WO2025049610A1 - Multi-action peptides - Google Patents

Abstract

The present disclosure relates to, inter alia, compositions and methods, including peptides or a derivatives thereof that find use, inter alia, in the treatment of diabetes, obesity, or metabolic syndrome.

Description

MULTI-ACTION PEPTIDES

TECHNICAL FIELD

The present disclosure relates to, inter alia, compositions and methods, including peptides, fusion protein comprising the peptides, and nucleic acids encoding the peptides or fusion proteins that find use, inter alia, in the treatment management of hyperglycemia, diabetes, including type II diabetes, obesity, metabolic syndrome and the reduction of cardiovascular risk.

PRIORITY

This application claims the benefit of, and priority to, U.S. Provisional Application No. 63/579,243, filed August 28, 2023, U.S. Provisional Application No. 63/606,414, filed December 5, 2023, and U.S. Provisional Application No. 63/656,436, filed June 5, 2024, the contents of each of which are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a sequence listing, which has been submitted in XML format via Patent Center. The contents of the XML copy named “SHK-095PC_116981 -5095_Sequence_Listing”, which was created on August 23, 2024, and is 61 ,868 bytes in size, are incorporated herein by reference in their entirety.

BACKGROUND

The worldwide prevalence of diabetes mellitus and obesity has been on the rise for past decades possibly because of stressful and sedentary lifestyles and unhealthy eating habits. Diabetes mellitus, obesity, diabesity, which is a term used to describe the combined harmful health outcomes of obesity and diabetes mellitus, are major health hazards affecting people worldwide. Ng et al., Diabesity: the combined burden of obesity and diabetes on heart disease and the role of imaging Nature Reviews Cardiology 2021 ; 18: 291 - 304. Based on an estimate from the World Health Organization (WHO), the people with diabetes rose from 108 million in 1980 to 422 million in 2014. According to the Center for Disease Control (CDC), diabetes affects about 37.3 million people in the US, which is 11.3% of the US population, and this number includes about 8.5 million people that have undiagnosed diabetes. Similarly, obesity has nearly tripled worldwide since 1975, with more than 1.9 billion overweight, and over 650 million obese adults worldwide as of 2016. likewise, US obesity prevalence increased from about 30.5% in year 2000 to about 41.9% in 2017 according to the CDC. Currently, over 20% children and over 40% adults suffer from obesity. Obesity is estimated to cost health services US $990 billion, which is 13% healthcare expenditure, per year globally. For example, the aggregate medical cost due to obesity among adults in the United States was $260.6 billion in 2016. Cawley et al. Direct medical costs of obesity in the United States and the most populous states, J Manag Care Spec Pharm 2021 ; 27(3): 354-366.

Diabetes is linked to a number of health problems, including microvascular complications, such as retinopathy, neuropathy, nephropathy, blindness in working-age adults, end-stage renal disease, peripheral artery disease (PAD), cardiovascular complications, and cardiovascular disease (CVD). Similarly, obesity itself increases risk for many serious diseases, including hypertension, dyslipidemia, type 2 diabetes, coronary heart disease, metabolic syndrome, fatty liver disease, stroke, gallstones, cholecystitis, osteoarthritis, kidney disease, sleep apnea and breathing problems, clinical depression, anxiety, and many types of cancers.

Therefore, there remains a need for more effective and accessible methods of treating diabetes, obesity, diabesity and related diseases.

SUMMARY

In various aspects, the present disclosure provides compositions and methods that are useful, inter alia, in the treatment or prevention of hyperglycemia, diabetes, including type II diabetes, obesity, metabolic syndrome and the reduction of cardiovascular risk.

Accordingly, in aspects, the present disclosure provides a peptide capable of modulating at least two of GLP- 1 receptor (GLP-1 R), GIP receptor (GIPR), glucagon receptor (GCGR) and GLP-2 receptor (GLP-2R), or a polynucleotide encoding the peptide. In embodiments, the dual- or triple-action peptide comprises an amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 60 to 69.

Accordingly, in aspects, the present disclosure provides a peptide or a derivative thereof, wherein the peptide comprises a general formula (I):

N-L1-[Aaa]1-[Aaa]2-[Aaa]3-Gly-Thr-Phe-Thr-Ser-Asp-[Aaa]4-Ser-[Aaa]5-[Aaa]6-[Aaa]7-[Aaa]8- [Aaa]9-[Aaa]10-[Aaa]11-[Aaa]12-[Aaa]13-[Aaa]14-Phe-[Aaa]15-[Aaa]16-[Aaa]17-Leu-[Aaa]18- [Aaa]19-[Aaa]20-Gly-Pro-[Aaa]21-[Aaa]22-[Aaa]23-[Aaa]24-Pro-Pro-Pro-[Aaa]25-L2-C

(I) wherein N is the N-terminus; L1 and/or L2 are independently present or absent; and L1 and/or L2, if present, are each independently selected from a carrier protein, a tag, and a polymer, optionally wherein the L1 and/or L2 further comprise a linker that adjoins the carrier protein, tag, or polymer with the peptide; [Aaa] 1 is His; [Aaa]2 is Ser or Gly; [Aaa]3 is His or Pro; [Aaa]4 is Phe or lie; [Aaa]5 is Leu or Vai; [Aaa]6 is Ala; [Aaa]7 is Leu or lie; [Aaa]8 is Asp or Glu; [Aaa]9 is Lys or Glu; [Aaa]10 is Gin; [Aaa]11 is Arg; [Aaa]12 is Gin; [Aaa]13 is Ala or Gin; [Aaa]14 is Glu; [Aaa]15 is lie or Leu; [Aaa]16 is Asp or Glu; [Aaa]17 is Trp; [Aaa]18 is Arg or Gly; [Aaa] 19 is Ala; [Aaa]20 is Gly or Ala; [Aaa]21 is Pro or Ser; [Aaa]22 is Ser or Thr; [Aaa]23 is Gly or Ala; [Aaa]24 is Arg or Lys; [Aaa]25 is Arg or Lys; and C is the C-terminus.

In aspects, the present disclosure provides a peptide or a derivative thereof, wherein the peptide comprises formula (II):

N-L1-[Aaa]1-[Aaa]2-[Aaa]3-Gly-Thr-Phe-Thr-Ser-Asp-[Aaa]4-Ser-[Aaa]5-[Aaa]6-[Aaa]7-[Aaa]8- [Aaa]9-[Aaa]10-[Aaa]11-[Aaa]12-[Aaa]13-[Aaa]14-Phe-[Aaa]15-[Aaa]16-[Aaa]17-Leu-[Aaa]18- [Aaa]19-[Aaa]20-Gly-Pro-[Aaa]21-[Aaa]22-[Aaa]23-[Aaa]24-Pro-Pro-Pro-[Aaa]25-L2-C

(II) wherein: N is the N-terminus; L1 and/or L2 are independently present or absent; and L1 and/or L2, if present, are each independently selected from a carrier protein, a tag, and a polymer; [Aaa]1 is a positively charged amino acid residue selected from His, Lys, and Arg; [Aaa]2 is a polar and neutral of charge hydrophilic amino acid selected from Ser, Asn, Gin, Thr, and Pro, or a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met; [Aaa]3 is a positively charged amino acid residue selected from His, Lys, and Arg, or a polar and neutral of charge hydrophilic amino acid selected from Pro Ser, Asn, Gin, and Thr; [Aaa]4 is a, hydrophobic, aromatic amino acid selected from Phe, Trp, and Tyr, or a hydrophobic, aliphatic amino acid selected from lie, Gly, Ala, Leu, Met, and Vai; [Aaa]5 is a hydrophobic, aliphatic amino acid selected from Leu, Vai, lie, Gly, Ala, and Met; [Aaa]6 is a hydrophobic, aliphatic amino acid selected from Ala, Gly, Vai, lie, Leu, and Met; [Aaa]7 is a hydrophobic, aliphatic amino acid selected from Leu, lie, Vai, Gly, Ala, and Met; [Aaa]8 is a polar and negatively charged hydrophilic amino acid selected from Asp and Glu; [Aaa]9 is a polar and positively charged hydrophilic amino acid selected from Lys, His, and Arg, or a polar and negatively charged hydrophilic amino acid selected from Glu, and Asp; [Aaa]10 is a polar and neutral of charge hydrophilic amino acid selected from Gin, Asn, Ser, Thr, and Pro; [Aaa] 11 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His; [Aaa]12 is a polar and neutral of charge hydrophilic amino acid selected from Gin, Asn, Ser, Thr, and Pro; [Aaa]13 is a hydrophobic, aliphatic amino acid selected from Ala, Vai, Gly, Leu, lie, and Met, or a polar and neutral of charge hydrophilic amino acid selected from Gin, Asn, Ser, Thr, and Pro; [Aaa]14 is a polar and negatively charged hydrophilic amino acid selected from Glu, and Asp; [Aaa]15 is a hydrophobic, aliphatic amino acid selected from lie, Leu, Vai, Ala, Gly, and Met; [Aaa]16 is a polar and negatively charged hydrophilic amino acid selected from Asp, and Glu; [Aaa]17 is hydrophobic, aromatic amino acid selected from Trp, Phe, and Tyr; [Aaa] 18 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His, or a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met; [Aaa] 19 is a hydrophobic, aliphatic amino acid selected from Ala, Gly, Vai, Leu, lie, and Met; [Aaa]20 is a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met; [Aaa]21 is a polar and neutral of charge hydrophilic amino acid selected from Pro, Ser, Gin, Asn, and Thr; [Aaa]22 is a polar and neutral of charge hydrophilic amino acid selected from Ser, Thr, Gin, Asn, and Pro; [Aaa]23 a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met; [Aaa]24 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His; [Aaa]25 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His; and C is the C-terminus.

In embodiments, [Aaa]1 is His. In embodiments, [Aaa]2 is Ser or Gly. In embodiments, [Aaa]3 is His or Pro.

In embodiments, [Aaa]4 is Phe or lie. In embodiments, [Aaa]5 is Leu or Vai. In embodiments, [Aaa]6 is Ala.

In embodiments, [Aaa]7 is Leu or lie. In embodiments, [Aaa]8 is Asp or Glu. In embodiments, [Aaa]9 is Lys or Glu. In embodiments, [Aaa]10 is Gin. In embodiments, [Aaa]11 is Arg. In embodiments, [Aaa]12 is Gin. In embodiments, [Aaa]13 is Ala or Gin. In embodiments, [Aaa]14 is Glu. In embodiments, [Aaa]15 is lie or Leu. In embodiments, [Aaa]16 is Asp or Glu. In embodiments, [Aaa]17 is Trp. In embodiments, [Aaa] 18 is Arg or Gly. In embodiments, [Aaa]19 is Ala. In embodiments, [Aaa]20 is Gly or Ala. In embodiments, [Aaa]21 is Pro or Ser. In embodiments, [Aaa]22 is Ser or Thr. In embodiments, [Aaa]23 is Gly or Ala. In embodiments, [Aaa]24 is Arg or Lys. In embodiments, and/or [Aaa]25 is Arg or Lys.

In embodiments, [Aaa]1 is His; [Aaa]2 is Ser or Gly; [Aaa]3 is His or Pro; [Aaa]4 is Phe or lie; [Aaa]5 is Leu or Vai; [Aaa]6 is Ala; [Aaa]7 is Leu or lie; [Aaa]8 is Asp or Glu; [Aaa]9 is Lys or Glu; [Aaa]10 is Gin; [Aaa]11 is Arg; [Aaa]12 is Gin; [Aaa]13 is Ala or Gin; [Aaa]14 is Glu; [Aaa]15 is lie or Leu; [Aaa]16 is Asp or Glu; [Aaa] 17 is Trp; [Aaa]18 is Arg or Gly; [Aaa]19 is Ala; [Aaa]20 is Gly or Ala; [Aaa]21 is Pro or Ser; [Aaa]22 is Ser or Thr; [Aaa]23 is Gly or Ala; [Aaa]24 is Arg or Lys; and/or [Aaa]25 is Arg or Lys.

In embodiments, the peptide or the derivative thereof comprises an amino acid sequence that is selected from the amino acid sequence of SEQ ID NOs: 60 to 69. In embodiments, the peptide or the derivative thereof comprises an amino acid sequence that is selected from the amino acid sequence of SEQ ID NOs: 60 to 69, or a variant thereof having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from SEQ ID NOs: 60 to 69. In embodiments, the peptide or the derivative thereof comprises an amino acid sequence that is selected from the amino acid sequence of SEQ ID NOs: 60, SEQ ID NO: 64, SEO ID NO: 65, SEO ID NO: 68 and SEQ ID NO: 69, or a variant thereof having 1 or 2 amino acid mutations with respect to an amino acid sequence selected from SEQ ID NOs: 60, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 68 and SEQ ID NO: 69. In embodiments, the peptide or the derivative thereof comprises an amino acid sequence that is selected from the amino acid sequence of SEQ ID NOs: 60, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 68 and SEQ ID NO: 69.

In embodiments, the peptide or the derivative thereof comprises a carrier protein selected from Fc domain, albumin, transferrin, or elastin-like protein, Keyhole Limpet Hemocyanin (KLH), ovalbulin, or a variant thereof. In embodiments, the carrier protein comprises an Fc domain selected from an IgG Fc domain, an IgA Fc domain, an IgM Fc domain, an IgE Fc domain, and an IgD Fc domain, or a variant thereof. In embodiments, the carrier protein comprises the IgG Fc domain selected from an lgG1 Fc domain, an lgG2 Fc domain, an lgG3 Fc domain, and an lgG4 Fc domain, or a variant thereof. In embodiments, the carrier protein comprises the IgG Fc domain is selected from a human lgG1 Fc domain, and a human lgG4 Fc domain, or a variant thereof. In embodiments, the human lgG1 Fc domain comprises a hinge-CH2-CH3-Fc domain derived from human lgG1 or a derivative thereof. In embodiments, the human I gG4 Fc domain comprises a hinge-CH2- CH3-Fc domain derived from human lgG4 or a derivative thereof.

In embodiments, the peptide or the derivative thereof further comprises a linker that adjoins the carrier protein with the peptide or the derivative thereof. In embodiments, the peptide linker is rigid. In embodiments, the peptide linker is flexible.

In embodiments, the peptide or the derivative thereof further comprises a glycosyl moiety. In embodiments, the glycosyl moiety is an N-linked glycosyl moiety. In embodiments, the glycosyl moiety is an O-linked glycosyl moiety. In embodiments, the peptide or the derivative thereof comprises one or more N-linked glycosylation consensus sites and/or O-linked glycosylation consensus sites.

In embodiments, the peptide or the derivative thereof is biosynthesized as a single polypeptide chain. In embodiments, the peptide or the derivative thereof is biosynthesized from a single open reading frame. In embodiments, the peptide or the derivative thereof is prepared using an expression system. In embodiments, the expression system is selected from bacterial, yeast, invertebrate, vertebrate, and plant expression system.

In embodiments, the peptide or the derivative thereof further comprises a non-natural amino acid selected from an amino isobutyric acid (Aib), a D-amino acid, and those comprising a modification selected from N- methylation (Nm), Ca-methylation (Cm), M^J(CH2NH) reduced amide bonds (Rd), and a peptoids (Pp)). In embodiments, the peptide or the derivative thereof comprises a polymer. In embodiments, the polymer is selected from poly(alkylene oxide) (e.g., polyethylene glycol (PEG)), poly(N-vinylpyrrolidone), poly(vinyl alcohol), poly(glycerol), poly(zwitterions), poly(carbonates), polyoxazoline, poly(acryloylmorpholine), poly(oxazolines), poly(sacharrides), and a combination thereof. In embodiments, the polymer is polyethylene glycol (PEG).

In embodiments, one or more amino acids present in the peptide or the derivative thereof is PEGylated. In embodiments, the one or more PEGylated amino acids are located inside the carrier protein. In embodiments, the PEGylation is conducted using a succinimidyl ester, an aldehyde, a maleimide, and/or a p-nitrophenyl carbonate ester reagent. In embodiments, one or more Lys residues, one or more Ser residues, one or more Tyr residues, one or more His residues, one or more Cys residues, the N-terminus and/or the C-terminus are PEGylated. In embodiments, at least about 1 , or at least about 3, or at least about 5, or at least about 8, or at least about 10, or more amino acid residues, the N-terminus and/or the C-terminus are PEGylated. In embodiments, the one or more PEGylated amino acids comprise a Lys. In embodiments, the PEGylation is conducted via amine conjugation. In embodiments, the one or more PEGylated amino acids comprise a Gin. In embodiments, the PEGylation is conducted via transglutaminase (TGase) mediated enzymatic conjugation. In embodiments, the one or more PEGylated amino acids comprise a Cys. In embodiments, the PEGylation is conducted via thiol conjugation.

In embodiments, the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on glucagon-like peptide- 1 receptor (GLP-1 R), glucagon-like peptide-2 receptor (GLP- 2R), gastric inhibitory peptide receptor (GIPR), and/or glucagon receptor (GCGR).

In embodiments, the peptide or the derivative thereof of any of the embodiments disclosed herein is dual active. In embodiments, the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GLP-1 R and GCGR. In embodiments, the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GLP-1 R and GIPR. In embodiments, the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GLP- 1 R and GLP-2R. In embodiments, the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GLP-2R and GIPR. In embodiments, the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GLP-2R and GCGR. In embodiments, the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GIPR and GCGR. In embodiments, the peptide or the derivative thereof of any of the embodiments disclosed herein is triple active. In embodiments, the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GLP-1 R, GIPR and GCGR. In embodiments, the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GLP-1 R, GLP-2R and GCGR. In embodiments, the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GLP-1 R, GLP-2R and GIPR. In embodiments, the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GLP-2R, GIPR and GCGR. In an illustrative embodiment, the peptide or the derivative thereof has an agonistic activity on GLP-1 R, an agonistic activity on GIPR, and an antagonistic activity on GCGR. In another illustrative embodiment, the peptide or the derivative thereof has an agonistic activity on GLP-1 R, an antagonistic activity on GIPR, and an agonistic activity on GCGR.

In embodiments, the peptide or the derivative thereof of any of the embodiments disclosed herein is quadruple active. In embodiments, the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GLP-1 R, GLP-2R, GIPR and GCGR. In an illustrative embodiment, the peptide or the derivative thereof has an agonistic activity on GLP-1 R, an agonistic activity on GLP-2R, an agonistic activity on GIPR, and an antagonistic activity on GCGR. In another illustrative embodiment, the peptide or the derivative thereof has an agonistic activity on GLP-1 R, an agonistic activity on GLP-2R, an antagonistic activity on GIPR, and an agonistic activity on GCGR.

In embodiments, the peptide or the derivative thereof exhibits an EC50 value for GLP-1 R in the range of 100 pM to 1000 nM, as determined by a cell-based assay. In embodiments, the peptide or the derivative thereof exhibits an EC50 value for GLP-1 R in the range of 2 nM to 100 nM, as determined by a cell-based assay.

In embodiments, the peptide or the derivative thereof exhibits an EC50 value for GIPR in the range of 100 pM to 1000 nM, as determined by a cell-based assay. In embodiments, the peptide or the derivative thereof exhibits an EC50 value for GIPR in the range of 100 pM to 2 nM, as determined by a cell-based assay.

In embodiments, the peptide or the derivative thereof exhibits an EC50 value for GCGR in the range of 100 pM to 1000 nM, as determined by a cell-based assay. In embodiments, the peptide or the derivative thereof exhibits an EC50 value for GCGR in the range of 2 nM to 100 nM, as determined by a cell-based assay. In embodiments, the peptide or the derivative thereof exhibits an I C50 value for GLP-1 R in the range of 10 pM to 10 pM, as determined by a cell-based assay. In embodiments, the peptide or the derivative thereof exhibits an I C50 value for GIPR in the range of 10 pM to 10 pM, as determined by a cell-based assay. In embodiments, the peptide or the derivative thereof exhibits an IC50 value for GCGR in the range of 10 pM to 10 pM, as determined by a cell-based assay. In embodiments, the peptide or the derivative thereof exhibits an EC50 value for GLP-2R in the range of 10 pM to 10 pM, as determined by a cell-based assay. In embodiments, the peptide or the derivative thereof exhibits an IC50 value for GLP-2R in the range of 10 pM to 10 pM, as determined by a cell-based assay.

In aspects, the present disclosure provides a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 60, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 60. In embodiments, the peptide comprises an amino acid sequence of SEQ ID NO: 60.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP- 2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 61 , or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 61. In embodiments, the peptide comprises an amino acid sequence of SEQ ID NO: 61 .

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP- 2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 62, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 62. In embodiments, the peptide comprises an amino acid sequence of SEQ ID NO: 62.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP- 2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 63, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 63. In embodiments, the peptide comprises an amino acid sequence of SEQ ID NO: 63.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP- 2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 64, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 64. In embodiments, the peptide comprises an amino acid sequence of SEQ ID NO: 64.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP- 2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 65, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 65. In embodiments, the peptide comprises an amino acid sequence of SEQ ID NO: 65.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP- 2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 66, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 66. In embodiments, the peptide comprises an amino acid sequence of SEQ ID NO: 66.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP- 2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 67, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 67. In embodiments, the peptide comprises an amino acid sequence of SEQ ID NO: 67.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP- 2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 68, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 68. In embodiments, the peptide comprises an amino acid sequence of SEQ ID NO: 68.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP- 2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 69, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 69. In embodiments, the peptide comprises an amino acid sequence of SEQ ID NO: 69.

In embodiments, the hinge CH2-CH3-Fc domain is derived from lgG1. In embodiments, the lgG1 is human lgG1. In embodiments, the hinge-CH2-CH3 Fc domain is derived from lgG4. In embodiments, the lgG4 is human lgG4. In embodiments, the hinge CH2-CH3-Fc domain comprises an amino acid sequence that is at least about 95%, or at least about 97%, or at least about 97%, or at least about 98% or at least 99% identical to the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In embodiments, the hinge CH2-CH3-Fc domain is fused to the peptide or a derivative thereof via a linker. In embodiments, the linker has an amino acid sequence selected from SEQ ID NOs: 5 to 53.

In aspects, the present disclosure provides an isolated polynucleotide encoding the peptide or the derivative thereof of any of the embodiments disclosed herein or the fusion proteins of any of the embodiments disclosed herein. In embodiments, the isolated polynucleotide is DNA. In embodiments, the nucleic acid is RNA.

In embodiments, the isolated polynucleotide is an mRNA. In embodiments, the mRNA a modified mRNA (mmRNA). In embodiments, the mmRNA comprises at least one nucleoside modification selected from pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio- 5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl- uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl- 2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1 -methylpseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1 -deazapseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2- methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5- aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5- hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio- cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1- methyl-1-deaza-pseudoisocytidine, 1 -methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl- cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2, 6- diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2- aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1 -methyladenosine, N6- methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2- methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio- adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7- deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7- methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6- thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, and combinations thereof. In embodiments, the mmRNA further comprises a 5'-cap and/or a poly A tail.

In aspects, the present disclosure provides an expression vector comprising the isolated polynucleotide of any of the embodiments disclosed herein. In aspects, the present disclosure provides an expression vector of any of the embodiments disclosed herein. In embodiments, the expression vector is a mammalian expression vector.

In aspects, the present disclosure provides a host cell comprising the isolated polynucleotide of the embodiments disclosed herein. In aspects, the present disclosure provides a host cell comprising the expression vector of the embodiments disclosed herein. In aspects, the present disclosure provides a pharmaceutical composition comprising the peptide or the derivative thereof of any of the embodiments disclosed herein. In aspects, the present disclosure provides a pharmaceutical composition comprising the fusion proteins of any of the embodiments disclosed herein. In aspects, the present disclosure provides a pharmaceutical composition comprising the isolated polynucleotide of any of the embodiments disclosed herein. In aspects, the present disclosure provides a pharmaceutical composition comprising the expression vector of any of the embodiments disclosed herein. In aspects, the present disclosure provides a pharmaceutical composition comprising the host cell of any of the embodiments disclosed herein.

In aspects, the present disclosure provides a method of treating or preventing hyperglycemia, diabetes, obesity or metabolic syndrome, nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), a cardiometabolic disease, or liver fibrosis or for reducing blood glucose, or for reducing fed and fasting blood glucose, or for reducing cardiovascular risk, or for decreasing body weight, decreasing food intake, decreasing blood glucose, decreasing liver adiposity, decreasing liver weight, decreasing subcutaneous white adipose tissue (sWAT), or for increasing glucose tolerance in a subject in need thereof, the method comprising administering to the subject the peptide or the derivative thereof of any of the embodiments disclosed herein, the fusion proteins of any of the embodiments disclosed herein, the isolated polynucleotide of any of the embodiments disclosed herein, or the expression vector of any of the embodiments disclosed herein, or the pharmaceutical composition of any of the embodiments disclosed herein.

Any aspect or embodiment disclosed herein can be combined with any other aspect or embodiment as disclosed herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows an alignment of sequences of the illustrative, non-limiting peptides disclosed herein with glucagon (GCG), glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP). The regions showing identity are shown in a bold-underlined font. FIG. 1B shows an alignment of sequences of the illustrative, non-limiting hybrid peptide SL-044. The regions derived from source peptide is shown in a bold or underlined font.

FIG. 2A to FIG. 2C show the characterization of GLP-1 R/GIPR/GCGR agonist activity of te SL_TriAg_V1 , SL_TriAg_V2_Pro3 peptides in comparison with a GIPR/GLP-1 R/GCGR co-agonist peptide (Comparator 1) and a dual GIPR/GLP-1 R co-agonist (Comparator 2). FIG. 2A demonstrates the activation of GLP-1 R receptor as measured a cell-based reporter assay. FIG. 2B demonstrates the activation of GIP receptor (GIPR) as measured a cell-based reporter assay. FIG. 20 demonstrates the activation of Glucagon Receptor (GCGR) as measured a cell-based reporter assay.

FIG. 3 shows a summary of activation of glucagon-like peptide 1 (GLP-1) receptor, glucagon (GCG) receptor, glucose-dependent insulinotropic polypeptide (GIP) receptor by illustrative, non-limiting peptides disclosed herein.

DETAILED DESCRIPTION

Disclosed herein peptides and derivatives thereof having agonistic or antagonistic activity on two or three of glucagon-like peptide-1 receptor (GLP-1 R), gastric inhibitory peptide receptor (GIPR), and glucagon receptor (GCGR) that are useful, inter alia, for treating or preventing disease, such as therapies for diseases/ disorders such as hyperglycemia, diabetes, obesity or metabolic syndrome, nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or liver fibrosis or for reducing blood glucose, or for reducing fed and fasting blood glucose, or for reducing cardiovascular risk, or for decreasing body weight, decreasing food intake, decreasing blood glucose, decreasing liver adiposity, decreasing liver weight, decreasing subcutaneous white adipose tissue (sWAT), or for increasing glucose tolerance. In embodiments, the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on at least one or at least two or all three of GLP-1 R, GIPR, and GCGR. In embodiments, the peptide or the derivative thereof exhibits an EC50 value for GLP-1 R in the range of 100 pM to 1000 nM, as determined by a cell-based assay. In embodiments, the peptide or the derivative thereof exhibits an EC50 value for GLP-1 R in the range of 2 nM to 100 nM, an EC50 value for GIPR in the range of 100 pM to 2 nM, and/or an EC50 value for GCGR in the range of 100 pM to 1000 nM, as determined by a cell-based assay.

GLP-1, GLP-2, GIP, and Glucagon

In aspects, the present disclosure provides a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), gastric inhibitory peptide (GIP), and glucagon (GCG). GLP-1, GLP-2, GIP, and/or glucagon play major roles in the obesity-diabetes-metabolic syndrome axis of metabolism, such as control of glucose, glycogenolysis, gluconeogenesis, appetite, food intake, satiety, insulin secretion, etc., that are at the base of the diabetesobesity axis. Receptors Glucagon-like peptide 1 (GLP-1) is a 30-amino acid peptide hormone produced in the intestine. GLP-1 is normally produced after meals and stimulates insulin secretion and inhibits glucagon secretion. It is also involved in the regulation of p-cell growth and survival, gastric emptying, and appetite. In the body, GLP-1 is degraded by dipeptidyl peptidase IV and has a short half-life of around 2 minutes. Reduced GLP-1 secretion is associated with type 2 diabetes and the development of obesity.

GLP-1, GLP-2 and glucagon are produced by the alpha cells of the pancreas and in the intestinal L cells in the distal ileum and colon in form of a precursor called preglucagon that is cleaved in different organs into glicentin, glicentin-related pancreatic polypeptide (GRPP), oxyntomodulin, glucagon (marked in a boldface- italicized font), glucagon-like peptide 1 (GLP-1, indicated in a boldface-underlined font), and glucagon-like peptide 2 (GLP-2, indicated in a italicized-underlined font). Preglucagon has the following sequence:

MKSlYFVAGLFVMLVQGSWQRSLQDTEEKSRSFSASQADPLSDPDQMNEDKRHSQGTFTSDySKYLDSR RAQDFVQWL/W/VTKRNRNNIAKRHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDFPEEVAI

\/EE\.GRRHADGSFSDEMNTILDNLAARDFINWLIQTKITDRK (SEQ ID NO: 54)

In embodiments, the GLP-1 has the following sequence:

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 55).

GLP-1 agonists and antagonists are disclosed in US Patent Nos. 5,188,666, 5,120,712, 5,523,549, 5,512,549, 5,977,071 , 6,191,102; 6,956,026; 6,506,724; 6,703,359; 6,858,576; 6,872,700; 6,902,744; 7,157,555; 7,223,725; 7,220,721 ; 9,161,953; PCT International Publication Nos: WO 1998/008871 ; WO 1998/05351; WO 1999/07404; WO 1999/25727; WO 1999/25728; WO 1999/40788; WO 2000/034331; WO 2000/41546; WO 2000/41548; WO 2000/069911; WO 2000/73331; WO 2001/004156; WO 2001/51078; WO 2003/018516; WO 2003/099314; U.S. Publication No. 2003/0036504; and U.S. Publication No. 2006/0094652, the entire contents of which are hereby incorporated by reference in their entirety.

Glucose-dependent insulinotropic polypeptide (GIP) is a biologically active 42-amino acid-long gastrointestinal peptide hormone, having a short half-life (2-5 min) in circulation, which is synthesized and secreted into the blood stream by intestinal endocrine K cells within minutes of ingesting a meal. GIP binds to a specific glucose-dependent insulinotropic polypeptide receptor (GIPR), which is a class B G-protein-coupled receptor (GPCR) expressed in the endocrine pancreas, gastrointestinal tract, brain, immune and cardiovascular systems, testis, pituitary, lung, kidney, thyroid, several regions of the central nervous system and adipose tissue. GIP is believed to induce insulin secretion, which is stimulated primarily by hyperosmolarity of glucose in the duodenum. The amount of insulin secreted is greater when glucose is administered orally than intravenously. GIP is also believed to reduce food intake upon signaling via the hypothalamic GIPR. Adriaenssens, et al., Glucose-Dependent Insulinotropic Polypeptide Receptor-Expressing Cells in the Hypothalamus Regulate Food Intake, Cell Metabolism 2019; 30(5): 987-996. GIP is known to inhibit apoptosis of the pancreatic beta cells and to promote their proliferation. It also stimulates glucagon secretion and fat accumulation. Both GIP receptor (GIPR) agonism and antagonism are effective strategies for inhibiting weight gain. Miyawaki et al., Inhibition of gastric inhibitory polypeptide signaling prevents obesity, Nat. Med., 2002; 8: 738-742; McClean et al., GIP receptor antagonism reverses obesity, insulin resistance, and associated metabolic disturbances induced in mice by prolonged consumption of high-fat diet, Am. J. Physiol. Endocrinol. Metab., 2007; 293: E1746-E1755. Boylan et al., Gastric inhibitory polypeptide immunoneutralization attenuates development of obesity in mice, Am. J. Physiol. Endocrinol. Metab., 309: E1008-E1018; Fulurija ef al., Vaccination against GIP for the treatment of obesity, PLoS One 2008; 3: e3163. Accordingly, in embodiments, the GIPR modulator is a GIPR agonist. In embodiments, the GIPR modulator is a GIPR antagonist.

GIP, which is encoded by the GIP gene, is derived from a 153-amino acid proprotein having the following sequence (GIP is shown in a boldface-underlined font):

MVATKTFALLLLSLFLAVGLGEKKEGHFSALPSLPVGSHAKVSSPQPRGPRYAEGTFISDYSIAMDKIHQQD FVNWLLAQKGKKNDWKHNITQREARALELASQANRKEEEAVEPQSSPAKNPSDEDLLRDLLIQELLACLL DQTNLCRLRSR (SEQ ID NO: 56)

In embodiments, the GIP has the following sequence:

YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ (SEQ ID NO: 57)

GIPR modulators (without limitations, e.g., GIP analogs, GIP agonists, and GIP antagonists) are disclosed in US Patent Nos 6,921 ,748; 8,497,240; 9,453,062; 10,253,078 and US Patent Application Publication Nos. 2003/0232761 ; 2008/0312157; 2011/0136737; 2014/0162945; 2015/0329611; 2017/0240609; 2017/0240609, the entire contents of which are hereby incorporated by reference in their entirety.

Glucagon is a peptide hormone, produced by alpha cells of the pancreas when the amount of glucose in the bloodstream is too low. Glucagon plays a critical role in maintaining glucose homeostasis as a counterregulatory hormone for insulin. Specifically, glucagon promotes hepatic glucose output by increasing glycogenolysis and gluconeogenesis in response to reduced blood levels of glucose. However, diabetic subjects have abnormal secretion of not only insulin but also glucagon compared with healthy subjects, leading to abnormalities in glucose homeostasis. Hyperglucagonemia and altered insulin-to-glucagon ratios play important roles in initiating and maintaining pathological hyperglycemic states.

Glucagon is produced by the alpha cells of the pancreas and in the intestinal L cells in the distal ileum and colon in form of a precursor called preglucagon that is cleaved in different organs by subtilisin-like proprotein convertases into glicentin, glicentin-related pancreatic polypeptide (GRPP), oxyntomodulin, glucagon (marked in a boldface-italicized font), glucagon-like peptide 1 (GLP-1 , indicated in a boldface-underlined font), and glucagon-like peptide 2 (GLP-2). Preglucagon has the following sequence:

MKSlYFVAGLFVMLVQGSWQRSLQDTEEKSRSFSASQADPLSDPDQMNEDKRHSQGTFTSDYSKyLDS/? RAQDFYQWL/ NTKRNRNNIAKRHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDFPEEVAI VEELGRRHADGSFSDEMNTILDNLAARDFINWLIQTKITDRK (SEQ ID NO: 54)

In embodiments, the glucagon has the following sequence:

HSQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID NO: 58).

GLP-2 receptor agonists and antagonists are disclosed in PCT International Publication Nos: WO 2007/056362, WO 2008/086086, WO 2008/152403, WO 2009/155257, WO 2010/070253, WO 2010/070251 , WO 2010/070252, WO 2010/070255, WO 2011/006497, WO 2011/094337, WO 2011/117417, WO 2011 /117416, WO 2011 /160633, WO 2011 /160630, WO 2012/130866, WO 2013/041678, WO 2013/092703, and WO 2014/016300 the entire contents of which are hereby incorporated by reference in their entirety.

GLP-2 is produced, along with GLP-1 and glucagon by the alpha cells of the pancreas and in the intestinal L cells in the distal ileum and colon in form of a precursor called preglucagon that is cleaved in different organs into glicentin, glicentin-related pancreatic polypeptide (GRPP), oxyntomodulin, glucagon (marked in a boldface-italicized font), glucagon-like peptide 1 (GLP-1 , indicated in a boldface-underlined font), and glucagon-like peptide 2 (GLP-2, indicated in a italicized-underlined font). Preglucagon has the following sequence:

MKSlYFVAGLFVMLVQGSWQRSLQDTEEKSRSFSASQADPLSDPDQMNEDKRHSQGTFTSDYSKyLDS/? RAQDFI/QI/I/L/W/VTKRNRNNIAKRHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDFPEEVAI \/EE\.GRRHADGSFSDEMNTILDNLAARDFINWLIQTKITDRK (SEQ ID NO: 54)

GLP-2 regulates gastric motility, gastric acid secretion, intestinal hexose transport, and increases the barrier function of the gut epithelium. GLP-2 is also known to enhance nutrient absorption and gut adaptation in rodents or humans with short bowel syndrome. GLP-2 acts via the GLP-2 receptor, a G protein-coupled receptor expressed in gut endocrine cells of the stomach, small bowel, and colon.

In embodiments, the GLP-2 has the following sequence:

HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ ID NO: 59).

GLP-2 receptor agonists and antagonists are disclosed in US Patent Nos. 6,051 ,557; 6,297,214; 6,943,151 ; 7,056,886; 7,745,403; 8,278,273; 8,263,552; 10,092,648 and U.S. Publication Nos. 2023/0295260, 2023/0057847; 2023/0057847 the entire contents of which are hereby incorporated by reference in their entirety.

Peptides having Actions on GLP-1R, GLP-2R, GIPR, and/or Glucagon Receptor (GCGR)

In aspects, the present disclosure provides a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, wherein the peptide comprises a general formula (I):

N-L1 -[Aaa]1-[Aaa]2-[Aaa]3-Gly-Thr-Phe-Thr-Ser-Asp-[Aaa]4-Ser-[Aaa]5-[Aaa]6-[Aaa]7-[Aaa]8- [Aaa]9-[Aaa]10-[Aaa]11-[Aaa]12-[Aaa]13-[Aaa]14-Phe-[Aaa]15-[Aaa]16-[Aaa]17-Leu-[Aaa]18- [Aaa]19-[Aaa]20-Gly-Pro-[Aaa]21-[Aaa]22-[Aaa]23-[Aaa]24-Pro-Pro-Pro-[Aaa]25-L2-C

(I) wherein N is the N-terminus; L1 and/or L2 are independently present or absent; and L1 and/or L2, if present, are each independently selected from a carrier protein, a tag, and a polymer, optionally wherein the L1 and/or L2 further comprise a linker that adjoins the carrier protein, tag, or polymer with the peptide; [Aaa] 1 is His; [Aaa]2 is Ser or Gly; [Aaa]3 is His or Pro; [Aaa]4 is Phe or lie; [Aaa]5 is Leu or Vai; [Aaa]6 is Ala; [Aaa]7 is Leu or lie; [Aaa]8 is Asp or Glu; [Aaa]9 is Lys or Glu; [Aaa]10 is Gin; [Aaa]11 is Arg; [Aaa]12 is Gin; [Aaa]13 is Ala or Gin; [Aaa]14 is Glu; [Aaa]15 is lie or Leu; [Aaa]16 is Asp or Glu; [Aaa]17 is Trp; [Aaa]18 is Arg or Gly; [Aaa] 19 is Ala; [Aaa]20 is Gly or Ala; [Aaa]21 is Pro or Ser; [Aaa]22 is Ser or Thr; [Aaa]23 is Gly or Ala; [Aaa]24 is Arg or Lys; [Aaa]25 is Arg or Lys; and C is the C-terminus. In embodiments, the peptide or the derivative thereof has dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR.

