Modified TFF2 polypeptides

Abstract

To provide modified TFF2 polypeptides.SOLUTION: Described herein are modified TFF2 polypeptides, compositions comprising these polypeptides, and their use to treat cancer and inflammation. In some embodiments, the improved properties of the disclosed modified TFF2 polypeptides are achieved using chemical modifications including PEGylation or poly (D,L-lactic-co-glycolic acid) (PLGA), and / or polysialylation (PSA), and / or fusion proteins, including fusion proteins with C-terminal peptide (CTP) of human chorionic gonadotropin β subunit, PASylation, homo-amino acid polymers (HAP), elastin-like peptides (ELPylation), XTENylated, and combinations of these modifications.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 892,520, filed August 27, 2019, U.S. Provisional Application No. 62 / 943,803, filed December 4, 2019, and U.S. Provisional Application No. 63 / 041,097, filed June 18, 2020, the contents of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application has been submitted electronically in ASCII format and includes a Sequence Listing, which is incorporated herein by reference in its entirety. The ASCII copy, created on August 27, 2020, is named 104545-0046-WO1_SL.txt and is 37,566 bytes in size.

[0003] Field of the Disclosure The present disclosure is in the field of using modified TFF2 polypeptides to treat subjects with cancer and / or inflammatory conditions. [Background technology]

[0004] Background of the Disclosure Trefoil family factor 2 (TFF2) (also known as pancreatic spasmolytic polypeptide, PSP, or spasmolytic peptide, SP) is a member of the trefoil factor family of peptides. Human TFF2 is a secreted protein of 106 amino acids. Mature human TFF2 is a 12 kDa protein containing two trefoil domains separated by seven residues that are highly conserved in other species, including pigs. The crystal structure of pig TFF2 has been solved (De A et al, (1994) Proc Natl Acad Sci USA 91(3):1084-8). The solution structure of pig TFF2 has been studied by NMR (Carr, MD et al. al, (1994) Proc Natl Acad Sci USA 91(6):2206-10). There are six conserved cysteine ​​residues in the trefoil domain that form three intramolecular disulfide bonds that result in three stacked loops in a three-loop structure (May FEB, et al. (2000), Gut, 46:454-459). A portion of human TFF2 in gastric juice is conserved at Asn(15). As previously described, glycosylated ribonucleotides are N-linked (May FEB et al., Gut 2000). 46(4):454-9).

[0005] TFF2 is primarily expressed in the duodenum and Brunner's glands in the human gastric pylorus and has been shown to have functional roles in the stomach and intestinal lumen (Jorgenson, KH, and Jacobsen HE, (1982) Regul Pept., 3:207-219). Gastrin has been shown to regulate the TFF2 promoter through a gastrin-responsive cis-acting element and through a signal transduction pathway (Tu, S. et al., (2007), Am J Physiol. Gastrointest Liver Physiol., 292(6):G1726-37). TFF2 has also been found at high concentrations in cells adjacent to mucosal ulcers (Wright NA, Poulsom R, Stamp GW (1990) J Pathol.;162:279-284).

[0006] TFF2 deficiency in knockout (KO) mice exacerbates dextran sulfate sodium (DSS)-induced colitis (Judd LM et al, Am J. Physiol Gatrointest Liver Physiol. (2015) 308(1):G12-24). TFF2 stabilizes It is believed that TFF2 protects the gastrointestinal mucosa from injury by supporting mucin gel, reducing inflammation, and stimulating epithelial regeneration. Cook et al. showed that TFF2 is expressed by and active on lymphocytes (Cook et al., (1999), FEBS Lett., 456(1):155-9). Dubeykovskaya et al. It has been shown that TFF2 is a lymphocyte-activating polypeptide and functions as an activating ligand for the CXCR4 receptor (CXC chemokine receptor type 4, also known as fusin or CD184) (Dubeykovskaya, Z. Dubeykovskaya, A., Wang, J., (2009), J Biol Chem., 284(6):3650-62). TFF2 is also expressed in the spleen. Circulating TFF2 appears to have an immunomodulatory role (Dubeykovskaya Z, et al. Nat Commun. (2016), 7:1-11).

[0007] Exogenous TFF2 has poor pharmacokinetics and is rapidly cleared from plasma. Modified TFF2 was generated by genetically fusing the C-terminus of TFF2 to the carboxyl terminal peptide (CTP) of the human chorionic gonadotropin beta subunit and further fusing a Flag tail (TFF2-CTP-Flag). Recombinant TFF2-CTP-Flag protein has been shown to suppress colon tumor growth (Dubeykovskaya, ZA et al., (2019), Cancer Gene Therapy, 26:48-57). Recombinant TFF2 has also been shown to inhibit pancreatic cancer. It has been reported to be immunosuppressive against rhesus monkeys (Sung, Gi-Ho, et al., (2018), Animal Cells and Systems, 22:6, 368-381). TFF2 is an attractive biological treatment for cancer because it is stable in harsh pH environments such as the stomach. The tumor microenvironment (TME) is known to have a low pH, which can reduce the binding of other cancer drugs, such as monoclonal antibodies. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] De A et al, (1994) Proc Natl Acad Sci USA 91(3):1084-8 [Non-patent document 2] Carr, MD et al, (1994) Proc Natl Acad Sci USA 91(6):2206-10 [Non-patent document 3] May FEB, et al. (2000), Gut, 46:454-459 [Non-patent document 4] Jorgenson, KH, and Jacobsen HE, (1982) Regul Pept., 3:207-219. [Non-patent document 5] Tu, S. et al., (2007), Am J Physiol. Gastrointest Liver Physiol., 292(6):G1726-37 [Non-patent document 6] Wright NA, Poulsom R., Stamp GW (1990) J Pathol.;162:279-284 [Non-Patent Document 7] Judd LM et al, Am J. Physiol Gatrointest Liver Physiol. (2015) 308(1):G12-24 [Non-patent document 8] Cook et al., (1999), FEBS Lett., 456(1):155-9 [Non-Patent Document 9] Dubeykovskaya, Z. Dubeykovskaya, A., Wang, J., (2009), J Biol Chem., 284(6):3650-62 [Non-Patent Document 10] Dubeykovskaya Z, et al. Nat Commun. (2016), 7:1-11 [Non-Patent Document 11] Dubeykovskaya, ZA et al., (2019), Cancer Gene Therapy, 26:48-57 [Non-Patent Document 12] Sung, Gi-Ho, et al., (2018), Animal Cells and Systems, 22:6, 368-381 Summary of the Invention [Means for solving the problem]

[0009] Summary of the Disclosure The present disclosure provides compositions of modified TFF2 polypeptides that have enhanced biological activity and pharmacokinetic properties, such as increased stability and / or in vivo efficacy.

[0010] In some embodiments, the improved properties of the disclosed modified TFF2 polypeptides are achieved using chemical modifications including PEGylation or poly(D,L-lactic-co-glycolic acid) (PLGA), and / or polysialylation (PSA), and / or fusion proteins including fusion proteins using the C-terminal peptide of human chorionic gonadotropin beta subunit (CTP), PASylation, homoamino acid polymer (HAP), elastin-like peptide (ELPylation), Xtenylation, and combinations of these modifications.

[0011] As used herein, TFF2 polypeptides modified by PEGylation, PASylation, PLGA conjugation and / or PSA conjugation, or fusion proteins using HAP, ELPylation, XTENylation or the CTP of human chorionic gonadotropin beta subunit, as well as combinations of these modifications, are referred to as modified TFF2 polypeptides.

[0012] The present disclosure provides compositions of modified TFF2 polypeptides, including PEGylated TFF2, PASylated TFF2, PLGA-modified TFF2 and / or PSA-modified TFF2, or TFF2 fusion proteins, such as fusion proteins with CTP peptide, fusion proteins with HAP, or ELPylated TFF2, and combinations of these modifications, and uses of these modified TFF2 polypeptides for treating cancer, hyperplasia, dysplasia, inflammatory conditions, inflammation of the digestive system, and / or any symptoms occurring in COVID-19.

[0013] As defined herein, the term "effective amount" means the amount of modified TFF2 polypeptide required to at least partially obtain the desired response, or to delay the onset of, inhibit the progression of, or completely stop the onset or progression of the particular condition being treated.

[0014] In some embodiments, the modified TFF2 polypeptide is homogeneous and has improved pharmacokinetic properties compared to unmodified or native human TFF2 polypeptide.

[0015] In some embodiments, the modified TFF2 polypeptide has an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:6.

[0016] In certain embodiments, the modified TFF2 polypeptide has a polypeptide sequence having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:6.

[0017] In some embodiments, the modified TFF2 polypeptide has at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:6.

[0018] In some embodiments, the modified TFF2 polypeptides described herein are PEGylated with a low molecular weight linear PEG.

[0019] In some embodiments, the modified TFF2 polypeptides described herein are PEGylated with high molecular weight branched PEGs.

[0020] In some embodiments, the modified TFF2 polypeptide has an increased serum half-life compared to an unmodified human TFF2 polypeptide, such as SEQ ID NO:6.

[0021] In one embodiment, the PEGylated TFF2 polypeptide has an increased serum half-life compared to unPEGylated human TFF2 polypeptide.

[0022] In some embodiments, the modified TFF2 polypeptides described herein are PEGylated at a specific site or sites.

[0023] In some embodiments, the modified TFF2 polypeptides described herein are PEGylated at the N-terminus.

[0024] In some embodiments, the modified TFF2 polypeptides described herein are PEGylated at the N-terminus via aldehyde-PEG chemistry.

[0025] In other embodiments, the PEGylated TFF2 polypeptides described herein are PEGylated at the C-terminus.

[0026] In some embodiments, PEGylation of the TFF2 polypeptides described herein involves neat exposed amines via NHS-PEG chemistry.

[0027] In some embodiments, the modified TFF2 polypeptide comprises a fusion protein, such as the C-terminal peptide (CTP) of the human chorionic gonadotropin beta subunit.

[0028] In some embodiments, the modified TFF2 polypeptide is a conjugated polypeptide, such as a conjugate of PLGA.

[0029] Disclosed herein, in some embodiments, is a TFF2 polypeptide fusion polypeptide selected from one or more of the group consisting of a TFF2 albumin fusion protein, a TFF2-IgG1 fusion protein, and a TFF2-affinity tag fusion protein.

[0030] In some embodiments, the modified TFF2 polypeptide is a fusion protein with a polyhistidine tag.In some embodiments, the histidine tag contains an amino acid cleavage site.In some embodiments, the histidine tag cleavage site is selected from SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23.

[0031] In some embodiments, the native TFF2 polypeptide is formed by removing the polyhistidine tag from a fusion protein of TFF2.

[0032] In some embodiments, the histidine tag is at either the N-terminus or C-terminus of the TFF2 polypeptide.

[0033] In other embodiments, after cleavage of the histidine tag, the modified TFF2 polypeptide is formed by 1) purifying the TFF2 peptide, and 2) preparing a conjugate of the purified modified TFF2 with PEGylation, polysialylation, and / or poly(D,L-lactic-co-glycolic acid) (PLGA).

[0034] In another embodiment of the disclosure are modified TFF2 polypeptides having alterations to those binding domains represented by SEQ ID NOs: 26-28 and FIG.

[0035] In another embodiment of the disclosure are modified TFF2 polypeptides having changes to the receptor binding site residues represented by SEQ ID NOs: 29-31 and FIG.

[0036] In some embodiments, the modified TFF2 peptides represented by SEQ ID NOs: 26-31 are further modified by one or more of PEGylation, polysialylation, conjugation to PLGA, and / or expression as a fusion protein comprising a fusion polypeptide selected from the group consisting of the C-terminal peptide of human chorionic gonadotropin beta subunit (CTP), a PAS-ylated fusion polypeptide, an XTEN-ylated fusion polypeptide, an ELP-ylated fusion polypeptide, and a HAP-ylated fusion polypeptide.

[0037] In some embodiments, the modified TFF2 peptides represented by SEQ ID NOs:29-31 are further modified by one or more of PEGylation, polysialylation, conjugation to PLGA, and / or expression as a fusion protein comprising a fusion polypeptide selected from the group consisting of the C-terminal peptide of human chorionic gonadotropin β subunit (CTP), a PAS-ylated fusion polypeptide, an XTEN-ylated fusion polypeptide, an ELP-ylated fusion polypeptide, and / or a HAP-ylated fusion polypeptide. In some embodiments, these modified TFF2 polypeptides have increased serum half-life and / or improved pharmacodynamic properties compared to unmodified human TFF2 of SEQ ID NO:6.

[0038] In some embodiments, when the modified TFF2 peptides represented by SEQ ID NOs: 26-31 are PEGylated, the modified TFF2 binding domain polypeptides are PEGylated with a low molecular weight linear PEG.

[0039] In some embodiments, when the modified TFF2 peptides represented by SEQ ID NOs: 26-31 are PEGylated, the modified TFF2 binding domain polypeptides are PEGylated with a high molecular weight branched PEG.

[0040] In some embodiments, when the modified TFF2 peptides represented by SEQ ID NOs: 26-31 are PEGylated, the modified TFF2 binding domain polypeptides are PEGylated at one or more specific sites.

[0041] In some embodiments, when the modified TFF2 peptides represented by SEQ ID NOs: 26-31 are PEGylated, the modified TFF2 binding domain polypeptide is PEGylated at its N-terminus.

[0042] In some embodiments, when the modified TFF2 peptides represented by SEQ ID NOs: 26-31 are PEGylated, the modified TFF2 binding domain polypeptides are PEGylated using N-terminal PEGylation via aldehyde-PEG chemistry.

[0043] In some embodiments, when the modified TFF2 peptides represented by SEQ ID NOs: 26-31 are PEGylated, the modified TFF2 binding domain polypeptide is PEGylated at its C-terminus.

[0044] In some embodiments, when the modified TFF2 peptides represented by SEQ ID NOs: 26-31 are PEGylated, the PEGylation involves neat exposed amines via NHS-PEG chemistry.

[0045] In some embodiments, the modified TFF2 peptides described herein are glycosylated.

[0046] In some embodiments, the modified TFF2 polypeptides described herein are in a homogeneous composition.

[0047] In some embodiments, the modified TFF2 polypeptides described herein are in a pharmaceutical composition, which may contain one or more excipients.

[0048] In some embodiments, the pharmaceutical composition is a homogeneous population of modified TFF2 polypeptides selected from the group consisting of modified TFF2 polypeptides that are PEGylated, polysialylated, conjugated to PLGA, or are fusion polypeptides using CTP-, PAS-, XTEN-, ELP-, or HAP-ylated versions of human chorionic gonadotropin beta subunit, or combinations of these modifications.

[0049] An aspect of the present disclosure is a method of treating cancer in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of one or more modified TFF2 polypeptides described herein, thereby treating the cancer.

[0050] In embodiments of the present disclosure, the cancer is a cancer of the digestive system, for example, but not limited to, oral cavity cancer, pharyngeal cancer, oropharyngeal cancer, esophageal cancer, stomach cancer, small intestine cancer, large intestine cancer, colon cancer, rectal cancer, anal cancer, gastric cancer, liver cancer, pancreatic cancer, gallbladder cancer, or colon cancer.

[0051] In some embodiments, the cancer being treated is oropharyngeal cancer.

[0052] In some embodiments, the cancer being treated is esophageal cancer.

[0053] In some embodiments, the cancer being treated is gastric cancer.

[0054] In some embodiments, the cancer being treated is pancreatic cancer.

[0055] In some embodiments, the cancer being treated is colon cancer.

[0056] In some embodiments, the cancer being treated is rectal cancer.

[0057] In some embodiments, the cancer being treated is anal cancer.

[0058] In some embodiments, the cancer being treated is liver cancer.

[0059] In some embodiments, the cancer being treated is a metastatic cancer.

[0060] In some embodiments, the cancer being treated is also treated with blocking antibodies against PD-1 (programmed cell death protein 1, CD279), PD-L1 (programmed death-ligand 1, CD274 or B7 homolog 1 [B7-H1]), and / or CTLA-4.

[0061] In yet another embodiment, disclosed herein is a method of treating cancer in a subject in need thereof, wherein the cancer is unresponsive to treatment with a blocking antibody against PD-1, PD-L1 or CTLA-4, the subject is treated with one or more of the modified TFF2 polypeptides described herein, and after treatment with the modified TFF2 polypeptide composition, the subject's cancer becomes sensitive to treatment with a blocking antibody against PD-1, PD-L1 or CTLA-4, and the subject is then treated with a blocking antibody against PD-1, PD-L1 or CTLA-4 within about 1 to about 60 days after treatment with the modified TFF2 polypeptide composition.

[0062] In some embodiments, the modified TFF2 peptides disclosed herein can be combined with standard therapy for treating cancer of the digestive system. In some embodiments, the modified TFF2 polypeptides are administered before, at the same time as, or after the standard therapy.

[0063] Another aspect of the present disclosure is a method for treating an inflammatory condition, such as inflammation of the digestive system, in a subject in need thereof, comprising administering a modified TFF2 polypeptide to the subject.

[0064] In one embodiment, the inflammation of the digestive system is inflammatory bowel disease (IBD), including but not limited to ulcerative colitis and Crohn's disease.

[0065] In some embodiments for treating an inflammatory condition, the modified TFF2 polypeptides disclosed herein are administered orally, intravenously, or intramuscularly.

[0066] Another aspect of the present disclosure provides a method for treating COVID-19 or any of the complications that have arisen in a subject in need thereof, comprising administering to the subject one or more of the compositions of the present disclosure or one or more of the modified TFF2 polypeptides of the present disclosure.

[0067] In some embodiments of any of the methods of the present disclosure, the modified TFF2 polypeptide may be given before, concurrently with, or after a standard therapy for treating the inflammatory disease.

[0068] The modified TFF2 polypeptide is preferably administered to an individual in a "therapeutically effective amount" or "desired amount," which is sufficient to show benefit to the individual.

