteins, and residues identified based on the search results can be used for amino acid substitutions to enhance the stability of therapeutic proteins.

[0296] First, a BLAST search of the GenBank nonredundant protein database (NCBI BLAST / default parameters / BLOSUM62 matrix) was performed to identify other prokaryotic proteins that may be homologous to SG-11. The identified protein sequences are shown in Figure 17. SEQ ID NO: 21 is a hypothetical protein from Roseburia intestinalis (GenBank: WP_006857001.1; BLAST E-score: 3e-90); SEQ ID NO: 22 is a hypothetical protein from Roseburia sp. 831b (GenBank: WP_075679733.1; BLAST E-score: 4e-58); and SEQ ID NO: 23 is a hypothetical protein from Roseburia inulinivorans (GenBank: WP_055301040.1; BLAST E-score: 1e-83).

[0297] Each of SEQ ID NOs:21, 22, and 23 is a predicted mature form of the indicated protein (lacking a signal peptide) and containing an N-terminal methionine. A multiple sequence alignment of these sequences with SG-11 (SEQ ID NO:7) was performed to identify conserved regions between these proteins. This alignment is shown in Figure 17. This alignment was used to identify the most conserved residues between different proteins to evaluate the potential impact of substituting specific amino acid(s). Some portions of SG-11 are somewhat or highly conserved, with amino acids at specific positions in the protein being identical in all four of the aligned proteins, or in at least two (multiple positions) or three (multiple positions) of the four proteins. The high sequence conservation between these SG-11 homologs suggests that SEQ ID NOs:21, 22, and 23 may also have important functions in maintaining a healthy epithelial barrier. Thus, the present disclosure provides methods of utilizing SEQ ID NOs:21-23 to treat the diseases of the present disclosure, and methods of making them.

[0298] Example 10 Analysis of post-translational modifications (PTMs) of SG-11 A study was conducted using LC / MS / MS to identify residues in SG-11 that are particularly susceptible to PTM. This analysis was performed by LakePharma (Belmont, CA) to 1) confirm the amino acid sequence of SG-11 (SEQ ID NO:9) and 2) investigate post-translational modifications, particularly deamidation and oxidation, that may result in reduced biological activity and immunogenicity.

[0299] For peptide mapping and PTM analysis, samples were treated with DTT and IAA followed by trypsin digestion, and then analyzed on a Waters ACQUITY UPLC coupled to a Xevo G2-XS QTOF mass spectrometer using a Protein BEH C18 column.

[0300] Peptide mapping and sequencing confirmed the predicted amino acid sequence and also revealed multiple deamidation sites and one oxidation site. Among them, 7.84% of N53 and 3.77% of N83 were deamidated. These results, shown in Table 6, indicate that N53 and N83 are the major sites of deamidation under non-stress conditions. N53 represents an asparagine (Asn; N) at position 53 of mature SG-11 (SEQ ID NO:7) with a methionine at the first position.

[0301] Table 6. Post-translational modifications of SG-11 TIFF0007789303000008.tif71146 1 Amino acid positions in SG-11 (SEQ ID NO:7) 2 Normalized to total peptide ion intensity 3 Normalized to the total intensity of the corresponding precursor with or without modification

[0302] Example 11 Forced disassembly of SG-11 SG-11 (SEQ ID NO:9) was also tested under a series of stress conditions, as shown in Table 7 below, to further characterize the stability of purified recombinant SG-11. Stressed samples were analyzed by SEC-HPLC for the presence of aggregates and / or degradants. LC / MS / MS was performed to determine the levels of deamidation and oxidation.

[0303] (Table 7) TIFF0007789303000009.tif139146

[0304] In this assay, SG-11 (SEQ ID NO:9) was present 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 tests 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, pH 9).

[0305] This analysis shows that SG-11 (SEQ ID NO:9) samples treated at 4°C contain low levels of aggregates. Increasing the temperature increased the aggregation. At 37°C, extensive aggregation occurred. In contrast, mechanical stress and repeated freezing and thawing do not cause protein aggregation or degradation.

[0306] Three samples, each treated with either 2 weeks of incubation at 40°C, oxidation (HO treatment), or high pH 9, were analyzed for PTMs by LC / MS / MS as described in Example 10. As shown in Table 8 below, significant deamidation of N83 occurred after sample treatment at 40°C, resulting in nearly 100% deamidation. In samples treated with hydrogen peroxide, significant deamidation of N83 (37%) and oxidation of M200 (63.9%) was observed. 7.84% of N53 was deamidated without treatment.

[0307] (Table 8) TIFF0007789303000010.tif109153TIFF0007789303000011.tif170153 1 Amino acid positions in SG-11 (SEQ ID NO:7)

[0308] After reduction, free cysteines were artificially carbamidomethylated with iodoacetamide to block oxidation of cysteine ​​residues in this assay.