In aspects, the present disclosure provides a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, wherein the peptide comprises formula (II): N-L1 -[Aaa]1-[Aaa]2-[Aaa]3-Gly-Thr-Phe-Thr-Ser-Asp-[Aaa]4-Ser-[Aaa]5-[Aaa]6-[Aaa]7-[Aaa]8- [Aaa]9-[Aaa]10-[Aaa]11-[Aaa]12-[Aaa]13-[Aaa]14-Phe-[Aaa]15-[Aaa]16-[Aaa]17-Leu-[Aaa]18- [Aaa]19-[Aaa]20-Gly-Pro-[Aaa]21-[Aaa]22-[Aaa]23-[Aaa]24-Pro-Pro-Pro-[Aaa]25-L2-C

(II) wherein: N is the N-terminus; L1 and/or L2 are independently present or absent; and L1 and/or L2, if present, are each independently selected from a carrier protein, a tag, and a polymer; [Aaa]1 is a positively charged amino acid residue selected from His, Lys, and Arg; [Aaa]2 is a polar and neutral of charge hydrophilic amino acid selected from Ser, Asn, Gin, Thr, and Pro, or a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met; [Aaa]3 is a positively charged amino acid residue selected from His, Lys, and Arg, or a polar and neutral of charge hydrophilic amino acid selected from Pro Ser, Asn, Gin, and Thr; [Aaa]4 is a, hydrophobic, aromatic amino acid selected from Phe, Trp, and Tyr, or a hydrophobic, aliphatic amino acid selected from lie, Gly, Ala, Leu, Met, and Vai; [Aaa]5 is a hydrophobic, aliphatic amino acid selected from Leu, Vai, lie, Gly, Ala, and Met; [Aaa]6 is a hydrophobic, aliphatic amino acid selected from Ala, Gly, Vai, lie, Leu, and Met; [Aaa]7 is a hydrophobic, aliphatic amino acid selected from Leu, lie, Vai, Gly, Ala, and Met; [Aaa]8 is a polar and negatively charged hydrophilic amino acid selected from Asp and Glu; [Aaa]9 is a polar and positively charged hydrophilic amino acid selected from Lys, His, and Arg, or a polar and negatively charged hydrophilic amino acid selected from Glu, and Asp; [Aaa]10 is a polar and neutral of charge hydrophilic amino acid selected from Gin, Asn, Ser, Thr, and Pro; [Aaa] 11 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His; [Aaa]12 is a polar and neutral of charge hydrophilic amino acid selected from Gin, Asn, Ser, Thr, and Pro; [Aaa]13 is a hydrophobic, aliphatic amino acid selected from Ala, Vai, Gly, Leu, lie, and Met, or a polar and neutral of charge hydrophilic amino acid selected from Gin, Asn, Ser, Thr, and Pro; [Aaa]14 is a polar and negatively charged hydrophilic amino acid selected from Glu, and Asp; [Aaa]15 is a hydrophobic, aliphatic amino acid selected from lie, Leu, Vai, Ala, Gly, and Met; [Aaa]16 is a polar and negatively charged hydrophilic amino acid selected from Asp, and Glu; [Aaa]17 is hydrophobic, aromatic amino acid selected from Trp, Phe, and Tyr; [Aaa]18 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His, or a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met; [Aaa] 19 is a hydrophobic, aliphatic amino acid selected from Ala, Gly, Vai, Leu, lie, and Met; [Aaa]20 is a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met; [Aaa]21 is a polar and neutral of charge hydrophilic amino acid selected from Pro, Ser, Gin, Asn, and Thr; [Aaa]22 is a polar and neutral of charge hydrophilic amino acid selected from Ser, Thr, Gin, Asn, and Pro; [Aaa]23 a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met; [Aaa]24 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His; [Aaa]25 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His; and C is the C-terminus. In embodiments, the peptide or the derivative thereof has dual, triple or quadruple actions on GLP-1 R, GLP- 2R, GIPR, and GCGR.

In embodiments, [Aaa]1 is His. In embodiments, [Aaa]2 is Ser or Gly. In embodiments, [Aaa]3 is His or Pro. In embodiments, [Aaa]4 is Phe or lie. In embodiments, [Aaa]5 is Leu or Vai. In embodiments, [Aaa]6 is Ala. In embodiments, [Aaa]7 is Leu or lie. In embodiments, [Aaa]8 is Asp or Glu. In embodiments, [Aaa]9 is Lys or Glu. In embodiments, [Aaa]10 is Gin. In embodiments, [Aaa]11 is Arg. In embodiments, [Aaa]12 is Gin. In embodiments, [Aaa]13 is Ala or Gin. In embodiments, [Aaa]14 is Glu. In embodiments, [Aaa]15 is lie or Leu. In embodiments, [Aaa]16 is Asp or Glu. In embodiments, [Aaa]17 is Trp. In embodiments, [Aaa] 18 is Arg or Gly. In embodiments, [Aaa]19 is Ala. In embodiments, [Aaa]20 is Gly or Ala. In embodiments, [Aaa]21 is Pro or Ser. In embodiments, [Aaa]22 is Ser or Thr. In embodiments, [Aaa]23 is Gly or Ala. In embodiments, [Aaa]24 is Arg or Lys. In embodiments, and/or [Aaa]25 is Arg or Lys.

In embodiments, the peptide or the derivative thereof comprises any 2 of, or any 3 of, or any 4 of, or any 5 of, or any 6 of, or any 7 of, or any 8 of, or any 9 of, or any 10 of, or any 11 of, or any 12 of, or any 13 of, or any 14 of, or any 15 of, or any 16 of, or any 17 of, or any 18 of, or any 19 of, or any 20 of, or any 21 of, or any 22 of, or any 23 of, or any 24 of, or all 25 of the following:

(1) [Aaa]1 is His;

(2) [Aaa]2 is Ser or Gly;

(3) [Aaa]3 is His or Pro;

(4) [Aaa]4 is Phe or lie;

(5) [Aaa]5 is Leu or Vai;

(6) [Aaa]6 is Ala;

(7) [Aaa]7 is Leu or lie;

(8) [Aaa]8 is Asp or Glu;

(9) [Aaa]9 is Lys or Glu;

(10) [Aaa]10 is Gin; (11) [Aaa]11 is Arg;

(12) [Aaa]12 is Gin;

(13) [Aaa]13 is Ala or Gin;

(14) [Aaa]14 is Glu;

(15) [Aaa]15 is lie or Leu;

(16) [Aaa]16 is Asp or Glu;

(17) [Aaa]17 is Trp;

(18) [Aaa]18 is Arg or Gly;

(19) [Aaa]19 is Ala;

(20) [Aaa]20 is Gly or Ala;

(21) [Aaa]21 is Pro or Ser;

(22) [Aaa]22 is Ser or Thr;

(23) [Aaa]23 is Gly or Ala; and

(24) [Aaa]24 is Arg or Lys; and/or

(25) [Aaa]25 is Arg or Lys.

In embodiments, [Aaa] 1 is His, [Aaa]2 is Ser or Gly; [Aaa]3 is His or Pro; [Aaa]4 is Phe or lie; [Aaa]5 is Leu or Vai; [Aaa]6 is Ala; [Aaa]7 is Leu or lie; [Aaa]8 is Asp or Glu; [Aaa]9 is Lys or Glu; [Aaa]10 is Gin; [Aaa]11 is Arg; [Aaa]12 is Gin; [Aaa]13 is Ala or Gin; [Aaa]14 is Glu; [Aaa]15 is lie or Leu; [Aaa]16 is Asp or Glu; [Aaa] 17 is Trp; [Aaa]18 is Arg or Gly; [Aaa]19 is Ala; [Aaa]20 is Gly or Ala; [Aaa]21 is Pro or Ser; [Aaa]22 is Ser or Thr; [Aaa]23 is Gly or Ala; [Aaa]24 is Arg or Lys; and [Aaa]25 is Arg or Lys.

In embodiments, the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GLP-1 R. In embodiments, the peptide or the derivative thereof has an antagonistic activity on GLP-1 R. In embodiments, the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GLP-2R. In embodiments, the peptide or the derivative thereof has an antagonistic activity on GLP-2R. In embodiments, the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GIPR. In embodiments, the peptide or the derivative thereof has an antagonistic activity on GIPR. In embodiments, the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GCGR. In embodiments, the peptide or the derivative thereof has an antagonistic activity on GCGR.

In embodiments, the peptide or the derivative thereof has an agonistic activity and an antagonistic activity on GLP-1R, GLP-2R, GIPR, and/or Glucagon Receptor (GCGR).

In embodiments, the peptide or the derivative thereof of any of the embodiments disclosed herein is dual active. In embodiments, the peptide or the derivative thereof an agonistic activity or an antagonistic activity on GLP-1R, and an agonistic activity or an antagonistic activity on GLP-2R. In embodiments, the peptide or the derivative thereof an agonistic activity or an antagonistic activity on GLP-1 R, and an agonistic activity or an antagonistic activity on GIPR. In embodiments, the peptide or the derivative thereof an agonistic activity or an antagonistic activity on GLP-1 R, and an agonistic activity or an antagonistic activity on GCGR. In embodiments, the peptide or the derivative thereof an agonistic activity or an antagonistic activity on GLP- 2R, and an agonistic activity or an antagonistic activity on GIPR. In embodiments, the peptide or the derivative thereof an agonistic activity or an antagonistic activity on GLP-2R, and an agonistic activity or an antagonistic activity on GCGR. In embodiments, the peptide or the derivative thereof an agonistic activity or an antagonistic activity on GIPR, and an agonistic activity or an antagonistic activity on GCGR.

In embodiments, the peptide or the derivative thereof of any of the embodiments disclosed herein is triple active. In embodiments, the peptide or the derivative thereof an agonistic activity or an antagonistic activity on GLP-1 R, an agonistic activity or an antagonistic activity on GLP-2R, and an agonistic activity or an antagonistic activity on GIPR. In embodiments, the peptide or the derivative thereof an agonistic activity or an antagonistic activity on GLP-1 R, an agonistic activity or an antagonistic activity on GLP-2R, and an agonistic activity or an antagonistic activity on GCGR. In embodiments, the peptide or the derivative thereof an agonistic activity or an antagonistic activity on GLP-1 R, an agonistic activity or an antagonistic activity on GIPR, and an agonistic activity or an antagonistic activity on GCGR. In embodiments, the peptide or the derivative thereof an agonistic activity or an antagonistic activity on GLP-2R, an agonistic activity or an antagonistic activity on GIPR, and an agonistic activity or an antagonistic activity on GCGR.

In embodiments, the peptide or the derivative thereof of any of the embodiments disclosed herein is triple active. In embodiments, the peptide or the derivative thereof an agonistic activity or an antagonistic activity on GLP-1 R, an agonistic activity or an antagonistic activity on GLP-2R, an agonistic activity or an antagonistic activity on GIPR, and an agonistic activity or an antagonistic activity on GCGR. In embodiments, the peptide or the derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, which is also referred to herein as SL_TriAg_V1 , comprises the following sequence:

HSHGTFTSDFSLALDKQRQAEFIDWLRAGGPPSGRPPPR (SEQ ID NO: 60).

In embodiments, the SL_TriAg_V1 or a derivative thereof has dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR. In embodiments, the peptide having dual, triple or quadruple actions on GLP- 1 R, GLP-2R, GIPR, and GCGR is a variant of SL_TriAg_V1 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 60. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR has an amino acid sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 60.

In embodiments, the peptide or the derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, which is also referred to herein as SL_TriAg_V2, comprises the following sequence:

HGHGTFTSDISVAIEEQRQQEFLEWLGAAGPSTAKPPPK (SEQ ID NO: 61).

In embodiments, the SL_TriAg_V2 or a derivative thereof has dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR. In embodiments, the peptide having dual, triple or quadruple actions on GLP- 1 R, GLP-2R, GIPR, and GCGR is a variant of SL_TriAg_V2 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 61. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR has an amino acid sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to an amino acid sequence of SEQ ID NO: 61.

In embodiments, the peptide or the derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, which is also referred to herein as SL_TriAg_V3, comprises the following sequence:

HSHGTFTSDVSRIKDKQRQKEFADWLRAQGPPSGRPPPR (SEQ ID NO: 62). In embodiments, the SL_TriAg_V3 or a derivative thereof has dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR. In embodiments, the peptide having dual, triple or quadruple actions on GLP- 1 R, GLP-2R, GIPR, and GCGR is a variant of SL_TriAg_V3 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 62. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1R, GLP-2R, GIPR, and GCGR has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to an amino acid sequence of SEQ ID NO: 62.

In embodiments, the peptide or the derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, which is also referred to herein as SL_TriAg_V4, comprises the following sequence:

HGHGTFTSDSSHGREEQRQREFGEWLGAGGPSTAKPPPK (SEQ ID NO: 63).

In embodiments, the SL_TriAg_V4 or a derivative thereof has dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR. In embodiments, the peptide having dual, triple or quadruple actions on GLP- 1 R, GLP-2R, GIPR, and GCGR is a variant of SL_TriAg_V4 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 63. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR has an amino acid sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 63.

In embodiments, the peptide or the derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, which is also referred to herein as SL_TriAg_V1_Pro3, comprises the following sequence:

HSPGTFTSDFSLALDKQRQAEFIDWLRAGGPPSGRPPPR (SEQ ID NO: 64).

In embodiments, the SL_Tri Ag_V1 _Pro3 or a derivative thereof has dual, triple or quadruple actions on GLP- 1 R, GLP-2R, GIPR, and GCGR. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR is a variant of SL_TriAg_V1_Pro3 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 64. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 64.

In embodiments, the peptide or the derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, which is also referred to herein as SL_TriAg_V2_Pro3, comprises the following sequence:

HGPGTFTSDISVAIEEQRQQEFLEWLGAAGPSTAKPPPK (SEQ ID NO: 65).

In embodiments, the SL_TriAg_V2_Pro3 or a derivative thereof has dual, triple or quadruple actions on GLP- 1 R, GLP-2R, GIPR, and GCGR. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR is a variant of SL_TriAg_V2_Pro3 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 65. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 65.

In embodiments, the peptide or the derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, which is also referred to herein as SL_TriAg_V3_Pro3, comprises the following sequence:

HSPGTFTSDVSRIKDKQRQKEFADWLRAQGPPSGRPPPR (SEQ ID NO: 66).

In embodiments, the SL_TriAg_V3_Pro3 or a derivative thereof has dual, triple or quadruple actions on GLP- 1 R, GLP-2R, GIPR, and GCGR. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR is a variant of SL_TriAg_V3_Pro3 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 66. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 66. In embodiments, the peptide or the derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, which is also referred to herein as SL_TriAg_V4_Pro3, comprises the following sequence:

HGPGTFTSDSSHGREEQRQREFGEWLGAGGPSTAKPPPK (SEQ ID NO: 67)

In embodiments, the SL_TriAg_V4_Pro3 or a derivative thereof has dual, triple or quadruple actions on GLP- 1 R, GLP-2R, GIPR, and GCGR. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR is a variant of SL_TriAg_V4_Pro3 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 67. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 67.

In embodiments, the peptide or the derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, which is also referred to herein as SL-045, comprises the following sequence:

HSHGTFTSDFSLALDKQRQAEFIDWLKAAGPPSAKPPPK (SEQ ID NO: 68)

In embodiments, the SL-045 or a derivative thereof has dual, triple or quadruple actions on GLP-1 R, GLP- 2R, GIPR, and GCGR. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR is a variant of SL-045 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 68. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP- 2R, GIPR, and GCGR has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 68.

In embodiments, the peptide or the derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, which is also referred to herein as SL-044, comprises the following sequence:

HSHGTFTSDFSLALDKQRQAEFIDWLGAAGPSTAKPPPK (SEQ ID NO: 69) In embodiments, the SL-044 or a derivative thereof has dual, triple or quadruple actions on GLP-1 R, GLP- 2R, GIPR, and GCGR. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1R, GLP-2R, GIPR, and GCGR is a variant of SL-044 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 69. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP- 2R, GIPR, and GCGR has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 69.

In embodiments, the peptide or the derivative thereof comprises an amino acid sequence that is selected from the amino acid sequence of SEQ ID NOs: 60 to 69. In embodiments, the peptide or the derivative thereof comprises an amino acid sequence that is selected from the amino acid sequence of SEQ ID NOs: 60 to 69, or a variant thereof having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from SEQ ID NOs: 60 to 69. In embodiments, the peptide or the derivative thereof comprises an amino acid sequence that is selected from the amino acid sequence of SEQ ID NOs: 60, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 68 and SEQ ID NO: 69, or a variant thereof having 1 or 2 amino acid mutations with respect to an amino acid sequence selected from SEQ ID NOs: 60, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 68 and SEQ ID NO: 69. In embodiments, the peptide or the derivative thereof comprises an amino acid sequence that is selected from the amino acid sequence of SEQ ID NOs: 60, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 68 and SEQ ID NO: 69.

In aspects, the present disclosure provides a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence that is selected from the amino acid sequence of SEQ ID NOs: 60 to 69. In embodiments, the peptide or the derivative thereof comprises an amino acid sequence that is selected from the amino acid sequence of SEQ ID NOs: 60 to 69, or a variant thereof having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations with respect to an amino acid sequence selected from SEQ ID NOs: 60 to 69.

In aspects, the present disclosure provides a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, wherein the peptide comprises the following sequence, or a variant thereof:

HSHGTFTSDFSLALDKQRQAEFIDWLRAGGPPSGRPPPR (SEQ ID NO: 60). In embodiments, the peptide or the derivative thereof is a variant of SEQ ID NO: 60 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 60. In embodiments, the peptide the peptide or the derivative thereof has a dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR, and has an amino acid sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 60.

In aspects, the present disclosure provides a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises the following sequence, or a variant thereof:

HGHGTFTSDISVAIEEQRQQEFLEWLGAAGPSTAKPPPK (SEQ ID NO: 61).

In embodiments, the peptide or the derivative thereof is a variant of SEQ ID NO: 61 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 61. In embodiments, the peptide the peptide or the derivative thereof has a dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR, and has an amino acid sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 61 .

In aspects, the present disclosure provides a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises the following sequence, or a variant thereof:

HSHGTFTSDVSRIKDKQRQKEFADWLRAQGPPSGRPPPR (SEQ ID NO: 62).

In embodiments, the peptide or the derivative thereof is a variant of SEQ ID NO: 62 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 62. In embodiments, the peptide the peptide or the derivative thereof has a dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR, and has an amino acid sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 62. In aspects, the present disclosure provides a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises the following sequence, or a variant thereof:

HGHGTFTSDSSHGREEQRQREFGEWLGAGGPSTAKPPPK (SEQ ID NO: 63).

In embodiments, the peptide or the derivative thereof is a variant of SEQ ID NO: 63 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 63. In embodiments, the peptide the peptide or the derivative thereof has a dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR, and has an amino acid sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 63.

In aspects, the present disclosure provides a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises the following sequence, or a variant thereof:

HSPGTFTSDFSLALDKQRQAEFIDWLRAGGPPSGRPPPR (SEQ ID NO: 64).

In embodiments, the peptide or the derivative thereof is a variant of SEQ ID NO: 64 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 64. In embodiments, the peptide the peptide or the derivative thereof has a dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR, and has an amino acid sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 64.

In aspects, the present disclosure provides a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises the following sequence, or a variant thereof:

HGPGTFTSDISVAIEEQRQQEFLEWLGAAGPSTAKPPPK (SEQ ID NO: 65).

In embodiments, the peptide or the derivative thereof is a variant of SEQ ID NO: 65 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 65. In embodiments, the peptide the peptide or the derivative thereof has a dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR, and has an amino acid sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 65.

In aspects, the present disclosure provides a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises the following sequence, or a variant thereof:

HSPGTFTSDVSRIKDKQRQKEFADWLRAQGPPSGRPPPR (SEQ ID NO: 66).

In embodiments, the peptide or the derivative thereof is a variant of SEQ ID NO: 66 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 66. In embodiments, the peptide the peptide or the derivative thereof has a dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR, and has an amino acid sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 66.

In aspects, the present disclosure provides a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises the following sequence, or a variant thereof:

HGPGTFTSDSSHGREEQRQREFGEWLGAGGPSTAKPPPK (SEQ ID NO: 67)

In embodiments, the peptide or the derivative thereof is a variant of SEQ ID NO: 67 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 67. In embodiments, the peptide the peptide or the derivative thereof has a dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR, and has an amino acid sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 67.

In aspects, the present disclosure provides a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises the following sequence, or a variant thereof: HSHGTFTSDFSLALDKQRQAEFIDWLKAAGPPSAKPPPK (SEQ ID NO: 68)

In embodiments, the peptide or the derivative thereof is a variant of SEQ ID NO: 68 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 68. In embodiments, the peptide the peptide or the derivative thereof has a dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR, and has an amino acid sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 68.

In aspects, the present disclosure provides a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises the following sequence, or a variant thereof:

HSHGTFTSDFSLALDKQRQAEFIDWLGAAGPSTAKPPPK (SEQ ID NO: 69)

In embodiments, the peptide or the derivative thereof is a variant of SEQ ID NO: 69 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 69. In embodiments, the peptide the peptide or the derivative thereof has a dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR, and has an amino acid sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 69.

In embodiments, the peptide or the derivative thereof comprises a carrier protein selected from Fc domain, albumin, transferrin, or elastin-like protein, Keyhole Limpet Hemocyanin (KLH), ovalbulin, or a variant thereof.

In embodiments, the peptide or the derivative thereof comprises a carrier protein selected from Fc domain, albumin, transferrin, or elastin-like protein, Keyhole Limpet Hemocyanin (KLH), ovalbulin, or a variant thereof. In embodiments, the carrier protein comprises an Fc domain selected from an IgG Fc domain, an IgA Fc domain, an IgM Fc domain, an IgE Fc domain, and an IgD Fc domain, or a variant thereof. In embodiments, the carrier protein comprises the IgG Fc domain selected from an lgG1 Fc domain, an lgG2 Fc domain, an lgG3 Fc domain, and an lgG4 Fc domain, or a variant thereof. In embodiments, the carrier protein comprises the IgG Fc domain is selected from a human lgG1 Fc domain, and a human lgG4 Fc domain, or a variant thereof. In embodiments, the human lgG1 Fc domain comprises a hinge-CH2-CH3-Fc domain derived from human lgG1 or a derivative thereof. In embodiments, the human I gG4 Fc domain comprises a hinge-CH2- CH3-Fc domain derived from human lgG4 or a derivative thereof.

Fusion Proteins Comprising Peptides having Actions on GLP-1R, GLP-2R, GIPR, and/or Glucagon Receptor (GCGR)

In aspects, the present disclosure provides a fusion protein comprising a carrier protein joined, optionally via a linker, to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP- 1 , GLP-2, GIP, and/or glucagon of any of the embodiments disclosed herein. In embodiments, the peptide comprises a general formula (I):

N-L1-[Aaa]1-[Aaa]2-[Aaa]3-Gly-Thr-Phe-Thr-Ser-Asp-[Aaa]4-Ser-[Aaa]5-[Aaa]6-[Aaa]7-[Aaa]8- [Aaa]9-[Aaa]10-[Aaa]11-[Aaa]12-[Aaa]13-[Aaa]14-Phe-[Aaa]15-[Aaa]16-[Aaa]17-Leu-[Aaa]18- [Aaa]19-[Aaa]20-Gly-Pro-[Aaa]21-[Aaa]22-[Aaa]23-[Aaa]24-Pro-Pro-Pro-[Aaa]25-L2-C

(I) wherein N is the N-terminus; one of L1 or L2 is present the carrier protein, and the other is a tag, and a polymer, optionally wherein the L1 and/or L2 further comprise a linker that adjoins the carrier protein, tag, or polymer with the peptide; [Aaa] 1 is His; [Aaa]2 is Ser or Gly; [Aaa]3 is His or Pro; [Aaa]4 is Phe or lie; [Aaa]5 is Leu or Vai; [Aaa]6 is Ala; [Aaa]7 is Leu or lie; [Aaa]8 is Asp or Glu; [Aaa]9 is Lys or Glu; [Aaa]10 is Gin; [Aaa] 11 is Arg; [Aaa]12 is Gin; [Aaa]13 is Ala or Gin; [Aaa] 14 is Glu; [Aaa]15 is lie or Leu; [Aaa]16 is Asp or Glu; [Aaa]17 is Trp; [Aaa] 18 is Arg or Gly; [Aaa]19 is Ala; [Aaa]20 is Gly or Ala; [Aaa]21 is Pro or Ser; [Aaa]22 is Ser or Thr; [Aaa]23 is Gly or Ala; [Aaa]24 is Arg or Lys; [Aaa]25 is Arg or Lys; and C is the C-terminus. In embodiments, the peptide or the derivative thereof has dual, triple or quadruple actions on GLP-1 R, GLP- 2R, GIPR, and GCGR. In embodiments, the carrier protein is selected from Fc domain, albumin, transferrin, or elastin-like protein, Keyhole Limpet Hemocyanin (KLH), ovalbulin, or a variant thereof. In embodiments, the carrier protein is a hinge CH2-CH3-Fc domain. In embodiments, the a hinge CH2-CH3-Fc domain is derived from I gG1 or lgG4 (e.g., human lgG1 or lgG4).

In aspects, the present disclosure provides a fusion protein comprising a carrier protein joined, optionally via a linker, to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP- 1 , GLP-2, GIP, and/or glucagon of any of the embodiments disclosed herein. In embodiments, the peptide comprises a general formula (II): N-L1 -[Aaa]1-[Aaa]2-[Aaa]3-Gly-Thr-Phe-Thr-Ser-Asp-[Aaa]4-Ser-[Aaa]5-[Aaa]6-[Aaa]7-[Aaa]8- [Aaa]9-[Aaa]10-[Aaa]11-[Aaa]12-[Aaa]13-[Aaa]14-Phe-[Aaa]15-[Aaa]16-[Aaa]17-Leu-[Aaa]18- [Aaa]19-[Aaa]20-Gly-Pro-[Aaa]21-[Aaa]22-[Aaa]23-[Aaa]24-Pro-Pro-Pro-[Aaa]25-L2-C

(II) wherein N is the N-terminus; one of L1 or L2 is present the carrier protein, and the other is a tag, and a polymer, optionally wherein the L1 and/or L2 further comprise a linker that adjoins the carrier protein, tag, or polymer with the peptide; [Aaa] 1 is a positively charged amino acid residue selected from His, Lys, and Arg; [Aaa]2 is a polar and neutral of charge hydrophilic amino acid selected from Ser, Asn, Gin, Thr, and Pro, or a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met; [Aaa]3 is a positively charged amino acid residue selected from His, Lys, and Arg, or a polar and neutral of charge hydrophilic amino acid selected from Pro Ser, Asn, Gin, and Thr; [Aaa]4 is a, hydrophobic, aromatic amino acid selected from Phe, Trp, and Tyr, or a hydrophobic, aliphatic amino acid selected from lie, Gly, Ala, Leu, Met, and Vai; [Aaa]5 is a hydrophobic, aliphatic amino acid selected from Leu, Vai, lie, Gly, Ala, and Met; [Aaa]6 is a hydrophobic, aliphatic amino acid selected from Ala, Gly, Vai, lie, Leu, and Met; [Aaa]7 is a hydrophobic, aliphatic amino acid selected from Leu, lie, Vai, Gly, Ala, and Met; [Aaa]8 is a polar and negatively charged hydrophilic amino acid selected from Asp and Glu; [Aaa]9 is a polar and positively charged hydrophilic amino acid selected from Lys, His, and Arg, or a polar and negatively charged hydrophilic amino acid selected from Glu, and Asp; [Aaa] 10 is a polar and neutral of charge hydrophilic amino acid selected from Gin, Asn, Ser, Thr, and Pro; [Aaa]11 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His; [Aaa]12 is a polar and neutral of charge hydrophilic amino acid selected from Gin, Asn, Ser, Thr, and Pro; [Aaa] 13 is a hydrophobic, aliphatic amino acid selected from Ala, Vai, Gly, Leu, lie, and Met, or a polar and neutral of charge hydrophilic amino acid selected from Gin, Asn, Ser, Thr, and Pro; [Aaa]14 is a polar and negatively charged hydrophilic amino acid selected from Glu, and Asp; [Aaa] 15 is a hydrophobic, aliphatic amino acid selected from lie, Leu, Vai, Ala, Gly, and Met; [Aaa]16 is a polar and negatively charged hydrophilic amino acid selected from Asp, and Glu; [Aaa] 17 is hydrophobic, aromatic amino acid selected from Trp, Phe, and Tyr; [Aaa]18 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His, or a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met; [Aaa]19 is a hydrophobic, aliphatic amino acid selected from Ala, Gly, Vai, Leu, lie, and Met; [Aaa]20 is a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met; [Aaa]21 is a polar and neutral of charge hydrophilic amino acid selected from Pro, Ser, Gin, Asn, and Thr; [Aaa]22 is a polar and neutral of charge hydrophilic amino acid selected from Ser, Thr, Gin, Asn, and Pro; [Aaa]23 a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met; [Aaa]24 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His; [Aaa]25 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His; and C is the C-terminus. In embodiments, the peptide or the derivative thereof present in the fusion protein has dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR. In embodiments, the carrier protein is selected from Fc domain, albumin, transferrin, or elastin-like protein, Keyhole Limpet Hemocyanin (KLH), ovalbulin, or a variant thereof. In embodiments, the carrier protein is a hinge CH2-CH3-Fc domain. In embodiments, the a hinge CH2-CH3-Fc domain is derived from lgG1 or lgG4 (e.g., human I gG1 or I gG4)

In embodiments, [Aaa]1 is His. In embodiments, [Aaa]2 is Ser or Gly. In embodiments, [Aaa]3 is His or Pro. In embodiments, [Aaa]4 is Phe or lie. In embodiments, [Aaa]5 is Leu or Vai. In embodiments, [Aaa]6 is Ala. In embodiments, [Aaa]7 is Leu or lie. In embodiments, [Aaa]8 is Asp or Glu. In embodiments, [Aaa]9 is Lys or Glu. In embodiments, [Aaa]10 is Gin. In embodiments, [Aaa]11 is Arg. In embodiments, [Aaa]12 is Gin. In embodiments, [Aaa]13 is Ala or Gin. In embodiments, [Aaa]14 is Glu. In embodiments, [Aaa]15 is lie or Leu. In embodiments, [Aaa]16 is Asp or Glu. In embodiments, [Aaa]17 is Trp. In embodiments, [Aaa] 18 is Arg or Gly. In embodiments, [Aaa]19 is Ala. In embodiments, [Aaa]20 is Gly or Ala. In embodiments, [Aaa]21 is Pro or Ser. In embodiments, [Aaa]22 is Ser or Thr. In embodiments, [Aaa]23 is Gly or Ala. In embodiments, [Aaa]24 is Arg or Lys. In embodiments, and/or [Aaa]25 is Arg or Lys.

In embodiments, the peptide or the derivative thereof present in the fusion protein comprises any 2 of, or any 3 of, or any 4 of, or any 5 of, or any 6 of, or any 7 of, or any 8 of, or any 9 of, or any 10 of, or any 11 of, or any 12 of, or any 13 of, or any 14 of, or any 15 of, or any 16 of, or any 17 of, or any 18 of, or any 19 of, or any 20 of, or any 21 of, or any 22 of, or any 23 of, or any 24 of, or all 25 of the following:

(1) [Aaa]1 is His;

(2) [Aaa]2 is Ser or Gly;

(3) [Aaa]3 is His or Pro;

(4) [Aaa]4 is Phe or lie;

(5) [Aaa]5 is Leu or Vai;

(6) [Aaa]6 is Ala;

(7) [Aaa]7 is Leu or lie; (8) [Aaa]8 is Asp or Glu;

(9) [Aaa]9 is Lys or Glu;

(10) [Aaa]10 is Gin;

(11) [Aaa]11 is Arg;

(12) [Aaa]12 is Gin;

(13) [Aaa]13 is Ala or Gin;

(14) [Aaa]14 is Glu;

(15) [Aaa]15 is lie or Leu;

(16) [Aaa]16 is Asp or Glu;

(17) [Aaa]17 is Trp;

(18) [Aaa]18 is Arg or Gly;

(19) [Aaa]19 is Ala;

(20) [Aaa]20 is Gly or Ala;

(21) [Aaa]21 is Pro or Ser;

(22) [Aaa]22 is Ser or Thr;

(23) [Aaa]23 is Gly or Ala; and

(24) [Aaa]24 is Arg or Lys; and/or

(25) [Aaa]25 is Arg or Lys.

In embodiments, [Aaa] 1 is His, [Aaa]2 is Ser or Gly; [Aaa]3 is His or Pro; [Aaa]4 is Phe or lie; [Aaa]5 is Leu or Vai; [Aaa]6 is Ala; [Aaa]7 is Leu or lie; [Aaa]8 is Asp or Glu; [Aaa]9 is Lys or Glu; [Aaa]10 is Gin; [Aaa]11 is Arg; [Aaa]12 is Gin; [Aaa]13 is Ala or Gin; [Aaa]14 is Glu; [Aaa]15 is lie or Leu; [Aaa]16 is Asp or Glu; [Aaa] 17 is Trp; [Aaa]18 is Arg or Gly; [Aaa]19 is Ala; [Aaa]20 is Gly or Ala; [Aaa]21 is Pro or Ser; [Aaa]22 is Ser or Thr; [Aaa]23 is Gly or Ala; [Aaa]24 is Arg or Lys; and [Aaa]25 is Arg or Lys.

In embodiments, the peptide or the derivative thereof present in the fusion protein has independently an agonistic activity or an antagonistic activity on GLP-1 R. In embodiments, the peptide or the derivative thereof present in the fusion protein has an antagonistic activity on GLP-1 R. In embodiments, the peptide or the derivative thereof present in the fusion protein has independently an agonistic activity or an antagonistic activity on GLP-2R. In embodiments, the peptide or the derivative thereof present in the fusion protein has an antagonistic activity on GLP-2R. In embodiments, the peptide or the derivative thereof present in the fusion protein has independently an agonistic activity or an antagonistic activity on GIPR. In embodiments, the peptide or the derivative thereof present in the fusion protein has an antagonistic activity on GIPR. In embodiments, the peptide or the derivative thereof present in the fusion protein has independently an agonistic activity or an antagonistic activity on GCGR. In embodiments, the peptide or the derivative thereof present in the fusion protein has an antagonistic activity on GCGR.

In embodiments, the peptide or the derivative thereof present in the fusion protein has an agonistic activity and an antagonistic activity on GLP-1 R, GLP-2R, GIPR, and/or Glucagon Receptor (GCGR).

In embodiments, the peptide or the derivative thereof present in the fusion protein of any of the embodiments disclosed herein is dual active. In embodiments, the peptide or the derivative thereof present in the fusion protein an agonistic activity or an antagonistic activity on GLP-1R, and an agonistic activity or an antagonistic activity on GLP-2R. In embodiments, the peptide or the derivative thereof present in the fusion protein an agonistic activity or an antagonistic activity on GLP-1 R, and an agonistic activity or an antagonistic activity on GIPR. In embodiments, the peptide or the derivative thereof present in the fusion protein an agonistic activity or an antagonistic activity on GLP-1 R, and an agonistic activity or an antagonistic activity on GCGR. In embodiments, the peptide or the derivative thereof present in the fusion protein an agonistic activity or an antagonistic activity on GLP-2R, and an agonistic activity or an antagonistic activity on GIPR. In embodiments, the peptide or the derivative thereof present in the fusion protein an agonistic activity or an antagonistic activity on GLP-2R, and an agonistic activity or an antagonistic activity on GCGR. In embodiments, the peptide or the derivative thereof present in the fusion protein an agonistic activity or an antagonistic activity on GIPR, and an agonistic activity or an antagonistic activity on GCGR.

In embodiments, the peptide or the derivative thereof present in the fusion protein of any of the embodiments disclosed herein is triple active. In embodiments, the peptide or the derivative thereof present in the fusion protein an agonistic activity or an antagonistic activity on GLP-1 R, an agonistic activity or an antagonistic activity on GLP-2R, and an agonistic activity or an antagonistic activity on GIPR. In embodiments, the peptide or the derivative thereof present in the fusion protein an agonistic activity or an antagonistic activity on GLP- 1 R, an agonistic activity or an antagonistic activity on GLP-2R, and an agonistic activity or an antagonistic activity on GCGR. In embodiments, the peptide or the derivative thereof present in the fusion protein an agonistic activity or an antagonistic activity on GLP-1 R, an agonistic activity or an antagonistic activity on GIPR, and an agonistic activity or an antagonistic activity on GCGR. In embodiments, the peptide or the derivative thereof present in the fusion protein an agonistic activity or an antagonistic activity on GLP-2R, an agonistic activity or an antagonistic activity on GIPR, and an agonistic activity or an antagonistic activity on GCGR.

In embodiments, the peptide or the derivative thereof present in the fusion protein of any of the embodiments disclosed herein is triple active. In embodiments, the peptide or the derivative thereof present in the fusion protein an agonistic activity or an antagonistic activity on GLP-1 R, an agonistic activity or an antagonistic activity on GLP-2R, an agonistic activity or an antagonistic activity on GIPR, and an agonistic activity or an antagonistic activity on GCGR.

In embodiments, the fusion protein comprises a carrier protein selected from an Fc domain, albumin, transferrin, or elastin-like protein, Keyhole Limpet Hemocyanin (KLH), ovalbulin, or a variant thereof. In embodiments, the fusion protein comprises a hinge CH2-CH3-Fc domain. In embodiments, the fusion protein comprises an Fc domain of an antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., lgG1 , lgG2, lgG3, and I gG4, and I gA1 and lgA2)). In embodiments, the fusion protein comprises a hinge-CH2-CH3 Fc domain derived from lgG4, optionally human I gG4. In embodiments, the fusion protein comprises a hinge- CH2-CH3 Fc domain derived from lgG1 , optionally human lgG1.

In embodiments, the fusion protein comprises a hinge-CH2-CH3 Fc domain derived from lgG4. In embodiments, the fusion protein comprises a hinge-CH2-CH3 Fc domain derived from a human lgG4. In embodiments, the hinge-CH2-CH3 Fc domain has an amino acid sequence that has at least about 95%, or at least about 97%, or at least about 97%, or at least about 98% sequence identity with the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 4. In embodiments, the hinge-CH2-CH3 Fc domain has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2. In embodiments, the hinge-CH2-CH3 Fc domain has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4.