[0069] In some embodiments of the methods for treating COVID-19, the methods further include administering an agent that inhibits or reduces replication of SARS-CoV-2.

[0070] In some embodiments of the methods for treating COVID-19, the methods further comprise administering an antiviral agent selected from the group consisting of ribavirin, interferon (Alfacon-1), chloroquine, hydroxychloroquine, EIDD-2801, EIDD-1931, GS-5734, GS-441524, ivermectin, favipiravir, indomethacin, chlorpromazine, penciclovir, nafomostat, camostat, nitazoxanide, remdesivir, famotidine, and dexamethasone. [Brief explanation of the drawings]

[0071] [Figure 1] FIG. 1 shows the chimeric recombinant modified TFF2 polypeptide domain (D) exchanged peptides disclosed in the present application.

[0072] [Figure 2] FIG. 2 shows the chimeric recombinant modified TFF2 polypeptide ligand binding domain (LBD) exchange peptides disclosed in the present application.

[0073] [Figure 3A-B] Figure 3A: Mice (C57BL / 6 WT) received azoxymethane (AOM; 10 mg / kg i.p.) followed one week later by 2.5% dextran sodium sulfate (DSS) in drinking water for 7 days. (SAC = sacrifice). Figure 3B-D: AOM / DSS-treated mice formed tumors at 10 weeks and developed adenocarcinomas 17 weeks after AOM. Figure 3B: Overall image. Scale bar, 5 mm. Tumors were more frequently observed in the distal colon. Figure 3C: Macroscopic tumors were counted, and tumor area was measured using ImageJ Fiji. Figure 3D: Hematoxylin and eosin (H&E) staining. Increased intramucosal immune cell infiltration was detected 10 weeks after AOM. [Figure 3C-D] Figure 3A: Mice (C57BL / 6 WT) received azoxymethane (AOM; 10 mg / kg i.p.) followed one week later by 2.5% dextran sodium sulfate (DSS) in drinking water for 7 days. (SAC = sacrifice). Figure 3B-D: AOM / DSS-treated mice formed tumors at 10 weeks and developed adenocarcinomas 17 weeks after AOM. Figure 3B: Overall image. Scale bar, 5 mm. Tumors were more frequently observed in the distal colon. Figure 3C: Macroscopic tumors were counted, and tumor area was measured using ImageJ Fiji. Figure 3D: Hematoxylin and eosin (H&E) staining. Increased intramucosal immune cell infiltration was detected 10 weeks after AOM.

[0074] [Figure 4A]Figure 4A: Immunostaining for CD45, CD11b, and PD-L1 in colon tissue from AOM / DSS-treated C57BL / 6 WT mice. CD11b+ myeloid cells and PD-L1 expression increased as tumors progressed. Figures 4B and 4C: Immunophenotyping of intratumoral myeloid cells (% of CD45+) by flow cytometry. CD11b+Gr-1+ MDSCs, as well as both granulocytic (CD11b+Ly6G+) and monocytic (CD11b+Ly6G-Ly6C+) MDSC subsets, were significantly increased in tumors (see Figure 4B). Macrophages (MQ; CD11b+Ly6C-F4 / 80+) and dendritic cells (DC; CD11c+F4 / 80-) were also significantly increased in tumors (see Figure 4C). [Figure 4B-C] Figure 4A: Immunostaining for CD45, CD11b, and PD-L1 in colon tissue from AOM / DSS-treated C57BL / 6 WT mice. CD11b+ myeloid cells and PD-L1 expression increased as tumors progressed. Figures 4B and 4C: Immunophenotyping of intratumoral myeloid cells (% of CD45+) by flow cytometry. CD11b+Gr-1+ MDSCs, as well as both granulocytic (CD11b+Ly6G+) and monocytic (CD11b+Ly6G-Ly6C+) MDSC subsets, were significantly increased in tumors (see Figure 4B). Macrophages (MQ; CD11b+Ly6C-F4 / 80+) and dendritic cells (DC; CD11c+F4 / 80-) were also significantly increased in tumors (see Figure 4C).

[0075] [Figure 5A] Figures 5A and 5B: Immunophenotyping of tumor-infiltrating T cells by flow cytometry (% CD45+). The proportion of T cells decreased with tumor development, driven by a reduction in CD8+ T cells (Figure 5A). CD4+CD25+Foxp3+ regulatory T cells (Tregs) increased in later tumor stages, resulting in a higher decrease in the ratio of CD8+ T cells to Tregs (Figure 5B). Figure 5C: Dynamics of immune cell subsets during CRC development. [Figure 5B-C]Figures 5A and 5B: Immunophenotyping of tumor-infiltrating T cells by flow cytometry (% CD45+). The proportion of T cells decreased with tumor development, driven by a reduction in CD8+ T cells (Figure 5A). CD4+CD25+Foxp3+ regulatory T cells (Tregs) increased in later tumor stages, resulting in a higher decrease in the ratio of CD8+ T cells to Tregs (Figure 5B). Figure 5C: Dynamics of immune cell subsets during CRC development.

[0076] [Figure 6A-C] Figures 6A-6C: Generation of R26-LSL-Pdl1-EGFP mice. The R26-LSL-Pdl1-IRES-EGFP gene construct was used (Figure 6A). Endogenous GFP expression by flow cytometry (Figure 6B) and Pdl1 gene expression by qPCR (Figure 6C) in splenic CD11b- and CD11b+ cells in R26-PD-L1 and LysM-Cre;R26-PD-L1 mice were analyzed. Figure 6D: Experimental scheme showing the induction of CRC by AOM / DSS. Figure 6E: Overall images of colorectal tumors 10 weeks after AOM. Scale bar, 5 mm. Figure 6F: Tumor numbers were counted and tumor area was measured. Note that LysM-Cre;R26-PD-L1 mice treated with AOM / DSS exhibited significantly enhanced early colorectal tumor development. [Figure 6D-F] Figures 6A-6C: Generation of R26-LSL-Pdl1-EGFP mice. The R26-LSL-Pdl1-IRES-EGFP gene construct was used (Figure 6A). Endogenous GFP expression by flow cytometry (Figure 6B) and Pdl1 gene expression by qPCR (Figure 6C) in splenic CD11b- and CD11b+ cells in R26-PD-L1 and LysM-Cre;R26-PD-L1 mice were analyzed. Figure 6D: Experimental scheme showing the induction of CRC by AOM / DSS. Figure 6E: Overall images of colorectal tumors 10 weeks after AOM. Scale bar, 5 mm. Figure 6F: Tumor numbers were counted and tumor area was measured. Note that LysM-Cre;R26-PD-L1 mice treated with AOM / DSS exhibited significantly enhanced early colorectal tumor development.

[0077] [Figure 7A-B] Figures 7A and 7B: TFF2 overexpression (CD2-Tff2 mice) (Figure 7A) and treatment with adenovirus Ad-Tff2 (Figure 7B) compared to control Ad-Fc conferred resistance to colon carcinogenesis by suppressing MDSCs. Figure 7C: Fusion construct Tff2-2CTP-3Flag. Figures 7D and 7E: TFF2-CTP-Flag extended its circulation time in the blood (Figure 7D) but retained its biological activity (Figure 7E). Dubeykovskaya et al. 2016 Nat Commun. (Figures 7A-B); 2019 Cancer Gene Ther. (Figures 7C-E). [Figure 7C-E] Figures 7A and 7B: TFF2 overexpression (CD2-Tff2 mice) (Figure 7A) and treatment with adenovirus Ad-Tff2 (Figure 7B) compared to control Ad-Fc conferred resistance to colon carcinogenesis by suppressing MDSCs. Figure 7C: Fusion construct Tff2-2CTP-3Flag. Figures 7D and 7E: TFF2-CTP-Flag extended its circulation time in the blood (Figure 7D) but retained its biological activity (Figure 7E). Dubeykovskaya et al. 2016 Nat Commun. (Figures 7A-B); 2019 Cancer Gene Ther. (Figures 7C-E).

[0078] [Figure 8] Panel A: R26-PD-L1 and LysM-Cre;R26-PD-L1 mice were given AOM / DSS and treated with fusion recombinant TFF2-CTP-Flag (300μg i.p.) and / or anti-PD-1 (RMP1-14; 200μg i.p.) three times weekly, starting at the indicated time points. Panel B: Tumors were counted and tumor area measured. Mice with a >50% reduction in tumor area compared to control animals were defined as responders. Note that LysM-Cre;R26-PD-L1 mice (5 / 5; 100%) showed a higher response rate to combined TFF2-CTP and anti-PD-1 treatment than control animals (2 / 5; 40%).

[0079] [Figure 9]Panel A: Percentage of CD3+CD8+ T cells among CD45+ cells and ratio of CD8+ T cells to Tregs in the tumor. Note that responders had a higher number of tumor-infiltrating CD8+ T cells and a higher ratio of CD8+ T cells to Tregs. Panel B: Immunophenotyping of intratumoral myeloid cells after different treatments. A significant reduction in MDSCs, especially M-MDSCs, was observed in responders. Responders also showed a lower ratio of monocytes to MQ.

[0080] [Figure 10] SDS-PAGE (non-reducing conditions) of Protein A purified different TFF2-HSA fusion proteins. Lane 1: Marker; Lane 2: TFF2-HSA[WT]; Lane 3: TFF2-HSA[DI / I]; Lane 4: TFF2-HSA[D II / I]; Lane 5: TFF2-HSA[D II / II]; Lane 6: TFF2-HSA[LBD I / I]; Lane 7: TFF2-HSA[LBD II / I]; Lane 8: TFF2-HSA[LBD II / II].

[0081] [Figure 11] Yield of purified TFF2-HSA fusion protein as described in FIG. DETAILED DESCRIPTION OF THE INVENTION

[0082] Detailed Description of the Embodiments The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The use of the words "a" or "an," when used in conjunction with the word "comprising" in the claims and / or specification, may mean "one," which can also be used in conjunction with "one or more," "at least one," and "one or more This also coincides with the meaning of "than one."

[0083] As used herein, the term "about" is used herein to mean approximately, roughly, around, or within the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the upper and lower boundaries of the numerical values ​​set forth. In general, the term "about" is used herein to modify numerical values ​​above and below the stated value by a variance (higher or lower) of 20 percent.

[0084] In one embodiment, the modified TFF2 polypeptide used for PEGylation, polysialylation (PSA), or conjugation with PLGA comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 6. SEQ ID NO: 1 represents a human TFF2 polypeptide. The displayed sequence is further processed to the mature form (SEQ ID NO: 6). SEQ ID NO: 2 represents a human nucleotide sequence encoding TFF2, where the underlined and bolded "ATG" represents the start codon. Sequence information for TFF2 is accessible in public databases under GenBank accession numbers NP_005414 (protein) and NM_005423 (nucleic acid). MGRRDAQLLA ALLVLGLCAL AGSEKPSPCQ CSRLSPHNRT NCGFPGITSD QCFDNGCCFD SSVTGVPWCF HPLPKQESDQ CVMEVSDRRN CGYPGISPEE CASRKCCFSN FIFEVPWCFF PKSVEDCHY (SEQ ID NO: 1)

[0085] With the signal peptide removed, the human TFF2 peptide has the following amino acid sequence: Native human TFF2 (106AA) EKPSPCQCSRLSPHNRTNCGFPGITSDQCFDNGCCFDSSVTGVPWCFHPLPKQESDQCVMEVSDRRNCGYPGISPEECASRKCCFSNFIFEVPWCFFPKSVEDCHY (SEQ ID NO: 6)

[0086] SEQ ID NO: 2 is the human wild-type nucleotide sequence corresponding to TFF2 (nucleotides 1-717), where the underlined and bolded "ATG" indicates the start of the open reading frame. [ka] [ka]

[0087] In the context of different aspects of the present disclosure, the term "polypeptide" refers to a single, linear chain of amino acids linked together by peptide bonds, preferably containing at least about 21 amino acids. A polypeptide can be a single-chain protein composed of more than one chain, or it can be the protein itself, when the protein is composed of a single chain. The term "polypeptide" includes glycosylated (i.e., glycoproteins) and non-glycosylated forms of such a linear chain of amino acids, as well as mixtures of glycosylated and non-glycosylated forms.

[0088] In another embodiment, the modified TFF2 polypeptide used for PEGylation, polysialylation or conjugation with PLGA comprises, consists of, or essentially consists of the amino acid sequence of SEQ ID NO: 3, which represents the mouse TFF2 polypeptide (accession number NP_033389).

[0089] SEQ ID NO: 3 shows the amino acid sequence of mouse TFF2, including the signal peptide: MRPRGAPLLA VVLVLGLHAL VEGEKPSPCR CSRLTPHNRK NCGFPGITSE QCFDLGCCFD SSVAGVPWCF HPLPNQESEQ CVMEVSARKN CGYPGISPED CASRNCCFSN LIFEVPWCFF PQSVEDCHY. (SEQ ID NO: 3)

[0090] SEQ ID NO: 4 represents the Mus muscle TFF2 nucleic acid sequence of accession number NM_009363. ATTCTGCAGGCTGCCCAGGTCCAGTGGAGCAGACATGCGACCTCGAGGTGCCCCCCTGCT GGCAGTGGTCCTGGTTTTGGGACTGCATGCTCTGGTAGAGGGCGAGAAACCTTCCCCCTG TCGGTGCTCCAGGCTGACACCCCACAACAGAAAGAACTGTGGCTTCCCGGGCATCACCAG TGAGCAGTGCTTTGATCTTGGATGCTGCTTTGACTCTAGCGTCGCTGGGGTCCCTTGGTG TTTCCACCCACTTCCAAACCAAGAATCGGAGCAGTGTGTCATGGAAGTGTCAGCTCGCAA GAATTGTGGGTACCCGGGCATCAGTCCCGAGGACTGTGCCAGTCGAAACTGCTGCTTTTC CAACCTGATCTTTGAAGTGCCCTGGTGTTTCTTCCCACAGTCTGTGGAAGATTGTCACTA CTGAGAGTTGCTACTGCCGAGCCACCCGTTCCCTGGGAGCTGCAAGCCAGAAGAAAGTTT CAACCAGACTTCATCAATCTCTGGGGTTTCTAAAACCATCTTGACCCTTAGCAGTGGCTA GACACAGCATTTTCCAAGTAAAGAAAAGTTG (SEQ ID NO: 4)

[0091] Methods for collecting, preparing, isolating and sequencing human TFF2 are described in May FEB et al. (2000), Gut, 46:454-459, which is incorporated herein by reference.

[0092] In some embodiments, the PEGylated, polysialylated or PLGA-conjugated protein / polypeptide has at least about 46% to about 50% identity to SEQ ID NO: 1, 3 or 10, or has at least about 50.1% to about 55% identity to SEQ ID NO: 1, 3 or 10, or has at least about 55.1% to about 60% identity to SEQ ID NO: 1, 3 or 10, or has at least about 60.1% to about 65% identity to SEQ ID NO: 1, 3 or 10, or has about 65.1% to about 70% identity to SEQ ID NO: 1, 3 or 10, or has at least about 70.1% to about 75% identity to SEQ ID NO: 1, 3 or 10. or a variant of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 6 having at least about 75.1% to about 80% identity to SEQ ID NO: 1, 3 or 10, or having at least about 80.1% to about 85% identity to SEQ ID NO: 1, 3 or 10, or having at least about 85.1% to about 90% identity to SEQ ID NO: 1, 3 or 10, or having at least about 90.1% to about 95% identity to SEQ ID NO: 1, 3 or 10, or having at least about 95.1% to about 97% identity to SEQ ID NO: 1, 3 or 10, or having at least about 97.1% to about 99% identity to SEQ ID NO: 1, 3 or 10.

[0093] In some embodiments, the modified TFF2 polypeptide is generated from codon-optimized DNA (see Examples 1-4).

[0094] In some embodiments, the PEGylated or PAS-modified TFF2 polypeptide is a hybrid peptide, for example, but not limited to, a modified TFF2 polypeptide with a His tag; TFF2-C-terminal HULG1 FC tag, TFF2-HSA, TFF2-CTP, TFF2-CTP-FLAG, TFF2-FLAG.

[0095] In some embodiments, the C-terminal peptide (CTP) of human chorionic gonadotropin is used to improve the pharmacokinetic (PK) and pharmacodynamic (PD) properties of the modified TFF2 polypeptides described herein (Calo, et al., (2015), Precision Medicine, 2:e989).

[0096] In some embodiments, the PEGylated or PASylated modified TFF2 polypeptide is truncated.

[0097] In other embodiments, the PEGylated or PASylated modified TFF2 polypeptide is glycosylated.

[0098] In some embodiments, the modified human PEGylated or PASylated TFF2 polypeptide contains conservative amino acid changes compared to wild-type.Conservative amino acid mutation or conservative amino acid substitution is the replacement of an amino acid in a polypeptide, changing the amino acid to a different amino acid with similar biochemical properties, such as charge, hydrophobicity and size.For example, an aliphatic amino acid can be replaced by another aliphatic amino acid (see Table 1).Conservative amino acid changes can also be determined using a matrix based on the Dayhoff matrix, see, for example, Altschul, SF, (1991), Journal of Molecular Biology 219 (3):555-65. [Table 1]

[0099] Modified TFF2 polypeptides with swapped domains (D) and ligand binding domains (LBD) The TFF2 structure contains two relatively symmetric domains (DI and D2), each containing two putative ligand-binding domains (LBDI in DI and LBDII in D2) (see, e.g., Carr et al., Proc. Natl. Acad. Sci. USA (1994), 91:2206-2210). Although the identity of all ligands is unknown, it is possible that they each bind to the same ligand, or that they each bind to different ligands. If they bind to the same ligand, it is possible that their affinities for this ligand will differ. One possible ligand for either or both of TFF2's LBDI and LBDII is the CXCR4 receptor. If TFF2 binds to the CXCR4 receptors of both LBDI and LBDII, it would result in a complex on the cell surface with effective dimerization of the two CXCR4 receptors. This type of dimerization would also be expected if LBDI and LBDII bind to a common receptor other than CXCR4. If LBDI and LBDII each bind to different ligands, it would result in effective heterodimerization of such receptors, one of which may be CXCR4.