[0309] Example 12 Cysteine ​​residues and SG-11 stability 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) at 37°C for 1 week and at 4°C for 3 weeks. Stability was assessed by monitoring aggregate formation by analytical size-exclusion chromatography (SEC) equilibrated with buffer D (100 mM sodium phosphate, pH 7.0, 10% glycerol). Compared to freshly thawed protein, no significant changes were observed after 3 weeks of storage at 4°C; both samples showed a single peak at 1.57 mL. However, after 1 week of incubation at 37°C, the samples clearly showed aggregate peaks at 1.29 and 1.41 mL, in addition to the smallest peak, the monomer peak at 1.57 mL. The cause of aggregation was considered to be as follows: SG-11 contains two cysteine ​​residues at positions 147 and 151 (relative to SEQ ID NO:7). Ellman's reagent assay revealed the presence of a free sulfhydryl group in SG-11 (SEQ ID NO:9), and Cys 147 and / or Cys 151 It was shown that Cys does not form stable disulfide bonds. Because free sulfhydryl groups can cause aggregation by forming unfavorable intermolecular disulfide bonds, we investigated whether the presence of a reducing agent such as β-mercaptoethanol could prevent aggregation. After 4 days of incubation at 37°C, aggregation was significantly suppressed in the presence of 2.5% (v / v) β-mercaptoethanol in buffer (50 mM sodium phosphate, 150 mM NaCl, and 10% glycerol), in contrast to the formation of aggregates without β-mercaptoethanol. These results suggest that Cys 147 and / or Cys 151 It has been suggested that this provides free sulfhydryl groups that cause aggregation.

[0310] Example 13 Post-translational modifications of SG-11 mutants Although the SG-11 protein is stable at elevated temperatures, the formation of aggregates at 37°C over a week can be problematic in downstream processing steps. Deamidation of asparagine residues, as detected by LC / MS / MS, is also a risk factor. To improve the manufacturability of proteins containing SEQ ID NO:3 or its variants, 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)) were designed to reduce the occurrence of deleterious PTMs, taking into account the results of Examples 10-12.

[0311] Examples 13-16 describe experiments performed to characterize the effects of amino acid substitutions on the stability and function of SG-11 mutant SG-11V5 (SEQ ID NO:19; containing N53S, N83S, C147V, and C151S relative to SEQ ID NO:7) SG-11V5, which was expressed and purified as described in Example 1.

[0312] Due to the PTMs observed when SG-11 (SEQ ID NO:9) was exposed to stress conditions (Example 11), SG-11V5 (SEQ ID NO:19) was analyzed for post-translational modifications by LC-MS / MS using the methods described in Example 11 and compared to the PTMs of SG-11 (SEQ ID NO:7).

[0313] This analysis compared the PTMs of wild-type SG-11 (SEQ ID NO:7) and SG-11V5 (SEQ ID NO:19). In the first analysis (results shown in Table 9 below), the proteins were incubated in Buffer 1 (50 mM NaPO4 -Proteins were stored at a concentration of 1 mg / ml in a 5% CO₂O₂O₂O₂O₁₄ ...

[0314] (Table 9) TIFF0007789303000012.tif148149

[0315] In a second analysis, SG-11 (SEQ ID NO:7) and SG-11V5 (SEQ ID NO:19) proteins were each stored at 40°C in various buffers. The results are shown in Table 10 below. The storage buffers used in this experiment were 100 mM NaPO4 with 10% sorbitol (+Sor) or without 10% sorbitol (-Sor) and with 10% glycerol (+Gly) or without 10% glycerol (-Gly), as shown in Table 10. - , pH 7. As the data in Table 10 show, oxidation of the first methionine was significantly reduced in SG-11V5 (SEQ ID NO:19) protein compared to SG-11 (SEQ ID NO:7) protein across all buffer conditions. There were also differences in the levels of N137 deamidation between these two proteins, with a significant reduction in N137 deamidation at least in the presence of glycerol, and even in the presence of both sorbitol and glycerol. These data indicate that amino acid substitutions in SG-11 protein can have significant beneficial effects on the PTMs of the protein in solution.

[0316] (Table 10) TIFF0007789303000013.tif149146

[0317] Example 14 Construction and stability analysis of SG-11 mutants Although the SG-11 protein is highly stable at elevated temperatures, the formation of aggregates at 37°C over a one-week period can be problematic in downstream processing steps. Deamidation of asparagine residues, as detected by LC / MS / MS, is also a risk factor. To improve the manufacturability of proteins containing SEQ ID NO:3 or its variants, the protein designated SG-11 (SEQ ID NO:7) was mutated to contain the following four substitutions: N53S, N83S, C147V, and C151S. This variant containing the four substitutions is designated 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) shares approximately 98.3% sequence identity with SEQ ID NO:7.

[0318] Stability analysis of SG-11 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 (Figures 18A, 18B, and 18C), pH 7.0 (Figures 18D, 18E, and 18F), and pH 8.0 (Figures 18G, 18H, and 18I). The effect of additives was also tested at three different pH conditions: 150 mM NaCl (Figures 18A, 18D, and 18G); 150 mM NaCl and 100 mM arginine (Figures 18B, 18E, and 18H); and 150 mM NaCl and 0.5 M sorbitol (Figures 18C, 18F, and 18I). Stability was analyzed by analytical SEC. Arrows indicate the retention time of the monomeric form.