In embodiments, the hinge-CH2-CH3 Fc domain is derived from a human lgG1 antibody. In embodiments, the Fc domain exhibits increased affinity for and enhanced binding to the neonatal Fc receptor (FcRn). In embodiments, the Fc domain includes one or more mutations that increases the affinity and enhances binding to FcRn. Without wishing to be bound by theory, it is believed that increased affinity and enhanced binding to FcRn increases the in vivo half-life of the present fusion proteins. In embodiments, the Fc domain contains one or more amino acid substitutions at amino acid residue 250, 252, 254, 256, 308, 309, 311, 416, 428, 433 or 434 (in accordance with Kabat numbering, as in as in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) expressly incorporated herein by reference), or equivalents thereof. In embodiments, the amino acid substitution at amino acid residue 250 is a substitution with glutamine. In embodiments, the amino acid substitution at amino acid residue 252 is a substitution with tyrosine, phenylalanine, tryptophan or threonine. In embodiments, the amino acid substitution at amino acid residue 254 is a substitution with threonine. In embodiments, the amino acid substitution at amino acid residue 256 is a substitution with serine, arginine, glutamine, glutamic acid, aspartic acid, or threonine. In embodiments, the amino acid substitution at amino acid residue 308 is a substitution with threonine. In embodiments, the amino acid substitution at amino acid residue 309 is a substitution with proline. In embodiments, the amino acid substitution at amino acid residue 311 is a substitution with serine. In embodiments, the amino acid substitution at amino acid residue 385 is a substitution with arginine, aspartic acid, serine, threonine, histidine, lysine, alanine or glycine. In embodiments, the amino acid substitution at amino acid residue 386 is a substitution with threonine, proline, aspartic acid, serine, lysine, arginine, isoleucine, or methionine. In embodiments, the amino acid substitution at amino acid residue 387 is a substitution with arginine, proline, histidine, serine, threonine, or alanine. In embodiments, the amino acid substitution at amino acid residue 389 is a substitution with proline, serine or asparagine. In embodiments, the amino acid substitution at amino acid residue 416 is a substitution with serine. In embodiments, the amino acid substitution at amino acid residue 428 is a substitution with leucine. In embodiments, the amino acid substitution at amino acid residue 433 is a substitution with arginine, serine, isoleucine, proline, or glutamine. In embodiments, the amino acid substitution at amino acid residue 434 is a substitution with histidine, phenylalanine, or tyrosine.

In embodiments, the Fc domain (e.g., comprising an IgG constant region) comprises one or more mutations such as substitutions at amino acid residue 252, 254, 256, 433, 434, or 436 (in accordance with Kabat numbering, as in as in Kabat, ef al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) expressly incorporated herein by reference). In embodiments, the Fc domain of the present fusion proteins comprise an IgG constant region includes a triple M252Y/S254T/T256E mutation or YTE mutation. In embodiments, the IgG constant region includes a triple H433K/N434F/Y436H mutation or KFH mutation. In embodiments, the IgG constant region includes an YTE and KFH mutation in combination. In embodiments, the IgG constant region that contains one or more mutations at amino acid residues 250, 253, 307, 310, 380, 428, 433, 434, and 435 (in accordance with Kabat numbering, as in as in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) expressly incorporated herein by reference). Illustrative mutations include T250Q, M428L, T307A, E380A, I253A, H310A, M428L, H433K, N434A, N434F, N434S, and H435A. In embodiments, the IgG constant region comprises a M428L/N434S mutation or LS mutation. In embodiments, the IgG constant region comprises a T250Q/M428L mutation or QL mutation. In embodiments, the IgG constant region comprises an N434A mutation. In embodiments, the IgG constant region comprises a T307A/E380A/N434A mutation or AAA mutation. In embodiments, the IgG constant region comprises an I253A/H310A/H435A mutation or y mutation. In embodiments, the IgG constant region comprises a H433K/N434F mutation. In embodiments, the IgG constant region comprises a M252Y/S254T/T256E and a H433K/N434F mutation in combination. In embodiments, the IgG constant region comprises mutations that decrease Fc effector function (e.g., L234A and L235A mutations (LALA) with or without G236R and/or P329G mutations).

Additional exemplary mutations in the IgG constant region are described, for example, in Robbie, et al., Antimicrobial Agents and Chemotherapy (2013), 57(12):6147-6153, Dall’Acqua et al., JBC (2006), 281 (33):23514-24, Dall’Acqua et al., Journal of Immunology (2002), 169:5171-80, Ko et al. Nature (2014) 514:642-645, Grevys etal. Journal of Immunology. (2015), 194(11):5497-508, and U.S. Patent No. 7,083,784, the entire contents of which are hereby incorporated by reference.

In embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 1 (see Table 1), or at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, mutations are made to SEQ ID NO: 1 to increase stability and/or half-life. For instance, in embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 2 (see Table 1), or at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. An illustrative Fc stabilizing mutant is S228P. Illustrative Fc half-life extending mutants are T250Q, M428L, V308T, L309P, and Q311 S and the Fc domains may comprise 1 , or 2, or 3, or 4, or 5 of these mutants. In embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 3 (see Table 1), or at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 4 (see Table 1), or at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

In embodiments, the fusion protein binds to FcRn with high affinity. In embodiments, the fusion protein may bind to FcRn with a KD of about 1 nM to about 80 nM. For example, the fusion protein may bind to FcRn with a KD of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM, about 70 nM, about 71 nM, about 72 nM, about 73 nM, about 74 nM, about 75 nM, about 76 nM, about 77 nM, about 78 nM, about 79 nM, or about 80 nM. In embodiments, the fusion protein may bind to FcRn with a KD of about 9 nM. In embodiments, the fusion protein does not substantially bind to other Fc receptors (/.e., other than FcRn) with effector function.

In embodiments, the Fc domain comprises a hinge region of an antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., lgG1 , lgG2, lgG3, and lgG4, and lgA1 , and lgA2)). The hinge region, found in IgG, IgA, IgD, and IgE class antibodies, acts as a flexible spacer, allowing the Fab portion to move freely in space. In contrast to the constant regions, the hinge domains are structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses. For example, the length and flexibility of the hinge region varies among the IgG subclasses. The hinge region of lgG1 encompasses amino acids 216-231 and, because it is freely flexible, the Fab fragments can rotate about their axes of symmetry and move within a sphere centered at the first of two inter-heavy chain disulfide bridges. I gG2 has a shorter hinge than lgG1 , with 12 amino acid residues and four disulfide bridges. The hinge region of lgG2 lacks a glycine residue, is relatively short, and contains a rigid poly-proline double helix, stabilized by extra inter-heavy chain disulfide bridges. These properties restrict the flexibility of the I gG2 molecule. lgG3 differs from the other subclasses by its unique extended hinge region (about four times as long as the I gG 1 hinge), containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible poly-proline double helix. In lgG3, the Fab fragments are relatively far away from the Fc fragment, giving the molecule a greater flexibility. The elongated hinge in I gG3 is also responsible for its higher molecular weight compared to the other subclasses. The hinge region of I gG4 is shorter than that of lgG1 and its flexibility is intermediate between that of lgG1 and lgG2. The flexibility of the hinge regions reportedly decreases in the order lgG3>lgG1>lgG4>lgG2. In embodiments, the linker may be derived from human lgG4 and contain one or more mutations to enhance dimerization (including S228P) or FcRn binding. According to crystallographic studies, the immunoglobulin hinge region can be further subdivided functionally into three regions: the upper hinge region, the core region, and the lower hinge region. See Shin et al., 1992 Immunological Reviews 130:87. The upper hinge region includes amino acids from the carboxyl end of CHI to the first residue in the hinge that restricts motion, generally the first cysteine residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the segmental flexibility of the antibody. The core hinge region contains the inter-heavy chain disulfide bridges, and the lower hinge region joins the amino terminal end of the CH2 domain and includes residues in CH2. Id. The core hinge region of wild-type human lgG1 contains the sequence CPPC (SEQ ID NO: 24) which, when dimerized by disulfide bond formation, results in a cyclic octapeptide believed to act as a pivot, thus conferring flexibility. In embodiments, the present linker comprises, one, or two, or three of the upper hinge regions, the core region, and the lower hinge region of any antibody (e.g., of IgG, IgA, I g D, and IgE, inclusive of subclasses (e.g., lgG1 , lgG2, lgG3, and lgG4, and lgA1 and lgA2)). The hinge region may also contain one or more glycosylation sites, which include a number of structurally distinct types of sites for carbohydrate attachment. For example, lgA1 contains five glycosylation sites within a 17-amino-acid segment of the hinge region, conferring resistance of the hinge region polypeptide to intestinal proteases, considered an advantageous property for a secretory immunoglobulin. In embodiments, the linker of the present disclosure comprises one or more glycosylation sites.

In embodiments, the fusion protein disclosed herein comprise a linker. In embodiments, the linker is a polypeptide selected from a flexible amino acid sequence or an IgG hinge region. In embodiments, the linker is derived from naturally occurring multi-domain proteins or is an empirical linker as described, for example, in Chichili et al., (2013), Protein Sci. 22(2):153-167, Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357- 1369, the entire contents of which are hereby incorporated by reference. In embodiments, the linker may be designed using linker designing databases and computer programs such as those described in Chen et al., (2013), Adv Drug Deliv Rev. 65(10): 1357-1369 and Crasto et. al., (2000), Protein Eng. 13(5):309-312, the entire contents of which are hereby incorporated by reference.

In embodiments, the linker is a synthetic linker such as PEG.

In embodiments, the linker is flexible.

In embodiments, the linker is rigid. In embodiments, the linker is substantially comprised of glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97%, or about 98%, or about 99%, or about 100% glycines and serines).

In embodiments, the , the linker comprises an amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 5-53 (or a variant thereof).

In embodiments, the present fusion proteins may comprise variants of the linkers disclosed in Table 1, below. For instance, a linker may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71 %, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81 %, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 5-53.

Without wishing to be bound by theory, including at least a part of an Fc domain in the fusion protein, helps avoid formation of insoluble and, likely, non-functional protein concatamers and/or aggregates. This is in part due to the presence of cysteines in the Fc domain which are capable of forming disulfide bonds between fusion proteins.

In embodiments, the first and/or second linkers are independently selected from the amino acid sequences of SEQ ID NOs: 5-53 and are provided in Table 1 below:

Table 1 : Illustrative linkers (Fc domains and linkers)

In embodiments, the linker substantially comprises glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97%, or about 98%, or about 99%, or about 100% glycines and serines). For example, in embodiments, the linker is (Gly4Ser)_n, where n is from about 1 to about 8, e.g., 1 , 2, 3, 4, 5, 6, 7, or 8 (SEQ ID NO: 26 to SEQ ID NO: 33, respectively). In embodiments, the linker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO: 34). Additional illustrative linkers include, but are not limited to, linkers having the sequence LE, (EAAAK)n (n=1 - 3) (SEQ ID NO: 37 to SEQ ID NO: 39), A(EAAAK)_nA (n = 2-5) (SEQ ID NO: 40 to SEQ ID NO: 43), A(EAAAK)₄ALEA(EAAAK)4A (SEQ ID NO: 44), PAPAP (SEQ ID NO: 45), KESGSVSSEQLAQFRSLD (SEQ ID NO: 46), GSAGSAAGSGEF (SEQ ID NO: 47), and (XP)_n, with X designating any amino acid, e.g., Ala, Lys, or Glu. In embodiments, a linker has the sequence (Gly)n where n is any number from 1 to 100, for example: (Gly)s (SEQ ID NO: 35) and (Gly)e (SEQ ID NO: 36). In embodiments, the linker has the amino acid sequence GGS (SEQ ID NO: 4), or GS or LE.

In embodiments, the linker is one or more of GGGSE (SEQ ID NO: 48), GSESG (SEQ ID NO: 49), GSEGS (SEQ ID NO: 50), GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 51), and a linker of randomly placed G, S, and E every 4 amino acid intervals. In embodiments, the linker has the amino acid sequence EPKSCDKTHTCP (SEQ ID NO: 52). In embodiments, the linker has the amino acid sequence EPKSVDKTHTCP (SEQ ID NO: 53).

In embodiments, the fusion protein comprises a hinge-CH2-CH3 Fc domain (e.g., comprising an amino acid sequence having at least 95% sequence identity with one of SEQ ID NOs: 1-4), joined to the peptide or a derivative thereof of any of the embodiments disclosed herein via a linker having an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused via a linker to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence selected from SEQ ID NOs: 60 to 69, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NOs: 60 to 69. In embodiments, the hinge CH2-CH3-Fc domain comprises an amino acid sequence that at least 95% sequence identical to the amino acid sequence of one of SEQ ID NOs: 1-4. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53, or an amino acid sequence having about 1, 2, 3 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NOs: 5-53. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused via a linker to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 60, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 60. In embodiments, the hinge CH2- CH3-Fc domain comprises an amino acid sequence that at least 95% sequence identical to the amino acid sequence of one of SEQ ID NOs: 1-4. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53, or an amino acid sequence having about 1 , 2, 3 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NOs: 5-53. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused via a linker to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 61 , or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 61. In embodiments, the hinge CH2- CH3-Fc domain comprises an amino acid sequence that at least 95% sequence identical to the amino acid sequence of one of SEQ ID NOs: 1-4. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53, or an amino acid sequence having about 1 , 2, 3 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NOs: 5-53. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53. In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused via a linker to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 62, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 62. In embodiments, the hinge CH2- CH3-Fc domain comprises an amino acid sequence that at least 95% sequence identical to the amino acid sequence of one of SEQ ID NOs: 1-4. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53, or an amino acid sequence having about 1 , 2, 3 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NOs: 5-53. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused via a linker to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 63, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 63. In embodiments, the hinge CH2- CH3-Fc domain comprises an amino acid sequence that at least 95% sequence identical to the amino acid sequence of one of SEQ ID NOs: 1-4. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53, or an amino acid sequence having about 1 , 2, 3 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NOs: 5-53. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused via a linker to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 64, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 64. In embodiments, the hinge CH2- CH3-Fc domain comprises an amino acid sequence that at least 95% sequence identical to the amino acid sequence of one of SEQ ID NOs: 1-4. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53, or an amino acid sequence having about 1 , 2, 3 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NOs: 5-53. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused via a linker to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 65, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 65. In embodiments, the hinge CH2- CH3-Fc domain comprises an amino acid sequence that at least 95% sequence identical to the amino acid sequence of one of SEQ ID NOs: 1-4. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53, or an amino acid sequence having about 1 , 2, 3 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NOs: 5-53. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused via a linker to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 66, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 66. In embodiments, the hinge CH2- CH3-Fc domain comprises an amino acid sequence that at least 95% sequence identical to the amino acid sequence of one of SEQ ID NOs: 1-4. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53, or an amino acid sequence having about 1 , 2, 3 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NOs: 5-53. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53. In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused via a linker to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 67, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 67. In embodiments, the hinge CH2- CH3-Fc domain comprises an amino acid sequence that at least 95% sequence identical to the amino acid sequence of one of SEQ ID NOs: 1-4. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53, or an amino acid sequence having about 1 , 2, 3 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NOs: 5-53. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused via a linker to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 68, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 68. In embodiments, the hinge CH2- CH3-Fc domain comprises an amino acid sequence that at least 95% sequence identical to the amino acid sequence of one of SEQ ID NOs: 1-4. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53, or an amino acid sequence having about 1 , 2, 3 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NOs: 5-53. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused via a linker to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 69, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 69. In embodiments, the hinge CH2- CH3-Fc domain comprises an amino acid sequence that at least 95% sequence identical to the amino acid sequence of one of SEQ ID NOs: 1-4. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53, or an amino acid sequence having about 1 , 2, 3 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NOs: 5-53. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53.

In aspects, the present disclosure provides a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 60, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 60. In embodiments, the peptide comprises an amino acid sequence of SEQ ID NO: 60.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP- 2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 61 , or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 61. In embodiments, the peptide comprises an amino acid sequence of SEQ ID NO: 61 .

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP- 2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 62, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 62. In embodiments, the peptide comprises an amino acid sequence of SEQ ID NO: 62.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP- 2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 63, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 63. In embodiments, the peptide comprises an amino acid sequence of SEQ ID NO: 63.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP- 2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 64, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 64. In embodiments, the peptide comprises an amino acid sequence of SEQ ID NO: 64.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP- 2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 65, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 65. In embodiments, the peptide comprises an amino acid sequence of SEQ ID NO: 65.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP- 2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 66, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 66. In embodiments, the peptide comprises an amino acid sequence of SEQ ID NO: 66.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP- 2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 67, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 67. In embodiments, the peptide comprises an amino acid sequence of SEQ ID NO: 67.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP- 2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 68, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 68. In embodiments, the peptide comprises an amino acid sequence of SEQ ID NO: 68.

In aspects, the present disclosure provides a fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP- 2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 69, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 69. In embodiments, the peptide comprises an amino acid sequence of SEQ ID NO: 69.

In embodiments, the hinge CH2-CH3-Fc domain is derived from IgG, IgM, IgA, IgD, or IgE. In embodiments, the IgG is selected from lgG1, lgG2, lgG3, and lgG4. In embodiments, the hinge CH2-CH3-Fc domain is derived from lgG1. In embodiments, the lgG1 is human lgG1. In embodiments, the hinge CH2-CH3-Fc domain comprises an amino acid sequence that is at least about 95%, or at least about 97%, or at least about 97%, or at least about 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 4.

In embodiments, the hinge-CH2-CH3 Fc domain is derived from lgG4. In embodiments, the lgG4 is human lgG4. In embodiments, the hinge CH2-CH3-Fc domain comprises an amino acid sequence that is at least about 95%, or at least about 97%, or at least about 97%, or at least about 98% or at least 99% identical to the amino acid sequence SEQ ID NO: 1 , SEQ ID NO: 2, or SEQ ID NO: 3. In embodiments, the hinge CH2- CH3-Fc domain is fused to the peptide or a derivative thereof via a linker. In embodiments, the linker has an amino acid sequence selected from SEQ ID NOs: 5 to 53.

In embodiments, the peptide or the derivative thereof or the fusion protein further comprises a glycosyl moiety. In embodiments, the glycosyl moiety is an N-linked glycosyl moiety. In embodiments, the glycosyl moiety is an O-linked glycosyl moiety. In embodiments, the peptide or the derivative thereof or the fusion protein comprises one or more N-linked glycosylation consensus sites and/or O-linked glycosylation consensus sites.

In embodiments, the peptide or the derivative thereof or the fusion protein is biosynthesized as a single polypeptide chain. In embodiments, the peptide or the derivative thereof or the fusion protein is biosynthesized from a single open reading frame. In embodiments, the peptide or the derivative thereof or the fusion protein is prepared using an expression system. In embodiments, the expression system is selected from bacterial, yeast, invertebrate, vertebrate, and plant expression system.

In embodiments, the peptide or the derivative thereof or the fusion protein or the fusion protein is chemically synthesized. In embodiments, the peptide or the derivative thereof or the fusion protein is produced by in vitro translation of a polynucleotide encoding the peptide or the derivative thereof or the fusion protein. In embodiments, the polynucleotide encoding the peptide or the derivative thereof or the fusion protein of any of the embodiments disclosed herein is used for in vitro translation.

In embodiments, the peptide or the derivative thereof or the fusion protein is then chemically modified. The chemical modifications include but are not limited to chemical modification of side chain of an amino acid, the N-terminus and/or the C-terminus. In embodiments, the chemical modification is carried out via conjugation of side chain of an amino acid, the N-terminus and/or the C-terminus to a chemical entity (without limitation, e.g., a polymer such as PEG). In embodiments, the chemical modification or the conjugation is performed directly on side chain of an amino acid, the N-terminus and/or the C-terminus. In embodiments, the chemical modification or the conjugation uses a suitable linker that adjoins the conjugated chemical entity with side chain of an amino acid, the N-terminus and/or the C-terminus.

In embodiments, the peptide or the derivative thereof or the fusion protein further comprises a non-natural amino acid selected from an amino isobutyric acid (Aib), a D-amino acid, and those comprising a modification selected from N-methylation (Nm), Ca-methylation (Cm), 4^J(CH2NH) reduced amide bonds (Rd), and a peptoids (Pp)).

In embodiments, the peptide or the derivative thereof or the fusion protein comprises a polymer. In embodiments, the polymer is selected from poly(alkylene oxide) (e.g., polyethylene glycol (PEG)), polyvinylpyrrolidone), poly(vinyl alcohol), poly(glycerol), poly(zwitterions), poly(carbonates), polyoxazoline, poly(acryloylmorpholine), poly(oxazolines), poly(sacharrides), and a combination thereof. In embodiments, the polymer is polyethylene glycol (PEG). In embodiments, one or more amino acids present in the peptide or the derivative thereof or the fusion protein is PEGylated. In embodiments, the one or more PEGylated amino acids are located inside the carrier protein. In embodiments, the PEGylation is conducted using a succinimidyl ester, an aldehyde, a maleimide, and/or a p-nitrophenyl carbonate ester reagent. In embodiments, one or more Lys residues, one or more Ser residues, one or more Tyr residues, one or more His residues, one or more Cys residues, the N-terminus and/or the C-terminus are PEGylated. In embodiments, at least about 1 , or at least about 3, or at least about 5, or at least about 8, or at least about 10, or more amino acid residues, the N-terminus and/or the C-terminus are PEGylated. In embodiments, the one or more PEGylated amino acids comprise a Lys. In embodiments, the PEGylation is conducted via amine conjugation. In embodiments, the one or more PEGylated amino acids comprise a Gin. In embodiments, the PEGylation is conducted via transglutaminase (TGase) mediated enzymatic conjugation. In embodiments, the one or more PEGylated amino acids comprise a Cys. In embodiments, the PEGylation is conducted via thiol conjugation.

In embodiments, the peptide or the derivative thereof or the fusion protein has independently an agonistic activity or an antagonistic activity on glucagon-like peptide- 1 receptor (GLP-1 R), glucagon-like peptide-2 receptor (GLP-2R), gastric inhibitory peptide receptor (GIPR), and/or glucagon receptor (GCGR).

In embodiments, the peptide or the derivative thereof or the fusion protein of any of the embodiments disclosed herein is dual active. In embodiments, the peptide or the derivative thereof or the fusion protein has independently an agonistic activity or an antagonistic activity on GLP-1 R and GCGR. In embodiments, the peptide or the derivative thereof or the fusion protein has independently an agonistic activity or an antagonistic activity on GLP-1 R and GIPR. In embodiments, the peptide or the derivative thereof or the fusion protein has independently an agonistic activity or an antagonistic activity on GLP-2R and GIPR. In embodiments, the peptide or the derivative thereof or the fusion protein has independently an agonistic activity or an antagonistic activity on GLP-2R and GCGR. In embodiments, the peptide or the derivative thereof or the fusion protein has independently an agonistic activity or an antagonistic activity on GLP-1 R and GLP-2R. In embodiments, the peptide or the derivative thereof or the fusion protein has independently an agonistic activity or an antagonistic activity on GIPR and GCGR.

In embodiments, the peptide or the derivative thereof or the fusion protein of any of the embodiments disclosed herein is triple active. In embodiments, the peptide or the derivative thereof or the fusion protein has independently an agonistic activity or an antagonistic activity on GLP-1 R, GIPR and GCGR. In embodiments, the peptide or the derivative thereof or the fusion protein has independently an agonistic activity or an antagonistic activity on GLP-1 R, GLP-2R and GCGR. In embodiments, the peptide or the derivative thereof or the fusion protein has independently an agonistic activity or an antagonistic activity on GLP-1R, GLP-2R and GIPR. In embodiments, the peptide or the derivative thereof or the fusion protein has independently an agonistic activity or an antagonistic activity on GLP-2R, GIPR and GCGR.

In embodiments, the peptide or the derivative thereof or the fusion protein of any of the embodiments disclosed herein is quadruple active. In embodiments, the peptide or the derivative thereof or the fusion protein has independently an agonistic activity or an antagonistic activity on GLP-1 R, GLP-2R, GIPR and GCGR.

In embodiments, the peptide or the derivative thereof or the fusion protein exhibits an ECso value for GLP- 1 R in the range of 100 pM to 1000 nM, as determined by a cell-based assay. In embodiments, the peptide or the derivative thereof or the fusion protein exhibits an EC50 value for GLP-1 R in the range of 2 nM to 100 nM, as determined by a cell-based assay.

In embodiments, the peptide or the derivative thereof or the fusion protein exhibits an EC50 value for GIPR in the range of 100 pM to 1000 nM, as determined by a cell-based assay. In embodiments, the peptide or the derivative thereof or the fusion protein exhibits an EC50 value for GIPR in the range of 100 pM to 2 nM, as determined by a cell-based assay.

In embodiments, the peptide or the derivative thereof or the fusion protein exhibits an EC50 value for GCGR in the range of 100 pM to 1000 nM, as determined by a cell-based assay. In embodiments, the peptide or the derivative thereof or the fusion protein exhibits an EC50 value for GCGR in the range of 2 nM to 100 nM, as determined by a cell-based assay. In embodiments, the peptide or the derivative thereof or the fusion protein exhibits an IC50 value for GLP-1 R in the range of 10 pM to 10 pM, as determined by a cell-based assay. In embodiments, the peptide or the derivative thereof or the fusion protein exhibits an IC50 value for GIPR in the range of 10 pM to 10 pM, as determined by a cell-based assay. In embodiments, the peptide or the derivative thereof or the fusion protein exhibits an I C50 value for GCGR in the range of 10 pM to 10 pM, as determined by a cell-based assay. In embodiments, the peptide or the derivative thereof or the fusion protein exhibits an EC50 value for GLP-2R in the range of 10 pM to 10 pM, as determined by a cell-based assay. In embodiments, the peptide or the derivative thereof or the fusion protein exhibits an I C50 value for GLP-2R in the range of 10 pM to 10 pM, as determined by a cell-based assay. Isolated Polynucleotide Encoding the Peptide, or the fusion protein, or the Derivative thereof

In aspects, the present disclosure provides an isolated polynucleotide encoding the peptide or the derivative thereof of any of the embodiments disclosed herein.

In aspects, the present disclosure provides an isolated polynucleotide encoding a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, wherein the peptide comprises a general formula (I):

N-L1 -[Aaa]1-[Aaa]2-[Aaa]3-Gly-Thr-Phe-Thr-Ser-Asp-[Aaa]4-Ser-[Aaa]5-[Aaa]6-[Aaa]7-[Aaa]8- [Aaa]9-[Aaa]10-[Aaa]11-[Aaa]12-[Aaa]13-[Aaa]14-Phe-[Aaa]15-[Aaa]16-[Aaa]17-Leu-[Aaa]18- [Aaa]19-[Aaa]20-Gly-Pro-[Aaa]21-[Aaa]22-[Aaa]23-[Aaa]24-Pro-Pro-Pro-[Aaa]25-L2-C

(I) wherein N is the N-terminus; L1 and/or L2 are independently present or absent; and L1 and/or L2, if present, are each independently selected from a carrier protein, a tag, and a polymer, optionally wherein the L1 and/or L2 further comprise a linker that adjoins the carrier protein, tag, or polymer with the peptide; [Aaa] 1 is His; [Aaa]2 is Ser or Gly; [Aaa]3 is His or Pro; [Aaa]4 is Phe or lie; [Aaa]5 is Leu or Vai; [Aaa]6 is Ala; [Aaa]7 is Leu or lie; [Aaa]8 is Asp or Glu; [Aaa]9 is Lys or Glu; [Aaa]10 is Gin; [Aaa]11 is Arg; [Aaa]12 is Gin; [Aaa]13 is Ala or Gin; [Aaa]14 is Glu; [Aaa]15 is lie or Leu; [Aaa]16 is Asp or Glu; [Aaa]17 is Trp; [Aaa]18 is Arg or Gly; [Aaa] 19 is Ala; [Aaa]20 is Gly or Ala; [Aaa]21 is Pro or Ser; [Aaa]22 is Ser or Thr; [Aaa]23 is Gly or Ala; [Aaa]24 is Arg or Lys; [Aaa]25 is Arg or Lys; and C is the C-terminus. In embodiments, the isolated polynucleotide encodes the peptide or the derivative thereof has dual, triple or quadruple actions on GLP- 1 R, GLP-2R, GIPR, and GCGR.

In aspects, the present disclosure provides an isolated polynucleotide encoding a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, wherein the peptide comprises formula (II):

N-L1 -[Aaa]1-[Aaa]2-[Aaa]3-Gly-Thr-Phe-Thr-Ser-Asp-[Aaa]4-Ser-[Aaa]5-[Aaa]6-[Aaa]7-[Aaa]8- [Aaa]9-[Aaa]10-[Aaa]11-[Aaa]12-[Aaa]13-[Aaa]14-Phe-[Aaa]15-[Aaa]16-[Aaa]17-Leu-[Aaa]18- [Aaa]19-[Aaa]20-Gly-Pro-[Aaa]21-[Aaa]22-[Aaa]23-[Aaa]24-Pro-Pro-Pro-[Aaa]25-L2-C

(II) wherein: N is the N-terminus; L1 and/or L2 are independently present or absent; and L1 and/or L2, if present, are each independently selected from a carrier protein, a tag, and a polymer; [Aaa] 1 is a positively charged amino acid residue selected from His, Lys, and Arg; [Aaa]2 is a polar and neutral of charge hydrophilic amino acid selected from Ser, Asn, Gin, Thr, and Pro, or a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met; [Aaa]3 is a positively charged amino acid residue selected from His, Lys, and Arg, or a polar and neutral of charge hydrophilic amino acid selected from Pro Ser, Asn, Gin, and Thr; [Aaa]4 is a, hydrophobic, aromatic amino acid selected from Phe, Trp, and Tyr, or a hydrophobic, aliphatic amino acid selected from lie, Gly, Ala, Leu, Met, and Vai; [Aaa]5 is a hydrophobic, aliphatic amino acid selected from Leu, Vai, lie, Gly, Ala, and Met; [Aaa]6 is a hydrophobic, aliphatic amino acid selected from Ala, Gly, Vai, lie, Leu, and Met; [Aaa]7 is a hydrophobic, aliphatic amino acid selected from Leu, lie, Vai, Gly, Ala, and Met; [Aaa]8 is a polar and negatively charged hydrophilic amino acid selected from Asp and Glu; [Aaa]9 is a polar and positively charged hydrophilic amino acid selected from Lys, His, and Arg, or a polar and negatively charged hydrophilic amino acid selected from Glu, and Asp; [Aaa]10 is a polar and neutral of charge hydrophilic amino acid selected from Gin, Asn, Ser, Thr, and Pro; [Aaa] 11 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His; [Aaa]12 is a polar and neutral of charge hydrophilic amino acid selected from Gin, Asn, Ser, Thr, and Pro; [Aaa]13 is a hydrophobic, aliphatic amino acid selected from Ala, Vai, Gly, Leu, lie, and Met, or a polar and neutral of charge hydrophilic amino acid selected from Gin, Asn, Ser, Thr, and Pro; [Aaa]14 is a polar and negatively charged hydrophilic amino acid selected from Glu, and Asp; [Aaa]15 is a hydrophobic, aliphatic amino acid selected from lie, Leu, Vai, Ala, Gly, and Met; [Aaa]16 is a polar and negatively charged hydrophilic amino acid selected from Asp, and Glu; [Aaa]17 is hydrophobic, aromatic amino acid selected from Trp, Phe, and Tyr; [Aaa] 18 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His, or a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met; [Aaa] 19 is a hydrophobic, aliphatic amino acid selected from Ala, Gly, Vai, Leu, lie, and Met; [Aaa]20 is a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met; [Aaa]21 is a polar and neutral of charge hydrophilic amino acid selected from Pro, Ser, Gin, Asn, and Thr; [Aaa]22 is a polar and neutral of charge hydrophilic amino acid selected from Ser, Thr, Gin, Asn, and Pro; [Aaa]23 a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met; [Aaa]24 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His; [Aaa]25 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His; and C is the C-terminus. In embodiments, the isolated polynucleotide encodes the peptide or the derivative thereof has dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR.

In embodiments, [Aaa]1 is His. In embodiments, [Aaa]2 is Ser or Gly. In embodiments, [Aaa]3 is His or Pro. In embodiments, [Aaa]4 is Phe or lie. In embodiments, [Aaa]5 is Leu or Vai. In embodiments, [Aaa]6 is Ala. In embodiments, [Aaa]7 is Leu or lie. In embodiments, [Aaa]8 is Asp or Glu. In embodiments, [Aaa]9 is Lys or Glu. In embodiments, [Aaa]10 is Gin. In embodiments, [Aaa]11 is Arg. In embodiments, [Aaa]12 is Gin. In embodiments, [Aaa]13 is Ala or Gin. In embodiments, [Aaa]14 is Glu. In embodiments, [Aaa]15 is lie or Leu. In embodiments, [Aaa]16 is Asp or Glu. In embodiments, [Aaa]17 is Trp. In embodiments, [Aaa] 18 is Arg or Gly. In embodiments, [Aaa]19 is Ala. In embodiments, [Aaa]20 is Gly or Ala. In embodiments, [Aaa]21 is Pro or Ser. In embodiments, [Aaa]22 is Ser or Thr. In embodiments, [Aaa]23 is Gly or Ala. In embodiments, [Aaa]24 is Arg or Lys. In embodiments, and/or [Aaa]25 is Arg or Lys.

In embodiments, the isolated polynucleotide encodes the peptide or the derivative thereof comprises any 2 of, or any 3 of, or any 4 of, or any 5 of, or any 6 of, or any 7 of, or any 8 of, or any 9 of, or any 10 of, or any 11 of, or any 12 of, or any 13 of, or any 14 of, or any 15 of, or any 16 of, or any 17 of, or any 18 of, or any 19 of, or any 20 of, or any 21 of, or any 22 of, or any 23 of, or any 24 of, or all 25 of the following:

(1) [Aaa]1 is His;

(2) [Aaa]2 is Ser or Gly;

(3) [Aaa]3 is His or Pro;

(4) [Aaa]4 is Phe or lie;

(5) [Aaa]5 is Leu or Vai;

(6) [Aaa]6 is Ala;

(7) [Aaa]7 is Leu or lie;

(8) [Aaa]8 is Asp or Glu;

(9) [Aaa]9 is Lys or Glu;

(10) [Aaa]10 is Gin;

(11) [Aaa]11 is Arg;

(12) [Aaa]12 is Gin;

(13) [Aaa]13 is Ala or Gin;

(14) [Aaa]14 is Glu;

(15) [Aaa] 15 is lie or Leu; (16) [Aaa]16 is Asp or Glu;

(17) [Aaa]17 is Trp;

(18) [Aaa]18 is Arg or Gly;

(19) [Aaa]19 is Ala;

(20) [Aaa]20 is Gly or Ala;

(21) [Aaa]21 is Pro or Ser;

(22) [Aaa]22 is Ser or Thr;

(23) [Aaa]23 is Gly or Ala; and

(24) [Aaa]24 is Arg or Lys; and/or

(25) [Aaa]25 is Arg or Lys.

In embodiments, [Aaa] 1 is His, [Aaa]2 is Ser or Gly; [Aaa]3 is His or Pro; [Aaa]4 is Phe or lie; [Aaa]5 is Leu or Vai; [Aaa]6 is Ala; [Aaa]7 is Leu or lie; [Aaa]8 is Asp or Glu; [Aaa]9 is Lys or Glu; [Aaa]10 is Gin; [Aaa]11 is Arg; [Aaa]12 is Gin; [Aaa]13 is Ala or Gin; [Aaa]14 is Glu; [Aaa]15 is lie or Leu; [Aaa]16 is Asp or Glu; [Aaa] 17 is Trp; [Aaa]18 is Arg or Gly; [Aaa]19 is Ala; [Aaa]20 is Gly or Ala; [Aaa]21 is Pro or Ser; [Aaa]22 is Ser or Thr; [Aaa]23 is Gly or Ala; [Aaa] 24 is Arg or Lys; and [Aaa]25 is Arg or Lys.

In embodiments, the isolated polynucleotide encodes a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, which is also referred to herein as SL_TriAg_V1 , comprises the following sequence:

HSHGTFTSDFSLALDKQRQAEFIDWLRAGGPPSGRPPPR (SEQ ID NO: 68).

In embodiments, the SL_TriAg_V1 or a derivative thereof has dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR. In embodiments, the peptide having dual, triple or quadruple actions on GLP- 1 R, GLP-2R, GIPR, and GCGR is a variant of SL_TriAg_V1 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 68. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR has an amino acid sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 68. In embodiments, the isolated polynucleotide encodes a peptide or a derivative thereof having agonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, which is also referred to herein as SL_TriAg_V2, comprises the following sequence:

HGHGTFTSDISVAIEEQRQQEFLEWLGAAGPSTAKPPPK (SEQ ID NO: 61).

In embodiments, the SL_TriAg_V2 or a derivative thereof has dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR. In embodiments, the peptide having dual, triple or quadruple actions on GLP- 1 R, GLP-2R, GIPR, and GCGR is a variant of SL_TriAg_V2 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 61. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR has an amino acid sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to an amino acid sequence of SEQ ID NO: 61.

In embodiments, the isolated polynucleotide encodes a peptide or a derivative thereof having agonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, which is also referred to herein as SL_TriAg_V3, comprises the following sequence:

HSHGTFTSDVSRIKDKQRQKEFADWLRAQGPPSGRPPPR (SEQ ID NO: 62).

In embodiments, the SL_TriAg_V3 or a derivative thereof has dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR. In embodiments, the peptide having dual, triple or quadruple actions on GLP- 1 R, GLP-2R, GIPR, and GCGR is a variant of SL_TriAg_V3 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 62. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR has an amino acid sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to an amino acid sequence of SEQ ID NO: 62.

In embodiments, the isolated polynucleotide encodes a peptide or a derivative thereof having agonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, which is also referred to herein as SL_TriAg_V4, comprises the following sequence:

HGHGTFTSDSSHGREEQRQREFGEWLGAGGPSTAKPPPK (SEQ ID NO: 63). In embodiments, the SL_TriAg_V4 or a derivative thereof has dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR. In embodiments, the peptide having dual, triple or quadruple actions on GLP- 1 R, GLP-2R, GIPR, and GCGR is a variant of SL_TriAg_V4 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 63. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1R, GLP-2R, GIPR, and GCGR has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 63.

In embodiments, the isolated polynucleotide encodes a peptide or a derivative thereof having agonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, which is also referred to herein as SL_TriAg_V1_Pro3, comprises the following sequence:

HSPGTFTSDFSLALDKQRQAEFIDWLRAGGPPSGRPPPR (SEQ ID NO: 64).

In embodiments, the SL_Tri Ag_V1 _Pro3 or a derivative thereof has dual, triple or quadruple actions on GLP- 1 R, GLP-2R, GIPR, and GCGR. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR is a variant of SL_TriAg_V1_Pro3 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 64. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 64.

In embodiments, the isolated polynucleotide encodes a peptide or a derivative thereof having agonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, which is also referred to herein as SL_TriAg_V2_Pro3, comprises the following sequence:

HGPGTFTSDISVAIEEQRQQEFLEWLGAAGPSTAKPPPK (SEQ ID NO: 65).

In embodiments, the SL_TriAg_V2_Pro3 or a derivative thereof has dual, triple or quadruple actions on GLP- 1 R, GLP-2R, GIPR, and GCGR. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR is a variant of SL_TriAg_V2_Pro3 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 65. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 65.

In embodiments, the isolated polynucleotide encodes a peptide or a derivative thereof having agonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, which is also referred to herein as SL_TriAg_V3_Pro3, comprises the following sequence:

HSPGTFTSDVSRIKDKQRQKEFADWLRAQGPPSGRPPPR (SEQ ID NO: 66).

In embodiments, the SL_TriAg_V3_Pro3 or a derivative thereof has dual, triple or quadruple actions on GLP- 1 R, GLP-2R, GIPR, and GCGR. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR is a variant of SL_TriAg_V3_Pro3 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 66. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 66.