[0100] Therefore, taking advantage of these structural features of TFF2 and potentially creating more potent or superpotent activators of target ligands, including CXCR4, the LBD and D exchanges were used to create new versions of the TFF2 protein, which are shown in Figures 1 and 2. Wild-type TFF2 is referred to as LBDI / II. LBDI and LBDII interact with the same counter-receptor, but to the extent that LBDI or LBDII has greater binding avidity for the counter-receptor, the LBD is then exchanged for the domain protein LBDI / I or LBDII / II, which interacts with the counter-receptor with higher affinity than wild-type LBDI / II, eliciting improved efficacy over wild-type LBDI / II. To the extent that LBDI or LBDII have a different counter-ligand (LBDIII or LBDI, respectively), such as a receptor other than the LBD, and LBDI / II induces a heterocomplex of the counter-receptor, then the LBD-exchanged version (such as LBDI / I or LBDII / II, see below and Figures 1 and 2) induces homodimerization of the counter-receptor, eliciting different and improved effects than wild-type LBDI / II. One possible counter-receptor for dimerization and oligomerization of LBDI and LBDII is CXCR4 (Ge B, et al., (2017) Sci Rep. 7(1):16873), and as a result, LBDI / I or LBDII / II are more potent functional ligands for CXCR4 than wild-type TFF2 (LBDI / II). CXCR4 also forms heterodimers with the membrane-bound chemokine receptors CCR5 and CCR2 (Gahbauer, S et al. (2018) PLoS Comput Biol. 14(3):e1006062). Certain modified TFF2 polypeptides encoded by LBD-exchange cDNA constructs, mimetics, and others inhibit the function of cognate and non-cognate ligands of TFF2 counter-receptors, including CXCR4. Examples of CXCR4 ligands include stromal-derived factor-1 alpha (SDF-1α or CXCL12), macrophage migration inhibitory factor (MIF), and extracellular ubiquitin.SDF-1α is the cognate ligand of CXCR4, which binds to and activates CXCR4. MIF is a non-cognate ligand of CXCR4 that triggers CXCR4 signaling (Bernhagen, J et al. (2007) Nature Medicine 13(5): 587-96). Extracellular ubiquitin is a ligand of CXCR4 (Saini, V et al. (2010) J Biol Chem 285(20) 15566; Scofield, SLC et al. (2018) Life Sci. 211:8).

[0101] In some embodiments, the modified TFF2 polypeptide contains one or more domain 1 (DI) regions of human TFF2.

[0102] In some embodiments, the modified TFF2 polypeptide contains one or more DII regions of human TFF2.

[0103] In some embodiments, the modified TFF2 polypeptide contains both the DI and DII regions of human TFF2.

[0104] In some embodiments, the modified TFF2 polypeptide contains a domain having the following sequence: Human TFF2 domain I (residues 8-46) [ka]

[0105] In some embodiments, the modified TFF2 polypeptide contains a domain having the following sequence (see Figure 1): Human TFF2 domain II (residues 58-95) [ka]

[0106] In some embodiments, the modified TFF2 polypeptide contains two DI regions having the following sequences: Human TFF2 domain I / I variant (DI / I, 107AA)—two domain I regions (see Figure 1) [ka]

[0107] In some embodiments, the modified TFF2 polypeptide contains two DII regions having the following sequences: Human TFF2 domain II / II variant (DII / II, 105AA)—two domain II regions (see Figure 1) [ka]

[0108] In some embodiments, the modified TFF2 polypeptide contains a DII and a DI variant, where the order of DI and DII are interchanged in the following sequence: Human TFF2 domain II / I variant (DII / I, 106AA)—interchanged domains I and II (see Figure 1) [ka]

[0109] In some embodiments, the modified TFF2 polypeptide contains amino acid substitutions at putative receptor binding site residues of the LBD having the following sequences (see Figure 2): Human TFF2-AA-substituted (106AA)-putative ligand-binding domain (LBD) site residues interchanged between DI and DII (LBD II / I) (see Figure 2). [ka]

[0110] In some embodiments, the modified TFF2 polypeptide contains amino acid substitutions at receptor binding site residues and comprises the sequence of SEQ ID NO: 29. In some embodiments, the modified TFF2 polypeptide contains amino acid substitutions at receptor binding site residues and has the sequence of SEQ ID NO: 29.

[0111] In some embodiments, the modified TFF2 polypeptide contains amino acid substitutions in receptor binding site residues of the LBD having the following sequence: A variant (LBD I / I) containing the putative receptor binding site residues of the LBD from human TFF2-AA-substituted (106AA)-DI only (see Figure 2). [ka]

[0112] In some embodiments, the modified TFF2 polypeptide contains amino acid substitutions at receptor binding site residues and comprises the sequence of SEQ ID NO: 30. In some embodiments, the modified TFF2 polypeptide contains amino acid substitutions at receptor binding site residues and has the sequence of SEQ ID NO: 30.

[0113] In some embodiments, the modified TFF2 polypeptide contains amino acid substitutions at receptor binding site residues of the LBD having the following sequences: Human TFF2-AA-substituted (106AA)-variant containing putative receptor binding site residues of the LBD from domain II only (LBD II / II) (see Figure 2). [ka]

[0114] In some embodiments, the modified TFF2 polypeptide contains amino acid substitutions at receptor binding site residues and comprises the sequence of SEQ ID NO: 31. In some embodiments, the modified TFF2 polypeptide contains amino acid substitutions at receptor binding site residues and has the sequence of SEQ ID NO: 31.

[0115] In some embodiments, modified TFF2 polypeptides having DI and DII regions have different binding affinities, ie, stronger to weaker binding affinities, to counter-receptors including CXCR4.

[0116] In some embodiments, the modified TFF2 polypeptides described herein, such as those described by SEQ ID NOs: 24-31, are modified by PEGylation, polysialylation (PSA), or conjugation with PLGA, or as fusion proteins modified by PASylation, HAPylation, ELPylation, CTP of human chorionic gonadotropin beta subunit, and / or by combinations of these modifications.

[0117] In some embodiments, the C-terminal peptide (CTP) of human chorionic gonadotropin is used to improve the pharmacokinetic (PK) and pharmacodynamic (PD) properties of the modified TFF2 polypeptides described herein, such as those described by SEQ ID NOs: 24-32.

[0118] In some embodiments, the modified TFF2 polypeptides, eg, those described by SEQ ID NOs: 24-32, are glycosylated.

[0119] The efficacy of modified TFF2 polypeptides with LBD and / or D-exchange regions is tested by measuring calcium flux, cell migration, and activation of extracellular signal-related kinases (ERKs) ERK1 and ERK2. The specificity of the effect on CXCR4 is investigated by using the CXCR4 inhibitor AMD3100 or mAb 12G5. The binding of LBD and D-exchange proteins is assessed by their ability to block the binding of mAb 2B11 (Dubeykovskaya, Z. Dubeykovskaya, A., Wang, J., (2009), J Biol Chem., 284(6):3650-62).

[0120] Assay for ERK1 / 2 phosphorylation In some embodiments, TFF2 activity is measured by ERK1 / ERK2 phosphorylation in Jurkat human acute T-cell leukemia cells using the AlphaLISA SureFire Ultra p-ERK 1 / 2 (Thr202 / Tyr204) Assay Kit from Perkin Elmer. Jurkat cells provided by ATCC are thawed and grown according to the instructions provided by ATCC. Cells are harvested by centrifugation and diluted to 10 mL in HBSS. 7 The cells are resuspended at 1000 cells / mL. The cells are seeded at 4 mL / well into 384-well white opaque culture plates (PerkinElmer) and incubated at 37°C for 1-2 hours. Recombinant wild-type and variant TFF2 proteins (4 μL) at a concentration of 10-30 mg / mL in HBSS containing 0.1% BSA are added to the plate to stimulate the cells and incubated at 37°C for 5-30 minutes. The cells are lysed with 2 μL / well of lysis buffer, followed by the addition of 5 mL of Acceptor Mix. The plate is then sealed with Topseal-A adhesive film and incubated at room temperature for 1 hour. 5 mL of Donor Mix is ​​then added to the wells under moderate light, sealed with Topseal-A adhesive film, covered with foil, and incubated in the dark at room temperature for 1 hour. The plate is read in an AlphaPlex-compatible plate reader using standard AlphaPlex settings. Inhibition of TFF2 stimulation of CXCR4 was performed using the CXCR4 small molecule antagonist AMD3100 (Sigma) or anti-CXCR4 mAbs 12G5 and 2B11 (eBioscience) at 37°C for 1 to 2 hours before the addition of recombinant TFF2.

[0121] PEGylation In some cases, protein-based drugs are problematic as therapeutic agents because they can be rapidly degraded and excreted from the patient, resulting in frequent dosing that can increase the immunogenic potential of the molecule and also increase the cost of treatment (Dozier, JK, and Distefano MD, (2015), Int, J. Mol. Sci., 16:25831-25864). The TFF2 protein is a circulating It has been shown to have poor pharmacokinetics due to its poor half-life in the blood (Dubeykovskaya, ZA et al., (2019), Cancer Gene Therapy, 26:48-57). Proteins chemically modified with ethylene glycol (PEG) exhibit improved pharmacological properties, including increased serum half-life, improved solubility, good physical and thermal stability, protection against enzymatic degradation, increased solubility, reduced toxicity and reduced immunogenicity.

[0122] In addition to the beneficial effects of PEGylation on pharmacokinetic parameters, PEGylation itself may enhance activity. For example, PEG-IL-10 has been shown to be more effective against certain cancers than non-PEGylated IL-10 (see, e.g., EP206636A2).

[0123] The present disclosure contemplates the use of other polymers, such as polypropylene glycol or polyoxyalkylenes.

[0124] An embodiment of the present disclosure is a modified TFF2 polypeptide, such as the polypeptide of SEQ ID NO: 1 or its variant, that is PEGylated compared to the full-length TFF2 polypeptide.Any suitable method of PEGylation can be used.PEGylation of polypeptides is known in the art, for example, see U.S. Patent Nos. 6,420,339; 7,610,156; 5,766,897; 7,052,686 and 7,947,473.Also see, for example, Fee, C., and Damodaran VB, Protein PEGylation: An overview of chemistry and process considerations, European Pharmaceutical Review, Issue 1 2010.

[0125] In embodiments of the present disclosure, the modified TFF2 polypeptide is PEGylated to increase its in vivo half-life, which may occur by prolonging its circulation in plasma by decreasing its renal clearance, and / or to reduce its immunogenicity. PEGylation can also increase the aqueous solubility of hydrophobic drugs and proteins.

[0126] The overall PEGylation processes used to date for protein conjugation can be broadly categorized into two types: solution-phase batch processes and fed-batch processes on a column (Fee, Conan J.; Van Alstine, James M. (2006), Chemical Engineering Science, 61 (3): 924). This involves mixing the reagents together in a suitable buffer, preferably at a temperature of 4-6°C, followed by isolation and purification of the desired product using a suitable technique based on its physicochemical properties, including size exclusion chromatography (SEC), ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC) and membrane or aqueous two-phase systems (Veronese, edited by Francesco M. (2009). "Protein conjugates purification and characterization". PEGylated protein drugs basic science and clinical applications (Online-Ausg. ed.). Basel: Birkhauser. pp. 113-125; and Fee, Conan J. (2003), Biotechnology and Bioengineering, 82 (2): 200-6).

[0127] The selection of suitable functional groups for PEG derivatives is based on the type of available reactive group on the molecule to be coupled to PEG. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine. N-terminal amino groups and C-terminal carboxylic acids can also be used as site-specific sites for conjugation with aldehyde-functional polymers (Fee, Conan J.; Damodaran, Vinod B. (2012), Biopharmaceutical Production Technology. p. 199).

[0128] In some embodiments, PEGylation occurs at one or both termini of the TFF2 polypeptide. PEG activated at each terminus with the same reactive moiety is known as "homobifunctional," whereas when the functional groups present are different, the resulting PEG derivative is referred to as "heterobifunctional" or "heterofunctional." Chemically active or activated derivatives of PEG polymers are prepared to attach PEG to desired molecules (Pasut, G.; Veronese, FM (2012), Journal of Controlled Release. 161 (2): 461-472).

[0129] The technique used to form first-generation PEG derivatives generally involves reacting PEG polymers with groups reactive with hydroxyl groups, typically anhydrides, acid chlorides, chloroformates, and carbonates. In second-generation PEGylation chemistries, more available functional groups, such as aldehydes, esters, and amides, became available for conjugation.

[0130] Heterobifunctional PEGs are useful in linking two entities when a hydrophilic, flexible, and biocompatible spacer is required. Preferred terminal groups for heterobifunctional PEGs are maleimide, vinyl sulfone, pyridyl disulfide, amine, carboxylic acid, and NHS ester (see WO 2011 / 008495).

[0131] Third generation PEGylated agents, where the polymer is branched, are available in Y- or comb-shaped configurations and exhibit reduced viscosity and lack of organ accumulation (Ryan, Sinead M; Mantovani, Giuseppe; Wang, Xuexuan; Haddleton, David M; Brayden, David J (2008), Expert Opinion on Drug Delivery, 5 (4): 371-83).

[0132] In one embodiment, PEG is covalently linked. In another embodiment, PEG is linked to the TFF2 polypeptide at a cysteine ​​or lysine residue. PEGylation can be achieved using several PEG-linking moieties, including, but not limited to, N-hydroxylsuccinimide active ester, succinimidyl propionate, maleimide, vinyl sulfone, or thiol. PEG polymers can be linked to the TFF2 polypeptide at any predetermined position, or can be linked randomly to the TFF2 polypeptide. PEGylation can also be mediated through a peptide linker attached to the TFF2 polypeptide. That is, the PEG moiety can be attached to a peptide linker fused to the TFF2 polypeptide, where the linker provides a site for PEG attachment (e.g., a free cysteine ​​or lysine).

[0133] PEGylation most frequently occurs at the alpha amino group at the N-terminus of a polypeptide, the epsilon amino acid on the side chain of a lysine residue, and the imidazole group on the side chain of a histidine residue. Because most recombinant polypeptides have a single alpha group and multiple epsilon amino and imidazole groups, a large number of possible isomers can arise depending on the linker chemistry. General PEGylation strategies known in the art can be applied herein. PEG can be attached to the polypeptides of the present disclosure via a terminal reactive group ("spacer") that mediates the bond between one or more free amino or carboxyl groups in the polypeptide sequence and polyethylene glycol. PEGs with spacers that can be attached to free amino groups include N-hydroxysuccinimide polyethylene glycol, which can be prepared by activating the succinate ester of polyethylene glycol with N-hydroxyhydroxysuccinimide. Another activated polyethylene glycol that can be attached to free amino groups is 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine, which can be prepared by reacting polyethylene glycol monomethyl ether with cyanuric chloride. Activated polyethylene glycols that bind to free carboxyl groups include polyoxyethylenediamines.

[0134] Conjugation of one or more of the polypeptide sequences of the present disclosure to a spacer-bearing PEG can be carried out by a variety of conventional methods. For example, the conjugation reaction can be carried out using a 4:1 to 30:1 molar ratio of reagent to protein in a solution with a pH of 5 to 10, at a temperature of 4°C to room temperature, for 30 minutes to 20 hours. Reaction conditions can be selected to primarily drive the reaction toward the production of the desired degree of substitution. Generally, low temperatures, low pH (e.g., about pH 5), and short reaction times tend to decrease the number of PEGs attached, while high temperatures, neutral to high pH (e.g., about pH 7), and longer reaction times tend to increase the number of PEGs attached. The reaction can be terminated using a variety of means known in the art. In some embodiments, the reaction is terminated by acidifying the reaction mixture and freezing, for example, at -20°C. PEGylation of various polypeptides is discussed, for example, in US Pat. Nos. 5,252,714; 5,643,575; 5,919,455; 5,932,462; and 5,985,263.

[0135] The present disclosure also contemplates the use of PEG mimetics. Recombinant PEG mimetics have been developed that retain the properties of PEG (e.g., enhanced serum half-life) but confer several additional advantageous properties. For example, simple polypeptide chains (e.g., containing Ala, Glu, Gly, Pro, Ser, and Thr) that can form extended configurations similar to PEG can be recombinantly produced already fused to the peptide or protein drug of interest (e.g., Amunix's XTEN technology; Mountain View, Calif.). This obviates the need for an additional conjugation step during the manufacturing process. Established molecular biology techniques also allow for control of the side chain composition of the polypeptide chain, enabling optimization of immunogenicity and manufacturing properties.

[0136] In certain embodiments, a hydrophilic polymer is attached to a TFF2 polypeptide. The hydrophilic polymer can be linked (directly or indirectly) to the modified TFF2 polypeptide. In specific embodiments, a linker (e.g., a 1-5, 5-10, or 1-10 amino acid linker, e.g., a glycine linker) is used to link the hydrophilic polymer to the modified TFF2 polypeptide. The hydrophilic polymer can be linked covalently or non-covalently to the modified TFF2 polypeptide. The hydrophilic polymer can be a hydrophilic amino acid polymer that is essentially unorganized and is a functional analog of PEG, poly(methacrylate), polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, polyacrylamide, N-(2-hydroxypropyl)methacrylamide (HPMA), divinyl ether-maleic anhydride (DIVEMA), polyoxazoline, polyphosphate, polyphosphazene, and a derivative of conventional PEG (e.g., hydroxy-PEG). Hydroxy-PEG is disclosed in U.S. Patent No. 8,129,300; and U.S. Patent Application Publication No. 20120114742. In certain embodiments, two, three or more hydrophilic polymers are linked to TFF2 peptide.The hydrophilic polymer can be linked to peptide at the C-terminus, N-terminus, or both C-terminus and N-terminus of modified TFF2 polypeptide.