[0319] Stability analysis of SG-11V5 Figure 19 shows the effect of conditions on the stability of SG-11V5 (SEQ ID NO:19). SG-11V5 (SEQ ID NO:19) was incubated at pH 5.2 (Figures 19A, 19B, and 19C), pH 7.0 (Figures 19D, 19E, and 19F), and pH 8.0 (Figures 19G, 19H, and 19I). The effect of additives was also tested at three different pH conditions: 150 mM NaCl (Figures 19A, 19D, and 19G); 150 mM NaCl and 100 mM Arg (Figures 19B, 19E, and 19H); and 150 mM NaCl and 0.5 M sorbitol (Figures 19C, 19F, and 19I). Stability was analyzed by analytical SEC. Arrows indicate the retention time of the monomeric form.

[0320] In the presence of 100 mM arginine at pH 7.0, aggregate formation of purified SG-11 (SEQ ID NO:7) protein was significantly suppressed. However, several small peaks were observed at early retention times, indicating the presence of different forms other than the monomeric form. Under all conditions tested in this example, SG-11V5 (SEQ ID NO:19) did not exhibit significant aggregation. Without additives, a distinct monomer peak was observed. The small aggregation peak at 1.34 mL was suppressed by 100 mM arginine or 0.5 M sorbitol. Purified SG-11 (SEQ ID NO:7) and SG-11V5 (SEQ ID NO:19) precipitated at pH 5.2.

[0321] Elevated temperature can increase the susceptibility of proteins to aggregation and degradation due to deamidation. To minimize potential instability associated with deamidation and aggregation, we introduced the mutations N53S, N83S, C147V, and C151S into SG-11. SG-11V5 thus exhibited improved stability at pH 7.0 and pH 8.0.

[0322] Example 15 In vitro functional analysis of SG-11V5 To demonstrate that SG-11 variants, such as SG-11V5, maintain the functionality associated with maintaining epithelial barrier function shown for the SG-11 protein (see, e.g., Example 2), an in vitro TEER assay was performed.

[0323] Cell culture was performed as described in Example 2. Briefly, after 8–10 days of culture, transwell plates containing intestinal cells were treated with 10 ng / ml IFN-γ added to the basolateral chamber of the transwell plate for 24 hours at 37°C + 5% CO2. After 24 hours, fresh cRPMI was added to the epithelial cell culture plate. Transepithelial electrical resistance (TEER) readings were measured after IFN-γ treatment and used as the pretreatment TEER value. Next, SG-11 (SEQ ID NO:9) or SG-11V5 (SEQ ID NO:19) was 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 at 50 nM as a positive control to prevent inflammation-induced barrier disruption (Zolotarevskky et al., 202, Gastroenterology, 123:163-172). Compounds were incubated on enterocytes for 6 h. After preincubation with test compounds, transwell inserts containing enterocytes were transferred on top of receiver plates containing U937 monocytes. Heat-killed Escherichia coli (HK E. coli) (bacteria heated to 80°C for 40 min) were then added to both the apical and basolateral chambers at a multiplicity of infection (MOI) of 10. Transwell plates were incubated at 37°C + 5% CO2 for 24 h, and post-treatment TEER measurements were performed. SG-11 (SEQ ID NO:9) increased TEER from 78.6% destruction by HK E. coli to 89.5% (p<0.0001), while SG-11V5 (SEQ ID NO:19) increased it to 89.1% (p<0.0001) (Figure 20). Statistical analysis was performed using one-way ANOVA compared to HK E. coli, followed by Fisher's LSD multiple comparison test. The graph in Figure 20 represents pooled data from four plates run in two separate experiments (n=12).

[0324] Example 16 In vivo functional analysis of SG-11V5 The DSS animal model experiments performed as described above in Examples 7 and 8 were then 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 either simultaneously with the initiation of DSS treatment (as in Example 7) or after prior 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.

[0325] In the first DSS mouse model (Example 16A; same method as Example 7), mice were treated intraperitoneally (ip) with test compounds on day 0, and DSS treatment was initiated 6 hours later. Administered doses included 50 nmol / kg (1.3 mg / ml) for SG-11 (SEQ ID NO:9) and Gly2-GLP2 (0.2 mg / kg), and dose-response for SG-11V5 (SEQ ID NO:19) including 16 nmol / kg (0.4 mg / ml), 50 nmol / kg (1.3 mg / ml), and 158 nmol / kg (4.0 mg / kg). Mice were treated with 2.5% DSS in drinking water for 6 days (days 0 to 6). Therapeutic protein treatment was administered twice daily during the DSS exposure period.

[0326] In the second experiment (Example 16B; same method as in Example 8), mice were given 2.5% DSS in drinking water for 7 days. On day 7, they were switched back to normal drinking water, and ip treatment with 50 nmol / kg of 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 initiated. Treatment was administered twice daily (bid) in the morning and evening (every 8 and 16 hours) for 4 days. In both DSS models (Examples 16A and 16B), fresh 2.5% DSS water was provided every 2 days during DSS administration.

[0327] At the end of each DSS experiment, mice were fasted for 4 hours and then gavaged with 600 mg / kg of fluorescein isothiocyanate (FITC)-labeled 4KDa dextran [4KDa-FITC]. One hour after gavage, mice were euthanized, blood was collected, and serum FITC signal was measured. In the first model, a significant increase in the translocation of 4KDa-FITC dextran across the epithelial barrier was observed in vehicle-treated DSS mice compared to untreated mice. These results are shown in Figure 21A: 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, but the difference did not reach statistical significance (p = 0.21). In Figure 21B, no effect of SG-11 or SG-11V5 treatment was observed, as no increase in 4KDa-FITC was observed, similar to the previous results in Example 8. Data in both graphs are plotted as mean ± SEM, and each figure represents data from a separate experiment (n = 10 / group).