In embodiments, the isolated polynucleotide encodes a peptide or a derivative thereof having agonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, which is also referred to herein as SL_TriAg_V4_Pro3, comprises the following sequence:

HGPGTFTSDSSHGREEQRQREFGEWLGAGGPSTAKPPPK (SEQ ID NO: 67)

In embodiments, the SL_TriAg_V4_Pro3 or a derivative thereof has dual, triple or quadruple actions on GLP- 1 R, GLP-2R, GIPR, and GCGR. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR is a variant of SL_TriAg_V4_Pro3 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 67. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 67. In embodiments, the peptide or the derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, which is also referred to herein as SL-045, comprises the following sequence:

HSHGTFTSDFSLALDKQRQAEFIDWLKAAGPPSAKPPPK (SEQ ID NO: 68)

In embodiments, the SL-045 or a derivative thereof has dual, triple or quadruple actions on GLP-1 R, GLP- 2R, GIPR, and GCGR. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR is a variant of SL-045 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 68. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP- 2R, GIPR, and GCGR has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 68.

In embodiments, the peptide or the derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, which is also referred to herein as SL-044, comprises the following sequence:

HSHGTFTSDFSLALDKQRQAEFIDWLGAAGPSTAKPPPK (SEQ ID NO: 69)

In embodiments, the SL-044 or a derivative thereof has dual, triple or quadruple actions on GLP-1 R, GLP- 2R, GIPR, and GCGR. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR is a variant of SL-044 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 69. In embodiments, the peptide having dual, triple or quadruple actions on GLP-1 R, GLP- 2R, GIPR, and GCGR has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 69.

In embodiments, the isolated polynucleotide encodes the peptide or the derivative thereof comprises an amino acid sequence that is selected from the amino acid sequence of SEQ ID NOs: 60 to 69. In embodiments, the isolated polynucleotide encodes the peptide or the derivative thereof comprises an amino acid sequence that is selected from the amino acid sequence of SEQ ID NOs: 60 to 69, or a variant thereof having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from SEQ ID NOs: 60 to 69. In embodiments, the isolated polynucleotide encodes the peptide or the derivative thereof comprises an amino acid sequence that is selected from the amino acid sequence of SEQ ID NOs: 60, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 68 and SEQ ID NO: 69, or a variant thereof having 1 or 2 amino acid mutations with respect to an amino acid sequence selected from SEQ ID NOs: 60, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 68 and SEQ ID NO: 69. In embodiments, the isolated polynucleotide encodes the peptide or the derivative thereof comprises an amino acid sequence that is selected from the amino acid sequence of SEQ ID NOs: 60, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 68 and SEQ ID NO: 69.

In aspects, the present disclosure provides an isolated polynucleotide encoding a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence that is selected from the amino acid sequence of SEQ ID NOs: 60 to 69. In embodiments, the isolated polynucleotide encodes the peptide or the derivative thereof comprises an amino acid sequence that is selected from the amino acid sequence of SEQ ID NOs: 60 to 69, or a variant thereof having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations with respect to an amino acid sequence selected from SEQ ID NOs: 60 to 69.

In aspects, the present disclosure provides an isolated polynucleotide encoding a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, wherein the peptide comprises the following sequence, or a variant thereof:

HSHGTFTSDFSLALDKQRQAEFIDWLRAGGPPSGRPPPR (SEQ ID NO: 68).

In embodiments, the isolated polynucleotide encodes the peptide or the derivative thereof is a variant of SEQ ID NO: 68 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 68. In embodiments, the isolated polynucleotide encodes the peptide the peptide or the derivative thereof that has a dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR, and has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 68.

In aspects, the present disclosure provides an isolated polynucleotide encoding a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, wherein the peptide comprises the following sequence, or a variant thereof: HGHGTFTSDISVAIEEQRQQEFLEWLGAAGPSTAKPPPK (SEQ ID NO: 61).

In embodiments, the isolated polynucleotide encodes the peptide or the derivative thereof is a variant of SEQ ID NO: 61 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 61 . In embodiments, the isolated polynucleotide encodes the peptide the peptide or the derivative thereof that has a dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR, and has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 61.

In aspects, the present disclosure provides an isolated polynucleotide encoding a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, wherein the peptide comprises the following sequence, or a variant thereof:

HSHGTFTSDVSRIKDKQRQKEFADWLRAQGPPSGRPPPR (SEQ ID NO: 62).

In embodiments, the isolated polynucleotide encodes the peptide or the derivative thereof is a variant of SEQ ID NO: 62 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 62. In embodiments, the isolated polynucleotide encodes the peptide the peptide or the derivative thereof that has a dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR, and has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 62.

In aspects, the present disclosure provides an isolated polynucleotide encoding a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, wherein the peptide comprises the following sequence, or a variant thereof:

HGHGTFTSDSSHGREEQRQREFGEWLGAGGPSTAKPPPK (SEQ ID NO: 63).

In embodiments, the isolated polynucleotide encodes the peptide or the derivative thereof is a variant of SEQ ID NO: 63 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 63. In embodiments, the isolated polynucleotide encodes the peptide the peptide or the derivative thereof that has a dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR, and has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 63.

In aspects, the present disclosure provides an isolated polynucleotide encoding a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, wherein the peptide comprises the following sequence, or a variant thereof:

HSPGTFTSDFSLALDKQRQAEFIDWLRAGGPPSGRPPPR (SEQ ID NO: 64).

In embodiments, the isolated polynucleotide encodes the peptide or the derivative thereof is a variant of SEQ ID NO: 64 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 64. In embodiments, the isolated polynucleotide encodes the peptide the peptide or the derivative thereof that has a dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR, and has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 64.

In aspects, the present disclosure provides an isolated polynucleotide encoding a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, wherein the peptide comprises the following sequence, or a variant thereof:

HGPGTFTSDISVAIEEQRQQEFLEWLGAAGPSTAKPPPK (SEQ ID NO: 65).

In embodiments, the isolated polynucleotide encodes the peptide or the derivative thereof is a variant of SEQ ID NO: 65 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 65. In embodiments, the isolated polynucleotide encodes the peptide the peptide or the derivative thereof that has a dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR, and has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 65.

In aspects, the present disclosure provides an isolated polynucleotide encoding a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, wherein the peptide comprises the following sequence, or a variant thereof:

HSPGTFTSDVSRIKDKQRQKEFADWLRAQGPPSGRPPPR (SEQ ID NO: 66). In embodiments, the isolated polynucleotide encodes the peptide or the derivative thereof is a variant of SEQ ID NO: 66 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 66. In embodiments, the isolated polynucleotide encodes the peptide the peptide or the derivative thereof that has a dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR, and has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 66.

In aspects, the present disclosure provides an isolated polynucleotide encoding a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, wherein the peptide comprises the following sequence, or a variant thereof:

HGPGTFTSDSSHGREEQRQREFGEWLGAGGPSTAKPPPK (SEQ ID NO: 67)

In embodiments, the isolated polynucleotide encodes the peptide or the derivative thereof is a variant of SEQ ID NO: 67 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 67. In embodiments, the isolated polynucleotide encodes the peptide the peptide or the derivative thereof that has a dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR, and has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 67.

In aspects, the present disclosure provides an isolated polynucleotide encoding a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, wherein the peptide comprises the following sequence, or a variant thereof:

HSHGTFTSDFSLALDKQRQAEFIDWLKAAGPPSAKPPPK (SEQ ID NO: 68)

In embodiments, the isolated polynucleotide encodes the peptide or the derivative thereof is a variant of SEQ ID NO: 68 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 68. In embodiments, the isolated polynucleotide encodes the peptide the peptide or the derivative thereof that has a dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR, and has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 68. In aspects, the present disclosure provides an isolated polynucleotide encoding a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, wherein the peptide comprises the following sequence, or a variant thereof:

HSHGTFTSDFSLALDKQRQAEFIDWLGAAGPSTAKPPPK (SEQ ID NO: 69)

In embodiments, the isolated polynucleotide encodes the peptide or the derivative thereof is a variant of SEQ ID NO: 69 having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 69. In embodiments, the isolated polynucleotide encodes the peptide the peptide or the derivative thereof that has a dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR, and has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NO: 69.

In aspects, the present disclosure provides an isolated polynucleotide encoding a fusion protein comprising a carrier protein joined, optionally via a linker, to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon of any of the embodiments disclosed herein. In embodiments, the peptide comprises a general formula (I):

N-L1-[Aaa]1-[Aaa]2-[Aaa]3-Gly-Thr-Phe-Thr-Ser-Asp-[Aaa]4-Ser-[Aaa]5-[Aaa]6-[Aaa]7-[Aaa]8- [Aaa]9-[Aaa]10-[Aaa]11-[Aaa]12-[Aaa]13-[Aaa]14-Phe-[Aaa]15-[Aaa]16-[Aaa]17-Leu-[Aaa]18- [Aaa]19-[Aaa]20-Gly-Pro-[Aaa]21-[Aaa]22-[Aaa]23-[Aaa]24-Pro-Pro-Pro-[Aaa]25-L2-C

(I) wherein N is the N-terminus; one of L1 or L2 is present the carrier protein, and the other is a tag, and a polymer, optionally wherein the L1 and/or L2 further comprise a linker that adjoins the carrier protein, tag, or polymer with the peptide; [Aaa] 1 is His; [Aaa]2 is Ser or Gly; [Aaa]3 is His or Pro; [Aaa]4 is Phe or lie; [Aaa]5 is Leu or Vai; [Aaa]6 is Ala; [Aaa]7 is Leu or lie; [Aaa]8 is Asp or Glu; [Aaa]9 is Lys or Glu; [Aaa]10 is Gin; [Aaa] 11 is Arg; [Aaa]12 is Gin; [Aaa]13 is Ala or Gin; [Aaa] 14 is Glu; [Aaa]15 is lie or Leu; [Aaa]16 is Asp or Glu; [Aaa]17 is Trp; [Aaa]18 is Arg or Gly; [Aaa]19 is Ala; [Aaa]20 is Gly or Ala; [Aaa]21 is Pro or Ser; [Aaa]22 is Ser or Thr; [Aaa]23 is Gly or Ala; [Aaa]24 is Arg or Lys; [Aaa]25 is Arg or Lys; and C is the C-terminus. In embodiments, the peptide or the derivative thereof has dual, triple or quadruple actions on GLP-1 R, GLP- 2R, GIPR, and GCGR. In embodiments, the carrier protein is selected from Fc domain, albumin, transferrin, or elastin-like protein, Keyhole Limpet Hemocyanin (KLH), ovalbulin, or a variant thereof. In embodiments, the carrier protein is a hinge CH2-CH3-Fc domain. In embodiments, the a hinge CH2-CH3-Fc domain is derived from I gG1 or lgG4 (e.g., human lgG1 or lgG4).

In aspects, the present disclosure provides an isolated polynucleotide encoding a fusion protein comprising a carrier protein joined, optionally via a linker, to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon of any of the embodiments disclosed herein. In embodiments, the peptide comprises a general formula (II):

N-L1-[Aaa]1-[Aaa]2-[Aaa]3-Gly-Thr-Phe-Thr-Ser-Asp-[Aaa]4-Ser-[Aaa]5-[Aaa]6-[Aaa]7-[Aaa]8- [Aaa]9-[Aaa]10-[Aaa]11-[Aaa]12-[Aaa]13-[Aaa]14-Phe-[Aaa]15-[Aaa]16-[Aaa]17-Leu-[Aaa]18- [Aaa]19-[Aaa]20-Gly-Pro-[Aaa]21-[Aaa]22-[Aaa]23-[Aaa]24-Pro-Pro-Pro-[Aaa]25-L2-C

(II) wherein N is the N-terminus; one of L1 or L2 is present the carrier protein, and the other is a tag, and a polymer, optionally wherein the L1 and/or L2 further comprise a linker that adjoins the carrier protein, tag, or polymer with the peptide; [Aaa] 1 is a positively charged amino acid residue selected from His, Lys, and Arg; [Aaa]2 is a polar and neutral of charge hydrophilic amino acid selected from Ser, Asn, Gin, Thr, and Pro, or a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met; [Aaa]3 is a positively charged amino acid residue selected from His, Lys, and Arg, or a polar and neutral of charge hydrophilic amino acid selected from Pro Ser, Asn, Gin, and Thr; [Aaa]4 is a, hydrophobic, aromatic amino acid selected from Phe, Trp, and Tyr, or a hydrophobic, aliphatic amino acid selected from lie, Gly, Ala, Leu, Met, and Vai; [Aaa]5 is a hydrophobic, aliphatic amino acid selected from Leu, Vai, lie, Gly, Ala, and Met; [Aaa]6 is a hydrophobic, aliphatic amino acid selected from Ala, Gly, Vai, lie, Leu, and Met; [Aaa]7 is a hydrophobic, aliphatic amino acid selected from Leu, lie, Vai, Gly, Ala, and Met; [Aaa]8 is a polar and negatively charged hydrophilic amino acid selected from Asp and Glu; [Aaa]9 is a polar and positively charged hydrophilic amino acid selected from Lys, His, and Arg, or a polar and negatively charged hydrophilic amino acid selected from Glu, and Asp; [Aaa] 10 is a polar and neutral of charge hydrophilic amino acid selected from Gin, Asn, Ser, Thr, and Pro; [Aaa]11 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His; [Aaa]12 is a polar and neutral of charge hydrophilic amino acid selected from Gin, Asn, Ser, Thr, and Pro; [Aaa] 13 is a hydrophobic, aliphatic amino acid selected from Ala, Vai, Gly, Leu, lie, and Met, or a polar and neutral of charge hydrophilic amino acid selected from Gin, Asn, Ser, Thr, and Pro; [Aaa]14 is a polar and negatively charged hydrophilic amino acid selected from Glu, and Asp; [Aaa] 15 is a hydrophobic, aliphatic amino acid selected from lie, Leu, Vai, Ala, Gly, and Met; [Aaa]16 is a polar and negatively charged hydrophilic amino acid selected from Asp, and Glu; [Aaa]17 is hydrophobic, aromatic amino acid selected from Trp, Phe, and Tyr; [Aaa]18 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His, or a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met; [Aaa]19 is a hydrophobic, aliphatic amino acid selected from Ala, Gly, Vai, Leu, lie, and Met; [Aaa] 20 is a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met; [Aaa]21 is a polar and neutral of charge hydrophilic amino acid selected from Pro, Ser, Gin, Asn, and Thr; [Aaa]22 is a polar and neutral of charge hydrophilic amino acid selected from Ser, Thr, Gin, Asn, and Pro; [Aaa]23 a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met; [Aaa]24 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His; [Aaa]25 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His; and C is the C-terminus. In embodiments, the peptide or the derivative thereof present in the fusion protein has dual, triple or quadruple actions on GLP-1 R, GLP-2R, GIPR, and GCGR. In embodiments, the carrier protein is selected from Fc domain, albumin, transferrin, or elastin-like protein, Keyhole Limpet Hemocyanin (KLH), ovalbulin, or a variant thereof. In embodiments, the carrier protein is a hinge CH2-CH3-Fc domain. In embodiments, the a hinge CH2-CH3-Fc domain is derived from lgG1 or lgG4 (e.g., human I gG1 or I gG4)

In embodiments, [Aaa]1 is His. In embodiments, [Aaa]2 is Ser or Gly. In embodiments, [Aaa]3 is His or Pro.

In embodiments, [Aaa]4 is Phe or lie. In embodiments, [Aaa]5 is Leu or Vai. In embodiments, [Aaa]6 is Ala.

In embodiments, [Aaa]7 is Leu or lie. In embodiments, [Aaa]8 is Asp or Glu. In embodiments, [Aaa]9 is Lys or Glu. In embodiments, [Aaa]10 is Gin. In embodiments, [Aaa]11 is Arg. In embodiments, [Aaa]12 is Gin. In embodiments, [Aaa]13 is Ala or Gin. In embodiments, [Aaa]14 is Glu. In embodiments, [Aaa]15 is lie or Leu. In embodiments, [Aaa]16 is Asp or Glu. In embodiments, [Aaa]17 is Trp. In embodiments, [Aaa] 18 is Arg or Gly. In embodiments, [Aaa]19 is Ala. In embodiments, [Aaa]20 is Gly or Ala. In embodiments, [Aaa]21 is Pro or Ser. In embodiments, [Aaa]22 is Ser or Thr. In embodiments, [Aaa]23 is Gly or Ala. In embodiments, [Aaa]24 is Arg or Lys. In embodiments, and/or [Aaa]25 is Arg or Lys.

In embodiments, the peptide or the derivative thereof present in the fusion protein comprises any 2 of, or any 3 of, or any 4 of, or any 5 of, or any 6 of, or any 7 of, or any 8 of, or any 9 of, or any 10 of, or any 11 of, or any 12 of, or any 13 of, or any 14 of, or any 15 of, or any 16 of, or any 17 of, or any 18 of, or any 19 of, or any 20 of, or any 21 of, or any 22 of, or any 23 of, or any 24 of, or all 25 of the following:

(1) [Aaa]1 is His;

(2) [Aaa]2 is Ser or Gly; (3) [Aaa]3 is His or Pro;

(4) [Aaa]4 is Phe or lie;

(5) [Aaa]5 is Leu or Vai;

(6) [Aaa]6 is Ala;

(7) [Aaa]7 is Leu or lie;

(8) [Aaa]8 is Asp or Glu;

(9) [Aaa]9 is Lys or Glu;

(10) [Aaa]10 is Gin;

(11) [Aaa]11 is Arg;

(12) [Aaa]12 is Gin;

(13) [Aaa]13 is Ala or Gin;

(14) [Aaa]14 is Glu;

(15) [Aaa]15 is lie or Leu;

(16) [Aaa]16 is Asp or Glu;

(17) [Aaa]17 is Trp;

(18) [Aaa]18 is Arg or Gly;

(19) [Aaa]19 is Ala;

(20) [Aaa]20 is Gly or Ala;

(21) [Aaa]21 is Pro or Ser;

(22) [Aaa]22 is Ser or Thr;

(23) [Aaa]23 is Gly or Ala; and

(24) [Aaa]24 is Arg or Lys; and/or

(25) [Aaa]25 is Arg or Lys.

In embodiments, [Aaa] 1 is His, [Aaa]2 is Ser or Gly; [Aaa]3 is His or Pro; [Aaa]4 is Phe or lie; [Aaa]5 is Leu or Vai; [Aaa]6 is Ala; [Aaa]7 is Leu or lie; [Aaa]8 is Asp or Glu; [Aaa]9 is Lys or Glu; [Aaa]10 is Gin; [Aaa]11 is Arg; [Aaa]12 is Gin; [Aaa]13 is Ala or Gin; [Aaa]14 is Glu; [Aaa]15 is lie or Leu; [Aaa]16 is Asp or Glu; [Aaa] 17 is Trp; [Aaa]18 is Arg or Gly; [Aaa]19 is Ala; [Aaa]20 is Gly or Ala; [Aaa]21 is Pro or Ser; [Aaa]22 is Ser or Thr; [Aaa]23 is Gly or Ala; [Aaa]24 is Arg or Lys; and [Aaa]25 is Arg or Lys.

In aspects, the present disclosure provides an isolated polynucleotide encoding a fusion protein comprising a hinge CH2-CH3-Fc domain fused via a linker to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence selected from SEQ ID NOs: 60 to 69, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NOs: 60 to 69. In embodiments, the hinge CH2-CH3-Fc domain comprises an amino acid sequence that at least 95% sequence identical to the amino acid sequence of one of SEQ ID NOs: 1-4. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53, or an amino acid sequence having about 1 , 2, 3 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NOs: 5-53. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53.

In aspects, the present disclosure provides an isolated polynucleotide encoding a fusion protein comprising a hinge CH2-CH3-Fc domain fused via a linker to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 60, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 60. In embodiments, the hinge CH2-CH3-Fc domain comprises an amino acid sequence that at least 95% sequence identical to the amino acid sequence of one of SEQ ID NOs: 1-4. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53, or an amino acid sequence having about 1, 2, 3 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NOs: 5-53. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53. In aspects, the present disclosure provides an isolated polynucleotide encoding a fusion protein comprising a hinge CH2-CH3-Fc domain fused via a linker to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 61 , or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO:

61. In embodiments, the hinge CH2-CH3-Fc domain comprises an amino acid sequence that at least 95% sequence identical to the amino acid sequence of one of SEQ ID NOs: 1-4. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53, or an amino acid sequence having about 1, 2, 3 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NOs: 5-53. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53.

In aspects, the present disclosure provides an isolated polynucleotide encoding a fusion protein comprising a hinge CH2-CH3-Fc domain fused via a linker to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 62, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO:

62. In embodiments, the hinge CH2-CH3-Fc domain comprises an amino acid sequence that at least 95% sequence identical to the amino acid sequence of one of SEQ ID NOs: 1-4. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53, or an amino acid sequence having about 1, 2, 3 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NOs: 5-53. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53.

In aspects, the present disclosure provides an isolated polynucleotide encoding a fusion protein comprising a hinge CH2-CH3-Fc domain fused via a linker to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 63, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO:

63. In embodiments, the hinge CH2-CH3-Fc domain comprises an amino acid sequence that at least 95% sequence identical to the amino acid sequence of one of SEQ ID NOs: 1-4. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53, or an amino acid sequence having about 1, 2, 3 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NOs: 5-53. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53.

In aspects, the present disclosure provides an isolated polynucleotide encoding a fusion protein comprising a hinge CH2-CH3-Fc domain fused via a linker to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 64, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO:

64. In embodiments, the hinge CH2-CH3-Fc domain comprises an amino acid sequence that at least 95% sequence identical to the amino acid sequence of one of SEQ ID NOs: 1-4. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53, or an amino acid sequence having about 1, 2, 3 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NOs: 5-53. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53.

In aspects, the present disclosure provides an isolated polynucleotide encoding a fusion protein comprising a hinge CH2-CH3-Fc domain fused via a linker to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 65, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO:

65. In embodiments, the hinge CH2-CH3-Fc domain comprises an amino acid sequence that at least 95% sequence identical to the amino acid sequence of one of SEQ ID NOs: 1-4. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53, or an amino acid sequence having about 1, 2, 3 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NOs: 5-53. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53.

In aspects, the present disclosure provides an isolated polynucleotide encoding a fusion protein comprising a hinge CH2-CH3-Fc domain fused via a linker to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 66, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO:

66. In embodiments, the hinge CH2-CH3-Fc domain comprises an amino acid sequence that at least 95% sequence identical to the amino acid sequence of one of SEQ ID NOs: 1-4. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53, or an amino acid sequence having about 1, 2, 3 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NOs: 5-53. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53.

In aspects, the present disclosure provides an isolated polynucleotide encoding a fusion protein comprising a hinge CH2-CH3-Fc domain fused via a linker to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 67, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO:

67. In embodiments, the hinge CH2-CH3-Fc domain comprises an amino acid sequence that at least 95% sequence identical to the amino acid sequence of one of SEQ ID NOs: 1-4. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53, or an amino acid sequence having about 1, 2, 3 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NOs: 5-53. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53. In aspects, the present disclosure provides an isolated polynucleotide encoding a fusion protein comprising a hinge CH2-CH3-Fc domain fused via a linker to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 68, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO:

68. In embodiments, the hinge CH2-CH3-Fc domain comprises an amino acid sequence that at least 95% sequence identical to the amino acid sequence of one of SEQ ID NOs: 1-4. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53, or an amino acid sequence having about 1, 2, 3 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NOs: 5-53. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53.

In aspects, the present disclosure provides an isolated polynucleotide encoding a fusion protein comprising a hinge CH2-CH3-Fc domain fused via a linker to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 69, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO:

69. In embodiments, the hinge CH2-CH3-Fc domain comprises an amino acid sequence that at least 95% sequence identical to the amino acid sequence of one of SEQ ID NOs: 1-4. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53, or an amino acid sequence having about 1, 2, 3 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence of SEQ ID NOs: 5-53. In embodiments, the linker has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5-53.

In embodiments, the isolated polynucleotide is RNA, optionally, an mRNA. In embodiments, the isolated polynucleotide is codon optimized.

In embodiments, the isolated polynucleotide is mRNA or a modified mRNA (mmRNA). In embodiments, the polypeptide includes a polynucleotide modification including, but not limited to, a nucleoside modification. In embodiments, the isolated polynucleotide is an mmRNA. In embodiments, the mmRNA comprises one or more nucleoside modifications. In embodiments, the nucleoside modifications are selected from pyridin-4- one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, pseudouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1 -carboxymethylpseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl- pseudouridine, 5-taurinomethyl-2-thio-uridine, 1 -taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl- pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1 -methyl-pseudouridine, 1 -methyl- 1 -deazapseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy- pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4- acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl- 1 -deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2- thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy- 1 -methyl-pseudoisocytidine, 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2,6- diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy- adenine, inosine, 1 -methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6- thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7- methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1 -methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2- methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, and combinations thereof.

In embodiments, the polypeptide the at least one chemically modified nucleoside is selected from pseudouridine ('+’), N1 -methylpseudouridine (m1 ^l), 2-thiouridine (s2U), 4’ -thiouridine, 5-methylcytosine, 2- th io- 1 -methyl- 1 -deaza-pseudouridine, 2-thio- 1 -methyl-pseudouridine, 2-th io-5-aza-uridi ne, 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4- methoxy-pseudouridine, 4-thio-1 -methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, 2’-O-methyl uridine, 1-methyl-pseudouridine (m14^J), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), .alpha.-thio-guanosine, .alpha.-thio-adenosine, 5-cyano uridine, 4’-thio uridine 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6- methyl-adenosine (m6A), and 2,6-Diaminopurine, (I), 1-methylinosine (ml I), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQO), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo- guanosine, and two or more combinations thereof.

In embodiments, the mmRNA does not cause a substantial induction of the innate immune response of a cell into which the mmRNA is introduced. In embodiments, the modification in the mmRNA enhance one or more of the efficiency of production of the peptide or the derivative thereof , intracellular retention of the mmRNA, and viability of contacted cells, and possess reduced immunogenicity.

In embodiments, the mmRNA has a length sufficient to include an open reading frame encoding the peptide or the derivative thereof of the present disclosure.

Modified mRNAs need not be uniformly modified along the entire length of the molecule. Different nucleotide modifications and/or backbone structures may exist at various positions in the nucleic acid. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of a nucleic acid such that the function of the nucleic acid is not substantially decreased. A modification may also be a 5' or 3' terminal modification. The nucleic acids may contain at a minimum one and at maximum 100% modified nucleotides, or any intervening percentage, such as at least about 50% modified nucleotides, at least about 80% modified nucleotides, or at least about 90% modified nucleotides.

In embodiments, the mmRNAs may contain a modified pyrimidine such as uracil or cytosine. In embodiments, at least about 5%, at least about 10%, at least about 25%, at least about 50%, In embodiments, the modified uracil may be replaced by a compound having a single unique structure or can be replaced by a plurality of compounds having different structures disclosed above (e.g., same mmRNA may contain 2, 3, 4 or more types of uniquely modified uracil). In embodiments, at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 80%, at least about 90% or 100% of the cytosine in the nucleic acid may be replaced with a modified cytosine. The modified cytosine can be replaced by a compound having a single unique structure or can be replaced by a plurality of compounds having different structures disclosed above (e.g., same mmRNA may contain 2, 3, 4 or more types of uniquely modified cytosine). In embodiments, the mmRNA comprises at least one chemically modified nucleoside. In embodiments, wherein the at least one chemically modified nucleoside is selected from pseudouridine ( ¹), N1- methylpseudouridine (ml ), 2-thiouridine (s2U), 4’-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1 -deazapseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1- methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5- methoxyuridine, 2’-O-methyl uridine, 1-methyl-pseudouridine (ml ), 5-methoxy-uridine (mo5U), 5-methyl- cytidine (m5C), .alpha. -thio-guanosine, .alpha.-thio-adenosine, 5-cyano uridine, 4’-thio uridine 7-deaza- adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), and 2,6- Diaminopurine, (I), 1-methylinosine (ml I), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7- cyano-7-deaza-guanosine (preQO), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G),

1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, and two or more combinations thereof. In embodiments, the mmRNA comprises at least one chemically modified nucleoside, wherein the at least one chemically modified nucleoside is selected from pseudouridine, N1 -methylpseudouridine, 5- methylcytosine, 5-methoxyuridine, and a combination thereof. In embodiments, the mmRNA comprises at least one chemically modified nucleoside is N1 -methylpseudouridine. In embodiments, the mmRNA is fully modified with chemically-modified uridines. In embodiments, the mmRNA is a fully modified N1- methylpseudouridine mRNA. Additional chemical modifications are disclosed in US Patent Application Publication No. 20190111003, the entire contents of which are hereby incorporated by reference.

In embodiments, modified nucleosides include pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza- uridine, 2-thiouridine, pseudouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3- methyluridine, 5-carboxymethyl-uridine, 1 -carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl- pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2- thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine,

2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine. In embodiments, modified nucleosides include 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5- formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1 -methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1- methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1 -methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy- cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl- pseudoisocytidine.

In embodiments, modified nucleosides include 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7- deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza- 2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6- (cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy- adenine.

In embodiments, modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza- guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza- guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1- methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo- guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

In embodiments, the nucleotide can be modified on the major groove face and can include replacing hydrogen on C-5 of uracil with a methyl group or a halo group.

In embodiments, a modified nucleoside is 5'-0-(1-Thiophosphate)-Adenosine, 5'-O-(1 -Thiophosphate)- Cytidine, 5'-O-(1-Thiophosphate)-Guanosine, 5'-O-(1-Thiophosphate)-Uridine or 5'-O-(1-Thiophosphate)- Pseudouridine.

Further examples of modified nucleotides and modified nucleotide combinations are disclosed in US Patent Nos. 8,710,200; 8,822,663; 8,999,380; 9,181,319; 9,254,311; 9,334,328; 9,464,124; 9,950,068; 10,626,400; 10,808,242; 11,020,477, and US Patent Application Publication Nos. 20220001026, 20210318817, 20210283262, 20200360481, 20200113844, 20200085758, 20170204152, 20190114089, 20190114090, 20180369374, 20180318385, 20190111003, and PCT International Application Publication Nos. WO/2017112943, WO 2014/028429, WO 2017/201325 the entire contents of which are hereby incorporated by reference. The methods for synthesizing the modified mRNA are disclosed, e.g., in US Patent Application Publication Nos. 20170204152, the entire contents of which are hereby incorporated by reference. In embodiments, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues of the mmRNA are replaced by modified cytosine residues. In embodiments, at least 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the uracil residues of the mmRNA are replaced by modified uracil residues.

In embodiments, the mmRNA further comprises a 5' untranslated region (UTR) and/or a 3'UTR, wherein either or both may independently contain one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present in the translatable region. In embodiments, the mmRNA further comprises a Kozak sequence. In embodiments, the mmRNA further comprises a internal ribosome entry site (IRES).

In embodiments, the mmRNA further comprises a 5'-cap and/or a poly A tail.

In embodiments, the 5'-cap contains a 5'-5'-triphosphate linkage between the 5'-most nucleotide and guanine nucleotide. In embodiments, the 5'-cap comprises a methylation of the ultimate and penultimate most 5'- nucleotides on the 2'-hydroxyl group. In embodiments, the 5'-cap facilitates binding the mRNA Cap Binding Protein (CBP), confers mRNA stability in the cell and/or confers translation competency.

In embodiments, the poly-A tail is greater than about 30 nucleotides, or greater than about 40 nucleotides in length. In embodiments, the poly-A tail at least about 40 nucleotides, or at least about 45 nucleotides, or at least about 55 nucleotides, or at least about 60 nucleotides, or at least about 80 nucleotides, or at least about 90 nucleotides, or at least about 100 nucleotides, or at least about 120 nucleotides, or at least about 140 nucleotides, or at least about 160 nucleotides, or at least about 180 nucleotides, or at least about 200 nucleotides, or at least about 250 nucleotides, or at least about 300 nucleotides, or at least about 350 nucleotides, or at least about 400 nucleotides, or at least about 450 nucleotides, or at least about 500 nucleotides, or at least about 600 nucleotides, or at least about 700 nucleotides, or at least about 800 nucleotides, or at least about 900 nucleotides, or at least about 1000 nucleotides in length.

In embodiments, the mmRNA comprises a 3’ untranslated region (UTR). In embodiments, the 3’ UTR comprises a nucleic acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence listed in Table 4 of US Patent Application Publication No. 20190114089, which is incorporated herein in its entirety. In embodiments, the 3’ UTR comprises at least one microRNA-122 (miR-122) binding site, wherein the miR-122 binding site is a miR-122-3p binding site or a miR-122-5-binding site. In embodiments, the mmRNA comprises a nucleic acid sequence comprising a miRNA binding site. In some embodiments, the miRNA binding site binds to miR-122. In a particular embodiment, the miRNA binding site binds to miR-122-3p or miR-122-5p. In embodiments, the mmRNA comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten miRNA binding sites.

In embodiments, the miRNA binding site is inserted within the 3’ UTR. In embodiments, the isolated polynucleotide is DNA. In embodiments, the further comprises a spacer sequence between the open reading frame and the miRNA binding site. In aspects, the spacer sequence comprises at least about 10 nucleotides, at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, at least about 80 nucleotides, at least about 90 nucleotides, or at least about 100 nucleotides.

In embodiments, the mmRNA further comprises a 5’ UTR. In embodiments, the 5’ UTR comprises a nucleic acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence listed in Table 3 of US Patent Application Publication No. 20190114089, or a sequence disclosed in PCT International Application Publication Nos. WO 2017/201325 and WO 2014/164253, each of which is incorporated herein in its entirety. In embodiments, the 5’ UTR bears features, which play roles in translation initiation. In embodiments, the 5’ UTR harbors signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. In embodiments, the 5’ UTR forms secondary structures which are involved in elongation factor binding. In embodiments, the 5’ UTR of mRNA known to be upregulated in cancers, such as c-myc, may be used to enhance expression of a nucleic acid molecule, such as a polynucleotides, in cancer cells. In embodiments, the 5’ UTR of mRNA known to be upregulated in liver and/or spleen may be used to enhance expression of a nucleic acid molecule, such as a polynucleotides, in liver and/or spleen.

In embodiments, at least one of the regions of linked nucleosides of A comprises a sequence of linked nucleosides which functions as a 5’ UTR and at least one of the regions of linked nucleosides of C comprises a sequence of linked nucleosides which functions as a 3’ UTR. In embodiments, the 5’ UTR and the 3’ UTR are from the same or different species. In embodiments, the 5’ UTR and the 3’ UTR may be the native untranslated regions from different proteins from the same or different species. In embodiments, the 5’ UTR and the 3’ UTR may have synthetic sequences.

In embodiments, the mmRNA further comprises a 3’ polyadenylation (polyA tail).

In embodiments, the mmRNA further comprises a 5’ terminal cap. In embodiments, the 5’ terminal cap is a CapO, Cap1 , ARCA, inosine, N1-methyl-guanosine, 2’fluoro-guanosine, 7-deaza-guanosine, 8-oxo- guanosine, 2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5’ methylG cap, or an analog thereof.

In embodiments, the isolated polynucleotide is in vitro transcribed (IVT). In embodiments, the isolated polynucleotide is chimeric. In embodiments, the isolated polynucleotide is circular.

In embodiments, the isolated polynucleotide is or comprises DNA. In embodiments, the isolated polynucleotide is or comprises a minicircle or a plasmid DNA. In embodiments, the plasmid DNA is devoid of any prokaryotic components. In embodiments, the isolated polynucleotide comprises a tissue-specific control element. In embodiments, the tissue-specific control element is a promoter or an enhancer. In embodiments, the plasmid DNA is an expression vector. In embodiments, the DNA is or comprises a minicircle. In embodiments, the minicircle is a circular molecule, which is optionally small. In embodiments, the minicircle utilizes a cellular transcription and translation machinery to produce an encoded gene product. In embodiments, the minicircle is devoid of any prokaryotic components. In embodiments, the minicircle only comprises substantially only sequences of mammalian origin (or those that have been optimized for mammalian cells). In embodiments, the minicircle lacks or has reduced amount of DNA sequence elements that are recognized by the innate immune system and/or toll-like receptors. In embodiments, the minicircle is produced by excising any bacterial components of from a parental plasmid, thereby making it smaller than a parental DNA sequence. In embodiments, the minicircle is of non-viral origin. In embodiments, the minicircle remains episomal. In embodiments, the minicircle does not replicate with a host cell. In embodiments, expression of the peptide or the derivative thereof in non-dividing cells harboring a minicircle lasts for at least 2 days, or at least 4 days, or at least 6 days, or at least 8 days, or at least 10 days, or at least 12 days, or at least 14 days, or at least 16 days, or at least 18 days, or at least 20 days, or at least 22 days, or at least 24 days, or longer in dividing cells. In embodiments, expression of the peptide or the derivative thereof in nondividing cells harboring a minicircle lasts for at least 4 days, or at least 6 days, or at least 8 days, or at least 10 days, or at least 1 week, or at least 2 weeks, or at least 3 weeks, or at least 4 weeks, or at least 5 weeks, or at least 6 weeks, or at least 1 month, or at least 2 months, or at least 3 months, or at least 4 months, or at least 5 months, or at least 6 months, or at least 8 months, or longer in dividing cells.

In embodiments, the mmRNAs of the present disclosure are produced by means available in the art, including but not limited to in vitro transcription (IVT) and synthetic methods. Enzymatic IVT, solid-phase, liquid-phase, combined synthetic methods, small region synthesis, and ligation methods may be utilized. In embodiments, mmRNAs are made using IVT enzymatic synthesis methods. Methods of making polynucleotides by IVT are known in the art and are described in International Application PCT International Patent Publication No. WO2013151666, the contents of which are incorporated herein by reference in their entirety. Accordingly, the present disclosure also includes polynucleotides, e.g., DNA, constructs and vectors that may be used to in vitro transcribe an mRNA described herein.

In embodiments, the isolated polynucleotide is DNA. In embodiments, the isolated polynucleotide comprises a liver-specific control element. In embodiments, the liver-specific control element is a liver-specific promoter selected from albumin promoter, thyroxine-binding globulin (TBG) promoter, hybrid liver-specific promoter (HLP), human a 1 -antitrypsin promoter, LP1 promoter, and hemopexin promoter. The gene therapy in accordance with the present disclosure can be performed using vector systems. In embodiments, the liverspecific promoter is an LP1 promoter. The LP1 promoter can be a human LP1 promoter, which can be constructed as described, e.g., in Nathwani et al. Blood vol. 107(7) (2006):2653-61 , which is incorporated herein by reference in its entirety.

In aspects, the present disclosure provides a vector comprising the isolated polynucleotide of any one of the embodiments disclosed herein. In embodiments, the peptide or the derivative thereof can be provided as an expression vector. In embodiments, the expression vector is a DNA expression vector or an RNA expression vector. In embodiments, the expression vector is a viral expression vector. In embodiments, the expression vector is a non-viral expression vector (without limitation, e.g., a plasmid).

In embodiments, the present non-viral vectors are linear or circular DNA molecules that comprise a polynucleotide encoding a polypeptide and is operably linked to control sequences, wherein the control sequences provide for expression of the isolated polynucleotide encoding the polypeptide. In embodiments, the non-viral vector comprises a promoter sequence, and transcriptional and translational stop signal sequences. In embodiments, the expression vector may include, among others, chromosomal and episomal vectors, e.g., vectors derived from bacterial plasmids, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, and vectors derived from combinations thereof. The present constructs may contain control regions that regulate as well as engender expression.