[0137] In embodiments of the present disclosure, modified TFF2 polypeptides can be PEGylated using a variety of methods, including 1) N-terminal PEGylation via aldehyde-PEG chemistry; and 2) PEGylation of exposed amines (lysines) in the absence of solvent via NHS-PEG chemistry. PEGylation via aldehyde chemistry is described in Tureck PL, et al., (2016), Journal of Pharmaceutical Sciences, 105:460-475. NHS-activated PEG derivatives can be used to The PEGylation used is based on the selectivity of NHS-activated esters for primary amine termini (see Fee, C. and Damodaran VB, (2010), European Pharmaceutical Review, Issue 1).

[0138] As used herein, the term "N-terminally modified" refers to a modification of a protein or peptide at its amino (N) terminal.For example, when the modification is PEGylation, a PEG moiety is added / linked / conjugated to one or more amino acid residues that form the first quarter of the modified TFF2 polypeptide at the N terminal.Amino acid residues include, but are not limited to, lysine, cysteine, serine, tyrosine, histidine, phenylalanine or arginine.

[0139] N-terminal modified PEG-modified TFF2 polypeptide conjugate can be obtained by reacting the N-terminal amine of modified TFF2 polypeptide with the aldehyde group of PEG in the presence of a reducing agent.Reducing agents can include NaCNBH3 and NaBH4.

[0140] PEG suitable for conjugation to polypeptide sequences is generally soluble in water at room temperature and has the general formula R(O-CH-CH) n OR (where R is hydrogen or a protecting group such as an alkyl or alkanol group, and n is an integer between 1 and 1000). When R is a protecting group, it generally has 1 to 8 carbons. PEG conjugated to a polypeptide sequence can be linear or branched. Branched PEG derivatives, "star-PEG" and multi-arm PEG, are contemplated by the present disclosure. The molecular weight of PEG used in the present disclosure is not limited to any particular range; examples are described elsewhere herein. For example, certain embodiments have a molecular weight between 5 kDa and 20 kDa, while other embodiments have a molecular weight between 4 kDa and 10 kDa.

[0141] As used herein, the term "branched" refers to the structure of a polymer molecule, wherein the polymer molecule is a linear polymer that serves as a backbone or main chain, with branches of the same basic polymer or other polymers extending from the main chain.This structure can be represented by a linear stretch of polymer molecule connected at one end of one or more functional groups of small molecules and a monomer that is polymerized into two or more of the linear stretches, wherein the small molecules have a molecular weight of less than 1000 Daltons.An example of a branched polymer molecule, for example, branched PEG, is shown in Roberts et al., Advanced Drug Delivery Reviews, 54:459-476 (2002).An exemplary small molecule with functional groups is: Examples include N-hydroxysuccinimide, maleimide, glycerin, pentaerythritol, or hexaglycerin.

[0142] The present disclosure also contemplates compositions of conjugates in which the PEG has different n values, and thus the various different PEGs are present in specific ratios. For example, some compositions contain a mixture of n=1, 2, 3, and 4 conjugates. In some compositions, the percentage of n=1 conjugates is 18-25%, the percentage of n=2 conjugates is 50-66%, the percentage of n=3 conjugates is 12-16%, and the percentage of n=4 conjugates is up to 5%. Such compositions can be produced by reaction conditions and purification methods known in the art. Exemplary reaction conditions are described throughout the specification. Cation exchange chromatography can be used to separate the conjugates, and fractions containing, for example, conjugates with the desired number of attached PEGs are then identified and purified away from unmodified protein sequences and conjugates with other numbers of attached PEGs.

[0143] In another embodiment, the modified TFF2 polypeptide is PEGylated with methoxyPEG (mPEG) (see, e.g., Poovi G., and Damodharan, N. (2018) European Journal of of Applied Sciences, 10(1):01-14).

[0144] In another embodiment, the modified TFF2 polypeptide is PEGylated with hydroxyPEG (hPEG). HydroxyPEG is described in U.S. Patent No. 8,129,300; and U.S. Patent Application Publication No. 20120114742.

[0145] In certain embodiments, PEGylation of a modified TFF2 polypeptide described herein, or the addition of a hydrophilic polymer to a modified TFF2 polypeptide described herein, increases the half-life of the peptide in vivo by 2-5 fold, 2-10 fold, 2-20 fold, 2-25 fold, 2-50 fold, 2-75 fold, or 2-100 fold compared to the unmodified TFF polypeptide, as assessed by techniques known to those of skill in the art. In some embodiments, PEGylation of a modified TFF2 polypeptide described herein, or the addition of a hydrophilic polymer to a modified TFF2 polypeptide described herein, increases the half-life of the peptide in vivo by 5-10 fold, 5-20 fold, 5-25 fold, 5-50 fold, 5-75 fold, or 5-100 fold compared to the unmodified TFF polypeptide, as assessed by techniques known to those of skill in the art. In certain embodiments, PEGylation of a modified TFF2 polypeptide described herein, or the addition of a hydrophilic polymer to a modified TFF2 polypeptide described herein, increases the half-life of the peptide in vivo by 10-20 fold, 10-25 fold, 10-50 fold, 10-75 fold, or 10-100 fold compared to the unmodified TFF polypeptide, as assessed by techniques known to those of skill in the art. In some embodiments, PEGylation of a modified TFF2 polypeptide described herein, or the addition of a hydrophilic polymer to a modified TFF2 polypeptide described herein, increases the half-life of the peptide in vivo by 25-50 fold, 25-75 fold, or 25-100 fold compared to the unmodified TFF polypeptide, as assessed by techniques known to those of skill in the art. In certain embodiments, PEGylation of a modified TFF2 polypeptide described herein, or the addition of a hydrophilic polymer to a modified TFF2 polypeptide described herein, increases the half-life of the peptide in vivo by 50-75 fold or 2-100 fold, as assessed by techniques known to those of skill in the art.

[0146] Other methods for increasing the stability and / or efficacy of therapeutic polypeptides are known in the art and are included as embodiments of the present disclosure, see, e.g., Strohl, WR, (2015), BioDrugs, 29(4):215-239.

[0147] CTP peptide In some embodiments, the conjugating moiety is the CTP peptide of the human chorionic gonadotropin beta subunit. The CTP peptide is the 31 amino acid residue peptide FQSSSS * KAPPPS * LPSPS * RLPGPS * DTPILPQ (SEQ ID NO: 11), wherein S * indicates an O-glycosylation site (see, e.g., Furuhashi et al., (1995) Mol Endocrinol., 9(1):54-63).

[0148] PAS (registered trademark) In some embodiments, the modified TFF2 polypeptides described herein are PASylated (Aghaabdollahian, S. et al., (2019) Scientific Reports, 9:2978; Payne et al. (2010) Pharm. Dev. Technol., 1-18;Pisal et al. (2010) J. Pharm. Sci. 99 (6), 2557-2575;Veronese. (2001) Biomaterials 22 (5), 405-417;Veronese (2009) Milestones in drug therapy (Parnham, MJ, and Bruinvels, J., Eds.) Birkhauser, Basel; U.S. Patent No. 9,221,882; U.S. (See Patent Nos. 9,260,494; 9,957,323; 10,081,657; 10,174,302; and 9,574,014). Each of these is incorporated herein by reference in its entirety. PASylation has been reported to increase in vivo and / or in vitro stability (U.S. Patent No. 9,260,494). PASylation is the genetic fusion of a nucleic acid encoding a polypeptide, such as a modified TFF2 polypeptide described herein, with a nucleic acid encoding a PAS polypeptide. PAS polypeptides are hydrophilic, uncharged polypeptides consisting of Pro, Ala, and Ser residues. In some embodiments, the PAS-modified TFF2 polypeptide consists of about 4, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500 or about 600 amino acids, or any range therebetween, for example, 4-600, 10-500 amino acids, etc.

[0149] XTEN conversion In some embodiments, the modified TFF2 polypeptides described herein are XTENized. The terms "XTEN™" (Amunix Operating Inc.) and / or "XTENized" refer to a large, unorganized recombinant polypeptide composed of the amino acids A, E, G, P, S, and T. The XTEN can have a length of approximately 864 amino acids, but can also be shorter (e.g., a fragment of the 864 amino acid-long polypeptide according to WO2010091122A1). The term XTENized refers to the fusion of the XTEN target with a therapeutic protein ("payload"). XTENization serves to increase the serum half-life of the therapeutic protein (i.e., as used herein, the fusion protein of the present disclosure). The terms "XTEN" and / or "XTENized" also refer to an unorganized recombinant polypeptide (URP) comprising at least 40 contiguous amino acids, wherein the sum of glycine (G), aspartic acid (D), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P) residues contained in the URP constitutes at least 80% of the total amino acids of the unorganized recombinant polypeptide, the remainder consisting of arginine or lysine, if present, and the remainder does not contain methionine, cysteine, asparagine, or glutamine.

[0150] ELP conversion In some embodiments, the modified TFF2 polypeptide is ELP-modified. The conjugated moiety is an elastin-like polypeptide (ELP). ELP-modification uses ELP, a repeating peptide unit containing a sequence commonly found in elastin (see Yeboah A, et al., (2016), Biotechnol Bioeng 113:1617-1627). ELP-modification involves the genetic fusion of a nucleic acid encoding a polypeptide of interest with a nucleic acid encoding an elastin-like polypeptide (ELP). ELP comprises a VPGxG repeat motif: Val Pro Gly Xaa Gly (SEQ ID NO: 12), where x is any amino acid except proline (see WO 2018 / 132768).

[0151] HAP (homo amino acid polymer) In some embodiments, the modified TFF2 polypeptide described herein is HAP-modified. HAP-modification is a genetic fusion of a nucleic acid encoding a polypeptide of interest with a nucleic acid encoding a glycine-rich homoamino acid polymer (HAP). In some examples, the HAP polymer is (Gly4Ser) n It contains a repeat motif (SEQ ID NO: 13) and may have a length of about 50, 100, 150, 200, 250, 300 or more residues (Schlapschy, M. et al. Protein Eng Des Sel 20, 273-284).

[0152] PSA (polysialylated) In some embodiments, the modified TFF2 polypeptide described herein can be polysialylated. Polysialic acid (PSA), also known as colominic acid (CA), is a naturally occurring polysaccharide. It is a homopolymer of N-acetylneuraminic acid with α(2→8) ketoside linkages or α(2→9) linkages, or a mixture of both, and contains a vicinal diol group at its non-reducing end. It is negatively charged and is a natural constituent of the human body. PSA can be produced in bacteria (U.S. Patent No. 5,846,951; U.S. Patent No. 9,018,166; U.S. Patent No. 10,414,793; Zhang et al., (2014), Asian Journal of Pharmaceutical Sciences, 9(2):75-81). Methods for polysialylating polypeptides are described in US Patent Application Publication No. 2012 / 0329127.

[0153] PLGA Conjugation with poly(D,L-lactic-co-glycolic acid) (PLGA). In some embodiments, the modified TFF2 polypeptides described herein can be conjugated with poly(D,L-lactic-co-glycolic acid) (PLGA). PLGA (PGLA) is charged and is a natural component of the human body. PLGA extends the plasma half-life of cyclic macrolide drugs, including, for example, Zilcoplan (Ra Pharmaceuticals technology).

[0154] Pharmaceutical Compositions and Methods of Administration The modified TFF2 polypeptides of the present disclosure can be administered in a variety of ways. For example, the modified TFF2 polypeptides can be administered using intravenous infusion, intramuscular administration, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump can be used (see Sefton (1987) Biomed. Eng. 14:201; Buchwald et al. (1980) Surgery 88:507; Saudek et al. (1989) N. Engl. J. Med. 321:574). In another embodiment, a polymeric material can be used. (See Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, (1983) J. Macromol. Sci. Rev. Macromol. Chem. 23:61; also see Levy et al. (1985) Science 228:190; During et al. (1989) Ann. Neurol. 25:351; Howard et al. (1989) J. Neurosurg. 71:105). In another embodiment, a controlled release system can be placed in proximity to the therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, vol. 2, pp. 115-138 (1984). Other controlled release systems are (Science (1990) 249:1527-1533). Proteins / peptides are poorly absorbed via oral administration, but delivery systems for oral administration are well known in the art, for example, Wu S. et al., (2019), Journal of Pharmaceutical Sciences, 108(6):2143-2152; and Renukunita, J. et al., (2013), Int. J. Pharm., 447:75-93.

[0155] In some embodiments, modified TFF2 polypeptide can be provided in the form of a pharmaceutical composition containing an isotonic excipient prepared under sufficiently sterile conditions for human administration.The selection of excipient and any additional components of the composition comprising PEGylated TFF2 are adapted according to the route and device used for administration.In some embodiments, the composition comprising PEGylated TFF2 polypeptide can also contain or be accompanied by one or more other components that facilitate the delivery or functional transfer of TFF2 peptide.

[0156] The methods described herein are not intended to be comprehensive, and those skilled in the art will recognize additional methods suitable for specific applications. Effective amounts of the compositions can also be further estimated by analogy with compounds known to exhibit the desired effect.

[0157] One aspect of the present disclosure provides a method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of any one of the compositions of the present disclosure or any one of the modified TFF2 polypeptides of the present disclosure.

[0158] Another aspect of the present disclosure provides a method for treating inflammatory bowel disease in a subject in need thereof, comprising administering to the subject an effective amount of any one of the compositions of the present disclosure or any one of the modified TFF2 polypeptides of the present disclosure.

[0159] Another aspect of the present disclosure provides a method of treating COVID-19 in a subject in need thereof, comprising administering to the subject an effective amount of any one of the compositions of the present disclosure or any one of the modified TFF2 polypeptides of the present disclosure.

[0160] In some embodiments, COVID-19 complications or symptoms treated by compositions or polypeptides of the present disclosure include, but are not limited to, fatigue, fever, shortness of breath, muscle pain, acute respiratory distress syndrome, acute respiratory failure, acute respiratory distress syndrome (ARD), pneumonia, liver damage, cardiovascular complications, neurological and neuropsychiatric complications, kidney damage, and the like.

[0161] In one embodiment, the modified TFF2 polypeptide can be administered in combination with an agent that inhibits or reduces the replication of SARS-CoV-2. In another embodiment, the modified TFF2 polypeptide can be administered in combination with an antiviral agent selected from the group consisting of ribavirin, interferon (Alfacon-1), chloroquine, hydroxychloroquine, EIDD-2801, EIDD-1931, GS-5734, GS-441524, ivermectin, favipiravir, indomethacin, chlorpromazine, penciclovir, nafomostat, camostat, nitazoxanide, remdesivir, famotidine, and dexamethasone.

[0162] In some embodiments, the modified TFF2 polypeptide can be given before, simultaneously with, or after an agent that inhibits or reduces the replication of SARS-CoV-2 or an antiviral agent.

[0163] According to the present disclosure, pharmaceutically acceptable carriers can include any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration.The use of such media and agents for pharmaceutically active substances is well known in the art.Any conventional media or agent that is compatible with active compounds can be used.Supplementary active compounds can also be incorporated into the composition.

[0164] The modified TFF2 polypeptide can be administered to a subject all at once (e.g., as a single injection or deposit). Alternatively, the modified TFF2 polypeptide can be administered once or twice daily to a subject in need of treatment for a period of about 2 to about 28 days, or about 7 to about 10 days, or about 7 to about 15 days. It can also be administered once or twice daily to a subject for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof. Furthermore, the modified TFF2 polypeptide can be co-administered with another therapeutic agent.

[0165] In one embodiment, the modified TFF2 polypeptide can be co-administered with a chemotherapeutic agent. Some non-limiting examples of conventional chemotherapy drugs include aminoglutethimide, amsacrine, asparaginase, bcg, anastrozole, bleomycin, buserelin, bicalutamide, busulfan, capecitabine, carboplatin, camptothecin, chlorambucil, cisplatin, carmustine, cladribine, colchicine, cyclophosphamide, cytarabine, dacarbazine, cyproterone, clodronate, daunorubicin, diethylstilbestrol, docetaxel, dactinomycin, doxorubicin, dienestrol, etoposide, exemestane, filgrastim, fluorouracil, fludarabine, fludrocortisone, epirubicin, estradiol, gemcitabine, genistein, estramustine, fluoxymesterone, flutamide, goserelin, leuprolide, hydroxyurea , idarubicin, levamisole, imatinib, lomustine, ifosfamide, megestrol, melphalan, interferon, irinotecan, letrozole, leucovorin, ironotecan, mitoxantrone, nilutamide, medroxyprogesterone, mechlorethamine, mercaptopurine, mitotane, nocodazole, octreotide, methotrexate, mitomycin, paclitaxel, oxaliplatin These include fluticasone, temozolomide, pentostatin, plicamycin, suramin, tamoxifen, porfimer, mesna, pamidronate, streptozocin, teniposide, procarbazine, titanocene dichloride, raltitrexed, rituximab, testosterone, thioguanine, vincristine, vindesine, thiotepa, topotecan, tretinoin, vinblastine, trastuzumab, and vinorelbine.

[0166] In one embodiment, the modified TFF2 polypeptide can be co-administered with a monoclonal antibody against PD-1, PD-L1, or CTLA-4. Examples of PD-1 blocking antibodies are pembrolizumab (Keytruda®), nivolumab (Opdivo®), and cemiplimab (Libtayo®). Examples of PD-L1 blocking antibodies are atezolizumab (Tecentriq®), avelumab (Bavencio®), and durvalumab (Imfinzi®). An example of a CTLA-4 blocking antibody is ipilimumab (Yervoy®).