[0328] Effect of SG-11V5 on inflammation-centric readout of barrier function in the DSS model of inflammatory bowel disease Upon completion of the DSS model, LBP levels were measured as an inflammation-centered readout of barrier function according to the protocol detailed in Example 7. Upon completion of both DSS models (Examples 16A and 16B), blood was collected and serum was separated. Serum LPS-binding protein (LBP) levels were measured using a commercially available ELISA kit (Enzo Life Sciences, Farmingdale, NY). The results are shown in Figure 22A (Example 16A) and Figure 22B (Example 16B). In the DSS model of Example 16A, a significant increase in LBP was observed in response to DSS exposure. Similar decreases in LBP were observed with 50 nmol / kg doses of SG-11 (SEQ ID NO:9) and SG-11V5 (SEQ ID NO:19), although neither was statistically significant. However, treatment with a high dose of 158 nmol / kg SG-11V5 (SEQ ID NO:19) resulted in a significant decrease in LBP production (p=0.003) (FIG. 22A). In the DSS model of Example 16B, exposure to DSS resulted in a significant increase in LBP production (FIG. 22B). However, no decrease in LBP was observed with any of the treatments, and similar results 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 due to the long half-life of LBP in the circulation (reportedly 12-24 hours), it may be difficult to observe a reduction in systemic LBP levels in models in which an LBP response is induced (DSS is administered) before treatment begins (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).

[0329] Effects of SG-11 and SG-11V5 on body weight in the DSS model of inflammatory bowel disease Body weight was measured throughout the experimental models in both Example 16A and Example 16B. In the DSS model of Example 16A (Figure 23A), similar trends in body weight were observed for 50 nmol / kg SG-11 (SEQ ID NO:9) and SG-11V5 (SEQ ID NO:19) treatment, with a significant improvement in body weight observed on day 6 with 158 nmol / kg SG-11V5 (SEQ ID NO:19). A similar pattern was observed in the therapeutic DSS model, with SG-11 (SEQ ID NO:9) and SG-11V5 (SEQ ID NO:19) showing similar body weight changes at the 50 nmol / kg dose, and both showing statistically improved body weight changes on day 11 (p<0.05). In Figures 23A and 23B, data are graphed as mean ± SEM, with each graph representing data from a separate experiment. Statistical analysis was performed using two-way ANOVA with Fisher's LSD multiple comparison test compared with the DSS+vehicle group.

[0330] Effects of SG-11 and SG-11V5 on macroscopic lesions in a DSS model of inflammatory bowel disease Macroscopic observation of colon tissue was performed for Example 16A as described in Example 7 above. Briefly, a scoring system based on the level of visible blood and fecal mass consistency was used. The scoring system used was as follows: (0) = no macroscopic lesions, (1) = blood streaks in the feces, (2) = completely bloody fecal masses, (3) bloody fecal material in the cecum, (4) bloody fecal material in the cecum and loose stool, and (5) = rectal bleeding. Similar results were obtained with SG-11 (SEQ ID NO:9) and SG-11V5 (SEQ ID NO:19) at a dose of 50 nmol / kg; a dose-dependent effect was observed for SG-11V5 (SEQ ID NO:19), with the 160 nmol / kg dose resulting in significant improvement (p<0.002). The data shown in Figure 24 are expressed as mean ± SEM and include data from individual experiments. Statistical analysis was performed using one-way ANOVA followed by Fisher's LSD multiple comparison test.

[0331] Effect of SG-11 and SG-11V5 on colon length in the DSS model of inflammatory bowel disease The DSS model from Example 16 was also analyzed for the effects of SG-11 and SG-11 mutant proteins on colon length. Colon length measurements were performed in the DSS model of Example 16A (FIG. 25A) or Example 16B (FIG. 25B). 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 on colon length was observed with SG-11V5 (SEQ ID NO:19). Data in both graphs are presented as mean ± SEM and represent data from individual experiments. Statistical analysis was performed using one-way ANOVA compared to DSS + vehicle, followed by Fisher's LSD multiple comparison test.

[0332] Effects of SG-11 and SG-11V5 on colon weight-to-length ratio in a DSS model of inflammatory bowel disease The DSS model from Example 16 was also analyzed for the effects of SG-11 and SG-11 mutant proteins on colon weight-to-length ratio. Colon weight-to-length ratio was similar between SG-11 (SEQ ID NO:9) and SG-11V5 (SEQ ID NO:19) in the DSS model treatment regimens of Example 16A (FIG. 26A) and Example 16B (FIG. 26B). In the treatments of Example 16A, all treatments and doses significantly improved 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 colon weight-to-length ratio (p<0.01), but the positive control Gly2-GLP2 did not. Statistical analysis was performed by one-way ANOVA compared to DSS + vehicle using Fisher's LSD multiple comparison test. Data are graphed as mean ± SEM, and each figure represents data from a single experiment.

[0333] Although the foregoing disclosure has been described in some detail by way of illustration and example for clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications can be made therein without departing from the spirit and scope of the present disclosure as set forth in the appended claims. Accordingly, the foregoing description should not be construed as limiting the scope of the present disclosure.