A vector generally comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. In embodiments, the expression vector is an autonomously replicating plasmid or a virus (e.g., AAV vectors). In embodiments, the expression vector is non-plasmid and non-viral compounds that facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

In embodiments, the isolated polynucleotide or cell therapy may employ expression vectors, which comprise the nucleic acid encoding the peptide or the derivative thereof operably linked to an expression control region that is functional in the host cell. The expression control region is capable of driving expression of the operably linked encoding nucleic acid such that the peptide or the derivative thereof is produced in a human cell transformed with the expression vector. Expression control regions are regulatory polynucleotides (sometimes referred to herein as elements), such as promoters and enhancers, that influence expression of an operably linked nucleic acid. An expression control region of an expression vector is capable of expressing operably linked encoding nucleic acid in a human cell. In an embodiment, the expression control region confers regulatable expression to an operably linked nucleic acid. A signal (sometimes referred to as a stimulus) can increase or decrease expression of a nucleic acid operably linked to such an expression control region. Such expression control regions that increase expression in response to a signal are often referred to as inducible. Such expression control regions that decrease expression in response to a signal are often referred to as repressible. In various embodiments, the peptide or the derivative thereof expression is inducible or repressible. Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal present; the greater the amount of signal, the greater the increase or decrease in expression.

Expression systems functional in human cells are well known in the art and include viral systems. Generally, a promoter functional in a human cell is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence into mRNA. A promoter will have a transcription-initiating region, which is usually placed proximal to the 5' end of the coding sequence, and typically a TATA box located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A promoter will also typically contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation. Of particular use as promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.

Where appropriate, gene delivery agents such as, e.g., integration sequences can also be employed. Numerous integration sequences are known in the art (see, e.g., Nunes-Duby et al., Nucleic Acids Res. 26:391-406, 1998; Sadwoski, J. Bacterio!., 165:341-357, 1986; Bestor, Cell, 122(3): 322-325, 2005; Plasterk et al., TIG 15:326-332, 1999; Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). These include recombinases and transposases. Examples include Cre (Sternberg and Hamilton, J. Mol. Biol., 150:467-486, 1981), lambda (Nash, Nature, 247, 543-545, 1974), Flp (Broach, et al., Cell, 29:227-234, 1982), R (Matsuzaki, et al., J. Bacteriology, 172:610-618, 1990), cpC31 (see, e.g., Groth et al., J. Mol. Biol. 335:667- 678, 2004), sleeping beauty, transposases of the mariner family, and components for integrating viruses such as AAV, retroviruses, and antiviruses having components that provide for virus integration such as the LTR sequences of retroviruses or lentivirus and the ITR sequences of AAV (Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). In addition, direct and targeted genetic integration strategies may be used to insert nucleic acid sequences including CRISPR/CAS9, zinc finger, TALEN, and meganuclease gene-editing technologies.

In aspects, the present disclosure provides a host cell comprising the vector of any of the embodiments disclosed herein. A host cell comprising the mmRNA of any of the embodiments disclosed herein.

Pharmaceutical Compositions

In aspects, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient or carrier, and the peptide or the derivative thereof of any of the embodiments disclosed herein, the isolated polynucleotide of any of the embodiments disclosed herein, the mmRNA of any of the embodiments disclosed herein, or the vector of any of the embodiments disclosed herein, or the host cell of any of the embodiments disclosed herein. In embodiments, the pharmaceutical composition comprises the mmRNA of any of the embodiments disclosed herein. In aspects, the present disclosure provides a pharmaceutical composition comprising the mmRNA of any of the embodiments disclosed herein, and a pharmaceutically acceptable carrier. In embodiments, the carrier is a lipidoid, a liposome, a lipoplex, a lipid nanoparticle, a polymeric nanoparticle, a peptide, a protein, a cell, a nanoparticle mimic, a nanotube, or a conjugate. In embodiments, the pharmaceutical composition is formulated as a lipid nanoparticle (LNPs), a lipoplex, or a liposome. In embodiments, the pharmaceutical composition is formulated as a lipid nanoparticle (LNPs). In embodiments, the lipid nanoparticles comprise lipids selected from an ionizable lipid e.g., an ionizable cationic lipid selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, and C12-200); a structural lipid (e.g., distearoylphosphatidylcholine (DSPC)); cholesterol, and a polyethyleneglycol (PEG)-lipid (e.g., a PEG- diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof, or a PEG-dilauryloxypropyl (C12, a PEG-dimyristyloxypropyl (C14), a PEG- dipalmityloxypropyl (C16), or a PEG-distearyloxypropyl (C18)); 1 ,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); dioleoylphosphatidylethanolamine (DOPE); and the mmRNA. In embodiments, the lipid nanoparticles comprise (a) a cationic lipid comprising from 50 mol % to 85 mol % of the total lipid present in the particle; (b) a non-cationic lipid comprising from 13 mol % to 49.5 mol % of the total lipid present in the particle; and (c) a conjugated lipid that inhibits aggregation of particles comprising from 0.5 mol % to 2 mol % of the total lipid present in the particle. In embodiments, the lipid nanoparticles comprise a lipid selected from SM-102, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, and C12-200; a cholesterol; and a PEG-lipid.

Suitable pharmaceutical compositions are disclosed in US Patent Nos. 8,710,200; 8,822,663; 8,999,380; 9,181 ,319; 9,254,311 ; 9,334,328; 9,464,124; 9,950,068; 10,626,400; 10,808,242; 11,020,477, US Patent Application Publication Nos. 20220001026, 20210318817, 20210283262, 20200360481, 20200113844, 20200085758, 20170204152, 20190114089, 20190114090, 20180369374, 20180318385, 20190111003, and PCT International Application Publication Nos. WO/2017112943, WO 2014/028429, WO 2017/201325 the entire contents of which are hereby incorporated by reference.

In aspects, the present disclosure relates to a pharmaceutical composition comprising an isolated modified mRNA (mmRNA) encoding a heterologous fusion protein having an amino acid sequence that has at least about 95% sequence identity with an amino acid sequence selected from SEQ ID NOs: 620-93.

In embodiments, the carrier is mmRNA comprises a modification (e.g., an RNA element), wherein the modification provides a desired translational regulatory activity. Such modifications are described in PCT Application No. PCT International Application Publication No. WO2018213789, the entire contents of which are herein incorporated by reference.

In embodiments, the mmRNA further comprises a 3’ untranslated region (UTR). In embodiments, the 3’ UTR comprises at least one microRNA-122 (miR-122) binding site. In embodiments, the miR-122 binding site is a miR-122-3p binding site or a miR-122-5-binding site. In embodiments, the mmRNA further comprises a spacer sequence between the open reading frame and the miRNA binding site. In embodiments, the spacer sequence comprises at least about 10 nucleotides, at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, at least about 80 nucleotides, at least about 90 nucleotides, or at least about 100 nucleotides.

In embodiments, the mmRNA further comprises a 5’ UTR. In embodiments, the 5’ UTR harbors a Kozak sequence and/or forms a secondary structure that stimulate elongation factor binding.

In embodiments, the mmRNA further comprises a 5’ terminal cap. In embodiments, the 5’ terminal cap is a CapO, Cap1 , ARCA, inosine, N1-methyl-guanosine, 2’fluoro-guanosine, 7-deaza-guanosine, 8-oxo- guanosine, 2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5’ methylG cap, or an analog thereof.

In any of the embodiments disclosed herein, the mmRNA may comprise one or more modifications. In any of the embodiments disclosed herein, the mmRNA may comprise at least one modification. In embodiments, the modification is nucleoside modification. In embodiments, the modification is a base modification. In embodiments, the modification is a sugar-phosphate backbone modification.

In embodiments, the modifications are selected from pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5- aza-uridine, 2-thiouridine, pseudouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3- methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl- pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2- thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5- hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio- cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1- methyl-1-deaza-pseudoisocytidine, 1 -methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl- cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2, 6- diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2- aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1 -methyladenosine, N6- methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2- methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio- adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7- deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7- methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6- thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, and a combination of any two or more thereof. In embodiments, the modifications are selected from pseudouridine ( ), N1- methylpseudouridine (mI ), 2-thiouridine (s2U), 4’ -thiouridine, 5-methylcytosine, 2-thio-1-methyl-1 -deazapseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1- methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5- methoxyuridine, 2’-O-methyl uridine, 1-methyl-pseudouridine (mi l¹), 5-methoxy-uridine (mo5U), 5-methyl- cytidine (m5C), .alpha. -thio-guanosine, .alpha.-thio-adenosine, 5-cyano uridine, 4’-thio uridine 7-deaza- adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), and 2,6- Diaminopurine, (I), 1-methylinosine (ml I), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7- cyano-7-deaza-guanosine (preQO), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1 G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, and a combination of any two or more thereof. In embodiments, modification is selected from pseudouridine, N1 -methylpseudouridine, 5- methylcytosine, 5-methoxyuridine, and a combination thereof.

In embodiments, the mmRNA comprises at least one N1 -methylpseudouridine. In embodiments, the mmRNA is fully modified with chemically-modified uridines. In embodiments, the mmRNA is a fully modified with N1- methylpseudouridine. In embodiments, the modifications are selected from pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5- aza-uridine, 2-thiouridine, pseudouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3- methyluridine, 5-carboxymethyl-uridine, 1 -carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl- pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2- thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine or a combination of any two or more thereof.

In embodiments, the modifications are selected from 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4- thio-pseudoisocytidine, 4-thio- 1 -methyl-pseudoisocytidine, 4-thio-1 -methyl- 1 -deaza-pseudoisocytidine, 1 - methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio- zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy- pseudoisocytidine, and 4-methoxy-1 -methyl-pseudoisocytidine.

In embodiments, the modifications are selected from 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza- 2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6- (cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy- adenine.

In embodiments, the modifications are selected from inosine, 1 -methyl-inosine, wyosine, wybutosine, 7- deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8- aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1- methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo- guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

In embodiments, the modifications are present on the major groove face. In embodiments, a hydrogen on C- 5 of uracil is replaced with a methyl group or a halo group. In embodiments, the mmRNA further comprises one or more modifications selected from 5’-O-(1- Thiophosphate)-Adenosine, 5’-O-(1 -Thiophosphate)-Cytidine, 5’-O-(1-Thiophosphate)-Guanosine, 5’-O-(1- Thiophosphate)-Uridine and 5’-O-(1-Thiophosphate)-Pseudouridine.

In embodiments, the pharmaceutical composition is formulated as a lipid nanoparticle (LNP), a lipoplex, or a liposome. In embodiments, the pharmaceutical composition is formulated as a lipid nanoparticle (LNP). In embodiments, the mmRNAs described herein may be formulated in a cationic oil-in-water emulsion where the emulsion particle comprises an oil core and a cationic lipid that can interact with the mRNA anchoring the molecule to the emulsion particle. In embodiments, the mRNAs described herein may be formulated in a water-in-oil emulsion comprising a continuous hydrophobic phase in which the hydrophilic phase is dispersed. Exemplary emulsions can be made by the methods described in PCT International Application Publication Nos. WO 2012006380 and WO 201087791, each of which is herein incorporated by reference in its entirety.

In some embodiments, nucleic acids of the invention (e.g., mRNA) are formulated in a lipid nanoparticle (LNP). Lipid nanoparticles comprise typically comprise ionizable cationic lipid, non-cationic lipid, sterol and PEG lipid components along with the nucleic acid cargo of interest. The lipid nanoparticles of the invention can be generated using components, compositions, and methods as are disclosed, e.g., in PCT International Application Publication Nos. WO 2021231854, WO 2021050986, WO 2021055833, WO 2021213924, WO2021055849, WO 2021214204, WO 2021188969, WO 2021055835, WO 2020061284, WO 2020061295, WO 2017049245, WO 2017031232, WO 2017112865, WO 2017218704, WO 2017218704, WO 2017099823, WO 2017049074, WO 2017117528, WO 2017180917, WO 2017075531, WO 2017223135, WO 2016118724, WO 2015164674, WO 2015038892, WO 2014152211, and WO 2013090648, the entire contents of each which are herein incorporated by reference. PEG-lipids selected from an ionizable lipid (e.g., as known in the art, such as those described in U.S. Pat. No. 8,158,601 and PCT International Application Publication Nos. WO 2012099755 and WO 2015/130584, which are incorporated herein by reference in their entirety. The ionizable lipid may be selected from, but not limited to, an ionizable lipid described in International Publication Nos. WO 2013086354 and WO 2013116126; the contents of each of which are herein incorporated by reference in their entirety. In embodiments, the lipid may be a cleavable lipid such as those described in PCT International Publication No. WO 2012170889, herein incorporated by reference in its entirety. In embodiments, the lipid may be synthesized by methods known in the art and/or as described in International Publication Nos. WO 2013086354; the contents of each of which are herein incorporated by reference in their entirety. In embodiments, the LNP formulations described herein can additionally comprise a permeability enhancer molecule. Non-limiting permeability enhancer molecules are described in U.S. Publication No. US 20050222064, herein incorporated by reference in its entirety.

In embodiments, the carrier is a lipidoid, a liposome, a lipoplex, a lipid nanoparticle, a polymeric nanoparticle, a peptide, a protein, a cell, a nanoparticle mimic, a nanotube, or a conjugate. In embodiments, the pharmaceutical composition is formulated as a lipid nanoparticle (LNP), a lipoplex, or a liposome. In embodiments, the pharmaceutical composition is formulated as a lipid nanoparticle (LNP). In embodiments, the lipid nanoparticles comprise lipids selected from an ionizable lipid (e.g., an ionizable cationic lipid selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, and C12-200); a structural lipid (e.g., distearoylphosphatidylcholine (DSPC)); cholesterol, and a polyethyleneglycol (PEG)-lipid (e.g., a PEG- diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof, or a PEG-dilauryloxypropyl (C12, a PEG-dimyristyloxypropyl (C14), a PEG- dipalmityloxypropyl (C16), or a PEG-distearyloxypropyl (C18)); 1 ,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); dioleoylphosphatidylethanolamine (DOPE).

In embodiments, the pharmaceutical composition is formulated as a lipid nanoparticle (LNP). In embodiments, the LNP comprises a molar ratio of about 20-60% ionizable amino lipid, about 5-25% phospholipid, about 25-55% structural lipid, and about 0.5-1.5% PEG lipid. In embodiments, the LNP comprises a molar ratio of about 50% ionizable amino lipid, about 8-12% phospholipid, about 37-40% structural lipid, and about 1-2% PEG lipid. In embodiments, the lipid nanoparticles comprise lipids selected from an ionizable lipid (e.g., an ionizable cationic lipid selected from DLin-DMA, DLin-K-DMA, DLin-KC2- DMA, DLin-MC3-DMA, 98N12-5, and C12-200); a structural lipid (e.g.., distearoylphosphatidylcholine (DSPC)); cholesterol, and a polyethyleneglycol (PEG)-lipid (e.g.., a PEG-diacylglycerol (DAG), a PEG- dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof, or a PEG- dilauryloxypropyl (C12, a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), or a PEG- distearyloxypropyl (C18)); 1 ,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); dioleoylphosphatidylethanolamine (DOPE).

In embodiments, the carrier is a lipidoid, a liposome, a lipoplex, a lipid nanoparticle, a polymeric nanoparticle, a peptide, a protein, a cell, a nanoparticle mimic, a nanotube, or a conjugate. In embodiments, the pharmaceutical composition is formulated as a lipid nanoparticle (LNP), a lipoplex, or a liposome. In embodiments, the pharmaceutical composition is formulated as a lipid nanoparticle (LNP). In embodiments, the lipid nanoparticles comprise lipids selected from an ionizable lipid (e.g., an ionizable cationic lipid selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, and C12- 200); a structural lipid (e.g., distearoylphosphatidylcholine (DSPC)); cholesterol, and a polyethyleneglycol (PEG)-lipid (e.g, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG- ceramide (Cer), or a mixture thereof, or a PEG-dilauryloxypropyl (C12, a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), or a PEG-distearyloxypropyl (C18)); 1 ,2-dioleoyl-3- trimethylammoniumpropane (DOTAP); dioleoylphosphatidylethanolamine (DOPE); and the mmRNA.

In embodiments, the lipid nanoparticles comprise lipids selected from an ionizable lipid; a structural lipid; cholesterol, and a polyethyleneglycol (PEG)-lipid; 1 ,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); dioleoylphosphatidylethanolamine (DOPE); and the mmRNA. In embodiments, the ionizable lipid is an ionizable cationic lipid selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, and C12-200. In embodiments, the polyethyleneglycol (PEG)-lipid is selected from a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof, or a PEG-dilauryloxypropyl (e.g, C12, a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), or a PEG-distearyloxypropyl (C18)).

In embodiments, the lipid nanoparticles comprise (a) a cationic lipid comprising from 50 mol % to 85 mol % of the total lipid present in the particle; (b) a non-cationic lipid comprising from 13 mol % to 49.5 mol % of the total lipid present in the particle; and (c) a conjugated lipid that inhibits aggregation of particles comprising from 0.5 mol % to 2 mol % of the total lipid present in the particle. In embodiments, the lipid nanoparticles comprise a lipid selected from SM-102, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12- 5, and C12-200; a cholesterol; and a PEG-lipid.

In any of the embodiments disclosed herein, the pharmaceutical composition is formulated for parenteral administration. In any of the embodiments disclosed herein, the pharmaceutical composition is formulated for topical administration

In aspects, the present disclosure provides a pharmaceutical composition comprising the mmRNA of any embodiment disclosed herein, or an LNP comprising an mmRNA of any embodiment disclosed herein. In embodiments, the pharmaceutical composition is formulated for parenteral administration.

In embodiments, the pharmaceutical composition comprises a modified mRNA (mmRNA) encoding a heterologous fusion protein having an amino acid sequence that has at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with the amino acid sequence selected from SEQ ID NOs: 620-93. In embodiments, the pharmaceutical composition is formulated as an LNP comprising an ionizable amino lipid, a phospholipid, a structural lipid and a PEG lipid.

In embodiments, the pharmaceutical composition is formulated for parenteral administration. In embodiments, the pharmaceutical composition is formulated for topical, dermal, intradermal, intramuscular, intraperitoneal, intraarticular, intravenous, subcutaneous, intraarterial or transdermal administration. In embodiments, the pharmaceutical composition is formulated for topical administration.

In aspects, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient or carrier, and the peptide or the derivative thereof of any one of the embodiments disclosed herein, the isolated polynucleotide of any one of the embodiments disclosed herein, the vector of the embodiments disclosed herein, or the host cell of any of the embodiments disclosed herein. In embodiments, the pharmaceutical composition comprises the nucleic acid, e.g., the mmRNA of any one of the embodiments disclosed herein.

In embodiments, the isolated polynucleotide is conjugated polynucleotide sequence that is introduced into cells by various transfection methods such as, e.g., methods that employ lipid particles. In embodiments, a composition, including a gene transfer construct, comprises a delivery particle. In embodiments, the delivery particle comprises a lipid-based particle (e.g., a lipid nanoparticle (LNP)), cationic lipid, or a biodegradable polymer). Lipid nanoparticle (LNP) delivery of gene transfer construct provides certain advantages, including transient, non-integrating expression to limit potential off-target events and immune responses, and efficient delivery with the capacity to transport large cargos. LNPs have been used for delivery of small interfering RNA (siRNA) and mRNA, and for in vitro and in vivo delivering CRISPR/Cas9 components to hepatocytes and the liver. For example, U.S. Pat. No. 10,195,291 describes the use of LNPs for delivery of RNA interference (RNAi) therapeutic agents.

In embodiments, the composition in accordance with embodiments of the present disclosure is in the form of a LNP. In embodiments, the LNP comprises one or more lipids selected from 1 ,2-dioleoyl-3- trimethylammonium propane (DOTAP); N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB), a cationic cholesterol derivative mixed with dimethylaminoethane-carbamoyl (DC-Chol), phosphatidylcholine (PC), triolein (glyceryl trioleate), and 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine- N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG), 1 ,2-dimyristoyl-rac-glycero-3- methoxypolyethyleneglycol - 2000 (DMG-PEG 2K), and 1 ,2 distearol-sn-glycerol-3phosphocholine (DSPC).

In embodiments, the composition can have a lipid and a polymer in various ratios, wherein the lipid can be selected from, e.g., DOTAP, DC-Chol, PC, Triolein, DSPE-PEG, and wherein the polymer can be, e.g., PEI or Poly Lactic-co-Glycolic Acid (PLGA). Any other lipid and polymer can be used additionally or alternatively. In embodiments, the ratio of the lipid and the polymer is about 0.5:1 , or about 1 :1 , or about 1 :1.5, or about 1 :2, or about 1 :2.5, or about 1 :3, or about 3:1, or about 2.5:1 , or about 2:1 , or about 1.5:1 , or about 1 :1 , or about 1 :0.5.

In embodiments, the LNP comprises a cationic lipid, non-limiting examples of which include N,N-dioleyl-N,N- dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(l-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2- Di Li noleyloxy-N, N-dimethylaminopropane (DLinDMA), 1 , 2-Dili nolenyloxy-N, N-dimethylami nopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1, 2-Dili noleyoxy-3- (dimethylamino)acetoxypropane (DLin-DAC), 1 ,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1 ,2- Dili noleoyl-3-dimethyl ami nopropane (DLinDAP), 1 , 2-Dili noleylthio-3-dimethylaminopropane (DLin-S-DMA), 1 -Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1 ,2-Dilinoleyloxy-3- trimethylaminopropane chloride salt (DLin-TMA.CI), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.CI), 1 ,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1 ,2- propanediol (DLinAP), 3-(N,N-Dioleylamino)-1 ,2-propanedio (DOAP), 1 ,2-Dilinolenyloxy-N,N- dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1 ,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH- cyclopenta[d][1 ,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate (MC3), 1 ,1'-(2-(4-(2-((2-(bis(2-‘)amino)ethyl)(2 hydroxydodecyl)amino)ethyl) piperazin-1 -yl)ethylazanediyl)didodecan-2-ol (Tech G1), 1 ,2-Dilinoleyloxo-3-(2-N,N-dimethylamino) ethoxypropane (DLin-EG-DMA), or a mixture thereof.

In embodiments, the LNP comprises one or more molecules selected from polyethylenimine (PEI) and poly(lactic-co-glycolic acid) (PLGA), and N-Acetylgalactosamine (GalNAc), which are suitable for hepatic delivery. In embodiments, the LNP comprises a hepatic-directed compound as described, e.g., in U.S. Pat. No. 5,985,826, which is incorporated by reference herein in its entirety. GalNAc is known to target Asialoglycoprotein Receptor (ASGPR) expressed on mammalian hepatic cells. See Hu et al. Protein Pept Lett. 2014;21 (10): 1025-30.

In some examples, the isolated polynucleotide can be formulated or complexed with PEI or a derivative thereof, such as polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives.

In embodiments, the lipid nanoparticles comprise lipids selected from an ionizable lipid (e.g., an ionizable cationic lipid selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, and C12- 200); a structural lipid (e.g., distearoylphosphatidylcholine (DSPC)); cholesterol, and a polyethyleneglycol (PEG)-lipid (e.g., a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG- ceramide (Cer), or a mixture thereof, or a PEG-dilauryloxypropyl (C12, a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), or a PEG-distearyloxypropyl (C18)); 1 ,2-dioleoyl-3- trimethylammoniumpropane (DOTAP); dioleoylphosphatidylethanolamine (DOPE); and the nucleic acid, e.g., the mmRNA.

In embodiments, the LNP comprises a molar ratio of about 20-60% ionizable amino lipid, about 5-25% phospholipid, about 25-55% structural lipid, and about 0.5-1.5% PEG lipid. In embodiments, the ionizable amino lipid comprises the following formula:

In embodiments, the lipid nanoparticles comprise lipids selected from an ionizable lipid; a structural lipid; cholesterol, and a polyethyleneglycol (PEG)-lipid; 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); dioleoylphosphatidylethanolamine (DOPE); and the nucleic acid, e.g., the mmRNA. In embodiments, the ionizable lipid is an ionizable cationic lipid selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin- MC3-DMA, 98N12-5, and C12-200. In embodiments, the polyethyleneglycol (PEG)-lipid is selected from a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof, or a PEG-dilauryloxypropyl (e.g., C12, a PEG-dimyristyloxypropyl (C14), a PEG- dipalmityloxypropyl (C16), or a PEG-distearyloxypropyl (C18)). In embodiments, the LNP is a conjugated lipid, non-limiting examples of which include a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG- phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (C12, a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), or a PEG- distearyloxypropyl (C18).

In embodiments, the LNP formulations may further contain a phosphate conjugate, which can increase in vivo circulation times and/or increase the targeted delivery of the nanoparticle. Phosphate conjugates can be made by the methods described in, e.g., PCT International Publication No. WO 2013033438 or U.S. Pub. No. US 20130196948. The LNP formulation can also contain a polymer conjugate (e.g., a water-soluble conjugate) as described in, e.g., U.S. Publication Nos. US 20130059360, US 20130196948, and US 20130072709, each of the references is herein incorporated by reference in its entirety.

In embodiments, the LNP formulations may comprise a carbohydrate carrier. As a non-limiting example, the carbohydrate carrier can include, but is not limited to, an anhydride-modified phytoglycogen or glycogen-type material, phytoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin (e.g., PCT International Publication No. WO 2012109121 , herein incorporated by reference in its entirety). In embodiments, the LNP formulations can be coated with a surfactant or polymer to improve the delivery of the particle. In some embodiments, the LNP can be coated with a hydrophilic coating such as, but not limited to, PEG coatings and/or coatings that have a neutral surface charge as described in U.S. Publication No. US 20130183244, herein incorporated by reference in its entirety. In embodiments, the LNP formulations can be engineered to alter the surface properties of particles so that the lipid nanoparticles can penetrate the mucosal barrier as described in U.S. Pat. No. 8,241 ,670 or PCT International Publication No. WO 2013110028, each of which is herein incorporated by reference in its entirety. In embodiments, the mucus penetrating LNP can be a hypotonic formulation comprising a mucosal penetration enhancing coating. The formulation can be hypotonic for the epithelium to which it is being delivered. Non-limiting examples of hypotonic formulations can be found in, e.g., PCT International Publication No. WO 2013110028, herein incorporated by reference in its entirety.

In embodiments, an mmRNA described herein is formulated as a solid lipid nanoparticle (SLN), which can be spherical with an average diameter between 10 to 1000 nm. SLN possess a solid lipid core matrix that can solubilize lipophilic molecules and can be stabilized with surfactants and/or emulsifiers. Exemplary SLN can be those as described in PCT International Publication No. WO 2013105101 , herein incorporated by reference in its entirety.

In embodiments, a nanoparticle is a particle having a diameter of less than about 1000 nm. In embodiments, nanoparticles of the present disclosure have a greatest dimension (e.g., diameter) of about 500 nm or less, or about 400 nm or less, or about 300 nm or less, or about 200 nm or less, or about 100 nm or less. In embodiments, nanoparticles of the present disclosure have a greatest dimension ranging between about 50 nm and about 150 nm, or between about 70 nm and about 130 nm, or between about 80 nm and about 120 nm, or between about 90 nm and about 110 nm. In embodiments, the nanoparticles of the present disclosure have a greatest dimension (e.g. , a diameter) of about 100 nm.

In embodiments, the peptide or the derivative thereof or the therapeutic nanoparticle comprising mRNA can be formulated for sustained release, which, as used herein, refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time. In embodiments, the period of time may include, but is not limited to, hours, days, weeks, months and years. As a non-limiting example, the sustained release nanoparticle of the mRNAs described herein can be formulated as disclosed in PCT International Publication No. WO 2010075072 and U.S. Publication Nos. US 20100216804, US 20110217377, US 20120201859 and US 20130150295, each of which is herein incorporated by reference in their entirety.

In embodiments, the peptide or the derivative thereof or the isolated polynucleotide or mmRNA (and/or additional agents) are included various formulations. Any fusion protein, or the isolated polynucleotide or mmRNA (and/or additional agents) described herein can take the form of solutions, suspensions, emulsion, drops, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. DNA or RNA constructs encoding the protein sequences may also be used. In embodiments, the composition is in the form of a capsule (see, e.g., U.S. Patent No. 5,698,155). Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), incorporated herein by reference.

In embodiments, the present disclosure provides an expression vector, comprising a nucleic acid encoding the peptide or the derivative thereof described herein. In embodiments, the expression vector comprises DNA or RNA. In embodiments, the expression vector is a mammalian expression vector. Both prokaryotic and eukaryotic vectors can be used for expression of the peptide or the derivative thereof. Prokaryotic vectors include constructs based on E. coll sequences (see, e.g., Makrides, Microbiol Rev 1996, 60:512-538). Non-limiting examples of regulatory regions that can be used for expression in E. coli include lac, trp, Ipp, phoA, recA, tac, T3, T7 and APL. Non-limiting examples of prokaryotic expression vectors may include the Agt vector series such as Agt11 (Huynh et al., in “DNA Cloning Techniques, Vol. I: A Practical Approach,” 1984, (D. Glover, ed.), pp. 49-78, IRL Press, Oxford), and the pET vector series (Studier et al., Methods Enzymol 1990, 185:60-89). Prokaryotic host-vector systems cannot perform much of the post- translational processing of mammalian cells, however. Thus, eukaryotic host- vector systems may be particularly useful. Avariety of regulatory regions can be used for expression of the peptides or the derivatives thereof in mammalian host cells. For example, the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter can be used. Inducible promoters that may be useful in mammalian cells include, without limitation, promoters associated with the metallothionein II gene, mouse mammary tumor virus glucocorticoid responsive long terminal repeats (MMTV-LTR), the p-interferon gene, and the hsp70 gene (see, Williams et al., Cancer Res 1989, 49:2735-42; and Taylor et al., Mol Cell Biol 1990, 10:165-75). Heat shock promoters or stress promoters also may be advantageous for driving expression of the peptides or the derivatives thereof in recombinant host cells.

In embodiments, expression vectors of the disclosure comprise a nucleic acid encoding the peptides or the derivatives thereof (and/or additional agents), or a complement thereof, operably linked to an expression control region, or complement thereof, that is functional in a mammalian cell. The expression control region is capable of driving expression of the operably linked blocking and/or stimulating agent encoding nucleic acid such that the blocking and/or stimulating agent is produced in a human cell transformed with the expression vector.

Expression control regions are regulatory polynucleotides (sometimes referred to herein as elements), such as promoters and enhancers, that influence expression of an operably linked nucleic acid. An expression control region of an expression vector of the disclosure is capable of expressing operably linked encoding nucleic acid in a human cell. In embodiments, the cell is a tumor cell. In embodiments, the cell is a non-tumor cell. In embodiments, the expression control region confers regulatable expression to an operably linked nucleic acid. A signal (sometimes referred to as a stimulus) can increase or decrease expression of a nucleic acid operably linked to such an expression control region. Such expression control regions that increase expression in response to a signal are often referred to as inducible. Such expression control regions that decrease expression in response to a signal are often referred to as repressible. Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal present; the greater the amount of signal, the greater the increase or decrease in expression.

In embodiments, the present disclosure contemplates the use of inducible promoters capable of effecting high level of expression transiently in response to a cue. For example, when in the proximity of a tumor cell, a cell transformed with an expression vector forthe peptide or the derivative thereof (and/or additional agents) comprising such an expression control sequence is induced to transiently produce a high level of the agent by exposing the transformed cell to an appropriate cue. Illustrative inducible expression control regions include those comprising an inducible promoter that is stimulated with a cue such as a small molecule chemical compound. Particular examples can be found, for example, in U.S. Patent Nos. 5,989,910, 5,935,934, 6,015,709, and 6,004,941, each of which is incorporated herein by reference in its entirety.

Expression control regions and locus control regions include full-length promoter sequences, such as native promoter and enhancer elements, as well as subsequences or polynucleotide variants which retain all or part of full-length or non-variant function. As used herein, the term "functional" and grammatical variants thereof, when used in reference to a nucleic acid sequence, subsequence or fragment, means that the sequence has one or more functions of native nucleic acid sequence (e.g., non-variant or unmodified sequence).

As used herein, “operable linkage’’ refers to a physical juxtaposition of the components so described as to permit them to function in their intended manner. In the example of an expression control element in operable linkage with a nucleic acid, the relationship is such that the control element modulates expression of the nucleic acid. Typically, an expression control region that modulates transcription is juxtaposed near the 5' end of the transcribed nucleic acid (/.e., “upstream”). Expression control regions can also be located at the 3’ end of the transcribed sequence (/.e., “downstream”) or within the transcript (e.g., in an intron). Expression control elements can be located at a distance away from the transcribed sequence (e.g., 100 to 500, 500 to 1000, 2000 to 5000, or more nucleotides from the nucleic acid). A specific example of an expression control element is a promoter, which is usually located 5' of the transcribed sequence. Another example of an expression control element is an enhancer, which can be located 5' or 3' of the transcribed sequence, or within the transcribed sequence.

Expression systems functional in human cells are well known in the art and include viral systems. Generally, a promoter functional in a human cell is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and typically a TATA box located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A promoter will also typically contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation. Of particular use as promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.

Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3’ terminus of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation. Examples of transcription terminator and polyadenylation signals include those derived from SV40. Introns may also be included in expression constructs.

There are a variety of techniques available for introducing nucleic acids into viable cells. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, polymer-based systems, DEAE-dextran, viral transduction, the calcium phosphate precipitation method, etc. For in vivo gene transfer, a number of techniques and reagents may also be used, including liposomes; natural polymer-based delivery vehicles, such as chitosan and gelatin; viral vectors are also suitable for in vivo transduction. In some situations, it is desirable to provide a targeting agent, such as an antibody or ligand specific for a tumor cell surface membrane protein. Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/orto facilitate uptake, e.g., capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins thattarget intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990).

Where appropriate, gene delivery agents such as, e.g., integration sequences can also be employed. Numerous integration sequences are known in the art (see, e.g., Nunes-Duby et al., Nucleic Acids Res. 26:391-406, 1998; Sadwoski, J. Bacterio!., 165:341-357, 1986; Bestor, Cell, 122(3): 322-325, 2005; Plasterk et al., TIG 15:326-332, 1999; Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). These include recombinases and transposases. Examples include Cre (Sternberg and Hamilton, J. Mol. Biol., 150:467-486, 1981), lambda (Nash, Nature, 247, 543-545, 1974), Flp (Broach, ef al., Cell, 29:227-234, 1982), R (Matsuzaki, et al., J. Bacteriology, 172:610-618, 1990), cpC31 (see, e.g., Groth et al., J. Mol. Biol. 335:667- 678, 2004), sleeping beauty, transposases of the mariner family (Plasterk et al., supra), and components for integrating viruses such as AAV, retroviruses, and antiviruses having components that provide for virus integration such as the LTR sequences of retroviruses or lentivirus and the ITR sequences of AAV (Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). In addition, direct and targeted genetic integration strategies may be used to insert nucleic acid sequences encoding the peptides or the derivatives thereof including CRISPR/CAS9, zinc finger, TALEN, and meganuclease gene-editing technologies.

In aspects, the disclosure provides expression vectors for the expression of the peptides or the derivatives thereof (and/or additional agents) that are viral vectors. Many viral vectors useful for gene therapy are known (see, e.g., Lundstrom, Trends Biotechnol. , 21 : 1 17, 122, 2003. Illustrative viral vectors include those selected from Antiviruses (LV), retroviruses (RV), adenoviruses (AV), adeno-associated viruses (AAV), and a viruses, though other viral vectors may also be used. For in vivo uses, viral vectors that do not integrate into the host genome are suitable for use, such as a viruses and adenoviruses. Illustrative types of a viruses include Sindbis virus, Venezuelan equine encephalitis (VEE) virus, and Semliki Forest virus (SFV). For in vitro uses, viral vectors that integrate into the host genome are suitable, such as retroviruses, AAV, and Antiviruses. In embodiments, the disclosure provides methods of transducing a human cell in vivo, comprising contacting a solid tumor in vivo with a viral vector of the disclosure.

In embodiments, the present disclosure provides a host cell, comprising the expression vector comprising the peptide or the derivative thereof described herein.

Expression vectors can be introduced into host cells for producing the present fusion proteins. Cells may be cultured in vitro or genetically engineered, for example. Useful mammalian host cells include, without limitation, cells derived from humans, monkeys, and rodents (see, for example, Kriegler in “Gene Transfer and Expression: A Laboratory Manual,” 1990, New York, Freeman & Co.). These include monkey kidney cell lines transformed by SV40 (e.g., COS-7, ATCC CRL 1651); human embryonic kidney lines (e.g., 293, 293- EBNA, or 293 cells subcloned for growth in suspension culture, Graham ef al., J Gen Virol 1977, 36:59); baby hamster kidney cells (e.g., BHK, ATCC CCL 10); Chinese hamster ovary-cells-DHFR (e.g., CHO, Urlaub and Chasin, Proc Natl Acad Sci USA 1980, 77:4216); DG44 CHO cells, CHO-K1 cells, mouse sertoli cells (Mather, Biol Reprod ] 980, 23:243-251); mouse fibroblast cells (e.g., NIH-3T3), monkey kidney cells (e.g., CV1 ATCC CCL 70); African green monkey kidney cells, (e.g., VERO-76, ATCC CRL-1587); human cervical carcinoma cells (e.g., HELA, ATCC CCL 2); canine kidney cells (e.g., MDCK, ATCC CCL 34); buffalo rat liver cells (e.g., BRL 3A, ATCC CRL 1442); human lung cells (e.g., W138, ATCC CCL 75); human liver cells (e.g., Hep G2, HB 8065); and mouse mammary tumor cells (e.g., MMT 060562, ATCC CCL51). Illustrative cancer cell types for expressing the peptides or the derivatives thereof described herein include mouse fibroblast cell line, NIH3T3, mouse Lewis lung carcinoma cell line, LLC, mouse mastocytoma cell line, P815, mouse lymphoma cell line, EL4 and its ovalbumin transfectant, E.G7, mouse melanoma cell line, B16F10, mouse fibrosarcoma cell line, MC57, and human small cell lung carcinoma cell lines, SCLC#2 and SCLC#7.

Host cells can be obtained from normal or affected subjects, including healthy humans, cancer patients, and patients with an infectious disease, private laboratory deposits, public culture collections such as the American Type Culture Collection, or from commercial suppliers.

Cells that can be used for production of the present fusion proteins in vitro, ex vivo, and/or in vivo include, without limitation, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells (e.g., as obtained from bone marrow), umbilical cord blood, peripheral blood, fetal liver, etc. The choice of cell type depends on the type of tumor or infectious disease being treated or prevented, and can be determined by one of skill in the art.

Where necessary, the formulations comprising the peptide or the derivative thereof, or the isolated polynucleotide (and/or additional agents) can also include a solubilizing agent. Also, the agents can be delivered with a suitable vehicle or delivery device as known in the art. Combination therapies outlined herein can be co-delivered in a single delivery vehicle or delivery device. Compositions for administration can optionally include a local anesthetic such as, for example, lignocaine to lessen pain at the site of the injection.