[0167] In one embodiment, the cancer is not responsive to a blocking anti-PD-1 or anti-PD-L1 monoclonal antibody, and treatment with the modified TFF2 polypeptide induces responsiveness to a blocking anti-PD-1, anti-PD-L1 or anti-CTLA-4 monoclonal antibody.

[0168] In one embodiment, the chemotherapeutic agent is an alkylating agent, a nitrosourea, an antimetabolite, a topoisomerase inhibitor, a mitotic inhibitor, an anthracycline, a corticosteroid hormone, a sex hormone, or a targeted anti-tumor compound.

[0169] In one embodiment, the modified TFF2 polypeptide can be co-administered with an anti-inflammatory agent, some non-limiting examples of which include anti-inflammatory steroids (corticosteroids) (e.g., prednisone), aminosalicylates (e.g., mesalazine, Asacol HD®, Delzicol®, etc.), balsalazide (Colazal®), and olsalazine (Dipentum), and / or nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., aspirin, ibuprofen, naproxen), and immunoselective anti-inflammatory derivatives (ImSAIDs). Anti-inflammatory drugs include, but are not limited to, anti-TNFα antibodies (e.g., infliximab (Remicade®), adalimumab (Humira®), certolizumab pegol (Cimzia®), golimumab (Simponi®), etanercept (Enbrel®), anti-IL12 antibodies, anti-IL2 antibodies (basiliximab (Simulect®), daclizumab (Zenapax®), azathioprine (Imuran®, Azasan®), 6-mercaptopurine (6-MP, Purinethol®), Antibodies or molecules that target cytokines and chemokines, including cyclosporine A (Sandimmune®, Neoral®), tacrolimus (Prograf®), and anti-GM-CSF antibodies, may also be included. In some embodiments, modified TFF2 polypeptides can be co-administered with natalizumab (Tysabri®), vedolizumab (Entyvio®), and ustekinumab (Stelara®). In some embodiments, modified TFF2 polypeptides are co-administered with inhibitors of Janus kinases 1-3, such as the small molecule tofacitinib.In some embodiments, modified TFF2 polypeptides can be administered with immune system suppressants used to treat IBD, such as azathioprine (Azasan®, Imuran®), mercaptopurine (Purinethol®, Purixan®), cyclosporine (Gengraf®, Neoral®, Sandimmune®), and methotrexate (Trexall®).

[0170] In one embodiment, the modified TFF2 polypeptide can be co-administered with radiation therapy. Some non-limiting examples of conventional radiation therapy include external beam radiation therapy, sealed source radiation therapy, unsealed source radiation therapy, particle therapy, and radioisotope therapy.

[0171] In one embodiment, modified TFF2 polypeptide can be co-administered with cancer immunotherapy.Cancer immunotherapy involves using the immune system of a subject to treat cancer.For example, the immune system of a subject can be stimulated to recognize and eliminate cancer cells.Some non-limiting examples of cancer immunotherapy include cancer vaccines, therapeutic antibodies, for example, monoclonal antibody therapy (for example, bevacizumab, cetuximab and panitumumab), cell-based immunotherapy and adoptive cell-based immunotherapy.

[0172] The modified TFF2 polypeptides may also be used in combination with surgical or other interventional treatment regimens used to treat diseases of the digestive system.

[0173] The compositions of the present disclosure can be formulated and administered to reduce symptoms associated with digestive system diseases by any means that allows the active ingredient to contact the site of action of the drug in the body of a human or non-human subject. For example, the compositions of the present disclosure can be formulated and administered to reduce symptoms associated with digestive system inflammatory diseases, digestive system cancer, or digestive system dysplasia, or to cause a reduction in cell proliferation or tumor growth. They can be administered by any conventional means available for use in combination with pharmaceuticals, either as individual therapeutic active ingredients or as a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected based on the selected route of administration and standard pharmaceutical practice.

[0174] Pharmaceutical compositions for use according to the present disclosure can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. Therapeutic compositions of the present disclosure can be formulated for a variety of routes of administration, including systemic and topical or localized administration. Techniques and formulations are generally described in Remington's Pharmaceutical Sciences, Meade, NY, pp. 111-115, 1997. Publishing Co., Easton, Pa. (20th ed., 2000), the entire disclosure of which is incorporated herein by reference. For systemic administration, injections, including intramuscular, intravenous, intraperitoneal, and subcutaneous, are useful. For injection, the therapeutic compositions of the present disclosure can be formulated in a liquid solution, such as PBS, Hank's solution, or Ringer's solution, or in a physiologically compatible buffer. In addition, the therapeutic compositions can be formulated in solid form and redissolved or suspended immediately upon use. Lyophilized forms are also included. The pharmaceutical compositions of the present disclosure are characterized as being at least sterile and pyrogen-free. These pharmaceutical formulations include those for human and veterinary use.

[0175] Any of the therapeutic applications described herein can be applied to any subject in need of such treatment, including, for example, a mammal such as a dog, cat, cow, horse, rabbit, monkey, pig, sheep, goat, or human.

[0176] The pharmaceutical composition of the present disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can contain the following components: a sterile diluent, e.g., water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent, e.g., benzyl alcohol or methylparaben; an antioxidant, e.g., ascorbic acid or sodium bisulfite; a chelating agent, e.g., ethylenediaminetetraacetic acid; a buffer, e.g., acetate, citrate, or phosphate, and an agent for adjusting isotonicity, e.g., sodium chloride or dextrose. pH can be adjusted using acids or bases, e.g., hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0177] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). The composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, glycerol, propylene glycol, a pharmaceutically acceptable polyol such as liquid polyethylene glycol, and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid and thimerosal.In many cases, it can be useful to include isotonic agents, for example, sugars, polyalcohols, for example, mannitol, sorbitol, sodium chloride in the composition.Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0178] Sterile injectable solutions can be prepared by incorporating modified TFF2 polypeptide in the required amount into a suitable solvent, optionally with one or a combination of the ingredients listed herein, followed by sterile filtration.Dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other ingredients required from those listed herein.In the case of sterile powders for preparing sterile injectable solutions, examples of useful methods include vacuum drying and freeze-drying, which produces a powder of the active ingredient and any additional desired ingredients from its previously sterile-filtered solution.

[0179] Systemic administration can also be by transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation.Such penetrants are known in the art, and for example, for transmucosal administration, detergents, bile salts and fusidic acid derivatives are included.Transmucosal administration can be achieved by using nasal sprays or suppositories.For transdermal administration, the active compound is formulated into ointments, salves, gels or creams known in the art.

[0180] The compositions of the present disclosure can be administered to a subject in need of treatment, which may include, but is not limited to, a mammal such as a dog, cat, cow, horse, rabbit, monkey, pig, sheep, goat, or human.

[0181] Compositions of the present disclosure can also be formulated as sustained and / or sustained release preparations.Such sustained and / or sustained release preparations can be carried out by sustained release means or delivery device well known to those skilled in the art, for example, those described in United States Patent No. 3,845,770; United States Patent No. 3,916,899; United States Patent No. 3,536,809; United States Patent No. 3,598,123; United States Patent No. 4,008,719; United States Patent No. 4,710,384; United States Patent No. 5,674,533; United States Patent No. 5,059,595; United States Patent No. 5,591,767; United States Patent No. 5,120,548; United States Patent No. 5,073,543; United States Patent No. 5,639,476; United States Patent No. 5,354,556; and United States Patent No. 5,733,566, the disclosures of which are incorporated herein by reference. The pharmaceutical compositions (e.g., having therapeutic effects) of the present disclosure can be used to provide delayed or sustained release of one or more active ingredients, for example, by using hydropropylmethylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, etc., or combinations thereof, to provide desired release profiles with various characteristics.Suitable sustained-release formulations known to those skilled in the art, including those described herein, can be easily selected for use with the pharmaceutical compositions of the present disclosure.Single-unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gel caps, caplets, or powders, that are adapted for sustained release, are encompassed by the present disclosure.

[0182] In the methods described herein, modified TFF2 polypeptides can be administered to a subject either as RNA in conjunction with a delivery reagent, or as a nucleic acid (e.g., recombinant plasmid or viral vector) containing a sequence that expresses a gene product. Suitable delivery reagents for administering modified TFF2 polypeptides include Mirus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), or liposomes.

[0183] The dosage administered may be a therapeutically effective amount of the composition sufficient to result in treatment of inflammatory diseases of the digestive system, treatment of any of the digestive system cancers, reduction in cell proliferation, reduction in tumor growth, or treatment of digestive system dysplasia, and may vary depending on known factors, such as the pharmacodynamic characteristics of the active ingredient and its mode and route of administration; the time of administration of the active ingredient; the age, sex, health and weight of the recipient; the nature and extent of the symptoms; the type of concomitant treatment, frequency of treatment and desired effect; and the rate of excretion.

[0184] In some embodiments, the effective amount of modified TFF2 polypeptide administered is at least about 0.01 μg / kg body weight, at least about 0.025 μg / kg body weight, at least about 0.05 μg / kg body weight, at least about 0.075 μg / kg body weight, at least about 0.1 μg / kg body weight, at least about 0.25 μg / kg body weight, at least about 0.5 μg / kg body weight, at least about 0.75 μg / kg body weight, at least about 1 μg / kg body weight, at least about 5 μg / kg body weight, at least about 10 μg / kg body weight, at least about 25 ... μg / kg body weight, at least about 50 μg / kg body weight, at least about 75 μg / kg body weight, at least about 100 μg / kg body weight, at least about 150 μg / kg body weight, at least about 200 μg / kg body weight, at least about 250 μg / kg body weight, at least about 300 μg / kg body weight, at least about 350 μg / kg body weight, at least about 400 μg / kg body weight, at least about 450 μg / kg body weight, at least about 500 μg / kg body weight, at least about 550 μg / kg body weight, at least about 600 μg / kg body weight, at least about 650 μg / kg body weight, at least about 700 μg / kg body weight, at least about 750 μg / kg body weight, at least about 800 μg / kg body weight, at least about 850 μg / kg body weight, at least about 900 μg / kg body weight, at least about 950 μg / kg body weight, at least about 1000 μg / kg body weight, at least about 1500 μg / kg body weight, at least about 2000 μg / kg body weight, at least about 2500 μg / kg body weight, at least about 3000 μg / kg body weight, at least about 3500 μg / kg body weight, at least about 40 00 μg / kg body weight, at least about 4500 μg / kg body weight, at least about 5000 μg / kg body weight, at least about 5500 μg / kg body weight, at least about 6000 μg / kg body weight, at least about 6500 μg / kg body weight, at least about 7000 μg / kg body weight, at least about 7500 μg / kg body weight, at least about 8000 μg / kg body weight, at least about 8500 μg / kg body weight, at least about 9000 μg / kg body weight, at least about 9500 μg / kg body weight or at least about 10000 μg / kg body weight.

[0185] In one embodiment, the modified TFF2 polypeptide is administered at least once a day. In another embodiment, the modified TFF2 polypeptide is administered at least twice a day. In some embodiments, the modified TFF2 polypeptide is administered for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 12 weeks, at least 18 weeks, at least 24 weeks, at least 36 weeks, at least 48 weeks, or at least 60 weeks. In a further embodiment, the modified TFF2 polypeptide is administered in combination with a second therapeutic agent.

[0186] The toxicity and therapeutic efficacy of the therapeutic compositions of the present disclosure can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Therapeutic agents that exhibit large therapeutic indices are useful. Therapeutic compositions that exhibit some toxic side effects can be used.

[0187] Experimental animals can be used as models for human diseases. For example, mice can be used as mammalian model systems. Physiological systems possessed by mammals can be found, for example, in mice and in humans. Certain diseases can be induced in mice by manipulating their environment, genome, or a combination of both. For example, the AOM / DSS mouse model is a model for human colon cancer. In another example, the DSS mouse model is a model for human colitis. Other mouse models of cancer development include the two-stage DMBA / TPA model of skin cancer, the DEN / CCL4 model of liver cancer, and the H. felis / MNU model of gastric cancer. In addition, there are many genetically engineered models of cancer, such as the KPC model of pancreatic cancer.

[0188] The administration of the modified TFF2 polypeptide is not limited to a single route, but can include administration by multiple routes. Multiple administrations can be sequential or simultaneous. Other modes of application by multiple routes will be apparent to those skilled in the art.

[0189] Recombinant proteins and techniques The present disclosure employs conventional molecular biology, microbiology, and recombinant DNA techniques available to those skilled in the art, which techniques are well known to those skilled in the art and are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, "DNA Cloning: A Practical Approach," Volumes I and II (DN Glover, ed., 1985); "Oligonucleotide Synthesis" (MJ Gait, ed., 1984); "Nucleic Acid Hybridization" (BD Hames & SJ Higgins, eds., 1985); "Transcription and Translation" (BD Hames & SJ Higgins, eds., 1984); "Animal Cell Culture" (RI Freshney, ed., 1986); "Immobilized Cells and Enzymes" (IRL Press, 1986); B. Perbal, "A Practical Guide to Molecular Cloning" (1984) and Sambrook, et al., "Molecular Cloning: a Laboratory Manual" (2001).

[0190] Those skilled in the art can obtain TFF2 protein in several ways, including, but not limited to, isolating the protein through biochemical means or expressing a nucleotide sequence encoding the protein of interest by genetic engineering methods. In some embodiments, the polynucleotide sequence in the host cell in which the TFF2 protein, e.g., human TFF2, is expressed can be optimized for expression while still encoding the protein of SEQ ID NO: 1 or 3. In some embodiments, the DNA encoding TFF2 can also encode a hybrid protein with amino acids useful for protein purification, e.g., human serum albumin (HSA), a His tag, or an Fc tag, as described herein.

[0191] The modified TFF2 polypeptide can be a fragment of a TFF2 protein, for example, a TFF2 protein fragment can encompass any portion of at least about 8 contiguous amino acids of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 6. The fragment can comprise at least about 10 contiguous amino acids, at least about 20 contiguous amino acids, at least about 30 contiguous amino acids, at least about 40 contiguous amino acids, at least about 50 contiguous amino acids, at least about 60 contiguous amino acids, at least about 70 contiguous amino acids, at least about 80 contiguous amino acids, at least about 90 contiguous amino acids, at least about 100 contiguous amino acids, at least about 110 contiguous amino acids, or at least about 120 contiguous amino acids of SEQ ID NO: 1, 3, or 6. Fragments include all possible amino acid lengths from about 8 to about 80 amino acids, for example, lengths of about 10 to about 80 amino acids, about 15 to about 80 amino acids, about 20 to about 80 amino acids, about 35 to about 80 amino acids, about 40 to about 80 amino acids, about 50 to about 80 amino acids, or about 70 to about 80 amino acids.

[0192] Modified TFF2 polypeptides can be obtained by expressing a nucleotide sequence encoding a protein of interest or a fragment thereof by several methods, including but not limited to, genetic engineering methods.

[0193] The nucleic acid encoding modified TFF2 polypeptide can be expressed in an expression cassette, for example, to achieve overexpression in cells.The nucleic acid can be in an expressible form, for example, the target RNA, cDNA, cDNA-like or DNA in an expression cassette, which can be expressed from a native promoter or a completely heterologous promoter.The target nucleic acid can code for a protein, and may or may not contain introns.Any recombinant expression system can be used, including but not limited to bacterial, mammalian, yeast, insect or plant cell expression systems.

[0194] Host cells transformed with nucleic acid sequences encoding modified TFF2 polypeptides can be cultured under suitable conditions for expression and protein recovery from cell culture.The polypeptide produced by transformed cells can be secreted or contained intracellularly depending on the sequence and / or the vector used.The expression vector containing nucleic acid sequences encoding modified TFF2 polypeptides can be designed to contain a signal sequence that directs the secretion of soluble polypeptide molecules encoding modified TFF2 polypeptides through the membrane of prokaryotic or eukaryotic cells.Examples of heterologous signal peptides include, but are not limited to, those shown in Table 2 below. [Table 2]

[0195] The nucleic acid sequence containing TFF2 encoding the polypeptide can be synthesized in whole or in part using chemical methods known in the art. Alternatively, the TFF2 protein can be produced using chemical methods to synthesize its amino acid sequence, for example, by direct peptide synthesis using solid-phase techniques. Protein synthesis can be performed either manually or automatically. Automated synthesis can be achieved, for example, using an Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). If necessary, fragments of TFF2 can be synthesized separately and combined using chemical methods to produce a full-length polypeptide.

[0196] Synthetic peptides can be substantially purified by high performance liquid chromatography (HPLC). The composition of synthetic modified TFF2 polypeptides can be confirmed by amino acid analysis or sequencing. In addition, any part of the TFF2 amino acid sequence can be altered during direct synthesis and / or during the use of sequences derived from other proteins in combination with chemical methods to produce variant modified TFF2 polypeptides or fusion proteins.

[0197] The present disclosure further encompasses methods for using proteins or modified TFF2 polypeptides encoded by TFF2 nucleic acid sequences, such as the sequences set forth in SEQ ID NOs: 2 and 3. In another embodiment, the polypeptides can be modified, for example, by glycosylation and / or acetylation and / or chemical reaction or coupling, and can contain one or several unnatural or synthetic amino acids. In certain embodiments, the present disclosure encompasses variants of TFF2.

[0198] Fusion proteins Those skilled in the art will understand that expression of a desired protein product can be based on a fusion protein. One embodiment of a modified TFF2 polypeptide is a fusion protein. One embodiment of a fusion protein is a TFF2-albumin protein. Another embodiment is a modified TFF2-IgG1 fusion protein. These fusion proteins increase the serum half-life of the modified TFF2 polypeptide compared to native or recombinant TFF2. Another type of fusion protein binds an affinity tag useful for purifying recombinant proteins. Fusion proteins can contain new sequences at either the N- or C-terminus of the TFF2 sequence. Fusion proteins can contain a portion of the TFF2 amino acid sequence, the entire amino acid sequence, or can contain new sequences linking the TFF2 sequence to the fusion protein.