[0334] Table 11 shows the SEQ ID NOs of the present disclosure with detailed information.

[0335] (Table 11) TIFF0007789303000014.tif50146TIFF0007789303000015.tif223146TIFF0007789303000016.tif184146

[0336] Numbered Embodiments of the Disclosure Without limiting the scope of the appended claims, the present disclosure sets forth the following numbered embodiments:

[0337] Treatment methods 1. 1. A method for treating gastrointestinal epithelial cell barrier dysfunction, comprising: a. To patients who need it, 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 ii. A pharmaceutically acceptable carrier administering a pharmaceutical composition comprising A method comprising: 2. Item 1. The method according to Item 1, wherein the gastrointestinal epithelial cell barrier dysfunction is a disease associated with a decrease in the integrity of the gastrointestinal wall. 3. Item 3. The method according to item 1 or 2, wherein the gastrointestinal epithelial cell barrier dysfunction is a disease associated with a decrease in the integrity of the gastrointestinal mucosal epithelium. 4. Item 4. The method according to any one of Items 1 to 3, wherein the gastrointestinal epithelial cell barrier dysfunction is a disease associated with a decrease in the integrity of the intestinal epithelium. 5. 5. The method of any one of paragraphs 1 to 4, wherein the gastrointestinal epithelial cell barrier dysfunction is at least one selected from the group consisting of inflammatory bowel disease, Crohn's disease, ulcerative colitis, pouchitis, irritable bowel syndrome, intestinal infection, Clostridium difficile infection, metabolic disease, obesity, type 2 diabetes, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, liver damage, alcoholic steatohepatitis, celiac disease, necrotizing enterocolitis, gastrointestinal disorders, short bowel syndrome, GI mucositis, chemotherapy-induced mucositis, radiation-induced mucositis, oral mucositis, interstitial cystitis, neurological disorders, cognitive disorders, Alzheimer's disease, Parkinson's disease, multiple sclerosis, autism, chemotherapy-associated steatohepatitis (CASH), and pediatric versions of the aforementioned diseases. 6. Item 6. The method according to any one of Items 1 to 5, wherein the gastrointestinal epithelial cell barrier dysfunction is inflammatory bowel disease. 7. Item 7. The method according to any one of Items 1 to 6, wherein the gastrointestinal epithelial cell barrier dysfunction is Crohn's disease. 8. Item 7. The method according to any one of Items 1 to 6, wherein the gastrointestinal epithelial cell barrier dysfunction is ulcerative colitis. 9. 9. The method of any one of paragraphs 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. 10. 10. The method of any one of paragraphs 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. 11. 11. The method of any one of paragraphs 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. 12. 12. The method of any one of paragraphs 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. 13. 13. The method of any one of paragraphs 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. 14. 14. The method of any one of paragraphs 1 to 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. 15. 15. The method of any one of paragraphs 1 to 14, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO:3. 16. 15. The method of any one of paragraphs 1 to 14, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO:7. 17. 15. The method of any one of paragraphs 1 to 14, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO:11. 18. 15. The method of any one of paragraphs 1 to 14, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO:13. 19. 15. The method of any one of paragraphs 1-14, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO:15. 20. 15. The method of any one of paragraphs 1-14, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO:17. twenty one. 15. The method of any one of paragraphs 1 to 14, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO:19. twenty two. 22. The method of any one of paragraphs 1 to 21, wherein the administering comprises rectal, parenteral, intravenous, topical, oral, cutaneous, transdermal, or subcutaneous administration. twenty three. 23. The method of any one of paragraphs 1 to 22, wherein the administering is to the patient's mouth, gastrointestinal lumen, and / or intestines. twenty four. 24. The method of any one of paragraphs 1 to 23, wherein the patient experiences a reduction in at least one symptom associated with gastrointestinal epithelial cell barrier dysfunction. twenty five. 25. The method of any one of paragraphs 1 to 24, wherein the patient experiences relief of at least one symptom associated with gastrointestinal epithelial cell barrier dysfunction selected from the group consisting of abdominal pain, bloody stool, pus-filled stool, fever, weight loss, frequent diarrhea, fatigue, loss of appetite, tenesmus, and rectal bleeding. 26. 26. The method of any one of paragraphs 1 to 25, wherein said administering reduces gastrointestinal inflammation in the patient. 27. 26. The method of any one of paragraphs 1 to 25, wherein the administering reduces inflammation of the intestinal mucosa in the patient. 28. 26. The method of any one of paragraphs 1 to 25, wherein the administering increases mucin production in the patient's intestinal tissue. 29. 26. The method of any one of paragraphs 1 to 25, wherein the administering increases intestinal epithelial wound healing in the patient. 30. 26. The method of any one of items 1 to 25, wherein the administering increases proliferation of intestinal epithelial cells in the patient. 31. 31. The method of any one of paragraphs 1 to 30, further comprising administering to the patient at least one second therapeutic agent. 32. 32. The method of any one of items 1 to 31, further comprising administering to the patient at least one second therapeutic agent, wherein the second therapeutic agent is 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.