The formulations comprising the peptide or the derivative thereof (and/or additional agents) of the present disclosure may conveniently be presented in unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing the therapeutic agents into association with a carrier, which constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation (e.g., wet or dry granulation, powder blends, etc., followed by tableting using conventional methods known in the art)

In embodiments, any fusion protein, or the isolated polynucleotide or mmRNA (and/or additional agents) described herein is formulated in accordance with routine procedures as a composition adapted for a mode of administration described herein.

Routes of administration include, for example: intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin. In embodiments, the administering is effected orally or by parenteral injection. In some instances, administration results in the release of any agent described herein into the bloodstream, or alternatively, the agent is administered directly to the site of active disease.

Any fusion protein, or the isolated polynucleotide (and/or additional agents) described herein can be administered orally. Such fusion proteins (and/or additional agents) can also be administered by any other convenient route, for example, by intravenous infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with another biologically active agent. Administration can be systemic or local. Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer.

In embodiments, the pharmaceutical composition is formulated for parenteral administration. In embodiments, the pharmaceutical composition is formulated for intradermal, intramuscular, intraperitoneal, intraarticular, intravenous, subcutaneous, intraarterial or transdermal administration.

Dosage forms suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous and intra-articular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g., lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art.

The dosage of any fusion protein, or the isolated polynucleotide or mmRNA (and/or additional agents) described herein as well as the dosing schedule can depend on various parameters, including, but not limited to, the disease being treated, the subject’s general health, and the administering physician’s discretion. Any fusion protein described herein, can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of an additional agent, to a subject in need thereof. In embodiments any fusion protein and additional agent described herein are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, 1 day apart, 2 days apart, 3 days part, 4 days apart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeks apart, or 4 weeks apart.

The dosage of any fusion protein, or the isolated polynucleotide or mmRNA (and/or additional agents) described herein can depend on several factors including the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the subject to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular subject may affect dosage used. Furthermore, the exact individual dosages can be adjusted somewhat depending on a variety of factors, including the specific combination of the agents being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the particular disease being treated, the severity of the disorder, and the anatomical location of the disorder. Some variations in the dosage can be expected.

In embodiments, delivery can be in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527- 1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989).

Any fusion protein, or the isolated polynucleotide (and/or additional agents) described herein can be administered by controlled-release or sustained-release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,556, each of which is incorporated herein by reference in its entirety. Such dosage forms can be useful for providing controlled- or sustained-release of one or more active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions. Controlled- or sustained- release of an active ingredient can be stimulated by various conditions, including but not limited to, changes in pH, changes in temperature, stimulation by an appropriate wavelength of light, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds.

In embodiments, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (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 ; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351 ; Howard et al., 1989, J. Neurosurg. 71 :105).

In embodiments, a controlled-release system can be placed in proximity of the target area to be treated, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled-release systems discussed in the review by Langer, 1990, Science 249:1527-1533) may be used.

Administration of any fusion protein, or the isolated polynucleotide or mmRNA (and/or additional agents) described herein can, independently, be one to four times daily or one to four times per month or one to six times per year or once every two, three, four or five years. Administration can be for the duration of one day or one month, two months, three months, six months, one year, two years, three years, and may even be for the life of the subject.

The dosage regimen utilizing any fusion protein, or the isolated polynucleotide or mmRNA (and/or additional agents) described herein can be selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the subject; the severity of the condition to be treated; the route of administration; the renal or hepatic function of the subject; the pharmacogenomic makeup of the individual; and the specific compound of the disclosure employed. Any fusion protein (and/or additional agents) described herein can be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three or four times daily. Furthermore, any fusion protein (and/or additional agents) described herein can be administered continuously rather than intermittently throughout the dosage regimen. Methods of Treatment or Prevention

In aspects, the present disclosure provides a method of treating or preventing hyperglycemia, diabetes, obesity or metabolic syndrome, nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), a cardiometabolic disease, or liver fibrosis or for reducing blood glucose, or for reducing fed and fasting blood glucose, or for reducing cardiovascular risk, or for decreasing body weight, decreasing food intake, decreasing blood glucose, decreasing liver adiposity, decreasing liver weight, decreasing subcutaneous white adipose tissue (sWAT), or for increasing glucose tolerance in a subject in need thereof, the method comprising administering to the subject the peptide or the derivative thereof of any of the embodiments disclosed herein.

In aspects, the present disclosure provides a method of treating or preventing hyperglycemia, diabetes, obesity or metabolic syndrome, nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), a cardiometabolic disease, or liver fibrosis or for reducing blood glucose, or for reducing fed and fasting blood glucose, or for reducing cardiovascular risk, or for decreasing body weight, decreasing food intake, decreasing blood glucose, decreasing liver adiposity, decreasing liver weight, decreasing subcutaneous white adipose tissue (sWAT), or for increasing glucose tolerance in a subject in need thereof, the method comprising administering to the subject the isolated polynucleotide of any of the embodiments disclosed herein.

In aspects, the present disclosure provides a method of treating or preventing hyperglycemia, diabetes, obesity or metabolic syndrome, nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), a cardiometabolic disease, or liver fibrosis or for reducing blood glucose, or for reducing fed and fasting blood glucose, or for reducing cardiovascular risk, or for decreasing body weight, decreasing food intake, decreasing blood glucose, decreasing liver adiposity, decreasing liver weight, decreasing subcutaneous white adipose tissue (sWAT), or for increasing glucose tolerance in a subject in need thereof, the method comprising administering to the subject the expression vector of any of the embodiments disclosed herein.

In aspects, the present disclosure provides a method of treating or preventing hyperglycemia, diabetes, obesity or metabolic syndrome, nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), a cardiometabolic disease, or liver fibrosis or for reducing blood glucose, or for reducing fed and fasting blood glucose, or for reducing cardiovascular risk, or for decreasing body weight, decreasing food intake, decreasing blood glucose, decreasing liver adiposity, decreasing liver weight, decreasing subcutaneous white adipose tissue (sWAT), or for increasing glucose tolerance in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of any of the embodiments disclosed herein.

In aspects, the present disclosure provides a method of treating or preventing hyperglycemia in a subject in need thereof, the method comprising administering to the subject the peptide or the derivative thereof of any of the embodiments disclosed herein, the isolated polynucleotide of any ofthe embodiments disclosed herein, or the expression vector of any of the embodiments disclosed herein, or the pharmaceutical composition of any of the embodiments disclosed herein.

In aspects, the present disclosure provides a method of treating or preventing diabetes in a subject in need thereof, the method comprising administering to the subject the peptide or the derivative thereof of any of the embodiments disclosed herein, the isolated polynucleotide of any of the embodiments disclosed herein, or the expression vector of any of the embodiments disclosed herein, or the pharmaceutical composition of any of the embodiments disclosed herein.

In aspects, the present disclosure provides a method of treating or preventing obesity in a subject in need thereof, the method comprising administering to the subject the peptide or the derivative thereof of any of the embodiments disclosed herein, the isolated polynucleotide of any of the embodiments disclosed herein, or the expression vector of any of the embodiments disclosed herein, or the pharmaceutical composition of any of the embodiments disclosed herein.

In aspects, the present disclosure provides a method of treating or preventing metabolic syndrome in a subject in need thereof, the method comprising administering to the subject the peptide or the derivative thereof of any of the embodiments disclosed herein, the isolated polynucleotide of any of the embodiments disclosed herein, or the expression vector of any ofthe embodiments disclosed herein, or the pharmaceutical composition of any of the embodiments disclosed herein.

In aspects, the present disclosure provides a method for reducing blood glucose in a subject in need thereof, the method comprising administering to the subject the peptide or the derivative thereof of any of the embodiments disclosed herein, the isolated polynucleotide of any of the embodiments disclosed herein, or the expression vector of any of the embodiments disclosed herein, or the pharmaceutical composition of any of the embodiments disclosed herein. In aspects, the present disclosure provides a method for reducing fed and fasting blood glucose in a subject in need thereof, the method comprising administering to the subject the peptide or the derivative thereof of any of the embodiments disclosed herein, the isolated polynucleotide of any of the embodiments disclosed herein, or the expression vector of any of the embodiments disclosed herein, or the pharmaceutical composition of any of the embodiments disclosed herein.

In aspects, the present disclosure provides a method for reducing cardiovascular risk in a subject in need thereof, the method comprising administering to the subject the peptide or the derivative thereof of any of the embodiments disclosed herein, the isolated polynucleotide of any of the embodiments disclosed herein, or the expression vector of any of the embodiments disclosed herein, or the pharmaceutical composition of any of the embodiments disclosed herein.

In aspects, the present disclosure provides a method for decreasing body weight in a subject in need thereof, the method comprising administering to the subject the peptide or the derivative thereof of any of the embodiments disclosed herein, the isolated polynucleotide of any of the embodiments disclosed herein, or the expression vector of any of the embodiments disclosed herein, or the pharmaceutical composition of any of the embodiments disclosed herein.

In aspects, the present disclosure provides a method for decreasing food intake in a subject in need thereof, the method comprising administering to the subject the peptide or the derivative thereof of any of the embodiments disclosed herein, the isolated polynucleotide of any of the embodiments disclosed herein, or the expression vector of any of the embodiments disclosed herein, or the pharmaceutical composition of any of the embodiments disclosed herein.

In aspects, the present disclosure provides a method for decreasing liver adiposity in a subject in need thereof, the method comprising administering to the subject the peptide or the derivative thereof of any of the embodiments disclosed herein, the isolated polynucleotide of any of the embodiments disclosed herein, or the expression vector of any of the embodiments disclosed herein, or the pharmaceutical composition of any of the embodiments disclosed herein.

In aspects, the present disclosure provides a method for decreasing liver weight in a subject in need thereof, the method comprising administering to the subject the peptide or the derivative thereof of any of the embodiments disclosed herein, the isolated polynucleotide of any of the embodiments disclosed herein, or the expression vector of any of the embodiments disclosed herein, or the pharmaceutical composition of any of the embodiments disclosed herein. In aspects, the present disclosure provides a method for decreasing subcutaneous white adipose tissue (sWAT) in a subject in need thereof, the method comprising administering to the subject the peptide or the derivative thereof of any of the embodiments disclosed herein, the isolated polynucleotide of any of the embodiments disclosed herein, or the expression vector of any of the embodiments disclosed herein, or the pharmaceutical composition of any of the embodiments disclosed herein.

In aspects, the present disclosure provides a method for increasing glucose tolerance in a subject in need thereof, the method comprising administering to the subject the peptide or the derivative thereof of any of the embodiments disclosed herein, the isolated polynucleotide of any of the embodiments disclosed herein, or the expression vector of any of the embodiments disclosed herein, or the pharmaceutical composition of any of the embodiments disclosed herein.

In embodiments, the subject selected for the treatment with the compositions comprising the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein based on diagnosis symptoms selected from one or more of the subject suffers from one or more symptoms selected from increased urination, increased thirst, increased hunger, increased food intake, increased weight, obesity, weight loss, blurry vision, numbing or tingling hands or feet, very dry skin, increased infections, and diabetic sores. In embodiments, the subject is asymptomatic. In embodiments, the patient has type 1 diabetes. In embodiments, the patient has type 2 diabetes. In embodiments, the patient has gestational diabetes or steroid-induced diabetes.

In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein is administered to a patient that has one or more of an average hemoglobin A1c value of more than about 10%, or more than about 11%, or more than about 12%, or more than about 13%, or more than about 14%, or more than about 15%) at the start of treatment with conventional diabetic therapy. In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein is administered to a patient that has an average glucose of more than about 200 mg/dl, or more than about 210 mg/dl, or more than about 220 mg/dl, or more than about 230 mg/dl, or more than about 240 mg/dl, or more than about 250 mg/dl at the start of treatment with conventional diabetic therapy. In various embodiments, the conventional diabetic therapy is any one of those described herein, including, for example, insulin therapy and non-insulin diabetes agent therapy. In embodiments, the treatment comprises one or more of a decrease of the blood glucose level, stimulation of peripheral glucose disposal, and inhibition of hepatic glucose production. These biological activities can be assayed in vitro using known methodologies. For example, the effect of the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein on glucose uptake in 3T3-L1 adipocytes can be measured and compared with that of insulin. Pretreatment of the cells with a biologically active analog may generally produce a dose-dependent increase in 2- deoxyglucose uptake. The ability of the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein to regulate glucose production may be measured in any number of cell types, for example, H4lle hepatoma cells. In this assay, pretreatment with a biologically active analog may generally result in a dose-dependent inhibition of the amount of glucose released.

In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein is administered as an anti-diabetic regimen that decreases blood glucose levels; stimulates peripheral glucose disposal; and/or inhibits hepatic glucose production. In embodiments, the treatment regimen comprises administering the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein on an insulinlike regimen. For example, in embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein is administered once in the morning or the evening (e.g., at bedtime), or in a twice-daily regimen (e.g., pre-breakfast and pre-evening meal, or as a continuous administration (e.g., analogously to administration of insulin via an insulin pump).

In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein administration is effective for providing glycemic control. Glycemic control refers to the typical levels of blood sugar (glucose) in a person with diabetes mellitus. Many of the long-term complications of diabetes, including microvascular complications, result from many years of hyperglycemia. Good glycemic control is an important goal of diabetes care. Because blood sugar levels fluctuate throughout the day and glucose records are imperfect indicators of these changes, the percentage of hemoglobin which is glycosylated is used as a proxy measure of long-term glycemic control in research trials and clinical care of people with diabetes. In this test, the hemoglobin A1c or glycosylated hemoglobin reflects average glucose values over the preceding 2-3 months. In non-diabetic persons with normal glucose metabolism glycosylated hemoglobin levels are usually about 4-6% by the most common methods (normal ranges may vary by method). “Perfect glycemic control” indicates that glucose levels are always normal (e.g., about 70-130 mg/dl, or about 3.9-7.2 mmol/L) and indistinguishable from a person without diabetes. In reality, because of the imperfections of treatment measures, even “good glycemic control” describes blood glucose levels that average somewhat higher than normal much of the time. It is noted that what is considered “good glycemic control” varies by age and susceptibility of the patient to hypoglycemia. The American Diabetes Association has advocated for patients and physicians to strive for average glucose and hemoglobin A1c values below 200 mg/dl (11 mmol/l) and 8%. “Poor glycemic control” refers to persistently elevated blood glucose and glycosylated hemoglobin levels, which may range from, e.g., about 200-500 mg/dl (about 11-28 mmol/L, e.g., about 200 mg/dl, or about 250 mg/dl, or about 300 mg/dl, or about 350 mg/dl, or about 400 mg/dl, or about 450 mg/dl, or about 500 mg/dl) and about 9-15% (e.g., about 9%, or about 10%, or about 11%, or about 12%, or about 13%, or about 14%, or about 15%) or higher over months and years before severe complications occur. In some aspects, the present disclosure provides for methods of treatment comprising administering the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein and/or uses of the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein in the treatment of or manufacture of a medicament for diabetes and/or glucose intolerance. In embodiments, the present disclosure provides for methods treating diabetes, prediabetes, and/or glucose intolerance, comprising administering an effective amount of the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein to a patient that suffers from poor glycemic control. In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein administration is effective for providing an average glucose of below about 200 mg/dl (11 mmol/l). In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein administration is effective for providing an average glucose of below about 190 mg/dl, or about 180 mg/dl, or about 170 mg/dl, or about 160 mg/dl, or about 150 mg/dl, or about 140 mg/dl, or about 130 mg/dl , or about 120 mg/dl, or about 120 mg/dl, or about 110 mg/dl, or about 100 mg/dl, or about 90 mg/dl, or about 80 mg/dl, or about 70 mg/dl. In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein administration is effective for providing an average glycosylated hemoglobin levels (hemoglobin A1c) value of about 8%, or about 7%, or about 6%, or about 5%, or about 4%. In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein administration is effective for providing average glycosylated hemoglobin levels (hemoglobin A1c) values of less than about 8%, or less than about 7%, or less than about 6%, or less than about 5%, or less than about 4%.

In various embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein administration does not cause a patient to experience an increase of insulin upon the administration of the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein administration. Accordingly, in embodiments, the anti-diabetic effects of the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein administration are insulin-independent.

In various embodiments, the patient is undergoing treatment with one or more of insulin or an insulin analog. In embodiments, the insulin analog is selected from a rapid acting (e.g., ispro, aspart and glulisine) or a long acting (e.g., glargine or detemir) insulin analog.

In various embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein is administered as an adjuvant therapy. For instance, a diabetic patient may receive treatment with insulin or an insulin analog, or any of the agents listed herein (e.g., sulfonylureas, biguanides, meglitinides, thiazolidinediones, DPP-4 inhibitors, SGLT2 Inhibitors, Alpha-glucosidase inhibitors, and bile acid sequestrants) and the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein is administered to supplement these treatments. For example, in embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein use an adjuvant therapy with a long-acting insulin offsets the high frequency of hypo- and hyperglycemic excursions and modest reduction in HbA1c seen with these agents.

Further, in embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein administration provides a sustained antidiabetic effect relative to insulin. There are two phases of insulin release in response to a rise in glucose. The first is an immediate release of insulin. This is attributable to the release of preformed insulin, which is stored in secretory granules. After a short delay, there is a second, more prolonged release of newly synthesized insulin. Once released, insulin is active for only a brief time before it is degraded by enzymes. Insulinase found in the liver and kidneys breaks down insulin circulating in the plasma, and as a result, insulin has a half-life of only about 2-5 minutes. This short duration of action results in rapid changes in the circulating levels of insulin. In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein solves this short duration of insulin problem by providing a substitute therapy for a diabetes patient.

Furthermore, insulin often multimerizes into dimers and hexamers, which slows its effect and/or reduces the amount of bioavailable agent. In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein solves this problem by providing a substitute therapy for a diabetes patient.

Furthermore, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein may be administered orally to diabetic patients and therefore improves patient quality of life/therapeutic adherence relative to injectable insulin regimens.

Further still, in embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein administration does not cause one or more of common side effects of standard diabetes care, such as hypoglycemia or hypokalemia.

In various aspects, the present methods provide for the treatment of diabetes with the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein in specific patient populations in need thereof. For example, in various embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein may supplement various agents in a treatment regimen for diabetes, including type 1 or type 2 diabetes, or may supplant various agents in a treatment regimen for diabetes, including type 1 or type 2 diabetes. For example, in embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein is an adjuvant therapy for type 1 or type 2 diabetes.

Type 1 diabetes, once known as juvenile diabetes or insulin-dependent diabetes, is a chronic condition in which the pancreas produces little or no insulin. Treatment is often via intensive insulin regimens, which attempt to mimic the body’s normal pattern of insulin secretion, and often involve basal and bolus insulin coverage. For example, one common regimen is the administration of a long-acting insulin (as described herein and including, for example, glargine/detemir) once or twice a day with rapid acting insulin (as described herein and including, for example, aspart, glulisine, lispro) preprandially or postprandially and as needed to correct high blood sugars (as monitored by a glucose meter, for example). Doses administered preprandially or postprandially or as needed to correct high blood sugars may be referred to as bolus administrations. Another common regimen involves dosing, including continuous dosing, via an insulin pump (or continuous subcutaneous insulin infusion device (CSI I)) of, for example a rapid acting insulin (as described herein and including, for example, aspart, glulisine, lispro). In various embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein may replace any of the insulins used in various regimens, including instances in which the insulins are not providing effective therapy in the patient. The peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein may cause an increase in patient compliance as it may allow for easier self-dosing relative to various forms of insulin, which must be administered as various doses throughout the day- even in the context of an insulin pump, which requires programming. Further, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein can offset common frustration of diabetic patient dosing, such as, for example, the dawn phenomenon. Alternatively, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein may be used adjuvant to any of the type 1 diabetes treatments described herein to, for example, normalize a patient’s regimen and avoid blood sugar “dips” (e.g., hypoglycemia, e.g., blood sugar of below about 70 mg/dL) and “spikes” (e.g., hyperglycemia, e.g., blood sugar of below about 200 mg/dL) that afflict many patients. Accordingly, In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein may treat or prevent symptoms associated with hypoglycemia, including for example, shakiness, anxiety, nervousness, palpitations, tachycardia, pallor, coldness, clamminess, dilated pupils (mydriasis), hunger, borborygmus, nausea, vomiting, abdominal discomfort, headache, abnormal mentation, impaired judgment, nonspecific dysphoria, paresthesia, negativism, irritability, belligerence, combativeness, rage, personality change, emotional lability, fatigue, weakness, apathy, lethargy, daydreaming, sleep, confusion, amnesia, lightheadedness or dizziness, delirium, staring, "glassy" look, blurred vision, double vision, flashes of light in the field of vision, automatism, difficulty speaking, slurred speech, ataxia, incoordination, focal or general motor deficit, paralysis, hemiparesis, paresthesia, headache, stupor, coma, abnormal breathing, generalized or focal seizures, memory loss, and amnesia. Accordingly, In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein may treat or prevent symptoms associated with hyperglycemia, including for example, polyphagia, polydipsia, polyuria, blurred vision, fatigue, weight loss, poor wound healing, dry mouth, dry or itchy skin, tingling in feet or heels, erectile dysfunction, recurrent infections, external ear infections (e.g., swimmer's ear), cardiac arrhythmia, stupor, coma, and seizures. In various regimens, a type 1 diabetes may receive additional agents to supplement insulin therapy. In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein are used in this manner. The peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein may provide additional therapeutic benefits in patients that are struggling to manage type 1 diabetes with insulin therapy alone. In embodiments, patients that are struggling to manage type 1 diabetes with insulin therapy alone have poor glycemic control as described herein.

Patients with type 2 diabetes may be instructed to manage their diabetes with healthy eating and exercise. However, certain non-insulin diabetes agents (e.g., selected from metformin (e.g., GLUCOPHAGE, GLUMETZA); sulfonylureas (e.g., glyburide (e.g., DIABETA, GLYNASE), glipizide (e.g., GLUCOTROL) and glimepiride (e.g., AMARYL)); thiazolidinediones (e.g., rosiglitazone (e.g., AVANDIA) and pioglitazone (e.g., ACTOS)); DPP-4 inhibitors (e.g., sitagliptin (e.g., JANUVIA), saxagliptin (e.g., ONGLYZA) and linagliptin (e.g., TRADJENTA)); GLP-1 receptor agonists (e.g., exenatide (e.g., BYETTA) and liraglutide (e.g., VICTOZA)); and SGLT2 inhibitors (e.g., canagliflozin (e.g., NVOKANA) and dapagliflozin (e.g., FARXIGA))) and/or insulin may be used in treatment. For example, certain patients may be able to manage diabetes with diet and exercise alone (e.g., along with glucose monitoring). However, often this is not the case and therapeutic agents are needed. A first line of treatment may be a non-insulin diabetes agent (e.g., selected from metformin (e.g., GLUCOPHAGE, GLUMETZA); sulfonylureas (e.g., glyburide (e.g., DIABETA, GLYNASE), glipizide (e.g., GLUCOTROL) and glimepiride (e.g., AMARYL)); thiazolidinediones (e.g., rosiglitazone (e.g., AVANDIA) and pioglitazone (e.g., ACTOS)); DPP-4 inhibitors (e.g., sitagliptin (e.g., JANUVIA), saxagliptin (e.g., ONGLYZA) and linagliptin (e.g., TRADJENTA)); GLP-1 receptor agonists (e.g., exenatide (e.g., BYETTA) and liraglutide (e.g., VICTOZA)); and SGLT2 inhibitors (e.g., canagliflozin (e.g., NVOKANA) and dapagliflozin (e.g., FARXIGA)). However, some of these agents provide side effects (e.g., in the case of metformin, abdominal or stomach discomfort, cough or hoarseness, decreased appetite, diarrhea, fast or shallow breathing, fever or chills, general feeling of discomfort, lower back or side pain, muscle pain or cramping, painful or difficult urination, and sleepiness) or negative drug interactions (e.g., in the case of metformin, certain imaging and contrast agents). In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein are used instead of a non-insulin diabetes agent or in combination with one or more non-insulin diabetes agents (e.g., to lower the dose of the non-insulin diabetes agents and increase their therapeutic windows). In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein is used to improve an ineffective treatment regimen. In certain embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein increase patient compliance and increase the likelihood of effective type 2 diabetes management. In certain embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein replaces a non-insulin diabetes agent in a patient’s treatment regimen in a patient whose diabetes is not wellmanaged by a non-insulin diabetes agent (e.g., those having uncontrolled, cardiovascular complications and/or blood glucose levels). In embodiments, a patient whose diabetes is not well-managed by a non-insulin diabetes agent has poor glycemic control as described herein.

In some type 2 diabetes patients, diet and exercise and/or non-insulin diabetes agents are insufficient for treatment of diabetes and treatment with insulin therapy is needed. In various embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein may prevent the need to turn to insulin therapy in type 2 diabetes patients or reduce the amount (e.g., frequency of administration) of insulin therapy in type 2 diabetes patients. For example, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein may be used in certain type 2 diabetes patient populations that are often at risk for needing insulin therapy, including patients afflicted having: acute infections or other serious illnesses, pregnancy, major surgery, congestive heart failure, kidney disease, liver disease, use of other drugs (e.g., prednisone and some psychiatric medications), overeating or excessive weight gain (including obesity), and progressive loss of beta cell function. In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein may be administered to patients having onset of diabetes prior to age thirty, or a duration over fifteen years to prevent the need for insulin therapy. Further, In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein is used to treat diabetes in patients at risk for uncontrolled or poorly controlled type 2 diabetes (overweight and/or obese patients, patients with high abdominal fat distribution, inactive patients, patients with a family history, of type 2 diabetes, patients of certain racial groups (e.g., blacks, Hispanics, American Indians and Asian-Americans), older patients (e.g., over the age of about 45), patients previously afflicted with gestational diabetes and/or who have birthed a baby weighing more than about 9 pounds, and patients having polycystic ovary syndrome).

In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein is used to treat type 2 diabetes patients that have uncontrolled or poorly controlled type 2 diabetes and are facing a nontraumatic lower extremity amputation (LEA). In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein is used to treat type 2 diabetes patients that have uncontrolled or poorly controlled type 2 diabetes and have some degree of vision loss and/or blindness (by way of non-limiting example, diabetic retinopathy, which may include one or more of non-proliferative diabetic retinopathy (including, for example, treating microanuerysms) and proliferative diabetic retinopathy (including, for example, treating vitreous, clouding vision, detachment of the retina and glaucoma). In embodiments, the determination of whether a patient is afflicted with or has a high risk for some degree of vision loss and/or blindness comprises diagnostic methods known in the art (e.g., ophthalmoscopy, fluorescein angiography). In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein is used to treat type 2 diabetes patients that have uncontrolled or poorly controlled type 2 diabetes and have end-stage renal disease (including, for example, end-stage renal disease).

In some aspects the present disclosure relates to a method for treating diabetes and/or prediabetes, glucose intolerance, comprising administering an effective amount of the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein to a patient in need thereof, wherein the patient is not receiving insulin therapy, optionally comprising treatment with one or more of insulin or an insulin analog. In embodiments, the patient is not receiving one or more of basal, pre- prandial, and postprandial insulin therapy. In embodiments, the patient is not receiving basal insulin therapy but is receiving pre-prandial or postprandial insulin therapy. For example, in embodiments, a patient does not receive basal insulin therapy but is receiving pre-prandial or postprandial insulin therapy and the basal insulin is replaced with the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein. Such patients, in embodiments, are those that present with at least a partial level of insulin resistance and/or those whose diabetes is not sufficiently controlled with basal insulin therapy. In embodiments, the patient is not receiving preprandial or postprandial insulin therapy but is receiving basal insulin therapy. For example, In embodiments, a patient does not receive preprandial or postprandial insulin therapy but is receiving basal insulin therapy and the preprandial or postprandial insulin therapy is replaced with the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein. Such patients, in embodiments, are those whose diabetes is not sufficiently controlled with preprandial or postprandial insulin therapy (e.g., experiencing bouts of hypoglycemia and/or hyperglycemia). In embodiments, the patient has not received insulin therapy in up to about 1 hour, or up to about 2 hours, or up to about 3 hours, or up to about 4 hours, or up to about 5 hours, or up to about 6 hours, or up to about 7 hours, or up to about 8 hours, or up to about 12 hours or up to about 16 hours or up to about 20 hours, or up to about 24 hours, up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, up to about 7 days.

In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein supplants insulin in the treatment of type 1 or type 2 diabetes. In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein is used as an insulin replacement therapy. In various embodiments, the patient has experienced one or more instances of lipodystrophy that is caused by injection (e.g., injection of insulin). In embodiments, the patient is afflicted with or is at risk of having hypokalemia. In embodiments, the patient is afflicted with or is at risk of having an insulin allergy or allergy to a an agent, such as zinc, commonly used to formulate insulin (e.g., a patient having or who has previously had an immediate hypersensitive reaction upon insulin injection (e.g., injection site swelling, redness and/or itching; local tender subcutaneous nodules which develop about 0.5 to about 6 hours after an insulin injection; inflammation of the lymph glands, a serum sickness reaction and arthralagia).

In embodiments, the patient is also receiving one or more non-insulin diabetes agents selected from metformin (e.g., GLUCOPHAGE, GLUMETZA); sulfonylureas (e.g., glyburide (e.g., DIABETA, GLYNASE), glipizide (e.g., GLUCOTROL) and glimepiride (e.g., AMARYL)); thiazolidinediones (e.g., rosiglitazone (e.g., AVANDIA) and pioglitazone (e.g., ACTOS)); DPP-4 inhibitors (e.g., sitagliptin (e.g., JANUVIA), saxagliptin (e.g., ONGLYZA) and linagliptin (e.g., TRADJENTA)); GLP-1 receptor agonists (e.g., exenatide (e.g., BYETTA) and liraglutide (e.g., VICTOZA)); and SGLT2 inhibitors (e.g., canagliflozin (e.g., NVOKANA) and dapagliflozin (e.g., FARXIGA)).

In embodiments, the patient is not receiving one or more non-insulin diabetes agents selected from metformin (e.g., GLUCOPHAGE, GLUMETZA); Sulfonylureas (e.g., glyburide (e.g., DIABETA, GLYNASE), glipizide (e.g., GLUCOTROL) and glimepiride (e.g., AMARYL)); thiazolidinediones (e.g., rosiglitazone (e.g., AVANDIA) and pioglitazone (e.g., ACTOS)); DPP-4 inhibitors (e.g., sitagliptin (e.g., JANUVIA), saxagliptin (e.g., ONGLYZA) and linagliptin (e.g., TRADJENTA)); GLP-1 receptor agonists (e.g., exenatide (e.g., BYETTA) and liraglutide (e.g., VICTOZA)); and SGLT2 inhibitors (e.g., canagliflozin (e.g., NVOKANA) and dapagliflozin (e.g., FARXIGA)). For example, insulin has negative interactions with thiazolidinediones (e.g., rosiglitazone (e.g., AVANDIA) and pioglitazone (e.g., ACTOS)), including, by way of illustration, adipogenesis and fluid retention. In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein is used in place of insulin to treat a patient with type 2 diabetes with one or more thiazolidinediones (e.g., rosiglitazone (e.g., AVANDIA) and pioglitazone (e.g., ACTOS)). Also, beta-blocker medications (such as, for example, metoprolol, propranolol, glaucoma eye drops, such as timolol) may prevent symptoms indicative of hypoglycemia (e.g., heartbeat) when used with insulin and therefore stifle quick treatment response to hypoglycemia. In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein is used in patients that are being treated with beta-blocker medications (such as, for example, metoprolol, propranolol, glaucoma eye drops, such as timolol) and, optionally, are experiencing have experienced a substantial number of hypoglycemic symptoms.

In some aspects the present disclosure provides a method for treating type 1 or type 2 diabetes, prediabetes, and/or glucose intolerance, comprising administering an effective amount of the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein to a patient in need thereof, wherein the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein stimulates glucose uptake in the patient. In embodiments, the glucose uptake in mediated by glucose transporter type 4 (GLUT4). In embodiments, the glucose uptake is in muscle or fat cells.

In some aspects the present disclosure provides a method for treating type 1 or type 2 diabetes, comprising administering an effective amount of the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein to a patient in need thereof, wherein the patient is afflicted with or has a high risk for a vascular disease.

In embodiments, the vascular disease is selected from stroke, deep vein thrombosis (DVT), myocardial infarction, coronary artery disease, cerebrovascular disease, peripheral arterial disease, diabetic retinopathy, atrial fibrillation, congestive heart failure, acute coronary syndrome (e.g., Unstable Angina/Non St-Elevation Myocardial Infarction (UA/NSTEMI)), stroke, pulmonary embolism, ischemic complications of peripheral vascular disease, atherosclerosis, and small vessel pathology. Often, aspirin or clopidogrel may be administered to a diabetic patient that is afflicted with or has a high risk for a vascular disease. However, certain patient populations are not suited for treatment with aspirin or clopidogrel because of, for example, various side effects to which they are susceptible or through various “resistance” presentations. In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein is used in a method of treating type 1 or type 2 diabetes in a patient that is afflicted with or has a high risk for a vascular disease and is not suited for treatment with aspirin or clopidogrel; for example, the patient presents with one or more of an aspirin allergy, asthma related to aspirin, bleeding or clotting disorder, bleeding tendency, anticoagulant therapy, recent gastrointestinal bleeding, and clinically active hepatic disease and/or the patient is less than about 21 years old and/or is a high risk for risk of Reye's syndrome. In embodiments, the determination of whether a patient is afflicted with or has a high risk for is afflicted with or has a high risk for a vascular disease comprises diagnostic methods known in the art (e.g., exercise treadmill testing, ECG stress testing, ankle/brachial index (ABI), duplex ultrasound, and various blood tests to measure, for example, cholesterol levels as well as the levels of other blood lipids).

In embodiments, one may determine whether a type 1 or type 2 diabetes has a high risk for a vascular disease by testing for expression of an early marker of vascular disease in a patient blood sample. In certain embodiments, the early marker is one or more of C-reactive peptide, myeloperoxidase, metalloproteinase-9, soluble CD40 ligand, pregnancy-associated plasma protein A, choline, ischemia-modified albumin, unbound free fatty acids, glycogen phosphorylase isoenzyme BB, placental growth factor and brain natriuretic peptide (BNP).

In some aspects the present disclosure provides a method for treating type 1 or type 2 diabetes, comprising administering an effective amount of the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein to a patient in need thereof, wherein the patient is afflicted with diabetes and has a high risk for cardiovascular disease or cardiovascular events. In embodiments, the high risk of cardiovascular disease or cardiovascular events is characterized by one or more of an age of greater than about 40 years, smoking, and a family history of cardiovascular disease, hypertension, dyslipidemia, albuminuria, history of myocardial infarction, vascular bypass procedure, stroke or transient ischemic attack, peripheral vascular disease, claudication, and/or angina.

Platelets, play a key role in atherogenesis, and its thrombotic complications and measures, which may lead to blockade of one or multiple pathways modulating platelet activation and aggregation processes, are pivotal in reducing ischemic risk in diabetic subjects. In some aspects the present disclosure provides a method for treating type 1 or type 2 diabetes, comprising administering an effective amount of the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein to a patient in need thereof, wherein the patient is afflicted with or has a high risk for a platelet dysfunction.

In embodiments, the platelet dysfunction is one or more of platelet hyperreactivity, increased baseline activation and/or reactivity (e.g., aggregation, accumulation, adhesion, and/or cohesion), increased platelet counts. In embodiments, the platelet dysfunction is determined by an increase in protein or nucleic acid levels of soluble P selectin and/or CD40-ligand. In embodiments, platelet counts and/or activity is assessed by methods known in the art. By way of illustration, Mean Platelet Component (MPC) value determination in patient blood samples- a low MPC value generally corresponds to increased platelet activation (a normal value of MPC for a healthy adult is about 25-30 g/d L. The ADVIA 120 Hematology System’s platelet analysis, or other similar automated methods that use one or more of volume and density measurements to derive an accurate platelet count, as well as a platelet density value may be used. In embodiments, the platelet dysfunction is caused by insulin therapy. In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein causes a reduction in platelet numbers or activation levels. In embodiments, the type 1 or type 2 diabetic patient that is afflicted with or has a high risk for a platelet dysfunction presents with stable or unstable atherosclerotic cardiovascular disease. In embodiments, the type 1 or type 2 diabetic patient that is afflicted with or has a high risk for a platelet dysfunction may not be suited for conventional anti-platelet agents (e.g., cyclooxygenase-1 (COX-1) inhibitors (aspirin), ADP P2Y12 receptor antagonists (thienopyridines), and platelet glycoprotein (GP) llb/llla inhibitors). In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein is used in place of a conventional anti-platelet agent or is used adjunctive to a conventional anti-platelet agent.

Further, insulin and clopidogrel combination therapy has been shown to increase platelet activation. In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein replace insulin in the insulin and clopidogrel combination therapy, especially in a type 1 or type 2 diabetic patient that is afflicted with or has a high risk for a platelet dysfunction.

In some aspects the present disclosure provides a method for treating type 1 or type 2 diabetes, comprising administering an effective amount of the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein to a patient in need thereof, wherein the patient his afflicted with or is at high risk for elevated hematocrit levels. In embodiments, the elevated hematocrit levels are about 0.35, or about 0.40, or about 0.45, or about 0.50, or about 0.55, or about 0.60. Hematocrit measurements are known in the art (e.g., centrifugation, cell counting, etc.).

In embodiments, the administration of the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein does not substantively change the patient’s hematocrit levels.

In some aspects the present disclosure provides a method for treating type 1 or type 2 diabetes, comprising administering an effective amount of the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein to a patient in need thereof, wherein the patient is afflicted with or is at risk for anemia. In embodiments, the type 1 or type 2 diabetes patient has hemochromatosis and therefore, cannot be treated for anemia by conventional iron supplementation.

In a number of embodiments, including those in which that fusion protein disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein prevents diabetes and/or treats a pre-diabetic condition, a patient is at risk of diabetes if the patient is characterized by one or more of: being physically inactive; having a parent or sibling with diabetes; having a family background associated with high incidence of diabetes, selected from that is African American, Alaska Native, American Indian, Asian American, Hispanic/Latino, or Pacific Islander American; giving birth to a baby weighing more than 9 pounds; being diagnosed with gestational diabetes; having high blood pressure of about 140/90 mmHg or above; being treated for high blood pressure; having HDL cholesterol level below about 35 mg/dL and/ or a triglyceride level above about 250 mg/dL; having polycystic ovary syndrome (PCOS); and having cardiovascular disease.

In some aspects the present disclosure provides methods for inducing weight loss or preventing weight gain, comprising administering an effective amount of the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein to a patient in need thereof; wherein the patient does not substantially change caloric intake. In some embodiments, the caloric intake is high, relative to guidelines, such as the USDA tables. In some embodiments, the patient's caloric intake is 2000-10000 calories/day, or greater than about 2000 calories/day, or about 2200 calories/day, or about 2400 calories/day, or about 2600 calories/day, or about 2800 calories/day, or about 3000 calories/day, or about 3200 calories/day, or about 3400 calories/day, or about 3600 calories/day, or about 3800 calories/day, or about 4000 calories/day, or about 5000 calories/day, or about 6000 calories/day. In various embodiments, the patient has a high caloric intake and does not gain weight or even loses weight. Therefore, the present disclosure provides for an effect without lifestyle changes that often reduce patient adherence (e.g., failed dieting). In some embodiments, the patient's caloric intake is not restricted by more than about 20%, or not by more than about 10%, or not by more than about 5% of the patient's caloric intake at the start of treatment. In some embodiments, a high proportion of the patient's caloric intake is "empty calories," i.e., calories from solid fats and/or added sugars. In some embodiments, greater than about 15%, or 20%, or 25%, or 30%, or 35%, or 50% of the patient's caloric intake is empty calories. Even in these embodiments, a patient may not gain weight or even lose weight.