[0199] A common fusion protein with an affinity tag uses a polyhistidine tag. The affinity tag is often linked to the TFF2 sequence by a target protease cleavage site sequence that can be cleaved with an appropriate protease (Waugh, DS. An Overview of Enzymatic Reagents for the Removal of Affinity Tags, Protein Expr Purif. 2011 Dec; 80(2): 283-293). Common target protease cleavage site sequences are listed in the sequence table. The affinity tag is a target for a thrombin cleavage site following the amino acid sequence of sequence number 20 (Leu-Val-Pro-Arg-Gly-Ser). Thrombin selectively cleaves between the arginine and glycine residues of the cleavage site. In other cases, the affinity tag is connected by a target sequence for enterokinase, which cleaves at the recognition site (Asp-Asp-Asp-Lys) (SEQ ID NO: 21). In another embodiment, the affinity tag is connected by a target protease cleavage site sequence for tobacco etch virus (TEV). TEV protease is a highly specific cysteine ​​protease that recognizes the amino acid sequence: Glu-Asn-Leu-Tyr-Phe-Gln-Gly (SEQ ID NO: 22) or Glu-Asn-Leu-Tyr-Phe-Gln-Ser (SEQ ID NO: 23) and cleaves between the Gln and Gly / Ser (P1') residues. The P1' residue can alternatively be Ala, Met or Cys (Kapust, RB et al. (2002). Biochem. and Biophysical Research Comm. 294, 949-955).

[0200] In other embodiments, after cleavage of the affinity tag, the resulting protein comprises one or more amino acid residues from the cleavage site.

[0201] In some embodiments, after cleavage of the affinity tag, the resulting protein is a native protein. For example, TAGZyme from Qiagen® is an enzyme system for affinity purification of recombinant proteins using his-tags and tag removal. It combines a dipeptidase (DAPase, or recombinant dipeptidyl peptidase I) for exonucleolytic cleavage from the N-terminus, and two potential accessory aminopeptidases (Qcyclase, or plant glutamine cyclotransferase, and pGAPase, or bacterial pyroglutamyl aminopeptidase) for complete removal of the his-tag. All three enzymes in TAGZyme exhibit non-cleavable his-tags for removal.

[0202] In certain embodiments, fusion proteins can be PEGylated to generate pharmaceutical products comprising fusion proteins with sequences that enhance half-life, such as albumin or IgG sequences, and sequences used as affinity tags, such as his tags, and sequences used as linker sequences for the affinity tags or for other aspects of production.

[0203] Bacterial Expression System Those skilled in the art will appreciate that expression of desired protein products in prokaryotes is most often achieved in E. coli using vectors containing constitutive or inducible promoters. Some non-limiting examples of bacterial cells for transformation include the bacterial cell line E. coli strains DH5α or MC1061 / p3 (Invitrogen Corp.®, San Diego, Calif.), which can be transformed using standard procedures practiced in the art, and colonies can then be screened for appropriate plasmid expression. Many expression vectors can be selected for bacterial systems. Non-limiting examples of such vectors include multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene®). Some E. coli expression vectors (also known in the art as fusion vectors) are designed to add a number of amino acid residues, usually to the N-terminus of the recombinant protein being expressed. Such fusion vectors can serve three functions: 1) increase the solubility of the desired recombinant protein; 2) increase the expression of the recombinant protein of interest; and 3) aid in recombinant protein purification by acting as a ligand in affinity purification. In some instances, vectors that direct the expression of high levels of fusion protein products that are easily purified can also be used. Some non-limiting examples of fusion expression vectors include pGEX, which fuses glutathione S-transferase (GST) to the desired protein; pcDNA 3.1 / V5-His AB & C (Invitrogen Corp.®, Carlsbad, Calif.), which fuses 6xHis (SEQ ID NO: 8) to the recombinant protein of interest; pMAL (New England Biolabs®, MA), which fuses maltose E-binding protein to the target recombinant protein; and the E. coli expression vector pUR278 (Ruther et al., (1983) EMBO 12:1791), where the coding sequence is the fusion protein. To produce fusion proteins, the lacZ coding region can be individually ligated into vectors in frame with the lacZ coding region; and the pIN vector (Inouye et al., (1985) Nucleic Acids Res. 13:3101-3109; Van Heeke et al., (1989) J. Biol. Chem. 24:5503-5509). Fusion proteins produced by vectors similar to those described above are generally soluble and can be easily purified from lysed cells via adsorption and binding of the fusion protein to an affinity matrix. For example, fusion proteins can be purified from lysed cells via adsorption and binding to a matrix of glutathione-agarose beads, followed by elution in the presence of free glutathione. For example, pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target can be released from the GST moiety.

[0204] Plant, insect and yeast expression systems In addition to microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the coding sequence for the TFF2 peptide, other suitable cell systems may alternatively be used to produce the molecule of interest. Non-limiting examples include plant cell systems infected with recombinant viral expression vectors (e.g., tobacco mosaic virus, TMV; cauliflower mosaic virus, CaMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the coding sequence for the modified TFF2 polypeptide. When a plant expression vector is used, expression of the sequence encoding the modified TFF2 polypeptide can be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from the tobacco mosaic virus, TMV. Alternatively, plant promoters such as the small subunit or heat shock promoter of RUBISCO can be used. These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection.

[0205] Insect systems can also be used to express modified TFF2 polypeptides or fusion proteins. Many methods for expressing recombinant proteins using insect systems are known in the art, for example, Bleckmann, M. et al., (2016), Biotechnol Bioeng. 113(9): 1975-1983; Zitzmann, J. et al., Process Optimization for Recombinant Protein Expression in Insect Cells, New Insights into See Cell Culture Technology; InTech; 2017; U.S. Patent No. 5,194,376; U.S. Patent No. 5,843,733; For example, in one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia virescens in Trichoplusia larvae. The sequence encoding the modified TFF2 polypeptide can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under the control of the polyhedrin promoter. Successful insertion of the nucleic acid sequence of the modified TFF2 polypeptide renders the polyhedrin gene inactive, resulting in the generation of a recombinant virus lacking coat protein. The recombinant virus is then used to infect, for example, Spodoptera frugiperda (S. frugiperda) cells, or Trichoplusia ni (in Trichoplusia) larvae in grass frugiperda (S. frugiperda) cells, or Trichoplusia night moth (Trichoplusia) larvae, thereby expressing the polypeptide of interest (see Engelhard, EK et al. (1994) in Proc. Natl. Acad. Sci. 3224).

[0206] In another embodiment, yeast, such as Schizosaccharomyces pombe (Schizosaccharomyces pombe); Kluyveromyces (Kluyveromyces) hosts, such as lactic acid bacteria, such as Lactobacillus glabra (Lactobacillus glabra), Kluyveromyces fragilis (K. fragilis) (ATCC 12424), K. bulgaricus (K. bulgaricus) (ATCC 16045), Clostridium Kluyveromyces (K. wickerhamii) (ATCC 24178), K. waltii (ATCC 56500), Drosophila Kluyveromyces (K. drosophilarum) (ATCC 36906), K. thermotolerans (K. thermotolerans), and Kluyveromyces marxianus (K. marxianus); Yarrowia (yarrowia) (EP 402226); Pichia yeast (Pichia pastoris) (EP 183070); Candida (Candida); Trichoderma reesei (Trichoderma reesei) (EP 244234); crude Tangmaiping-carrying bacteria (Neurospora crassa); Schwanniomyces (Schwanniomyces), for example, Schwanniomyces occidentalis; and filamentous fungi, for example, Neurospora strains (Neurospora), Penicillium (Penicillium), cyclosporine (Tolypocladium), and Aspergillus (Aspergillus) hosts, for example, Aspergillus nidulans (A. nidulans) and Niger (A. niger). Yeast can be transformed with a recombinant yeast expression vector containing a coding sequence for a modified TFF2 polypeptide.A preferred embodiment uses S. cerevisiae because yeast has the ability to glycosylate recombinant proteins and a significant proportion of human TFF2 in gastric juice is glycosylated via N-linkage on the putative Asn(15), which may be functionally important for intravascular TFF2 and may increase plasma half-life (May FE et al., Gut 2000 46(4):454-9). When recombinant human TFF2 is expressed in S. cerevisiae, a significant proportion of the recombinant protein is glycosylated via an N-linkage at Asn(15) (Thim L et al. FEBS Lett 1993: 318:345-52).

[0207] Mammalian Expression Systems Mammalian cells (e.g., BHK cells, VERO cells, CHO cells, HEK293 cells, etc.) can also contain an expression vector (e.g., one carrying a nucleotide sequence encoding a modified TFF2 polypeptide) for expression of a desired product. Expression vectors containing such nucleic acid sequences linked to at least one regulatory sequence in a manner allowing expression of the nucleotide sequence in the host cell can be introduced via methods known in the art. Many viral expression systems can be used to express modified TFF2 polypeptides in mammalian host cells. The vector can be a recombinant DNA or RNA vector, including a DNA plasmid or a viral vector. For example, when an adenovirus is used as an expression vector, the sequence encoding the modified TFF2 polypeptide can be ligated into an adenovirus transcription / translation complex containing a late promoter and a tripartite leader sequence. Insertion into the nonessential E1 or E3 region of the viral genome can be used to obtain a live virus capable of expressing the modified TFF2 polypeptide in infected hosts. Transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, can also be used to increase expression in mammalian host cells. Additionally, viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, lentivirus, or alphavirus.

[0208] Regulatory sequences are well known in the art and can be used to express a protein or polypeptide of interest in a suitable host cell, as described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences can be selected to direct expression of a gene (such as a modified TFF2 polypeptide). Non-limiting examples of regulatory sequences include polyadenylation signals, promoters (such as CMV, ASV, SV40 or other viral promoters, e.g., bovine papilloma, polyoma, and adenovirus 2 viral (Fiers, et al., 1973, Nature 273:113; Hager GL, et al., Curr Opin Genet Dev, 2002, 12(2):137-41) enhancers, and and other expression control elements. Those skilled in the art will understand that the design of the expression vector can depend on such factors as the choice of the host cell to be transfected and / or the type and / or amount of protein desired to be expressed.

[0209] It is known in the art that enhancer regions, which are sequences found upstream or downstream of promoter regions in non-coding DNA regions, are also important in optimizing expression.If necessary, origin of replication from virus sources can be used, for example, when prokaryotic hosts are used to introduce plasmid DNA.However, in eukaryotes, chromosomal integration is the general mechanism for DNA replication.

[0210] For stable transfection of mammalian cells, only a small number of cells can integrate the introduced DNA into their genome.The expression vector and transfection method used can be factors that contribute to successful integration events.For stable amplification and expression of desired proteins, a vector containing DNA encoding a protein of interest (e.g., a modified TFF2 polypeptide) is stably integrated into the genome of eukaryotic cells (e.g., mammalian cells such as HEK293 cells), resulting in stable expression of the transfected gene.Exogenous nucleic acid sequences can be introduced into cells (e.g., either primary or secondary cells, such as mammalian cells) by homologous recombination, as disclosed in U.S. Patent No. 5,641,670, the contents of which are incorporated herein by reference.

[0211] A gene encoding a selection marker (e.g., resistance to antibiotics or drugs such as ampicillin, neomycin, G418, and hygromycin) can be introduced into host cells together with the gene of interest to identify and select clones that stably express the gene encoding the protein of interest. The gene encoding the selection marker can be introduced into host cells on the same plasmid as the gene of interest, or can be introduced on a separate plasmid. Cells containing the gene of interest can be identified by drug selection, in which cells that have incorporated the selection marker gene survive in the presence of the drug. Cells that have not incorporated the gene for the selection marker die. Surviving cells can then be screened for the production of the desired protein molecule (e.g., modified TFF2 polypeptide).

[0212] Host cell lines can be selected for their ability to modulate the expression of inserted sequences or process the expressed modified TFF2 polypeptide in the desired manner. Such modifications of polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing, which cleaves the "prepro" form of the polypeptide, can also be used to promote correct insertion, folding, and / or function. Different host cells (e.g., CHO, HeLa, MDCK, HEK293, and WI38) with specific cellular mechanisms and characteristic mechanisms for post-translational activity are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, Va. 20110-2209) and can be selected to ensure the correct modification and processing of foreign proteins.

[0213] Exogenous nucleic acid can be introduced into cells through various techniques known in the art, such as lipofection, microinjection, calcium phosphate or calcium chloride precipitation, DEAE-dextrin-mediated transfection or electroporation. Electroporation is carried out under appropriate voltage and capacitance to cause the DNA construct to be transferred into the target cell. Other methods used to transfect cells can also include modified calcium phosphate precipitation, polybrene precipitation, liposome fusion and receptor-mediated gene delivery.

[0214] Animal or mammalian host cells capable of harboring, expressing, and secreting large amounts of the TFF2 peptide of interest into the culture medium for subsequent isolation and / or purification include, but are not limited to, human embryonic kidney 293 cells (HEK-293) (ATCC CRL-1573); Chinese hamster ovary cells (CHO), e.g., CHO-K1 (ATCC CCL-61), DG44 (Chasin et al., (1986) Som. Cell Molec. Genet, 12:555-556; Kolkekar et al., (1997) Biochemistry, 36:10901-10909; and No. WO 01 / 92337A2), dihydrofolate reductase-negative CHO cells (CHO / dhfr-, Urlaub et al., (1980) Proc. Natl. Acad. Sci. USA, 77:4216), and dp12.CHO cells (U.S. Pat. No. 5,721,121); monkey kidney CV1 cells transformed with COS cells (COS-7, ATCC CRL-1651); human embryonic kidney cells (e.g., 293 cells, or 293 cells subcloned for growth in suspension culture, Graham et al., (1977) J. Gen. Virol., 36:59); baby hamster kidney cells (BHK, ATCC CCL-10); monkey kidney cells (CV1, ATCC CCL-70); African green monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); mouse Sertoli cells (TM4; Mather (1980) Biol. Reprod., 23:243-251); human cervical carcinoma cells (HELA , ATCC CCL-2); dog kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL-51); buffalo rat hepatocytes (BRL 3A, ATCC CRL-1442); TRI cells (Mather (1982) Annals NY Acad. Sci., 383:44-68);MCR 5 cells;FS4 The cell line transformed to produce modified TFF2 polypeptide can also be an immortalized mammalian cell line of lymphoid origin, including but not limited to, myeloma, hybridoma, trioma or quadroma cell line. The cell line can also include normal lymphoid cells such as B cells, which are immortalized by transformation with a virus such as Epstein-Barr virus (such as myeloma cell line or its derivative).

[0215] A host cell line can also be selected that modulates the expression of the inserted sequence or modifies and processes the nucleic acid in a desired specific manner. Such modifications (e.g., glycosylation and other post-translational modifications) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cell lines have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Therefore, an appropriate host system or cell line can be selected to ensure the correct modification and processing of expressed foreign proteins, such as modified TFF2 polypeptides. Therefore, eukaryotic host cells that possess the cellular machinery for proper processing, glycosylation, and phosphorylation of the primary transcript of the gene product can be used. Non-limiting examples of mammalian host cells include HEK-293, 3T3, W138, BT483, Hs578T, CHO, VERY, BHK, Hela, COS, BT2O, T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, MDCK, 293, HTB2, and HsS78Bst cells.

[0216] A variety of culture parameters can be used for the cultured host cells. Suitable culture conditions for mammalian cells are well known in the art (Cleveland W. L, et al., J Immunol Methods, 1983, 56(2): 221-234), or can be determined by one skilled in the art (see, for example, Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D. and Hames, B.D., eds. (Oxford University Press: New York, 1992). Cell culture conditions may vary depending on the type of host cell selected. Commercially available media may be used.

[0217] Cells suitable for culturing can contain an introduced expression vector, such as a plasmid or virus. The expression vector construct can be introduced via transformation, microinjection, transfection, lipofection, electroporation, or infection. The expression vector can contain a coding sequence or a portion thereof that encodes a protein for expression and production. Expression vectors containing sequences encoding the proteins and polypeptides to be produced and appropriate transcriptional and translational control elements can be generated using methods well known and practiced by those skilled in the art. These methods include synthetic techniques, in vitro recombinant DNA techniques, and methods such as those described in J. Sambrook et al., 201, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, and F. M. Ausubel et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY.

[0218] Recombinant protein purification Modified TFF2 polypeptides can be purified from any human or non-human cells that express the polypeptide, including those transfected with an expression construct that expresses the modified TFF2 polypeptide. Purified modified TFF2 polypeptides can be separated from other compounds normally associated with TFF2, such as certain proteins, carbohydrates, or lipids, using methods known in the art. For protein recovery, isolation, and / or purification, cell culture medium or cell lysate is centrifuged to remove particulate cells and cell debris. The desired modified TFF2 polypeptide is isolated or purified from contaminating soluble proteins and polypeptides by a suitable purification technique. Non-limiting purification methods for proteins include size exclusion chromatography; affinity chromatography; ion exchange chromatography; ethanol precipitation; reverse-phase HPLC; chromatography on resins such as silica or cation exchange resins, for example, DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75, Sepharose®; Protein A Sepharose chromatography for removing immunoglobulin contaminants; etc. Other additives, such as protease inhibitors (e.g., PMSF or proteinase K), can be used to inhibit proteolysis during purification. Purification procedures that can be selected for carbohydrates can also use, for example, ion-exchange soft gel chromatography or HPLC using cation or anion exchange resins, where the more acidic fractions are collected. [Example]

[0219] Examples are provided below to facilitate a more complete understanding of the present disclosure. The following examples describe exemplary modes of making and practicing the present invention. However, since alternative methods can be utilized to achieve similar results, the scope of the present disclosure is not limited to the specific embodiments disclosed in these examples, which are for illustrative purposes only.