[0338] Pharmaceutical Compositions 1. 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 b. A pharmaceutically acceptable carrier 10. A pharmaceutical composition comprising: 2. Item 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. 3. 3. The pharmaceutical composition of paragraph 1 or 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. 4. 4. The pharmaceutical composition of any one of paragraphs 1 to 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. 5. 5. The pharmaceutical composition of any one of paragraphs 1 to 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. 6. 6. The pharmaceutical composition of any one of paragraphs 1 to 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. 7. 7. The pharmaceutical composition of any one of paragraphs 1 to 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. 8. 8. The pharmaceutical composition of any one of paragraphs 1 to 7, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO:3. 9. 8. The pharmaceutical composition of any one of paragraphs 1 to 7, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO:7. 10. 8. The pharmaceutical composition of any one of paragraphs 1 to 7, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO:11. 11. 8. The pharmaceutical composition of any one of paragraphs 1 to 7, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO:13. 12. 8. The pharmaceutical composition of any one of paragraphs 1 to 7, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO:15. 13. 8. The pharmaceutical composition of any one of paragraphs 1 to 7, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO:17. 14. 8. The pharmaceutical composition of any one of paragraphs 1 to 7, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO:19. 15. 15. The pharmaceutical composition of any one of paragraphs 1 to 14, formulated for rectal, parenteral, intravenous, topical, oral, dermal, transdermal, or subcutaneous administration. 16. 16. The pharmaceutical composition of any one of paragraphs 1 to 15, wherein the therapeutic protein is formulated to be active in the gastrointestinal lumen and / or intestine of a patient.

[0339] Expression vector 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. 2. 2. The expression vector of paragraph 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. 3. 3. The expression vector of paragraph 1 or 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. 4. 4. The expression vector of any one of paragraphs 1 to 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. 5. 5. The expression vector of any one of paragraphs 1 to 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. 6. 6. The expression vector of any one of paragraphs 1 to 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. 7. 7. The expression vector of any one of paragraphs 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. 8. 8. The expression vector of any one of paragraphs 1 to 7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO:3. 9. 8. The expression vector of any one of paragraphs 1 to 7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO:5. 10. 8. The expression vector of any one of paragraphs 1 to 7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO:7. 11. 8. The expression vector of any one of paragraphs 1 to 7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO:9. 12. 8. The expression vector of any one of paragraphs 1 to 7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO:11. 13. 8. The expression vector of any one of paragraphs 1 to 7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO:13. 14. 8. The expression vector of any one of paragraphs 1 to 7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO:15. 15. 8. The expression vector of any one of paragraphs 1 to 7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO:17. 16. 8. The expression vector of any one of paragraphs 1 to 7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO:19.

[0340] host cell 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. 2. The host cell of paragraph 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. 3. 3. The host cell of paragraph 1 or 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. 4. 4. The host cell of any one of paragraphs 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. 5. 5. The host cell of any one of paragraphs 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. 6. 6. The host cell of any one of paragraphs 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. 7. 7. The host cell of any one of paragraphs 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. 8. 8. The host cell of any one of paragraphs 1 to 7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO:3. 9. 8. The host cell of any one of paragraphs 1-7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO:5. 10. The host cell of any one of paragraphs 1 to 7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO:7. 11. 8. The host cell of any one of paragraphs 1-7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO:9. 12. 8. The host cell of any one of paragraphs 1-7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO:11. 13. 8. The host cell of any one of paragraphs 1 to 7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO:13. 14. 8. The host cell of any one of paragraphs 1-7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO:15. 15. The host cell of any one of paragraphs 1 to 7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO:17. 16. 8. The host cell of any one of paragraphs 1-7, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO:19. 17. 17. The host cell of any one of paragraphs 1 to 16, wherein the exogenous polynucleotide further encodes a host cell-specific signal sequence. 18. 18. The host cell of any one of paragraphs 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 a nucleic 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. 19. 19. The host cell of any one of paragraphs 1 to 18, which is a prokaryotic cell. 20. 20. The host cell of any one of paragraphs 1 to 19, which is an Escherichia coli cell. twenty one. 19. The host cell of any one of paragraphs 1 to 18, which is a eukaryotic cell. twenty two. The host cell of any one of paragraphs 1 to 18 and 21, which is a Chinese hamster ovary cell. twenty three. 23. A method of producing a protein, comprising culturing a host cell of any one of paragraphs 1 to 22 under conditions sufficient for expression of the encoded protein.