In various embodiments, the patient of the present disclosure is overweight or obese. In some embodiments, the patient of the present disclosure suffers from central obesity. In some embodiments, the obesity of one of simple obesity (alimentary obesity; usually resulting from consumption of more calories than the body can utilize), secondary obesity (usually resulting from an underlying medical condition, such as, for example, Gushing' s syndrome and polycystic ovary syndrome), and childhood obesity. In some embodiments, the obesity is classified as: Class I, which includes a BMI between 30 and 34.99; Class II, which includes BMIs of 35 to 39.99; and Class III, which includes a BMI of over 40. Further, the present disclosure provides for obesity of any of classes I, II, or II I that is further classified as severe, morbid, and super obesity. In some embodiments, the patient is at risk of further weight gain, as assessed by, for example, daily caloric intake.

In various embodiments, the weight management/ weight loss/anti-obesity effects of the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein can be assessed using various techniques and indices. In various embodiments, assessment before, during, and after treatment is undertaken. In some embodiments, body mass index (BMI), a measure of a person's weight taking into account height, may be used. In various embodiments, a patient described herein has a BMI that provides an "overweight" classification, i.e., 25-29.9, such as, for example, about 25, or about 25.5, or about 26, or about 26.5, or about 27, or about 27.5, or about 28, or about 28.5, or about 29, or about 29.5. In various embodiments, a patient described herein has a BMI that provides an "obese" classification, i.e., greater than 30, such as, for example, about 30, or about 31 , or about 32, or about 33, or about 34, or about 35, or about 36, or about 37, or about 38, or about 39, or about 40, or about 50. In some embodiments, body volume index (BVI) is used. BVI uses 3D software to create an 3D image of a person so BVI can differentiate between people with the same BMI rating, but who have a different shape and different weight distribution. BVI measures where a person's weight and the fat are located on the body, rather than total weight or total fat content and places emphasis on the weight carried around the abdomen, commonly known as central obesity. In some embodiments, whole-body air displacement plethysmography (ADP) is used to assess the weight management/weight loss/anti-obesity effects of the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein. In some embodiments, simple weighing is used in the present disclosure. In some embodiments, skinfold calipers or "pinch test," bioelectrical impedance analysis, hydrostatic weighing, or dual-energy X-ray absorptiometry (DEXA) may be used.

In some embodiments, simple circumferential measurement of the body may be used. In some embodiments, a patient of the present disclosure has a waist circumference exceeding about 35 inches, or about 36 inches, or about 37 inches, or about 38 inches, or about 39 inches, or about 40 inches, or about 41 inches, or about 42 inches, or about 43 inches, or about 44 inches, or about 45 inches, or about 46 inches, or about 47 inches, or about 48 inches, or about 50 inches, or about 55 inches, or about 60 inches. In some embodiments, the patient is male human with a waist circumference exceeding 40 inches. In some embodiments, the patient is a female human with a waist circumference exceeding 35 inches.

The methods of the disclosure may be used to treat humans having a body fat percentage above the recommended body fat percentage, i.e., at least in the "overweight" range, or at least in the "obese" range. The body fat percentage will differ between women and men. Specifically, for women, the methods of the disclosure may be used to treat a female human having a body fat percentage of at least about 25%, above 25%, at least about 32%, or above 32%. For men, the methods of the disclosure may be used to treat a male human having a body fat percentage of at least about 14%, above 14%, at least about 18%, above 18%, at least about 25%, or above 25%. Body fat percentage may be estimated using any method accepted in the art, including, for example, near infrared interactance, dual energy X-ray absorptiometry, body density measurement, bioelectrical impedance analysis, and the like.

The methods of the disclosure may be used to treat a patient who is a man that is greater than 100 pounds overweight and/or has waist circumference exceeding 40 inches. The methods of the disclosure may be used to treat a patient who is a woman that is greater than 80 pounds overweight" and/or waist circumference exceeding 35 inches.

In some embodiments, the disclosure provides for the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein being used to treat and/or prevent certain disorders associated with being overweight. For example, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein find use in cardiovascular diseases (e.g., high cholesterol, hypercholesterolemia, low HDL, high HDL, hypertension, coronary artery-disease, heart failure), sleep apnea (including obstructive sleep apnea), osteoarthritis, thyroid problems, dementia, gout, asthma, gastroesophageal reflux disease, and chronic renal failure.

In various embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein administration and/or use prevents or reduces the growth of adipose tissue. In some embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein effects one or more of white adipose tissue (WAT) and brown adipose tissue (BAT), including, for example, visceral adipose tissue (VAT), abdominal subcutaneous adipose tissue (ASAT), or ectopic fat. Such an effect may be assessed by, for example, using any of the techniques described herein (e.g., BMI, weight for-stature indexes, skinfold measures, electrical bioimpedance analysis, etc.), as well as various imaging techniques, including computed tomography (CT), magnetic-resonance imaging (MR], including transverse body scans), dual energy X-ray absorptiometry (DXA).

The peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein may also be used in combination with dietary therapy, behavioral therapy, physical therapy, exercise, and weight loss surgery, or a combination of two or more such therapies. In some embodiments, the subject is on a calorie restricted diet. In some embodiments, the subject engages in or is to engage in a physical exercise or physical therapy regimen. In some embodiments, the subject has undergone, or will undergo, weight loss surgery. In some embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein may be in combination with additional agents or may be administered to patient undergoing treatment with various agents.

For example, including, but not limited to, embodiments pertaining to obesity and/or weight reduction/loss, the additional agents may include one or more of orlistat (e.g., ALL1, XENICAL), loracaserin (e.g., BELVIQ), phentermine-topiramate (e.g., QSYMIA), sibutramme (e.g., REDUCTIL or MERJDIA), rimonabant (ACOMPLLA), exenatide (e.g., BYETTA), pramlintide (e.g., SYMLIN) phentermine, benzphetamine, diethylpropion, phendimetrazme, bupropion, and metformin. Agents that interfere with the body's ability to absorb specific nutrients in food are among the additional agents, e.g., orlistat (e.g., ALU, XENICAL), glucomannan, and guar gum. Agents that suppress appetite are also among the additional agents, e.g., catecholamines and their derivatives (such as phenteimine and other amphetamine-based drugs), various antidepressants and mood stabilizers (e.g., bupropion and topiramate), anorectics (e.g., dexedrine, digoxin). Agents that increase the body's metabolism are also among the additional agents.

In some embodiments, additional agents may be selected from among appetite suppressants, neurotransmitter reuptake inhibitors, dopaminergic agonists, serotonergic agonists, modulators of GABAergic signaling, anticonvulsants, antidepressants, monoamine oxidase inhibitors, substance P (NK1) receptor antagonists, melanocortin receptor agonists and antagonists, lipase inhibitors, inhibitors of fat absorption, regulators of energy intake or metabolism, cannabinoid receptor modulators, agents for treating addiction, agents for treating metabolic syndrome, peroxisome proliferator-activated receptor (PPAR) modulators; dipeptidyl peptidase 4 (DPP- 4) antagonists, agents for treating cardiovascular disease, agents for treating elevated triglyceride levels, agents for treating low HDL, agents for treating hypercholesterolemia, and agents fortreating hypertension. Some agents for cardiovascular disease include statins (e.g., lovastatin, atorvastatin, fluvastatin, rosuvastatin, simvastatin and pravastatin) and omega-3 agents (e.g., LOVAZA, EPANQVA, VASCEPA, esterified omega-3's in general, fish oils, krill oils, algal oils). In some embodiments, additional agents may be selected from among amphetamines, benzodiazepines, sulfonyl ureas, meglitinides, thiazolidinediones, biguanides, beta-blockers, XCE inhibitors, diuretics, nitrates, calcium channel blockers, phentermine, sibutramine, lorcaserin, cetilistat, rimonabant, taranabant, topiramate, gabapentin, valproate, vigabatrin, bupropion, tiagabine, sertraline, fluoxetine, trazodone, zonisamide, methylphenidate, varenicline, naltrexone, diethylpropion, phendimetrazine, repaglinide, nateglinide, glimepiride, metformin, pioglitazone, rosiglilazone, and sitagliptin.

In various embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein may be used to treat diabetes in the context of hospitalization. For example, in embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein may be administered to a patient that is in a diabetic coma. In embodiments, the patient may be administered to a patient that has one or more of a severe diabetic hypoglycemia, advanced diabetic ketoacidosis (e.g., advanced enough to result in unconsciousness, contributing factors may include one or more of hyperglycemia, dehydration, shock, and exhaustion), hyperosmolar nonketotic coma (e.g., with one or more of hyperglycemia and dehydration are contributing factors). In these embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein may be used in conjunction with standard treatment regimens of diabetic comas, including administering one or more of glucose, glucagon, insulin, fluids (e.g., saline with potassium and/or other electrolytes), any of which, optionally, are administered intravenously. In embodiments, the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein may replace insulin in these treatment regimens and, optionally, is administered intravenously.

Further, in, but not limited to, embodiments pertaining to diabetes, the additional agents described herein may be used in the context of combination therapies. Further, any of the methods of treatment described herein may comprise administering the peptide or the derivative thereof disclosed herein or the polynucleotides encoding the peptide or the derivative thereof disclosed herein to a patient that is receiving one or more additional agents and/or non-insulin diabetes agents. Additional agents include one or more of a sulfonylurea (e.g., DYMELOR (aceto hexami de), DIABINESE (chlorpropamide), ORINASE (tolbutamide), and TOLINASE (tolazamide), GLUCOTROL (glipizide), GLUCOTROL XL (extended release), DIABETA (glyburide), MICRONASE (glyburide), GLYNASE PRESTAB (glyburide), and AMARYL (glimepiride)); a Biguanide (e.g., metformin (GLUCOPHAGE, GLUCOPHAGE XR, RIOMET, FORTAMET, and GLUMETZA)); a thiazolidinedione (e.g., ACTOS (pioglitazone) and AVANDIA (rosiglitazone); an alpha-glucosidase inhibitor (e.g., PRECOSE (acarbose) and GLYSET (miglitol); a Meglitinide (e.g., PRANDIN (repaglinide) and STARLIX (nateglinide)); a Dipeptidyl peptidase IV (DPP-IV) inhibitor (e.g., JANUVIA (sitagliptin), NESINA (alogliptin), ONGLYZA (saxagliptin), and TRADJENTA (linagliptin)); Sodium-glucose co-transporter 2 (SGLT2) inhibitor (e.g., INVOKANA (canaglifozin)); and a combination pill (e.g., GLUCOVANCE, which combines glyburide (a sulfonylurea) and metformin, METAGLIP, which combines glipizide (a sulfonylurea) and metformin, and AVANDAMET, which uses both metformin and rosiglitazone (AVANDIA) in one pill, KAZANO (alogliptin and metformin), and OSENI (alogliptin plus pioglitazone).

Other additional agents include METFORMIN oral, ACTOS oral, BYETTA subcutaneous, JANUVIA oral, WELCHOL oral, JANUMET oral, glipizide oral, glimepiride oral, GLUCOPHAGE oral, LANTUS subcutaneous, glyburide oral, ONGLYZA oral, AMARYI oral, LANTUS SOLOSTAR subcutaneous, BYDUREON subcutaneous, LEVEMIR FLEXPEN subcutaneous, ACTOPLUS MET oral, GLUMETZA oral, TRADJENTA oral, bromocriptine oral, KOMBIGLYZE XR oral, INVOKANA oral, PRANDIN oral, LEVEMIR subcutaneous, PARLODEL oral, pioglitazone oral, NOVOLOG subcutaneous, NOVOLOG FLEXPEN subcutaneous, VICTOZA 2-PAK subcutaneous, HUMALOG subcutaneous, STARLIX oral, FORTAMET oral, GLUCOVANCE oral, GLUCOPHAGE XR oral, NOVOLOG Mix 70-30 FLEXPEN subcutaneous, GLYBURIDE-METFORMIN oral, acarbose oral, SYMLINPEN 60 subcutaneous, GLUCOTROI XL oral, NOVOLIN R inj, GLUCOTROL oral, DUETACT oral, sitagliptin oral, SYMLINPEN 120 subcutaneous, HUMALOG KWIKPEN subcutaneous, JANUMET XR oral, GLIPIZIDE-METFORMIN oral, CYCLOSET oral, HUMALOG MIX 75-25 subcutaneous, nateglinide oral, HUMALOG Mix 75-25 KWIKPEN subcutaneous, HUMULIN 70/30 subcutaneous, PRECOSE oral, API DRA subcutaneous, Humulin R inj, Jentadueto oral, Victoza 3-Pak subcutaneous, Novolin 70/30 subcutaneous, NOVOLIN N subcutaneous, insulin detemir subcutaneous, glyburide micronized oral, GLYNASE oral, HUMULIN N subcutaneous, insulin glargine subcutaneous, RIOMET oral, pioglitazone-metformin oral, APIDRA SOLOSTAR subcutaneous, insulin lispro subcutaneous, GLYSET oral, HUMULIN 70/30 Pen subcutaneous, colesevelam oral, sitagliptin-metformin oral, DIABETA oral, insulin regular human inj, HUMULIN N Pen subcutaneous, exenatide subcutaneous, HUMALOG Mix 50-50 KWIKPEN subcutaneous, liraglutide subcutaneous, KAZANO oral, repaglinide oral, chlorpropamide oral, insulin aspart subcutaneous, NOVOLOG Mix 70-30 subcutaneous, HUMALOG Mix SOSO subcutaneous, saxagliptin oral, ACTOPLUS Met XR oral, miglitol oral, NPH insulin human recomb subcutaneous, insulin NPH and regular human subcutaneous, tolazamide oral, mifepristone oral, insulin aspart protam-insulin aspart subcutaneous, repaglinide-metformin oral, saxagliptin-metformin oral, linagliptin-metformin oral, NESI NA oral, OSENI oral, tolbutamide oral, insulin lispro protamine and lispro subcutaneous, pramlintide subcutaneous, insulin glulisine subcutaneous, pioglitazone-glimepiride oral, PRANDIMET oral, NOVOLOG PenFill subcutaneous, linagliptin oral, exenatide microspheres subcutaneous, KORLYM oral, alogliptin oral, alogliptin-pioglitazone oral, alogliptin-metformin oral, and canagliflozin oral,.

Other additional agents include Lispro (HUMALOG); Aspart (NOVOLOG); Glulisine (APIDRA); Regular (NOVOLIN R or HUMULIN R); NPH (NOVOLIN N or HUMULIN N); Glargine (LANTUS); Detemir (LEVEMIR); HUMULIN or NOVOLIN 70/30; and NOVOLOG Mix 70/30 HUMALOG Mix 75/25 or 50/50.

In aspects, the present disclosure provides a method of treating nonalcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH) in a subject in need thereof, the method comprising administering to the subject in need thereof the peptide or the derivative thereof of any of the embodiments disclosed herein, the fusion protein of any of the embodiments disclosed herein, the isolated polynucleotide of any of the embodiments disclosed herein, the expression vector of any of the embodiments disclosed herein, or the pharmaceutical composition of the embodiments disclosed herein. NAFLD is characterized by hepatic steatosis with no secondary causes of hepatic steatosis including excessive alcohol consumption, other known liver diseases, or long-term use of a steatogenic medication (Chalasani et al., The diagnosis and management of nonalcoholic fatty liver disease: Practice guidance from the American Association for the Study of Liver Diseases, Hepatology 2018, 67(1 ): 328-357, which is hereby incorporated by reference in its entirety). In embodiments, the subject has NAFLD selected from nonalcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). In embodiments, the subject has NAFL, as indicated by the presence of > 5% hepatic steatosis without evidence of hepatocellular injury in the form of hepatocyte ballooning. In embodiments, the subject has NASH as indicated by the presence of > 5% hepatic steatosis and inflammation with hepatocyte injury (e.g., ballooning), with or without any liver fibrosis. In embodiments, the subject has NASH, which is associated with hepatic inflammation and liver fibrosis, which optionally has progressed to cirrhosis, end-stage liver disease, and/or hepatocellular carcinoma. In embodiments, the subject has NASH without liver fibrosis. In embodiments, the subject has fibrosis of very low severity of fibrosis.

There are many approaches used to assess and evaluate whether a subject has NAFLD and if so, the severity of the disease including differentiating whether the NAFLD is NAFL or NASH. For example, these approaches include determining one or more of hepatic steatosis (e.g., accumulation of fat in the liver); the NAFLD Activity Score (NAS); hepatic inflammation; biomarkers indicative of one or more of liver damage, hepatic inflammation, liver fibrosis, and/or liver cirrhosis (e.g., serum markers and panels); and liver fibrosis and/or cirrhosis. Accordingly, in embodiments, the subject selected for the treatment with the compositions comprising the peptide, a derivatives thereof, or the fusion protein disclosed herein or the polynucleotides encoding the peptide, a derivatives thereof, or the fusion protein disclosed herein based on diagnosis by determining one or more of hepatic steatosis (e.g., accumulation of fat in the liver); the NAFLD Activity Score (NAS); hepatic inflammation; biomarkers indicative of one or more of liver damage, hepatic inflammation, liver fibrosis, and/or liver cirrhosis (e.g., serum markers and panels); and liver fibrosis and/or cirrhosis. In embodiments, the subject selected for the treatment with the peptide, a derivatives thereof, or the fusion protein and/or compositions disclosed herein based on diagnosis of a physiological indicator of NAFLD selected from liver morphology, liver stiffness, and the size or weight of the subject’s liver. In some embodiments, NAFLD in the subject is evidenced by an accumulation of hepatic fat and detection of a biomarker indicative of liver damage. For example, elevated serum ferritin and low titers of serum autoantibodies can be common features of NAFLD. In embodiments, the subject selected for the treatment with the peptide, a derivatives thereof, or the fusion proteins or compositions disclosed herein based on diagnosis of NAFLD using a technique including, but not limited to, magnetic resonance imaging, either by spectroscopy or by proton density fat fraction (MRI-PDFF) to quantify steatosis, transient elastography (FIBROSCAN®), hepatic venous pressure gradient (HPVG), hepatic stiffness measurement with MRE for diagnosing significant liver fibrosis and/or cirrhosis, and assessing histological features of liver biopsy. In some embodiments, magnetic resonance imaging is used to detect one or more of steatohepatitis (NASH- MRI), liver fibrosis (Fibro-MRI), and steatosis see, for example, U.S. Application Publication Nos. 2016/146715 and 2005/0215882, each of which are incorporated herein by reference in their entireties.

In embodiments, the subject selected for the treatment with the compositions comprising the peptide, a derivatives thereof, or the fusion protein disclosed herein or the polynucleotides encoding the peptide, a derivatives thereof, or the fusion protein disclosed herein based on diagnosis symptoms selected from one or more of an enlarged liver, fatigue, pain in the upper right abdomen, abdominal swelling, enlarged blood vessels just beneath the skin's surface, enlarged breasts in men, enlarged spleen, red palms, jaundice, and pruritus. In some embodiments, the subject is asymptomatic.

In embodiments, hepatic steatosis is determined by one or more methods selected from ultrasonography, computed tomography (CT), magnetic resonance imaging, magnetic resonance spectroscopy (MRS), magnetic resonance elastography (MRE), transient elastography (TE) (e.g., FIBROSCAN®), measurement of liver size or weight, or by liver biopsy (see, e.g., Di Lascio et al, Ultrasound Med Biol. 2018; 44(8): 1585- 1596; Lv et al, J Clin Transl Hepatol. 2018 Jun 28; 6(2): 217-221 ; Reeder, et ah, JMagn Re son Imaging. 2011 Oct; 34(4): 848-855; and de Ledinghen V, et ah, J Gastroenterol Hepatol. 2016 Apr;31 (4):848-55, each of which are incorporated herein by reference in their entireties). In embodiments, a subject diagnosed with NAFLD may have more than about 5% hepatic steatosis, for example, about 5% to about 25%, about 25% to about 45%, about 45% to about 65%, or greater than about 65% hepatic steatosis. In embodiments, a subject with about 5% to about 33% hepatic steatosis has stage 1 hepatic steatosis, a subject with about 33% to about 66% hepatic steatosis has stage 2 hepatic steatosis, and a subject with greater than about 66% hepatic steatosis has stage 3 hepatic steatosis.

In embodiments, the amount of hepatic steatosis is determined prior to administration of the combination of

(a) the compositions comprising the peptide, a derivatives thereof, or the fusion protein disclosed herein or the polynucleotides encoding the peptide, a derivatives thereof, or the fusion protein disclosed herein, and

(b) an additional therapeutic agent. In embodiments, the amount of hepatic steatosis is determined during the period of time or after the period of time of administration of the combination of (a) and (b). In embodiments, a reduction in the amount of hepatic steatosis during the period of time or after the period of time of administration of the combination of (a) and (b) compared to prior to administration of the combination of (a) and (b) indicates treatment of NAFLD. For example, a reduction in the amount of hepatic steatosis by about 1 % to about 50%, about 25% to about 75%, or about 50% to about 100% indicates treatment of NAFLD. In embodiments, a reduction in the amount of hepatic steatosis by about 5%, bout 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% indicates treatment of NAFLD.

In embodiments, treatment of NAFLD can be assessed by measuring hepatic steatosis. In embodiments, treatment of NAFLD comprises a reduction in hepatic steatosis following administration of the peptide, a derivatives thereof, or the fusion proteins described herein, and/or the a nucleic acid encoding the peptide, a derivatives thereof, or the fusion proteins described herein. In embodiments, the treatment of NAFLD with the peptide, a derivatives thereof, or the fusion proteins or compositions disclosed herein comprises one or more of a decrease in symptoms; a reduction in the amount of hepatic steatosis; a decrease in the NAS; a decrease in hepatic inflammation; a decrease in the level of biomarkers indicative of one or more of liver damage, inflammation, liver fibrosis, and/or liver cirrhosis; and a reduction in fibrosis and/or cirrhosis, a lack of further progression of fibrosis and/or cirrhosis, or a slowing of the progression of fibrosis and/or cirrhosis. In embodiments, treatment of NAFLD comprises a decrease of one or more symptoms associated with NAFLD in the subject. In embodiments, the total body weight of the subject does not increase. In embodiments, the total body weight of the subject decreases. In embodiments, the body mass index (BMI) of the subject does not increase. In embodiments, the body mass index (BMI) of the subject decreases. In embodiments, the waist and hip (WTH) ratio of the subject does not increase. In embodiments, the waist and hip (WTH) ratio of the subject decreases.

In embodiments, the severity of NALFD can be assessed using the NAS. In embodiments, treatment of NAFLD can be assessed using the NAS. In embodiments, treatment of NAFLD comprises a reduction in the NAS following administration of one or more compounds described herein. In embodiments, the NAS can be determined as described in Kleiner et al., Hepatology. 2005, 41 (6): 1313-1321 , which is hereby incorporated by reference in its entirety.

In embodiments, the NAS following administration is determined non-invasively, for example, as described in U.S. Application Publication No. 2018/0140219, which is incorporated by reference herein in its entirety. In embodiments, the NAS following administration is determined for a sample from the subject prior to administration of the compositions comprising the peptide, a derivatives thereof, or the fusion protein disclosed herein or the polynucleotides encoding the peptide, a derivatives thereof, or the fusion protein disclosed herein. In embodiments, the NAS following administration is determined during the period of time or after the period of time of administration of the compositions comprising the peptide, a derivatives thereof, or the fusion protein disclosed herein or the polynucleotides encoding the peptide, a derivatives thereof, or the fusion protein disclosed herein. In embodiments, a lower NAS score during the period of time or after the period of time of administration of the compositions comprising the peptide, a derivatives thereof, or the fusion protein disclosed herein or the polynucleotides encoding the peptide, a derivatives thereof, or the fusion protein disclosed herein compared to prior to administration of the compositions comprising the peptide, a derivatives thereof, or the fusion protein disclosed herein or the polynucleotides encoding the peptide, a derivatives thereof, or the fusion protein disclosed herein indicates treatment of NAFLD. For example, a decrease in the NAS by 1, by 2, by 3, by 4, by 5, by 6, or by 7 indicates treatment of NAFLD. In embodiments, the NAS following administration of the compositions comprising the peptide, a derivatives thereof, or the fusion protein disclosed herein or the polynucleotides encoding the peptide, a derivatives thereof, or the fusion protein disclosed herein is 7 or less. In embodiments, the NAS during the period of time of administration of the compositions comprising the peptide, a derivatives thereof, or the fusion protein disclosed herein or the polynucleotides encoding the peptide, a derivatives thereof, or the fusion protein disclosed herein is 5 or less, 4 or less, 3 or less, or 2 or less. In embodiments, the NAS during the period of time of administration of the compositions comprising the peptide, a derivatives thereof, or the fusion protein disclosed herein or the polynucleotides encoding the peptide, a derivatives thereof, or the fusion protein disclosed herein is 7 or less. In embodiments, the NAS during the period of time of administration of the compositions comprising the peptide, a derivatives thereof, or the fusion protein disclosed herein or the polynucleotides encoding the peptide, a derivatives thereof, or the fusion protein disclosed herein is 5 or less, 4 or less, 3 or less, or 2 or less. In embodiments, the NAS after the period of time of administration of the compositions comprising the peptide, a derivatives thereof, or the fusion protein disclosed herein or the polynucleotides encoding the peptide, a derivatives thereof, or the fusion protein disclosed herein is 7 or less. In embodiments, the NAS after the period of time of administration of the compositions comprising the peptide, a derivatives thereof, or the fusion protein disclosed herein or the polynucleotides encoding the peptide, a derivatives thereof, or the fusion protein disclosed herein is 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the presence of hepatic inflammation is determined by one or more methods selected from the group consisting of biomarkers indicative of hepatic inflammation and a liver biopsy sample(s) from the subject. In some embodiments, the severity of hepatic inflammation is determined from a liver biopsy sample(s) from the subject. For example, hepatic inflammation in a liver biopsy sample can be assessed as described in Kleiner et al., Hepatology. 2005, 41 (6): 1313-1321 and Brunt et al., Am J Gastroenterol 1999, 94:2467-2474, each of which are hereby incorporated by reference in their entireties.

In some embodiments, the severity of hepatic inflammation is determined prior to administration of the compositions comprising the peptide, a derivatives thereof, or the fusion protein disclosed herein or the polynucleotides encoding the peptide, a derivatives thereof, or the fusion protein disclosed herein. In some embodiments, the severity of hepatic inflammation is determined prior to administration of the compositions comprising the peptide, a derivatives thereof, or the fusion protein disclosed herein or the polynucleotides encoding the peptide, a derivatives thereof, or the fusion protein disclosed herein. In some embodiments, the severity of hepatic inflammation is determined during the period of time or after the period of time of administration of the compositions comprising the peptide, a derivatives thereof, or the fusion protein disclosed herein or the polynucleotides encoding the peptide, a derivatives thereof, or the fusion protein disclosed herein. In some embodiments, a decrease in the severity of hepatic inflammation during the period of time or after the period of time of administration of the compositions comprising the peptide, a derivatives thereof, or the fusion protein disclosed herein or the polynucleotides encoding the peptide, a derivatives thereof, or the fusion protein disclosed herein compared to prior to administration of the compositions comprising the peptide, a derivatives thereof, or the fusion protein disclosed herein or the polynucleotides encoding the peptide, a derivatives thereof, or the fusion protein disclosed herein indicates treatment of NAFLD. For example, a decrease in the severity of hepatic inflammation by about 1 % to about 50%, about 25% to about 75%, or about 50% to about 100% indicates treatment of NAFLD. In some embodiments, a decrease in the severity of hepatic inflammation by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% indicates treatment of NAFLD.

In some embodiments, treatment of NAFLD comprises treatment of fibrosis and/or cirrhosis, e.g., a decrease in the severity of fibrosis, a lack of further progression of fibrosis and/or cirrhosis, or a slowing of the progression of fibrosis and/or cirrhosis. In some embodiments, the presence of fibrosis and/or cirrhosis is determined by one or more methods selected from the group consisting of transient elastography (e.g., FIBROSCAN®), non-invasive markers of hepatic fibrosis, and histological features of a liver biopsy. In some embodiments, the severity (e.g., stage) of fibrosis is determined by one or more methods selected from the group consisting of transient elastography (e.g., FIBROSCAN®), a fibrosis-scoring system, biomarkers of hepatic fibrosis (e.g., non-invasive biomarkers), and hepatic venous pressure gradient (HVPG). Non-limiting examples of fibrosis scoring systems include the NAFLD fibrosis scoring system (see, e.g., Angulo, et al., Hepatology. 2007; 45(4):846-54), the fibrosis scoring system in Brunt et al., Am J Gastroenterol. 1999, 94:2467-2474, the fibrosis scoring system in Kleiner et al., Hepatology. 2005, 41 (6): 1313- 1321 , and the ISHAK fibrosis scoring system (see Ishak et al., J Hepatol. 1995; 22:696-9), the contents of each of which are incorporated by reference herein in their entireties.

In some embodiments, the severity of fibrosis is determined prior to administration of the compositions comprising the peptide, a derivatives thereof, or the fusion protein disclosed herein or the polynucleotides encoding the peptide, a derivatives thereof, or the fusion protein disclosed herein. In some embodiments, the severity of fibrosis is determined prior to administration of a combination of (a) the compositions comprising the peptide, a derivatives thereof, or the fusion protein disclosed herein or the polynucleotides encoding the peptide, a derivatives thereof, or the fusion protein disclosed herein, and (b) an additional therapeutic agent. In some embodiments, the severity of fibrosis is determined during the period of time or after the period of time of administration of the combination of (a) and (b). In some embodiments, a decrease in the severity of fibrosis during the period of time or after the period of time of administration of the combination of (a) and (b) compared to prior to administration of the combination of (a) and (b) indicates treatment of NAFLD. In some embodiments, a decrease in the severity of fibrosis, a lack of further progression of fibrosis and/or cirrhosis, or a slowing of the progression of fibrosis and/or cirrhosis indicates treatment of NAFLD. In some embodiments, the severity of fibrosis is determined using a scoring system such as any of the fibrosis scoring systems described herein, for example, the score can indicate the stage of fibrosis, e.g., stage 0 (no fibrosis), stage 1 , stage 2, stage 3, and stage 4 (cirrhosis) (see, e.g., Kleiner et al). In some embodiments, a decrease in the stage of the fibrosis is a decrease in the severity of the fibrosis. For example, a decrease by 1 , 2, 3, or 4 stages is a decrease in the severity of the fibrosis. In some embodiments, a decrease in the stage, e.g., from stage 4 to stage 3, from stage 4 to stage 2, from stage 4 to stage 1 , from stage 4 to stage 0, from stage 3 to stage 2, from stage 3 to stage 1 , from stage 3 to stage 0, from stage 2 to stage 1 , from stage 2 to stage 0, or from stage 1 to stage 0 indicates treatment of NAFLD. In some embodiments, the stage of fibrosis decreases from stage 4 to stage 3, from stage 4 to stage 2, from stage 4 to stage 1 , from stage 4 to stage 0, from stage 3 to stage 2, from stage 3 to stage 1 , from stage 3 to stage 0, from stage 2 to stage 1 , from stage 2 to stage 0, or from stage 1 to stage 0 following administration of the combination of (a) and (b) compared to prior to administration of the combination of (a) and (b). In some embodiments, the stage of fibrosis decreases from stage 4 to stage 3, from stage 4 to stage 2, from stage 4 to stage 1 , from stage 4 to stage 0, from stage 3 to stage 2, from stage 3 to stage 1 , from stage 3 to stage 0, from stage 2 to stage 1, from stage 2 to stage 0, or from stage 1 to stage 0 during the period of time of administration of the combination of (a) and (b) compared to prior to administration of the combination of (a) and (b). In some embodiments, the stage of fibrosis decreases from stage 4 to stage 3, from stage 4 to stage 2, from stage 4 to stage 1 , from stage 4 to stage 0, from stage 3 to stage 2, from stage 3 to stage 1 , from stage 3 to stage 0, from stage 2 to stage 1 , from stage 2 to stage 0, or from stage 1 to stage 0 after the period of time of administration of the combination of (a) and (b) compared to prior to administration of the combination of (a) and (b).

In some embodiments, the presence of NAFLD is determined by one or more biomarkers indicative of one or more of liver damage, inflammation, liver fibrosis, and/or liver cirrhosis or scoring systems thereof. In some embodiments, the severity of NAFLD is determined by one or more biomarkers indicative of one or more of liver damage, inflammation, liver fibrosis, and/or liver cirrhosis or scoring systems thereof. The level of the biomarker can be determined by, for example, measuring, quantifying, and monitoring the expression level of the gene or mRNA encoding the biomarker and/or the peptide or protein of the biomarker. Non-limiting examples of biomarkers indicative of one or more of liver damage, inflammation, liver fibrosis, and/or liver cirrhosis and/or scoring systems thereof include the aspartate aminotransferase (AST) to platelet ratio index (APRI); the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ratio (AAR); the FIB-4 score, which is based on the APRI, alanine aminotransferase (ALT) levels, and age of the subject (see, e.g., McPherson et ah, Gut. 2010 Sep;59(9): 1265-9, which is incorporated by reference herein in its entirety); hyaluronic acid; pro-inflammatory cytokines; a panel of biomarkers consisting of a2-macroglobulin, haptoglobin, apolipoprotein Al, bilirubin, gamma glutamyl transpeptidase (GGT) combined with a subject’s age and gender to generate a measure of fibrosis and necroinflammatory activity in the liver (e.g., FIBROTEST®, FIBROSURE®), a panel of biomarkers consisting of bilirubin, gamma-glutamyltransferase, hyaluronic acid, a2 -macroglobulin combined with the subject’s age and sex (e.g., HEPASCORE®; see, e.g., Adams et al., Clin Chem. 2005, 51 (10): 1867-73), and a panel of biomarkers consisting of tissue inhibitor of metalloproteinase- 1 , hyaluronic acid, and a2-macroglobulin (e.g., FIBROSPECT®); a panel of biomarkers consisting of tissue inhibitor of metalloproteinases 1 (TIMP-1), amino-terminal propeptide of type III procollagen (PIIINP) and hyaluronic acid (HA) (e.g., the Enhanced Liver Fibrosis (ELF) score, see, e.g., Lichtinghagen R, et al., J Hepatol. 2013 Aug; 59(2): 236-42, which is incorporated by reference herein in its entirety). In some embodiments, the presence of fibrosis is determined by one or more of the FIB-4 score, a panel of biomarkers consisting of a2-macroglobulin, haptoglobin, apolipoprotein Al, bilirubin, gamma glutamyl transpeptidase (GGT) combined with a subject’s age and gender to generate a measure of fibrosis and necroinflammatory activity in the liver (e.g., FIBROTEST®, FIBROSURE®), a panel of biomarkers consisting of bilirubin, gamma-glutamyltransferase, hyaluronic acid, a2- macroglobulin combined with the subject’s age and sex (e.g., HEPASCORE®; see, e.g., Adams et al., Clin Chem. 2005 Oct;51 (10): 1867-73), and a panel of biomarkers consisting of tissue inhibitor of metalloproteinase- 1 , hyaluronic acid, and a2-macroglobulin (e.g., FIBROSPECT®); and a panel of biomarkers consisting of tissue inhibitor of metalloproteinases 1 (TIMP-1), amino- terminal propeptide of type III procollagen (PIIINP) and hyaluronic acid (HA) (e.g., the Enhanced Liver Fibrosis (ELF) score).

In some embodiments, the level of aspartate aminotransferase (AST) does not increase. In some embodiments, the level of aspartate aminotransferase (AST) decreases. In some embodiments, the level of alanine aminotransferase (ALT) does not increase. In some embodiments, the level of alanine aminotransferase (ALT) decreases. In some embodiments, the “level” of an enzyme refers to the concentration of the enzyme, e.g., within blood. For example, the level of AST or ALT can be expressed as Units/L.

In some embodiments, the severity of fibrosis is determined by one or more of the FIB-4 score, a panel of biomarkers consisting of a2-macroglobulin, haptoglobin, apolipoprotein Al, bilirubin, gamma glutamyl transpeptidase (GGT) combined with a subject’s age and gender to generate a measure of fibrosis and necroinflammatory activity in the liver (e.g., FIBROTEST®, FIBROSURE®), a panel of biomarkers consisting of bilirubin, gamma-glutamyltransferase, hyaluronic acid, a2 -macroglobulin combined with the subject’s age and sex (e.g., HEPASCORE®; see, e.g., Adams et al., Clin Chem. 2005 Oct;51 (10): 1867-73, which is incorporated by reference herein in its entirety), and a panel of biomarkers consisting of tissue inhibitor of metalloproteinase- 1 , hyaluronic acid, and a2-macroglobulin (e.g., FIBROSPECT®); and a panel of biomarkers consisting of tissue inhibitor of metalloproteinases 1 (TIMP-1), amino-terminal propeptide of type III procollagen (PIIINP) and hyaluronic acid (HA) (e.g., the Enhanced Liver Fibrosis (ELF) score). In some embodiments, hepatic inflammation is determined by the level of liver inflammation biomarkers, e.g., pro- inflammatory cytokines. Non-limiting examples of biomarkers indicative of liver inflammation include interleukin-(IL) 6, interleukin-(IL) 1 b, tumor necrosis factor (TNF)-a, transforming growth factor (TGF)-P, monocyte chemotactic protein (MCP)-I, C- reactive protein (CRP), PAI-1, and collagen isoforms such as Collal, Colla2, and Col4al (see, e.g., Neuman, et ah, Can J Gastroenterol Hepatol. 2014 Dec; 28(11): 607- 618 and U.S. Patent No. 9,872,844, each of which are incorporated by reference herein in their entireties). Liver inflammation can also be assessed by change of macrophage infiltration, e.g., measuring a change of CD68 expression level. In some embodiments, liver inflammation can be determined by measuring or monitoring serum levels or circulating levels of one or more of interleukin-(IL) 6, interleukin-(IL) 1 b, tumor necrosis factor (TNF)-a, transforming growth factor (TGF-b, monocyte chemotactic protein (MCP)-I, and C- reactive protein (CRP).