[0220] Example 1 Codon optimization of human modified TFF2 polypeptide with His_Strep tag The codon optimization of the human modified TFF2 polypeptide with a His_Strep tag is shown below in SEQ ID NO: 32. [ka]

[0221] The deduced amino acid sequence generated from the optimized DNA sequence is shown below in SEQ ID NO:33. [ka]

[0222] Example 2 Codon optimization of human modified TFF2-C-terminal HULG1 Fc tag polypeptide The codon-optimized DNA sequence of TFF2-C-terminal HULG1 FC tag is shown below as SEQ ID NO: 34. [ka] [ka]

[0223] The deduced amino acid sequence generated from the optimized DNA sequence is shown below in SEQ ID NO:35. [ka]

[0224] Example 3 Codon optimization of human TFF2-HSA The codon-optimized DNA of human TFF2-HSA is shown below (SEQ ID NO: 36). >Human TFF2-HSA codon-optimized DNA [ka] [ka]

[0225] The deduced TFF2-HSA amino acid sequence is shown below in SEQ ID NO:37. [ka] [ka]

[0226] Example 4 Codon optimization of human TFF2-CTPX2-FLAG X3 Human TFF2-CTPX2-FLAG X3_codon-optimized DNA (SEQ ID NO: 38) [ka] [ka]

[0227] The deduced TFF2-HSA amino acid sequence is shown below (SEQ ID NO: 39). [ka]

[0228] All constructs from Examples 1 to 4 are expressed in the CHO-S transient system. Expression of the three variants is analyzed using Western blot and anti-huTFF2.

[0229] Example 5 Measurement of modified TFF2 polypeptide activity by calcium mobilization Jurkat cells, KATO-III and / or AsPC-1 cells (2.5 × 10 6 The cells (100 cells / ml) were resuspended in RPMI 1640 medium containing 0.5% BSA and incubated in the dark with stirring at a final concentration of 5 mM Ca. 2+The cells are incubated with the binding dye Indo-1 AM for 1 hour at 37°C. The loaded cells are washed and resuspended in Hank's balanced salt solution medium containing 2 mM CaCl2 and 1 mM MgCl2 and left at room temperature for 20 minutes. The cells are aliquoted into fluorescence-activated cell sorter tubes, which are immediately transferred to a 37°C water bath for an additional 5 minutes before measurement. The equilibrated cells are then analyzed using an LSRII instrument (BD Biosciences) using CaCl2. 2+ Use for flow cytometry analysis of baseline intracellular Ca levels 2+ Levels are recorded for the first 25-30 seconds, followed by stimulation with the indicated concentrations of SDF-1a, TFF2, gastrin, ionomycin, or diluent (phosphate-buffered saline). Data collection continues for an additional 4-10 minutes at a speed of 2000 events / second. Cytosolic Ca levels are recorded for the first 25-30 seconds, followed by stimulation with the indicated concentrations of SDF-1a, TFF2, gastrin, ionomycin, or diluent (phosphate-buffered saline). 2+ The increase in binding of Ca to Indo-1 was observed from 510 nm (free form) to 420 nm (Ca 2+ This results in a change in the emission spectrum of Indo-1 to the cytosolic form (the bound form). Thus, the blue (4',6-diamidino-2-phenyl-indole channel, 420 nm) and purple (Indo channel, 510 nm) fluorescence of the cells is measured, and the data are plotted using FlowJo software (version 6.4; Tree Star, Inc.). Thus, after preincubating cells with AMD3100 or anti-CXCR4 mAbs 12G5 or 2B11 (eBioscience) for 40 minutes at 37°C, intracellular calcium mobilization in response to SDF-1a or TFF2 in the presence of AMD3100 or anti-CXCR4 antibodies is measured.

[0230] Example 6 Measurement of modified TFF2 polypeptide activity by ERK1 / 2 phosphorylation The activity of the modified TFF2 polypeptide is measured by ERK1 / ERK2 phosphorylation in Jurkat human acute T-cell leukemia cells, KATO-III human gastric cancer cells, and / or AsPC-1 human pancreatic cells (all cell lines provided by ATCC) using the AlphaLISA SureFire Ultra p-ERK1 / 2 (Thr202 / Tyr204) Assay Kit from Perkin Elmer. Cell lines are thawed and grown according to the instructions provided by ATCC. Cells are harvested by centrifugation and diluted to 10 mL in HBSS. 7 The cells are resuspended at 1000 cells / mL. The cells are seeded at 4 mL / well into 384-well white opaque culture plates (PerkinElmer) and incubated at 37°C for 1-2 hours. Recombinant TFF2 wild-type and variants are added to the plate at a concentration of 10-30 mg / mL in HBSS containing 0.1% BSA in 4 mL to stimulate the cells and incubated at 37°C for 5-30 minutes. The cells are lysed with 2 mL / well of lysis buffer, followed by the addition of 5 mL of Acceptor Mix. The plate is then sealed with Topseal-A adhesive film and incubated at room temperature for 1 hour. 5 mL of Donor Mix is ​​then added to the wells under moderate light, sealed with Topseal-A adhesive film, covered with foil, and incubated in the dark at room temperature for 1 hour. The plate is read in an AlphaPlex-compatible plate reader using standard AlphaPlex settings. Inhibition of TFF2 stimulation of CXCR4 was performed using the CXCR4 small molecule antagonist AMD3100 (Sigma) or anti-CXCR4 mAbs 12G5 and 2B11 (eBioscience) at 37°C for 1 to 2 hours before the addition of recombinant TFF2.

[0231] Example 7 colorectal adenocarcinoma A 51-year-old man presented with no family history of early-onset colorectal cancer or other malignancies consistent with Lynch syndrome. He was in normal health until his initial routine screening evaluation with colonoscopy, which revealed a partially obstructive mass in the transverse colon. A biopsy confirmed the presence of moderately differentiated adenocarcinoma with lymphovascular invasion. Reflectance molecular testing was notable for a KRAS exon 2 mutation (+) and a BRAF mutation (-). However, the patient was identified as metastatic microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) or dMMR / MSI-H (+). A subsequent staging CT scan identified both small volumes of liver and peritoneal disease, with no extra-abdominal metastases. The patient was classified as having asymptomatic stage IV colorectal cancer and underwent a successful transverse colectomy. Six weeks later, the patient begins a regimen of levofolinic acid (FOL or Fusilev®), 5-fluorouracil (5-FU or F), and oxaliplatin (OX or Eloxatin®, a platinum cytotoxic agent that forms both interstrand and intrastrand crosslinks in DNA), or "FOLFOX" plus bevacizumab (Avastin®). Recurrence after cycle #6 of FOLFOX-bevacizumab is consistent with near-complete remission (nCR). Despite well-tolerated therapy, the patient's health declines while "maintenance" bevacizumab / fluoropyrimidine is continued, and the patient is placed on an observation program. The patient's health improves for 14 months, at which time the patient notices the onset of respiratory distress. A CT scan is notable for a new, large right pleural effusion, ascites, and progressive liver metastases with significant liver function test abnormalities. A large-volume thoracentesis confirms the presence of a malignant pleural effusion with cytologic evidence of adenocarcinoma. The patient underwent chest tube placement followed by successful pleurodesis. The patient began second-line treatment with FOLFIRI-bevacizumab. The patient again tolerated the therapy well, and a repeat CT scan after cycle #4 was consistent with a partial response (PR).The patient continues FOLFIRI-bevacizumab with levofolinic acid (FOL), 5-FU (F), and irinotecan (IRI or Camptosar®, an inhibitor of topoisomerase I), with a treatment plan to treat progression tolerated by toxicity. A relapse after cycle #10 of FOLFIRI-Avastin demonstrates recurrence of progressive liver metastases and ascites. The patient begins treatment with single-agent pembrolizumab (Keytruda®) but does not respond. Subsequent treatment is initiated with a modified TFF2 polypeptide, and the patient achieves a partial objective response. Combination treatment is initiated with the modified TFF2 polypeptide and pembrolizumab, which results in a complete response and regression of the tumor and metastases.

[0232] Example 9 Esophageal squamous cell carcinoma The patient was a 58-year-old man with a history of tobacco abuse who was in normal health until he presented with dysphagia and intermittent subxiphoid discomfort. After the failure of several symptomatic interventions, including both histamine receptor type 2 (H2) blockers and proton pump blockers, the patient underwent a formal gastroenterology consultation and upper endoscopy, at which time he was informed of a 3.2 cm exophytic mid-esophageal mass that was partially obstructing. Pathology review revealed moderately differentiated squamous cell carcinoma. PD-L1 combined positive score (CPS): 20%. Staging PET / CT scan and endoscopic ultrasound were consistent with T4aN0 disease and confirmed the tumor was suitable for resection. The patient successfully completed concurrent low-dose weekly neoadjuvant carboplatinum / paclitaxel (a tubulin inhibitor, Taxol®) and radiation therapy, followed by definitive surgical resection. Surgical pathology review did not reveal any evidence of residual malignancy. Nine months later, the patient exhibits anorexia and weight loss. A CT scan demonstrated the presence of both liver and lung metastases. A CT-guided core needle biopsy confirmed the presence of metastatic squamous cell carcinoma. PD-L1 combined positive score (CPS): 16%. Because the progression-free survival (PFS) was >6 months after first-line chemotherapy (in this case, neoadjuvant), the patient had a good performance status (ECOG <1), particularly if the patient exhibited either rapidly progressive disease or highly symptomatic disease requiring an early meaningful response simply to stabilize the situation, resulting in initiation of second-line therapy. The patient received six cycles of FOLFIRI, achieved a quiescent partial response (PR), and entered an observation program. Four months later, a routine surveillance CT scan demonstrated progression of metastatic disease. The patient maintained an ECOG 1 performance status and requested further treatment. The patient initiated single-agent pembrolizumab, but did not respond. Treatment is then initiated with a modified TFF2 polypeptide and the patient achieves a partial objective response. Combination treatment is initiated with a modified TFF2 polypeptide and pembrolizumab, which results in a complete response and regression of the tumor and metastases.

[0233] Example 10 Gastric / esophageal adenocarcinoma The patient was a 47-year-old woman in normal health until 18 months ago, when she presented with dysphagia and intermittent subxiphoid discomfort. After the failure of several symptomatic interventions, including both H2 and proton pump blockers, the patient underwent a formal gastroenterology consultation and upper endoscopy, at which time she was informed of a 2.2 cm exophytic mass (at the gastric cardia / lower esophagus). Pathology review revealed a poorly differentiated adenocarcinoma. There was no evidence of H. pylori infection, and Her2 immunohistochemical (IHC) staining was 0. PD-L1 combined positive score (CPS): 12%. A staging CT scan demonstrated the presence of both regional lymphadenopathy and low-volume liver metastases. The patient was classified as having unresectable, low-volume, stage IV poorly differentiated gastric / esophageal adenocarcinoma. Based on low-volume disease with minimal symptoms and a CPS >10, the patient begins single-agent pembrolizumab as first-line treatment based on findings from the KEYNOTE-062 trial, in which patients with a CPS >10 had improved OS (17.4 months vs. 10.8 months) with fewer all-grade or grade 3 / 4 toxicities (vs. CDDP / fluoropyrimidines). However, the patient's condition progresses, and the patient develops large, symptomatic tumors (primarily 6.0 cm, extensive liver metastases) and a PD-L1 CPS <10. The patient then receives five cycles of FOLFOX, achieving a quiescent partial response (PR). The treatment is generally well tolerated, although the patient experiences grade 2 peripheral neuropathy. The patient is placed on an observation program. Seven months later, a routine surveillance CT scan reveals progressive liver metastases and new lung metastases. The patient maintains an ECOG 1 performance status and treatment is initiated with ramucirmab (Cyramza®, a direct VEGFR2 antagonist) and paclitaxel. A repeat CT scan after cycle #4 is consistent with stable disease. However, the patient's peripheral neuropathy worsens and paclitaxel is discontinued. The patient remains on single-agent ramucirumab but progresses after 3 months. Based on the results of the KEYNOTE-059 study (failure to second-line or more chemotherapy), the patient is switched to single-agent pembrolizumab. The patient begins single-agent pembrolizumab but does not respond.Treatment is then initiated with a modified TFF2 polypeptide and the patient achieves a partial objective response. Combination treatment is initiated with a modified TFF2 polypeptide and pembrolizumab, which results in a complete response and regression of the tumor and metastases.

[0234] Example 11 Pancreatic cancer The patient was a 39-year-old woman in good, generally excellent health until she reported the onset of vague mid-back pain, which was controlled with discontinuous use of nonsteroidal anti-inflammatory drugs (NSAIDs). The patient presented with night sweats and a 1-week history of scleral icterus and increasingly dark urine. Clinical evaluation confirmed the presence of jaundice, and chemistry identified a pattern of cholestatic liver dysfunction with a total bilirubin of 12.2 mg / dL. CT scan revealed an 8.4 cm mass in the head of the pancreas, as well as lymphadenopathy at the hilum of the liver, scattered small bilateral hepatic masses, and significant dilation of the common bile duct. Endoscopic retrograde cholangiopancreatography (ERCP) with hepatic stent placement was successful, and bilirubin returned to normal levels. CT-guided liver biopsy confirmed the presence of poorly differentiated KRAS:(+)TP53:(+) adenocarcinoma. CA 19-9 was markedly elevated (710). Symptoms are most consistent with unresectable stage IV adenocarcinoma of the pancreas. There is no family history of pancreatic, breast, or ovarian cancer, or known BRCA2 mutation. The patient undergoes next-generation sequencing (NGS). There is no evidence of germline mutations for either BRCA2 or PALB2. However, the patient is dMMR / MSI-H. The patient begins a modified FOLFIRINOX regimen (FOL+F+irinotecan or "IRIN"+OX) and successfully completes six cycles of treatment, which is generally well tolerated. Restaging CT scans after cycles 4 and 6 are consistent with a stable, quiescent partial response. The patient enters an observation program and remains well until 4 months later, when routine surveillance CT scans confirm the presence of progressive, asymptomatic, low-volume liver metastases. The patient begins single-agent nivolumab (Opdivo®) but does not respond. Treatment is then initiated with a modified TFF2 polypeptide, and the patient achieves a partial objective response. Combination treatment is initiated with a modified TFF2 polypeptide and nivolumab, which results in a complete response and tumor regression.

[0235] Example 12 Stabilized recombinant TFF2 (TFF2-CTP) enhances the antitumor activity of PD-1 blockade in a mouse model of colorectal cancer Despite significant responses to immune checkpoint blockade across multiple tumor types, clinical benefit in colorectal cancer (CRC) is limited to microsatellite-unstable tumors. PD-L1 expression is a negative prognostic marker in CRC but correlates with a favorable response to PD-1 blockade. In this example, we investigated the role of PD-L1 in colorectal tumorigenesis and evaluated the utility of targeting myeloid-derived suppressor cells (MDSCs) in combination with PD-1 blockade in a mouse model of colorectal cancer (CRC). We generated knock-in mice (R26-LSL-Pdl1-EGFP) that conditionally express the mouse Pdl1 gene and crossed them with LysM-Cre mice to overexpress PD-L1 specifically in the myeloid lineage. AOM / DSS-treated mice formed tumors within 10 weeks and developed adenocarcinomas 17 weeks after AOM. See Figures 3A-3D. AOM / DSS treatment resulted in significant expression of myeloid cells, particularly CD11b+Gr-1+MDSCs, compared with untreated mice (see Figures 4A-4C). Furthermore, there was a significant decrease in intratumoral CD8+ T cells, indicating attenuated antitumor immunity (see Figures 5A-5C). AOM / DSS-treated LysM-Cre;R26-PD-L mice overexpressing PD-L1 exhibited significantly enhanced early colorectal tumor development, with a significant increase in tumor number and size (see Figures 6A-6F). The secreted anti-inflammatory peptide TFF2 inhibits colon tumor growth by suppressing the expression of CD11b+Gr-1+MDSCs. TFF2 fused with two carboxyl-terminal peptides and three Flag motifs (TFF2-CTP-Flag) extended its circulation time in the blood while retaining its biological activity (see Figures 7A-7E). We induced tumors in R26-PD-L1 and LysM-Cre;R26-PD-L mice with AOM / DSS and administered fusion recombinant TFF2-CTP-Flag and / or anti-PD-1 antibody. Anti-PD-1 antibody in combination with TFF2-CTP significantly reduced tumor growth, whereas anti-PD-1 monotherapy failed to suppress growth. Interestingly, the combined treatment showed greater anti-tumor activity in mice overexpressing PD-L1 than in control animals.See Figure 8. Treatment responders showed a significant increase in tumor-infiltrating CD8+ T cells and a concomitant decrease in CD11b+Gr-1+ myeloid cells. See Figure 9. These early findings suggest that TFF2 increases the response rate of CRC to PD-1 blockade, possibly by suppressing MDSC proliferation, and support the potential of TFF2-CTP in IO treatment combinations for CRC.

[0236] Thus, anti-PD-1 monotherapy failed to induce antitumor immunity in CRC, whereas TFF2-CTP enhanced the efficacy of anti-PD-1 therapy. Anti-PD-1 in combination with TFF2-CTP exhibited greater antitumor activity in mice overexpressing PD-L1. Responders to TFF2-CTP alone or in combination with PD-1 blockade had increased tumor-infiltrating CD8+ T cells along with decreased MDSCs.

[0237] Example 13 Expression and purification of TFF2-human serum albumin (HSA) fusions Gene synthesis TFF-2-HSA proteins were codon-optimized and synthesized using Codex gene synthesis. The synthesized TFF-2 HSA proteins were TFF2-HSA[WT]; TFF2-HSA[DI / I]; TFF2-HSA[DII / I]; TFF2-HSA[DII / II]; TFF2-HSA[LBDI / I]; TFF2-HSA[LBDII / I], and TFF2-HSA[LBDII / II]. Oligonucleotides were synthesized by Codex, and the genes were assembled using SGI / Codex Assembler. The synthesized genes were subcloned into the expression vector pAB2 (digested with XbaI and BamHI) using SGI. An overlapping 30-bp sequence was used for Gibson assembly of the gene of interest into pAB2. The vector carrying the gene of interest was transformed into NEB® 5-alpha Competent E. coli [(High Efficiency); NEB; C2987H]. Three colonies were collected and scaled up for DNA isolation via mini-prep. Three colonies were then sent for sequencing. Upon sequence confirmation, positive clones were scaled up and plasmid DNA was isolated.