[0341] Isolated proteins 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. 2. The isolated therapeutic protein of paragraph 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. 3. The isolated therapeutic protein of paragraph 1 or 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. 4. 4. The isolated therapeutic protein of any one of paragraphs 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. 5. 5. The isolated therapeutic protein of any one of paragraphs 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. 6. 6. The isolated therapeutic protein of any one of paragraphs 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. 7. 7. The isolated therapeutic protein of any one of paragraphs 1 to 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. 8. 8. The isolated therapeutic protein of any one of paragraphs 1 to 7, comprising the amino acid sequence of SEQ ID NO:3. 9. 8. The isolated therapeutic protein of any one of paragraphs 1 to 7, comprising the amino acid sequence of SEQ ID NO:5. 10. 8. The isolated therapeutic protein of any one of paragraphs 1 to 7, comprising the amino acid sequence of SEQ ID NO:7. 11. 8. The isolated therapeutic protein of any one of paragraphs 1 to 7, comprising the amino acid sequence of SEQ ID NO:9. 12. 8. The isolated therapeutic protein of any one of paragraphs 1 to 7, comprising the amino acid sequence of SEQ ID NO:11. 13. 8. The isolated therapeutic protein of any one of paragraphs 1 to 7, comprising the amino acid sequence of SEQ ID NO:13. 14. 8. The isolated therapeutic protein of any one of paragraphs 1 to 7, comprising the amino acid sequence of SEQ ID NO:15. 15. 8. The isolated therapeutic protein of any one of paragraphs 1 to 7, comprising the amino acid sequence of SEQ ID NO:17. 16. 8. The isolated therapeutic protein of any one of paragraphs 1 to 7, comprising the amino acid sequence of SEQ ID NO:19. 17. 17. The isolated therapeutic protein of any one of paragraphs 1 to 16, wherein the protein increases electrical resistance in an in vitro transepithelial electrical resistance assay. 18. 18. The isolated therapeutic protein of any one of paragraphs 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 the assay performed in the absence of the protein. 19. 19. The isolated therapeutic protein of any one of paragraphs 1 to 18, wherein the protein increases electrical resistance in an in vitro transepithelial electrical resistance assay compared to a kinase inhibitor control. 20. 20. The isolated therapeutic protein of any one of paragraphs 1 to 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.

[0342] Synthetic Therapeutic Proteins 1. A synthetic therapeutic protein comprising an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:19. 2. The protein of item 1, comprising an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:19. 3. The protein of paragraph 1 or 2, comprising an amino acid sequence having at least about 97% sequence identity to SEQ ID NO:19. 4. The protein of any one of paragraphs 1 to 3, comprising an amino acid sequence having at least about 98% sequence identity to SEQ ID NO:19. 5. 5. The protein of any one of paragraphs 1 to 4, comprising an amino acid sequence having at least about 99% sequence identity to SEQ ID NO:19. 6. The protein of any one of paragraphs 1 to 5, comprising the amino acid sequence of SEQ ID NO:19. 7. The protein of any one of paragraphs 1 to 6, wherein the amino acid at position 147 is valine. 8. The protein of any one of items 1 to 6, wherein the amino acid at position 151 is serine. 9. 7. The protein of any one of Items 1 to 6, wherein the amino acid at position 147 is valine and the amino acid at position 151 is serine. 10. 6. The protein of any one of items 1 to 5, wherein the amino acid at position 84 is aspartic acid. 11. 6. The protein of any one of Items 1 to 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. 12. 7. The protein of any one of items 1 to 6, wherein the amino acid at position 83 is serine. 13. 7. The protein of any one of Items 1 to 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. 14. 7. The protein of any one of items 1 to 6, wherein the amino acid at position 53 is serine. 15. 6. The protein of any one of Items 1 to 5, wherein the amino acid at position 53 is serine, 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. 16. 7. The protein of any one of Items 1 to 6, wherein the amino acid at position 53 is serine, 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. 17. 7. The protein of any one of items 1 to 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. 18. 18. The protein of any one of paragraphs 1 to 17, wherein the protein increases electrical resistance in an in vitro transepithelial electrical resistance assay. 19. A pharmaceutical composition comprising the protein of any one of items 1 to 17 and a pharmaceutically acceptable carrier. 20. 1. A method for treating gastrointestinal epithelial cell barrier dysfunction, comprising: a. To patients who need it, 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 ii. A pharmaceutically acceptable carrier administering a pharmaceutical composition comprising: A method comprising:

[0343] Incorporation by Reference All references, articles, publications, patents, patent publications, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0344] However, mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be construed as, an admission or in any way an indication that they constitute valid prior art or form part of the common general knowledge in any country throughout the world.

[0345] References TIFF0007789303000017.tif218146TIFF0007789303000018.tif218147TIFF0007789303000019.tif225147TIFF0007789303000020.tif178146

Claims

1. 1. Use of a therapeutic protein in the manufacture of a medicament for treating gastrointestinal epithelial cell barrier dysfunction in a patient, comprising: The therapeutic protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:19 and has the function of increasing the barrier function of an epithelial cell layer by at least 5% in an in vitro transepithelial electrical resistance (TEER) assay.

2. The use according to claim 1, wherein the gastrointestinal epithelial cell barrier dysfunction is a disease associated with a decrease in the integrity of the gastrointestinal wall, a decrease in the integrity of the gastrointestinal mucosal epithelium, or a decrease in the integrity of the intestinal epithelium.

3. 2. The use of claim 1, wherein the gastrointestinal epithelial cell barrier dysfunction is associated with at least one of inflammatory bowel disease, Crohn's disease, ulcerative colitis, pouchitis, irritable bowel syndrome, celiac disease, necrotizing enterocolitis, short bowel syndrome, GI mucositis, chemotherapy-induced mucositis, radiation-induced mucositis, oral mucositis, and pediatric versions of the aforementioned diseases.

4. 2. The use according to claim 1, wherein the gastrointestinal epithelial cell barrier dysfunction is inflammatory bowel disease, Crohn's disease, or ulcerative colitis.

5. 2. The use of claim 1, wherein the therapeutic protein comprises an amino acid sequence having at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:

19.

6. 2. The use of claim 1, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO:

19.

7. 10. The use of claim 1, wherein the medicament is administered by rectal, parenteral, intravenous, topical, oral, cutaneous, transdermal, or subcutaneous administration.