[0238] Transfection HEK293 cells were seeded into flasks the day before transfection. On the day of transfection, cell counts and cell viability were determined and the cultures reached 1.8 x 10 with >96% viability. 6 ~2.2×10 6 Once the cell density reached 100 cells / mL, transfection proceeded. The DNA was then resuspended in FectoPro (Polyplus) transfection reagent, diluted in serum-free medium, and incubated at room temperature. The transfection complex was then gently added to the HEK293 cells while swirling the flask, which was then returned to the 37°C incubator. The cell culture was then fed with fresh medium 4-5 hours after transfection. Six days after transfection, the cell supernatant was harvested and clarified by centrifugation.

[0239] Protein purification HSA-tagged human TFF2 protein was purified using an AlbuPure® (product code 3151, Prometic Bioseparations®, Ltd.) selective affinity chromatography adsorption column. The column was first washed with 5 column volumes (CV) of 0.5 N NaOH, followed by 5 CV of autoclaved EDTA. The column was then equilibrated with 10 CV of 50 mM sodium citrate, pH 5.5 (Buffer A). The protein fraction was then loaded onto the column, which was then washed with 10 CV of Buffer A. The purified protein was then eluted from the column with 5 CV of 50 mM ammonium acetate, 10 mM sodium octanoate, pH 7.0.

[0240] SDS-PAGE Samples were run on NuPAGE gels 4-12% Bis-Tris 1.0 mm, 12-well (Invitrogen®, catalog no. NP0302BOX). Samples (2 μg) were loaded in NuPAGE LDS sample buffer (4×) and run at 200 V for 30 minutes in MES buffer (Invitrogen®, catalog no. 002-02). Accurate Plus MW standards were used as molecular weight standards (Bio-Rad®, catalog no. 161-0374). Gels were stained with Simply Blue Stain (Invitrogen®, catalog no. LC6060). Clarified harvest, flow-through, wash, and Protein A elution samples were run on the gel. See Figure 10. The yields obtained for each of the purified TFF2-HAS variants are shown in Figure 11.

[0241] All patents, patent applications and publications, and non-patent publications, cited herein are hereby incorporated by reference in their entirety.

[0242] A number of embodiments of the present disclosure have been described. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of the disclosure. Accordingly, other embodiments are within the scope of the following claims. In certain embodiments, for example, the following are provided: (Item 1) A composition comprising a modified TFF2 polypeptide, wherein the TFF2 polypeptide is modified by one or more of PEGylation, polysialylation, poly(D,L-lactic-co-glycolic acid) (PLGA) conjugation, and / or a fusion protein comprising the C-terminal peptide (CTP) of the human chorionic gonadotropin beta subunit, PASylation, XTENylation, ELPylation or HAPylation. (Item 2) The composition of item 1, wherein the modified TFF2 polypeptide has a polypeptide sequence having at least 90% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 6. (Item 3) The modified TFF2 polypeptide has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 6. 3. The composition of claim 2, having at least 95% amino acid sequence identity with the amino acid sequence of (Item 4) The composition of item 1, wherein the modified TFF2 polypeptide has the polypeptide sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 6. (Item 5) 5. The composition of any one of items 1 to 4, wherein the modified TFF2 polypeptide is PEGylated with a low molecular weight linear PEG. (Item 6) 5. The composition of any one of items 1 to 4, wherein the modified TFF2 polypeptide is PEGylated with a high molecular weight branched PEG. (Item 7) 7. The composition according to any one of items 1 to 6, which is a pharmaceutical composition. (Item 8) 8. The composition of claim 7, wherein the modified TFF2 polypeptide is a homogeneous population selected from the group consisting of PEGylated, polysialylated, PLGA conjugated, and fusion proteins using CTP, PAS, XTEN, ELP, and HAP of the human chorionic gonadotropin β subunit, or combinations thereof. (Item 9) 9. The composition of any one of items 1 to 8, wherein the modified TFF2 polypeptide has an increased half-life in blood compared to an unmodified human TFF2 polypeptide. (Item 10) 10. The composition of any one of items 1 to 9, wherein the modified TFF2 polypeptide is PEGylated at one or more specific sites. (Item 11) 11. The composition of claim 10, wherein the modified TFF2 peptide is PEGylated at its N-terminus. (Item 12) 12. The composition of claim 11, wherein the modified TFF2 polypeptide is PEGylated using N-terminal PEGylation via aldehyde-PEG chemistry. (Item 13) Item 11. The composition of item 10, wherein the modified TFF2 polypeptide is PEGylated at its C-terminus. (Item 14) 11. The composition of claim 10, wherein the PEGylation comprises a solvent-free exposed amine via NHS-PEG chemistry. (Item 15) 15. The composition of any one of items 1 to 14, wherein the modified TFF2 PEGylated polypeptide is a homogeneous composition. (Item 16) 16. A method of treating cancer in a subject in need thereof, comprising administering to said subject an effective amount of the composition of any one of items 1 to 15. (Item 17) Item 17. The method of item 16, wherein the cancer is a cancer of the digestive system. (Item 18) Item 18. The method of item 17, wherein the gastrointestinal cancer is selected from one or more of oral cavity cancer, pharyngeal cancer, oropharyngeal cancer, esophageal cancer, gastric cancer, stomach cancer, small intestine cancer, large intestine cancer, colon cancer, rectal cancer, anal cancer, liver cancer, pancreatic cancer, and gallbladder cancer. (Item 19) Item 19. The method of item 18, wherein the cancer is colon cancer. (Item 20) Item 19. The method of item 18, wherein the cancer is oropharyngeal cancer. (Item 21) Item 19. The method of item 18, wherein the cancer is esophageal cancer. (Item 22) Item 19. The method of item 18, wherein the cancer is gastric cancer. (Item 23) Item 19. The method of item 18, wherein the cancer is pancreatic cancer. (Item 24) Item 19. The method of item 18, wherein the cancer is rectal cancer. (Item 25) Item 19. The method of item 18, wherein the cancer is liver cancer. (Item 26) 26. The method according to any one of items 16 to 25, wherein the cancer is a metastatic cancer. (Item 27) 27. The method of any one of items 16 to 26, further comprising treating the cancer with a blocking antibody against PD-1, PD-L1 or CTLA-4. (Item 28) 16. A method of treating cancer in a subject in need thereof, wherein the cancer is unresponsive to treatment with a blocking antibody against PD-1, PD-L1, or CTLA-4, and the subject is treated with the composition of any one of items 1 to 15, wherein after treatment with the modified TFF2 polypeptide composition, the subject's cancer becomes susceptible to treatment with a blocking antibody against PD-1, PD-L1, or CTLA-4, and the subject is then treated with a blocking antibody against PD-1, PD-L1, or CTLA-4 within about 1 to about 60 days after treatment with the modified TFF2 polypeptide. (Item 29) Modified TFF2 polypeptides, including fusion proteins. (Item 30) 30. The modified TFF2 polypeptide of item 29, wherein the fusion protein is selected from one or more of the group consisting of a TFF2-albumin protein, a TFF2-IgG1 fusion protein, and a TFF2-polyhistidine tag. (Item 31) 31. The modified TFF2 polypeptide of item 30, wherein the fusion protein is polyhistidine tagged. (Item 32) 32. The modified TFF2 polypeptide of claim 31, wherein the histidine tag contains an amino acid cleavage site. (Item 33) 33. The modified TFF2 polypeptide of item 32, wherein the amino acid cleavage site is selected from SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23. (Item 34) 34. The modified TFF2 polypeptide according to any one of items 32 to 33, wherein after cleavage a native TFF2 polypeptide is formed. (Item 35) 35. The modified TFF2 polypeptide of any one of items 31 to 34, wherein the histidine tag is at either the N-terminus or the C-terminus of TFF2. (Item 36) 36. The method according to any one of items 29 to 35, further comprising: 1) purifying the TFF2 peptide; and 2) preparing a conjugate of the purified modified TFF2 with PEGylation, polysialylation, and / or poly(D,L-lactic-co-glycolic acid) (PLGA). A modified TFF2 polypeptide described in any one of claims 1 to 4. (Item 37) A method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of one or more of the modified TFF2 polypeptides described in any one of items 29 to 36. (Item 38) 38. The method of claim 37, wherein the cancer is a cancer of the digestive system. (Item 39) 39. The method of claim 38, wherein the gastrointestinal cancer is selected from one or more of oral cavity cancer, pharyngeal cancer, oropharyngeal cancer, esophageal cancer, stomach cancer, small intestine cancer, large intestine cancer, colon cancer, gastric cancer, rectal cancer, anal cancer, liver cancer, pancreatic cancer, and gallbladder cancer. (Item 40) 40. The method of claim 39, wherein the cancer is colon cancer. (Item 41) 40. The method of claim 39, wherein the cancer is oropharyngeal cancer. (Item 42) Item 39. The method of item 39, wherein the cancer is esophageal cancer. (Item 43) Item 39. The method of item 39, wherein the cancer is gastric cancer. (Item 44) Item 39. The method of item 39, wherein the cancer is pancreatic cancer. (Item 45) Item 39. The method of item 39, wherein the cancer is rectal cancer. (Item 46) 40. The method of claim 39, wherein the cancer being treated is liver cancer. (Item 47) 47. The method according to any one of items 37 to 46, wherein the cancer is a metastatic cancer. (Item 48) 48. The method of any one of items 37 to 47, further comprising treating the cancer with a blocking antibody against PD-1, PD-L1 or CTLA-4. (Item 49) 37. A method of treating cancer in a subject in need thereof, wherein the cancer is unresponsive to treatment with a blocking antibody against PD-1, PD-L1, or CTLA-4, and the subject is treated with a modified TFF2 polypeptide of any one of Items 29 to 36, wherein after treatment with the modified TFF2 polypeptide, the subject's cancer becomes susceptible to treatment with a blocking antibody against PD-1, PD-L1, or CTLA-4, and the subject is then treated with a blocking antibody against PD-1, PD-L1, or CTLA-4 within about 1 to about 60 days after treatment with the modified TFF2 polypeptide. (Item 50) A method for treating inflammatory bowel disease (IBD) in a subject in need thereof, wherein the subject is treated with one or more compositions described in any one of items 1 to 15 or one or more modified TFF2 polypeptides described in any one of items 29 to 36. (Item 51) 51. The method of item 50, wherein the IBD is Crohn's disease or ulcerative colitis. (Item 52) 52. The method of claim 50 or 51, wherein the composition or the modified TFF2 polypeptide is administered orally, intravenously, or intramuscularly. (Item 53) A modified TFF2 polypeptide comprising one or more domain I binding domains, wherein the one or more domain I binding domains comprise sequence number 24, and the polypeptide does not contain a domain II binding domain. (Item 54) A modified TFF2 polypeptide comprising one or more domain II binding domains, wherein the one or more domain II binding domains comprise sequence number 25, and the polypeptide does not contain a domain I binding domain. (Item 55) A modified TFF2 polypeptide comprising two Domain I binding domains as set forth in SEQ ID NO:26. (Item 56) A modified TFF2 polypeptide comprising two Domain II binding domains as set forth in SEQ ID NO:27. (Item 57) A modified TFF2 polypeptide comprising Domain I and Domain II binding domains interchangeable with each other and comprising the sequence set forth in SEQ ID NO:28. (Item 58) A modified TFF2 polypeptide containing amino acid substitutions at receptor binding site residues and having the sequence of SEQ ID NO:29. (Item 59) A modified TFF2 polypeptide containing amino acid substitutions at receptor binding site residues and having the sequence of SEQ ID NO:30. (Item 60) A modified TFF2 polypeptide containing amino acid substitutions at receptor binding site residues and having the sequence of SEQ ID NO:31. (Item 61) 61. The modified TFF2 polypeptide of any one of items 53 to 60, wherein the TFF2 binding domain is further modified by one or more of PEGylation, polysialylation, conjugation with poly(D,L-lactic-co-glycolic acid) (PLGA), and / or expression as a fusion protein comprising a fusion polypeptide selected from the group consisting of the C-terminal peptide of human chorionic gonadotropin beta subunit (CTP), a PAS-ylated fusion polypeptide, an XTEN-ylated fusion polypeptide, an ELP-ylated fusion polypeptide, and a HAP-ylated fusion polypeptide. (Item 62) 62. The modified TFF2 polypeptide of claim 61, wherein the modified TFF2 binding domain is PEGylated with a low molecular weight linear PEG. (Item 63) 62. The modified TFF2 polypeptide of claim 61, wherein the modified TFF2 binding domain is PEGylated with a high molecular weight branched PEG. (Item 64) 62. The modified TFF2 polypeptide of claim 61, wherein the modified TFF2 binding domain is PEGylated at one or more specific sites. (Item 65) 62. The modified TFF2 polypeptide of item 61, wherein the modified TFF2 binding domain is PEGylated at its N-terminus. (Item 66) 62. The modified TFF2 polypeptide of claim 61, wherein the modified TFF2 binding domain is PEGylated using N-terminal PEGylation via aldehyde-PEG chemistry. (Item 67) 62. The modified TFF2 binding domain according to item 61, wherein the modified TFF2 binding domain is PEGylated at its C-terminus. A modified TFF2 polypeptide. (Item 68) 62. The modified TFF2 polypeptide of item 61, wherein the PEGylation comprises a solvent-free exposed amine via NHS-PEG chemistry. (Item 69) 69. The modified TFF2 polypeptide of any one of items 53 to 68, which is part of a homogeneous composition. (Item 70) 70. The modified TFF2 polypeptide according to any one of items 53 to 69, which is part of a pharmaceutical composition. (Item 71) 71. The modified TFF2 polypeptide of any one of items 53 to 70, which has an increased serum half-life compared to the human wild-type TFF2 polypeptide of SEQ ID NO:6. (Item 72) 69. The modified TFF2 polypeptide of any one of items 53 to 68, wherein the C-terminal peptide (CTP) of human chorionic gonadotropin is used to improve the pharmacokinetic (PK) and pharmacodynamic (PD) properties of the modified TFF2 polypeptide. (Item 73) 73. The modified TFF2 polypeptide of any one of items 53 to 72, which is glycosylated. (Item 74) A method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of one or more of the modified TFF2 polypeptides described in any one of items 53 to 73. (Item 75) Item 75. The method of item 74, wherein the cancer is a cancer of the digestive system. (Item 76) 76. The method of claim 75, wherein the gastrointestinal cancer is selected from one or more of oral cavity cancer, pharyngeal cancer, oropharyngeal cancer, esophageal cancer, stomach cancer, gastric cancer, small intestine cancer, large intestine cancer, colon cancer, rectal cancer, anal cancer, liver cancer, pancreatic cancer, and gallbladder cancer. (Item 77) Item 77. The method of item 76, wherein the cancer is colon cancer. (Item 78) 77. The method of item 76, wherein the cancer is oropharyngeal cancer. (Item 79) Item 77. The method of item 76, wherein the cancer is esophageal cancer. (Item 80) Item 77. The method of item 76, wherein the cancer is gastric cancer. (Item 81) Item 77. The method of item 76, wherein the cancer is pancreatic cancer. (Item 82) Item 77. The method of item 76, wherein the cancer is rectal cancer. (Item 83) 77. The method of claim 76, wherein the cancer being treated is liver cancer. (Item 84) 84. The method of any one of items 74 to 83, wherein the cancer is a metastatic cancer. (Item 85) 85. The method of any one of items 74 to 84, further comprising treating the cancer with a blocking antibody against PD-1, PD-L1 or CTLA-4. (Item 86) 74. A method of treating cancer in a subject in need thereof, wherein the cancer is unresponsive to treatment with a blocking antibody against PD-1, PD-L1, or CTLA-4, and the subject is treated with one or more modified TFF2 polypeptides of any one of paragraphs 53 to 73, wherein after treatment with the modified TFF2 polypeptides, the subject's cancer becomes susceptible to treatment with a blocking antibody against PD-1, PD-L1, or CTLA-4, and the subject is then treated with a blocking antibody against PD-1, PD-L1, or CTLA-4 within about 1 to about 60 days after treatment with the modified TFF2 polypeptides. (Item 87) A method for treating inflammatory bowel disease (IBD) in a subject in need thereof, wherein the subject is treated with one or more modified TFF2 polypeptides described in any one of items 53 to 73. (Item 88) 88. The method of item 87, wherein the IBD is Crohn's disease or ulcerative colitis. (Item 89) 89. The method of item 87 or 88, wherein the modified TFF2 polypeptide is administered orally, intravenously, or intramuscularly. (Item 90) 1. A method for treating COVID-19 in a subject in need thereof, comprising administering to the subject one or more compositions of any one of items 1 to 15, one or more modified TFF2 polypeptides of any one of items 29 to 36, or one or more modified TFF2 polypeptides of any one of items 53 to 73. (Item 91) 91. The method of claim 90, wherein the modified TFF2 polypeptide is administered orally, intravenously, or intramuscularly. (Item 92) 92. The method of item 90 or 91, further comprising administering an agent that inhibits or reduces replication of SARS-CoV-2. (Item 93) 93. The method of any one of items 90 to 92, further comprising administering an antiviral agent selected from the group consisting of ribavirin, interferon (Alfacon-1), chloroquine, hydroxychloroquine, EIDD-2801, EIDD-1931, GS-5734, GS-441524, ivermectin, favipiravir, indomethacin, chlorpromazine, penciclovir, nafomostat, camostat, nitazoxanide, remdesivir, famotidine, and dexamethasone.

Claims

[Claim 1] The invention described in the specification.

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