8. The use of claim 1 , wherein the medicament is administered to the patient's mouth, gastrointestinal lumen, and / or intestines.

9. 10. The use of claim 1, wherein the patient experiences a reduction in at least one symptom associated with gastrointestinal epithelial cell barrier dysfunction.

10. 10. The use of claim 1, wherein the patient experiences relief of at least one symptom associated with gastrointestinal epithelial cell barrier dysfunction selected from the group consisting of abdominal pain, bloody stools, pus-filled stools, fever, weight loss, frequent diarrhea, fatigue, loss of appetite, tenesmus, and rectal bleeding.

11. 2. The use of claim 1, wherein the medicament reduces gastrointestinal inflammation or reduces inflammation of the intestinal mucosa in a patient.

12. 2. The use of claim 1, wherein the medicament increases mucin production in the patient's intestinal tissue, increases intestinal epithelial wound healing, or increases intestinal epithelial cell proliferation.

13. 10. The use of claim 1, wherein the medicament is administered to the patient in combination with at least one second therapeutic agent.

14. 2. The use of claim 1, wherein the medicament is administered to the patient in combination with at least one second therapeutic agent, and the second therapeutic agent is 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.

15. a. a therapeutic protein comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:19 and having the function of increasing the barrier function of an epithelial cell layer by at least 5% in an in vitro transepithelial electrical resistance (TEER) assay; and b. A pharmaceutically acceptable carrier 1. A pharmaceutical composition comprising: A pharmaceutical composition, wherein the pharmaceutical composition is for use in treating gastrointestinal epithelial cell barrier dysfunction in a patient.

16. 16. The pharmaceutical composition of claim 15, wherein the therapeutic protein comprises an amino acid sequence having at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:

19.

17. 16. The pharmaceutical composition of claim 15, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO:

19.

18. 16. The pharmaceutical composition of claim 15, formulated for rectal, parenteral, intravenous, topical, oral, dermal, transdermal, or subcutaneous administration.

19. 16. The pharmaceutical composition of claim 15, wherein the therapeutic protein is formulated to be active in the gastrointestinal lumen and / or intestine of a patient.

20. An expression vector comprising a polynucleotide encoding a protein comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:19, wherein the protein has the function of increasing the barrier function of an epithelial cell layer by at least 5% in an in vitro transepithelial electrical resistance (TEER) assay.

21. 21. The expression vector of claim 20, wherein the encoded protein comprises an amino acid sequence having at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:

19.

22. 21. The expression vector of claim 20, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO:

19.

23. A host cell comprising an exogenous polynucleotide encoding a protein comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:19, wherein the protein has the function of increasing the barrier function of an epithelial cell layer by at least 5% in an in vitro transepithelial electrical resistance (TEER) assay.

24. 24. The host cell of claim 23, wherein the encoded protein comprises an amino acid sequence having at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:

19.

25. 24. The host cell of claim 23, wherein the encoded protein comprises the amino acid sequence of SEQ ID NO:

19.

26. 24. The host cell of claim 23, wherein the exogenous polynucleotide further encodes a host cell-specific signal sequence.

27. 24. The host cell of claim 23, wherein the exogenous polynucleotide comprises a nucleic acid sequence having at least 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:20, and wherein the exogenous polynucleotide is capable of increasing the barrier function of an epithelial cell layer by at least 5% in an in vitro transepithelial electrical resistance (TEER) assay.

28. 24. The host cell of claim 23, which is a prokaryotic cell.

29. 24. The host cell of claim 23, which is an Escherichia coli cell.

30. 24. The host cell of claim 23, which is a eukaryotic cell.

31. 24. The host cell of claim 23, which is a Chinese hamster ovary cell.

32. 24. A method for producing a protein, comprising culturing the host cell of claim 23 under conditions sufficient for expression of the encoded protein, wherein the host cell is not cultured within a human body.

33. An isolated therapeutic protein comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:19, wherein the isolated therapeutic protein does not comprise an amino acid sequence identical to SEQ ID NO:3, and wherein the isolated therapeutic protein has the function of increasing the barrier function of an epithelial cell layer by at least 5% in an in vitro transepithelial electrical resistance (TEER) assay.

34. 34. The isolated therapeutic protein of claim 33, comprising an amino acid sequence having at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:

19.

35. 34. The isolated therapeutic protein of claim 33, comprising the amino acid sequence of SEQ ID NO:

19.

36. 34. The isolated therapeutic protein of claim 33, wherein the protein increases electrical resistance in an in vitro transepithelial electrical resistance assay compared to a kinase inhibitor control.

37. 34. The isolated therapeutic protein of claim 33, wherein the protein increases electrical resistance in an in vitro transepithelial electrical resistance assay compared to a staurosporine or myosin light chain kinase control.

38. 24. The host cell of claim 23, which is Lactococcus lactis.

39. The host cell of claim 23; and Pharmaceutically acceptable carrier 10. A pharmaceutical composition comprising:

40. A host cell for use in treating gastrointestinal epithelial cell barrier dysfunction, the host cell comprising an exogenous polynucleotide encoding a protein comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:19, and the exogenous polynucleotide having the function of increasing the barrier function of an epithelial cell layer by at least 5% in an in vitro transepithelial electrical resistance (TEER) assay.

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