In some embodiments, the level of one or more biomarkers indicative of one or more of liver damage, inflammation, liver fibrosis, and/or liver cirrhosis is determined for a sample from the subject prior to administration of the combination of (a) the compositions comprising the peptide, a derivatives thereof, or the fusion protein disclosed herein or the polynucleotides encoding the peptide, a derivatives thereof, or the fusion protein disclosed herein, and (b) an additional therapeutic agent. In some embodiments, the level of one or more biomarkers indicative of one or more of liver damage, inflammation, liver fibrosis, and/or liver cirrhosis is determined during the period of time or after the period of time of administration of the combination of (a) and (b). In some embodiments, a decrease in the level of one or more biomarkers indicative of one or more of liver damage, inflammation, liver fibrosis, and/or liver cirrhosis during the period of time or after the period of time of administration of the combination of (a) and (b) compared to prior to administration of the combination of (a) and (b) indicates treatment of NAFLD. For example, a decrease in the level of one or more biomarkers indicative of one or more of liver damage, inflammation, liver fibrosis, and/or liver cirrhosis by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% indicates treatment of NAFLD. In some embodiments, the decrease in the level of one or more biomarkers indicative of one or more of liver damage, inflammation, liver fibrosis, and/or liver cirrhosis following administration of the combination of (a) and (b) is by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%. In some embodiments, the level of one or more biomarkers indicative of one or more of liver damage, inflammation, liver fibrosis, and/or liver cirrhosis during the period of time of administration of the combination of (a) and (b) is by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%. In some embodiments, the level of one or more biomarkers indicative of one or more of liver damage, inflammation, liver fibrosis, and/or liver cirrhosis after the period of time of administration of the combination of (a) and (b) is by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%.

In some embodiments, the treatment of NAFLD decreases the level of serum bile acids in the subject. In some embodiments, the level of serum bile acids is determined by, for example, an ELISA enzymatic assay or the assays for the measurement of total bile acids as described in Danese et ah, PLoS One. 2017; 12(6): e0179200, which is incorporated by reference herein in its entirety. In some embodiments, the level of serum bile acids can decrease by, for example, 10% to 40%, 20% to 50%, 30% to 60%, 40% to 70%, 50% to 80%, or by more than 90% of the level of serum bile acids prior to administration of (a) and (b). In some embodiments, the NAFLD is NAFLD with attendant cholestasis. In cholestasis, the release of bile, including bile acids, from the liver is blocked. Bile acids can cause hepatocyte damage (see, e.g., Perez MJ, Briz O. World J Gastroenterol. 2009 Apr 14; 15(14): 1677-89) likely leading to or increasing the progression of fibrosis (e.g., cirrhosis) and increasing the risk of hepatocellular carcinoma (see, e.g., Sorrentino P et ah. Dig Dis Sci. 2005 Jun;50(6): 1130-5 and Satapathy SK and Sanyal AJ. Semin Liver Dis. 2015, 35(3):221-35, each of which are incorporated by reference herein in their entireties). In some embodiments, the NAFLD with attendant cholestasis is NASH with attendant cholestasis. In some embodiments, the treatment of NAFLD comprises treatment of pruritus. In some embodiments, the treatment of NAFLD with attendant cholestasis comprises treatment of pruritus. In some embodiments, a subject with NAFLD with attendant cholestasis has pruritus.

In some embodiments, treatment of NAFLD comprises an increase in adiponectin. It is thought that the compound of Formula (I) may be a selective activator of a highly limited number of PPARy pathways including pathways regulated by adiponectin. Adiponectin is an anti-fibrotic and anti-inflammatory adipokine in the liver (see e.g., Park et ah, Curr Pathobiol Rep. 2015 Dec 1 ; 3(4): 243-252.). In some embodiments, the level of adiponectin is determined by, for example, an ELIS A enzymatic assay. In some embodiments, the adiponectin level in the subject is increased by at least about 30%, at least about 68%, at least about 175%, or at least about 200%. In some embodiments, the increase is by at least about 175%. In some embodiments, the level of adiponectin is determined for a sample from the subject prior to administration of the combination of (a) the compositions comprising the peptide, a derivatives thereof, or the fusion protein disclosed herein or the polynucleotides encoding the peptide, a derivatives thereof, or the fusion protein disclosed herein, and (b) an additional therapeutic agent. In some embodiments, the level of adiponectin is determined for a sample from the subject prior to administration of the combination of (a) the compositions comprising the peptide, a derivatives thereof, or the fusion protein disclosed herein or the polynucleotides encoding the peptide, a derivatives thereof, or the fusion protein disclosed herein, and (b) an additional therapeutic agent. In some embodiments, the level of adiponectin is determined during the period of time or after the period of time of administration of the combination of (a) and (b). In some embodiments, an increase in the level of adiponectin during the period of time or after the period of time of administration of the combination of (a) and (b) compared to prior to administration of the combination of (a) and (b) indicates treatment of NAFLD. For example, an increase in the level of adiponectin by at least about 30%, at least about 68%, at least about 175%, or at least about 200% indicates treatment of NAFLD. In some embodiments, the increase in the level of adiponectin following administration of the combination of (a) and (b) is at least about 200%.

In aspects, the present disclosure provides a method of treating a cardiometabolic disease in a subject in need thereof, the method comprising administering to the subject in need thereof the peptide or the derivative thereof of any of the embodiments disclosed herein, the fusion protein of any of the embodiments disclosed herein, the isolated polynucleotide of any of the embodiments disclosed herein, the expression vector of any of the embodiments disclosed herein, or the pharmaceutical composition of the embodiments disclosed herein. Cardiometabolic disease include a group of chronic conditions, which affect the cardiovascular system and metabolic health, and are characterized by insulin resistance, impaired glucose tolerance, dyslipidemia, hypertension, and central adiposity. These conditions include heart attack, stroke, diabetes, insulin resistance, non-alcoholic fatty liver disease, heart failure and pulmonary arterial hypertension (PAH), and metabolic diseases, such as type 2 diabetes. They also include the endocrine, nutritional, and metabolic (ENM) diseases (e.g., thyroid conditions, diabetes, hyperlipidemia, obesity); hypertensive heart disease (e.g., heart disease caused by prolonged exposure to high blood pressure); and ischemic heart disease and other diseases of the circulatory system (e.g., reduced blood supply to the heart, including atherosclerosis and coronary heart disease (CHD), stroke, and other cardiovascular conditions). Subjects and/or Animals

In embodiments, the subject and/or animal is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon. In embodiments, the subject and/or animal is a non-mammal, such, for example, a zebrafish. In embodiments, the subject and/or animal may comprise fluorescently-tagged cells (with e.g., GFP). In embodiments, the subject and/or animal is a transgenic animal comprising a fluorescent cell.

In embodiments, the subject and/or animal is a human. In embodiments, the human is a pediatric human. In embodiments, the human is an adult human. In embodiments, the human is a geriatric human. In embodiments, the human may be referred to as a patient.

In certain embodiments, the human has an age in a range of from about 0 months to about 6 months old, from about 6 to about 12 months old, from about 6 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old.

In embodiments, the subject is a non-human animal, and therefore the disclosure pertains to veterinary use. In a specific embodiment, the non-human animal is a household pet. In another specific embodiment, the non-human animal is a livestock animal.

Kits

The disclosure provides kits that can simplify the administration of any agent described herein. An illustrative kit of the disclosure comprises any composition described herein in unit dosage form. In embodiments, the unit dosage form is a container, such as a pre-filled syringe, which can be sterile, containing any agent described herein and a pharmaceutically acceptable carrier, diluent, excipient, or vehicle. The kit can further comprise a label or printed instructions instructing the use of any agent described herein. The kit may also include a lid speculum, topical anesthetic, and a cleaning agent for the administration location. The kit can also further comprise one or more additional agent described herein. In embodiments, the kit comprises a container containing an effective amount of a composition of the disclosure and an effective amount of another composition, such those described herein.

Any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.

The disclosure will be further described in the following examples, which do not limit the scope of the disclosure described in the claims.

The disclosure will be further described in the following examples, which do not limit the scope of the disclosure described in the claims.

EXAMPLES

The examples herein are provided to illustrate advantages and benefits of the present technology and to further assist a person of ordinary skill in the art with preparing or using the peptides, fusion proteins and nucleic acids of the present technology. The examples herein are also presented in order to more fully illustrate the preferred aspects of the present technology. The examples should in no way be construed as limiting the scope of the present disclosure, as exemplified by the appended claims. The examples can include or incorporate any of the variations, aspects or embodiments of the present technology described above. The variations, aspects or embodiments described above may also further each include or incorporate the variations of any or all other variations, aspects or embodiments of the present technology.

Example 1. Activation ofGLP-1 Receptor (GLP-1R), GIP Receptor (GIPR), and Glucagon Receptor (GCGR) by the Dual- or triple-action peptides

The activity of the dual- or triple-action peptides disclosed herein on GLP-1 receptor (GLP-1 R) was assayed using a cell-based assay performed using CHO-K1 cells engineered to express GLP-1 R and a cyclic AMP (cAMP) luciferase reporter. This assay measures the reduction of cellular ATP levels because the consumption of ATP upon the activation of adenylate cyclase caused by the signaling via GLP-1 R. Without wishing to be bound by theory, the activation of GLP-1 R leads to temporary depletion of ATP because of consumption of ATP in cyclization reaction to produce cAMP as well as by phosphorylation by protein kinase A of its substrate. Without wishing to be bound by theory, the temporary depletion of ATP leads to an inhibition of luciferase activity, which needs ATP as a substrate.

Briefly, CHO-K1 cells expressing GLP-1 R and a cAMP luciferase reporter were treated with increasing amounts of SL_TriAg_V1 , and SL_TriAg_V2_Pro3. Two comparator peptides, a GIPR/GLP-1 R/GCGR co- agonist peptide (Comparator 1 ) and a dual GIPR/GLP-1 R co-agonist (Comparator 2), were used as a positive controls. ATP levels in the cells, which are decreased as more cAMP is produced in response to the activation of GLP-1 R, were detected following treatment by measuring the level of luciferase activity using a luminometer. In this assay, the inhibition of luciferase activity indicates GLP-1 R agonism. As shown in FIG. 2A, each of SL_TriAg_V1 , SL_TriAg_V2_Pro3, and the comparator peptides produced a dose-dependent, and saturable decrease in the levels of luciferase, consistent with the activation of the GLP-1 R. These results demonstrate, inter alia, that the dual- or triple-action peptides disclosed herein activate the GLP-1 receptor.

The activity of the dual- or triple-action peptides disclosed herein on GIP receptor (GIPR) activation was assayed using a cell-based assay performed using HEK293 cells engineered to express GIPR and a cyclic AMP (cAMP) luciferase reporter. This assay measures the reduction of cellular ATP levels because the consumption of ATP upon the activation of adenylate cyclase caused by the signaling via GIPR. Without wishing to be bound by theory, the activation of GIPR leads to temporary depletion of ATP because of consumption of ATP in cyclization reaction to produce cAMP as well as by phosphorylation by protein kinase A of its substrate. Without wishing to be bound by theory, the temporary depletion of ATP leads to an inhibition of luciferase activity, which needs ATP as a substrate. SL_TriAg_V1 was designed as a triple-action peptide capable of activating GLP-1 receptor (GLP-1 R), GIP receptor (GIPR), and glucagon receptor (GCGR). SL_TriAg_V2_Pro3 was designed as a triple-action peptide capable of modulating GLP-1 receptor (GLP- 1 R), GIP receptor (GIPR), and glucagon receptor (GCGR): activating GLP-1 receptor (GLP-1 R) and glucagon receptor (GCGR) and blocking GIP receptor (GIPR), by virtue of the Pro residue present at position 3.

Briefly, HEK293 cells expressing GIPR and a cAMP luciferase reporter were treated with increasing amounts of SL_Tri Ag_V1 , SL_TriAg_V2_Pro3, or the comparator peptides. The comparator peptides were used as a positive controls. ATP levels in the cells, which are decreased as more cAMP is produced in response to the activation of GIPR, were detected following treatment by measuring the level of luciferase activity using a luminometer. In this assay, the inhibition of luciferase activity indicates GIPR agonism. As shown in FIG. 2B, each of SL_TriAg_V1 , or the comparator peptides produced a dose-dependent, and saturable decrease in the levels of luciferase, consistent with the activation of the GIPR. SL_TriAg_V2_Pro3 did not show inhibition of luciferase activity, consistent with expected GIPR antagonistic, rather than agonistic activity. These results demonstrate, inter alia, that the dual- or triple-action peptides disclosed herein activate the GIP receptor. It is anticipated that the SL_TriAg_V2_Pro3 peptide will block the GIP receptor. The activity of the dual- or triple-action peptides disclosed herein on glucagon receptor (GCGR) was assayed using a cell-based assay performed using CHO-K1 cells engineered to express GCGR and a cyclic AMP (cAMP) luciferase reporter. This assay measures the reduction of cellular ATP levels because the consumption of ATP upon the activation of adenylate cyclase caused by the signaling via GCGR. Without wishing to be bound by theory, the activation of GCGR leads to temporary depletion of ATP because of consumption of ATP in cyclization reaction to produce cAMP as well as by phosphorylation by protein kinase A of its substrate. Without wishing to be bound by theory, the temporary depletion of ATP leads to an inhibition of luciferase activity, which needs ATP as a substrate.

Briefly, CHO-K1 cells expressing GCGR and a cAMP luciferase reporter were treated with increasing amounts of SL_TriAg_V1, SL_TriAg_V2_Pro3, or the comparator peptides. The comparator peptides were used as a positive controls. ATP levels in the cells, which are decreased as more cAMP is produced in response to the activation of GCGR, were detected following treatment by measuring the level of luciferase activity using a luminometer. In this assay, the inhibition of luciferase activity indicates GCGR agonism. As shown in FIG. 2C, each of SL_TriAg_V1 , SL_TriAg_V2_Pro3 or the Comparator 1 peptide produced a dosedependent, and saturable decrease in the levels of luciferase, consistent with the activation of the GCGR. As expected, the Comparator 2 peptide did not inhibit GCPR. These results demonstrate, inter alia, that the triple-action peptides disclosed herein activate the glucagon receptor.

Collectively, these results indicate, inter alia, that the SL_TriAg_V1 peptide is a triple-action peptide capable of agonizing the GLP-1 receptor, the GIP receptor, and the glucagon receptor. These results also indicate, inter alia, that the SL_TriAg_V2_Pro3 peptide is a triple-action peptide capable of agonizing the GLP-1 receptor and the glucagon receptor, but blocking the GIP receptor.

Example 2. Identification and Characterization of Dual and Triple Modulators of Glucagon-Like Peptide-1 Receptor (GLP-1 R), Gastric Inhibitory Peptide Receptor (GIP R), and Glucagon Receptor (GCGR)

A number of peptides from a library were evaluated for agonism or blockade of the glucagon-like peptide-1 receptor (GLP-1 R), the gastric inhibitory peptide receptor (GIPR), or the glucagon receptor (GCGR) using genetically engineered cell lines.

GIPR agonism was studied as described above in Example 1. Briefly, the HEK293 cells expressing GIPR and a cAMP luciferase reporter were treated with increasing concentrations of SL_TriAg_V1 , SL_TriAg_V1_Pro3, SL_TriAg_V2, SL_TriAg_V2_Pro3, SL_TriAg_V3, SL_TriAg_V3_Pro3, SL_TriAg_V4, and SL_TriAg_V4_Pro3. GIP was used as a positive control. Immediately after the addition, the luminescence of the cells was measured using a luminometer, and normalized using the basal luminescence.

GLP-1R agonism was studied as described above in Example 1. Briefly, CHO-K1 cells engineered to express GLP-1R and a cyclic AMP (cAMP) luciferase reporter were treated with increasing concentrations of SL_TriAg_V1 , SL_TriAg_V1 _Pro3, SL_TriAg_V2, SL_TriAg_V2_Pro3, SL_TriAg_V3, SL_TriAg_V3_Pro3,

SL_TriAg_V4, and SL_TriAg_V4_Pro3. GLP-1 was used as a positive control. Immediately after the addition, the luminescence of the cells was measured using a luminometer, and normalized using the basal luminescence.

GCGR agonism was studied as described above in Example 1. Briefly, CHO-K1 derivative engineered to express GCGR and a cyclic AMP (cAMP) luciferase reporter were treated with increasing concentrations of SL_TriAg_V1, SL_TriAg_V1 _Pro3, SL_TriAg_V2, SL_TriAg_V2_Pro3, SL_TriAg_V3, SL_TriAg_V3_Pro3, SL_TriAg_V4, and SL_TriAg_V4_Pro3. glucagon was used as a positive control. Immediately after the addition, the luminescence of the cells was measured using a luminometer, and normalized using the basal luminescence. The Table below shows results from these assays:

Key: -, EC5o > 1 pm; +, EC50 in the range of 500 nM to 1000 nM; ++, EC50 in the range of 101 nM to 499 nM;

+++ EC50 in the range of 2.1 nM to 100 nM; and ++++, EC50 in the range of 101 pM to 2 nM. These assays showed that the SL_TriAg_V1 peptide had ECso values for GLP-1 R agonism, GIPR agonism, and GCGR agonism of 118.9 nM, 1.72 nM, and 72.7 nM, respectively. The assay further showed that the SL_TriAg_V1_Pro3 peptide had EC50 values for GLP-1 R agonism, GIPR agonism, and GCGR agonism of 536 nM, 3.8 nM and 811 nM, respectively. The assay further showed that the SL_TriAg_V2_Pro3 peptide had EC50 values for GLP-1 R agonism and GCGR agonism of 60 nM and 778 nM, respectively.

The sequences of SL_TriAg_V1, SL_TriAg_V2, SL_TriAg_V3, SL_TriAg_V4, SL_TriAg_V1 _Pro3, SL_TriAg_V2_Pro3, SL_TriAg_V3_Pro3, and SL_TriAg_V4_Pro3 peptides disclosed herein with glucagon (GCG), glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP). The alignment is shown in FIG. 1A. Based on the alignment, the structural determinants required for the activity were identified.

These results show, inter alia, that the SL_TriAg_V1 peptide was the most active, and stimulated all 3 receptors. These results show, inter alia, that, without wishing to be bound by theory, the Pro3 mutant of SL_TriAg_V1 (SL_TriAg_V1 _Pro3) was produced hypothesizing that it would decrease activation of GIPR, and as expected, the Pro3 mutant exhibited a decrease in the potency to GIPR compared to the potency to GIPR of the SL_TriAg_V1 peptide.

Example 3. Identification and Characterization of Additional Dual and Triple Modulators of Glucagon-Like Peptide-1 Receptor (GLP-1 R), Gastric Inhibitory Peptide Receptor (GIPR), and Glucagon Receptor (GCGR)

Two hybrid peptides were synthesized based on the sequences of peptides disclosed in Example 1 . Hybrid peptide SL-044 comprises 26 N-terminal amino acids of peptide SLTriAgVI fused with 13 C-terminal amino acids of peptide SLTriAgV2 (FIG. 1 B). The activities of peptides disclosed in Example 1 on GLP-1 R, GIPR, and GCGR using genetically engineered cell lines is shown.

To assay for GIPR agonism, HEK293 cells expressing GIPR and a cAMP luciferase reporter were treated with increasing concentrations of SL_TriAg_V1, SL_TriAg_V1_Pro3, SL_TriAg_V2, SL_TriAg_V2_Pro3, SL_TriAg_V3, SL_TriAg_V3_Pro3, SL_TriAg_V4, SL_TriAg_V4_Pro3, SL-044, and SL-045. GIP was used as a positive control, and GLP-1 and GCG were used as negative controls. Immediately after the addition, the luminescence of the cells was measured using a luminometer, and normalized using the basal luminescence.

To assay for GLP-1 R agonism, the CHO-K1 cells engineered to express GLP-1 R and a cyclic AMP (cAMP) luciferase reporter were treated with increasing concentrations of SL_TriAg_V1, SL_TriAg_V1_Pro3, SL_TriAg_V2, SL_TriAg_V2_Pro3, SL_TriAg_V3, SL_TriAg_V3_Pro3, SL_TriAg_V4, SL_TriAg_V4_Pro3, SL-044, and SL-045. GLP-1 was used as a positive control, and GIP and GCG were used as negative controls. Immediately after the addition, the luminescence of the cells was measured using a luminometer, and normalized using the basal luminescence.

To assay for GCGR agonism, the CHO-K1 cells expressing GCGR and a cAMP luciferase reporter were treated with increasing concentrations of SL_TriAg_V1 , SL_TriAg_V1_Pro3, SL_TriAg_V2, SL_TriAg_V2_Pro3, SL_TriAg_V3, SL_TriAg_V3_Pro3, SL_TriAg_V4, SL_TriAg_V4_Pro3, SL-044, and SL- 045. GCG was used as a positive control, and GLP-1 and GIP were used as negative controls. Immediately after the addition, the luminescence of the cells was measured using a luminometer, and normalized using the basal luminescence.

The results from these assays are shown in FIG. 3.

The assay showed, inter alia, that the SL-044 peptide had EC50 values for GLP-1 R agonism, GIPR agonism, and GCGR agonism of 621 nM, 147 nM and 550 nM, respectively. In addition, the assay further showed that the SL-045 peptide had EC50 values for GLP-1 R agonism, GIPR agonism, and GCGR agonism of 13.2 nM, 1.5 nM and 79.2 nM, respectively.

These results show, inter alia, that the SL_TriAg_V1 , SL-044 and SL-045 peptides were the most active, and stimulated all 3 receptors.

Example 4. Activation of GLP-2 Receptor (GLP-2R) by the Dual- or triple-action peptides

The activity of the dual- or triple-action peptides disclosed herein on GLP-2 receptor (GLP-2R) is assayed using a cell-based assay performed cells engineered to express GLP-2R and a cyclic AMP (cAMP) luciferase reporter. This assay measures the reduction of cellular ATP levels because the consumption of ATP upon the activation of adenylate cyclase caused by the signaling via GLP-2R. Without wishing to be bound by theory, the activation of GLP-2R leads to temporary depletion of ATP because of consumption of ATP in cyclization reaction to produce cAMP as well as by phosphorylation by protein kinase A of its substrate. Without wishing to be bound by theory, the temporary depletion of ATP leads to an inhibition of luciferase activity, which needs ATP as a substrate.

Briefly, cells expressing GLP-2R and a cAMP luciferase reporter were treated with increasing amounts of SL_TriAg_V1, SL_TriAg_V1_Pro3, SL_TriAg_V2, SL_TriAg_V2_Pro3, SL_TriAg_V3, SL_TriAg_V3_Pro3, SL_TriAg_V4, SL_TriAg_V4_Pro3, SL-044, and SL-045. A vehicle only control is used as a negative control. ATP levels in the cells, which are decreased as more cAMP is produced in response to the activation of GLP- 2R, were detected following treatment by measuring the level of luciferase activity using a luminometer. In this assay, the inhibition of luciferase activity indicates GLP-2R agonism. The SL_TriAg_V1 , SL_TriAg_V1_Pro3, SL_TriAg_V2, SL_TriAg_V2_Pro3, SL_TriAg_V3, SL_TriAg_V3_Pro3, SL_TriAg_V4, SL_TriAg_V4_Pro3, SL-044, and/or SL-045 are expected to produce a dose-dependent, and saturable decrease in the levels of luciferase, consistent with the activation of the GLP-2R.

Example 5. In Vivo Control of Obesity and Type 2 Diabetes by the Purified Peptide or the Derivative thereof and Modified mRNA (mmRNA) Encoding the Peptide or the Derivative thereof Disclosed Herein

The SL_TriAg_V1, SL-044, SL-045, SL_TriAg_V1_Pro3 and SL_TriAg_V2_Pro3 peptides and mRNA constructs encoding the peptides are assessed in commonly used models of obesity/type 2 diabetes. Briefly, mice of approximately 13 weeks of age are introduced to 60% kcal HFD (e.g., Research Diets D12492). Weekly body weights and non-fasted blood glucose are collected. Upon induction of obesity using high fat diet, the mice are treated with vehicle only, the S L_Tri Ag_V1 peptide, the SL-044 peptide, the SL-045 peptide, the SL_TriAg_V1_Pro3 peptide, the SL_TriAg_V2_Pro3 peptide, mmRNA encoding the SL_TriAg_V1 peptide, mmRNA encoding the SL_TriAg_V1_Pro3 peptide, the SL-044 peptide, the SL-045 peptide, or mmRNA encoding the SL_TriAg_V2_Pro3 peptide. Body weights, insulin levels, food intake, blood glucose levels are measured during course of the experiment. It is expected that the treatment of mice with the SL_TriAg_V1 peptide, the SL-044 peptide, the SL-045 peptide, the SL_TriAg_V1_Pro3 peptide, the SL_TriAg_V2_Pro3 peptide, the mmRNA encoding the SL_TriAg_V1 peptide, the mmRNA encoding the SL_TriAg_V1_Pro3 peptide, the SL-044 peptide, the SL-045 peptide, or the mmRNA encoding the SL_TriAg_V2_Pro3 peptide would reduce body weights, insulin levels, food intake and blood glucose levels compared to the mice that are treated with vehicle only control.

Example 6. In Vivo Control of Obesity and Type 2 Diabetes by the Purified Fusion protein comprising an Fc domain and the Peptide or the Derivative thereof and Modified mRNA (mmRNA) Encoding the Fusion Protein or the Derivative thereof Disclosed Herein

Fc-based Fusion proteins are created by fusing a hinge CH2-CH3-Fc domain derived from lgG1 or lgG4 derivatives and one of the SL_TriAg_V1 , SL-044, SL-045, SL_TriAg_V1_Pro3 and SL_TriAg_V2_Pro3 peptides via a linker (e.g., a glycine and serine-rich flexible linker) are constructed. These constructs are called the SL_TriAg_V1-Fc, SL-044-Fc, SL-045-Fc, SL_TriAg_V1_Pro3-Fc and SL_TriAg_V2_Pro3-Fc fusion proteins. The fusion proteins and the mRNA constructs encoding the fusion proteins are assessed in commonly used models of obesity/type 2 diabetes. Briefly, mice of approximately 13 weeks of age are introduced to 60% kcal HFD (e.g., Research Diets D12492). Weekly body weights and non-fasted blood glucose are collected. Upon induction of obesity using high fat diet, the mice are treated with vehicle only, the SL_TriAg_V1-Fc, SL-044-Fc, SL-045-Fc, SL_TriAg_V1_Pro3-Fc or SL_TriAg_V2_Pro3-Fc fusion protein, or mmRNA encoding the SL_TriAg_V1-Fc, SL-044-Fc, SL-045-Fc, SL_TriAg_V1_Pro3-Fc or SL_TriAg_V2_Pro3-Fc fusion protein. Body weights, insulin levels, food intake, blood glucose levels are measured during course of the experiment. It is expected that the treatment of mice with the SL_TriAg_V1 - Fc, SL-044-Fc, SL-045-Fc, SL_TriAg_V1_Pro3-Fc or SL_TnAg_V2_Pro3-Fc fusion protein, or the mmRNA encoding SL_TriAg_V1-Fc, SL-044-Fc, SL-045-Fc, SL_TriAg_V1_Pro3-Fc and SL_TriAg_V2_Pro3-Fc fusion protein would reduce body weights, insulin levels, food intake and blood glucose levels compared to the mice that are treated with vehicle only control.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporated by reference in their entireties.

The publications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present technology is not entitled to antedate such publication by virtue of prior disclosure.

As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.

EQUIVALENTS

While the disclosure has been disclosed in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure as come within known or customary practice within the art to which the disclosure pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments disclosed specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

Claims

CLAIMS What is claimed is:

1 . A peptide or a derivative thereof, wherein the peptide comprises a general formula (I):

N-L1-[Aaa]1-[Aaa]2-[Aaa]3-Gly-Thr-Phe-Thr-Ser-Asp-[Aaa]4-Ser-[Aaa]5-[Aaa]6-[Aaa]7-[Aaa]8- [Aaa]9-[Aaa]10-[Aaa]11-[Aaa]12-[Aaa]13-[Aaa]14-Phe-[Aaa]15-[Aaa]16-[Aaa]17-Leu-[Aaa]18- [Aaa]19-[Aaa]20-Gly-Pro-[Aaa]21-[Aaa]22-[Aaa]23-[Aaa]24-Pro-Pro-Pro-[Aaa]25-L2-C

(I) wherein:

N is the N-terminus;

L1 and/or L2 are independently present or absent; and L1 and/or L2, if present, are each independently selected from a carrier protein, a tag, and a polymer, optionally wherein the L1 and/or L2 further comprise a linker that adjoins the carrier protein, tag, or polymer with the peptide;

[Aaa]1 is His;

[Aaa]2 is Ser or Gly;

[Aaa]3 is His or Pro;

[Aaa]4 is Phe or lie;

[Aaa]5 is Leu or Vai;

[Aaa]6 is Ala;

[Aaa]7 is Leu or lie;

[Aaa]8 is Asp or Glu;

[Aaa]9 is Lys or Glu;

[Aaa]10 is Gin;

[Aaa]11 is Arg;

[Aaa]12 is Gin;

[Aaa]13 is Ala or Gin;

[Aaa]14 is Glu;

[Aaa]15 is lie or Leu;

[Aaa]16 is Asp or Glu;

[Aaa]17 is Trp;

[Aaa]18 is Arg or Gly;

[Aaa]19 is Ala; [Aaa]20 is Gly or Ala;

[Aaa]21 is Pro or Ser;

[Aaa]22 is Ser or Thr;

[Aaa]23 is Gly or Ala;

[Aaa]24 is Arg or Lys;

[Aaa]25 is Arg or Lys; and

C is the C-terminus.

2. A peptide or a derivative thereof, wherein the peptide comprises formula (II):

N-L1 -[Aaa]1-[Aaa]2-[Aaa]3-Gly-Thr-Phe-Thr-Ser-Asp-[Aaa]4-Ser-[Aaa]5-[Aaa]6-[Aaa]7-[Aaa]8-

[Aaa]9-[Aaa]10-[Aaa]11-[Aaa]12-[Aaa]13-[Aaa]14-Phe-[Aaa]15-[Aaa]16-[Aaa]17-Leu-[Aaa]18-

[Aaa]19-[Aaa]20-Gly-Pro-[Aaa]21-[Aaa]22-[Aaa]23-[Aaa]24-Pro-Pro-Pro-[Aaa]25-L2-C

(II) wherein:

N is the N-terminus;

L1 and/or L2 are independently present or absent; and L1 and/or L2, if present, are each independently selected from a carrier protein, a tag, and a polymer;

[Aaa] 1 is a positively charged amino acid residue selected from His, Lys, and Arg;

[Aaa]2 is a polar and neutral of charge hydrophilic amino acid selected from Ser, Asn, Gin, Thr, and

Pro, or a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met;

[Aaa] 3 is a positively charged amino acid residue selected from His, Lys, and Arg, or a polar and neutral of charge hydrophilic amino acid selected from Pro Ser, Asn, Gin, and Thr;

[Aaa]4 is a hydrophobic, aromatic amino acid selected from Phe, Trp, and Tyr, or a hydrophobic, aliphatic amino acid selected from lie, Gly, Ala, Leu, Met, and Vai;

[Aaa]5 is a hydrophobic, aliphatic amino acid selected from Leu, Vai, lie, Gly, Ala, and Met;

[Aaa]6 is a hydrophobic, aliphatic amino acid selected from Ala, Gly, Vai, lie, Leu, and Met;

[Aaa]7 is a hydrophobic, aliphatic amino acid selected from Leu, lie, Vai, Gly, Ala, and Met; [Aaa]8 is a polar and negatively charged hydrophilic amino acid selected from Asp and Glu;

[Aaa]9 is: a polar and positively charged hydrophilic amino acid selected from Lys, His, and Arg, or a polar and negatively charged hydrophilic amino acid selected from Glu, and Asp;

[Aaa] 10 is a polar and neutral of charge hydrophilic amino acid selected from Gin, Asn, Ser, Thr, and Pro;

[Aaa] 11 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His;

[Aaa] 12 is a polar and neutral of charge hydrophilic amino acid selected from Gin, Asn, Ser, Thr, and Pro;

[Aaa] 13 is a hydrophobic, aliphatic amino acid selected from Ala, Vai, Gly, Leu, lie, and Met, or a polar and neutral of charge hydrophilic amino acid selected from Gin, Asn, Ser, Thr, and Pro;

[Aaa] 14 is a polar and negatively charged hydrophilic amino acid selected from Glu, and Asp;

[Aaa] 15 is a hydrophobic, aliphatic amino acid selected from lie, Leu, Vai, Ala, Gly, and Met;

[Aaa] 16 is a polar and negatively charged hydrophilic amino acid selected from Asp, and Glu;

[Aaa] 17 is hydrophobic, aromatic amino acid selected from Trp, Phe, and Tyr;

[Aaa] 18 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His, or a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met;

[Aaa] 19 is a hydrophobic, aliphatic amino acid selected from Ala, Gly, Vai, Leu, lie, and Met;

[Aaa]20 is a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met;

[Aaa]21 is a polar and neutral of charge hydrophilic amino acid selected from Pro, Ser, Gin, Asn, and Thr;

[Aaa]22 is a polar and neutral of charge hydrophilic amino acid selected from Ser, Thr, Gin, Asn, and Pro;

[Aaa]23 a hydrophobic, aliphatic amino acid selected from Gly, Ala, Vai, Leu, lie, and Met;

[Aaa]24 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His;

[Aaa]25 is a polar and positively charged hydrophilic amino acid selected from Arg, Lys, and His; and

C is the C-terminus.

3. The peptide or the derivative thereof of claim 2, wherein:

[Aaa]1 is His;

[Aaa]2 is Ser or Gly;

[Aaa]3 is His or Pro;

[Aaa]4 is Phe or lie;

[Aaa]5 is Leu or Vai;

[Aaa]6 is Ala;

[Aaa]7 is Leu or lie;

[Aaa]8 is Asp or Glu;

[Aaa]9 is Lys or Glu;

[Aaa]10 is Gin;

[Aaa]11 is Arg;

[Aaa]12 is Gin;

[Aaa]13 is Ala or Gin;

[Aaa]14 is Glu;

[Aaa]15 is lie or Leu;

[Aaa]16 is Asp or Glu;

[Aaa]17 is Trp;

[Aaa]18 is Arg or Gly;

[Aaa]19 is Ala;

[Aaa]20 is Gly or Ala;

[Aaa]21 is Pro or Ser;

[Aaa]22 is Ser or Thr;

[Aaa]23 is Gly or Ala;

[Aaa]24 is Arg or Lys; and/or

[Aaa]25 is Arg or Lys.

4. The peptide or the derivative thereof of any one of claim 1 to 3, wherein the peptide or the derivative thereof comprises an amino acid sequence that is selected from the amino acid sequence of SEQ ID NOs: 60 to 69.

5. The peptide or the derivative thereof of claim 4, wherein the peptide or the derivative thereof comprises an amino acid sequence that is selected from the amino acid sequence of SEQ ID NOs: 60 to 69, or a variant thereof having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from SEQ ID NOs: 60 to 69.

6. The peptide or the derivative thereof of claim 5, wherein the peptide or the derivative thereof comprises an amino acid sequence that is selected from the amino acid sequence of SEQ ID NOs: 60, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 68 and SEQ ID NO: 69, or a variant thereof having 1 or 2 amino acid mutations with respect to an amino acid sequence selected from SEQ ID NOs: 60, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 68 and SEQ ID NO: 69.

7. The peptide or the derivative thereof of claim 6, wherein the peptide or the derivative thereof comprises an amino acid sequence that is selected from the amino acid sequence of SEQ ID NOs: 60, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 68 and SEQ ID NO: 69.

8. The peptide or the derivative thereof any one of claims 1 to 7, wherein the peptide or the derivative thereof comprises a carrier protein selected from Fc domain, albumin, transferrin, or elastin-like protein, Keyhole Limpet Hemocyanin (KLH), ovalbulin, or a variant thereof.

9. The peptide or the derivative thereof of claim 8, wherein the carrier protein comprises an Fc domain selected from an IgG Fc domain, an IgA Fc domain, an IgM Fc domain, an IgE Fc domain, and an IgD Fc domain, or a variant thereof.

10. The peptide or the derivative thereof of claim 9, wherein the carrier protein comprises the IgG Fc domain selected from an lgG1 Fc domain, an I gG2 Fc domain, an I gG3 Fc domain, and an I gG4 Fc domain, or a variant thereof.

11. The peptide or the derivative thereof of claim 10, wherein the carrier protein comprises the IgG Fc domain is selected from a human I gG1 Fc domain, and a human I gG4 Fc domain, or a variant thereof.

12. The peptide or the derivative thereof of claim 11, wherein the human lgG1 Fc domain comprises a hinge-CH2-CH3-Fc domain derived from human I gG1 or a derivative thereof.

13. The peptide or the derivative thereof of claim 11, wherein the human lgG4 Fc domain comprises a hinge-CH2-CH3-Fc domain derived from human I gG4 or a derivative thereof.

14. The peptide or the derivative thereof any one of claims 7 to 13, wherein the peptide or the derivative thereof further comprises a linker that adjoins the carrier protein with the peptide or the derivative thereof.

15. The peptide or the derivative thereof of claim 14, wherein the peptide linker is rigid or flexible.

16. The peptide or the derivative thereof any one of claims 1 to 15, wherein the peptide or the derivative thereof further comprises a glycosyl moiety.

17. The peptide or the derivative thereof of claim 16, wherein the glycosyl moiety is an N-linked glycosyl moiety.

18. The peptide or the derivative thereof of claim 16, wherein the glycosyl moiety is an O-linked glycosyl moiety.

19. The peptide or the derivative thereof any one of claims 1 to 18, wherein the peptide or the derivative thereof comprises one or more N-linked glycosylation consensus sites and/or O-linked glycosylation consensus sites.

20. The peptide or the derivative thereof any one of claims 1 to 19, wherein the peptide or the derivative thereof is biosynthesized as a single polypeptide chain.

21 . The peptide or the derivative thereof any one of claims 1 to 20, wherein the peptide or the derivative thereof is biosynthesized from a single open reading frame.

22. The peptide or the derivative thereof any one of claims 1 to 21 , wherein the peptide or the derivative thereof is prepared using an expression system.

23. The peptide or the derivative thereof of claim 22, wherein the expression system is selected from bacterial, yeast, invertebrate, vertebrate, and plant expression system.

24. The peptide or the derivative thereof any one of claims 1 to 23, wherein the peptide or the derivative thereof further comprises a non-natural amino acid selected from an amino isobutyric acid (Aib), a D-amino acid, and those comprising a modification selected from N-methylation (Nm), Ca-methylation (Cm), (CH2NH) reduced amide bonds (Rd), and a peptoids (Pp)).

25. The peptide or the derivative thereof any one of claims 1 to 24, wherein the peptide or the derivative thereof comprises a polymer.

26. The peptide or the derivative thereof of claim 25, wherein the polymer is selected from poly (alkyl ene oxide) (e.g., polyethylene glycol (PEG)), poly(N-vinylpyrrolidone), poly(vinyl alcohol), poly(glycerol), poly(zwitterions), poly(carbonates), polyoxazoline, poly(acryloylmorpholine), poly(oxazolines), poly(sacharrides), and a combination thereof.

27. The peptide or the derivative thereof of claim 26, wherein the polymer is polyethylene glycol (PEG).

28. The peptide or the derivative thereof of claim 27, wherein one or more amino acids present in the peptide or the derivative thereof is PEGylated.

29. The peptide or the derivative thereof of claim 28, wherein the one or more PEGylated amino acids are located inside the carrier protein.

30. The peptide or the derivative thereof of any one of claims 27 to 29, wherein the PEGylation is conducted using a succinimidyl ester, an aldehyde, a maleimide, and/or a p-nitrophenyl carbonate ester reagent.

31 . The peptide or the derivative thereof of any one of claims 27 to 30, wherein one or more Lys residues, one or more Ser residues, one or more Tyr residues, one or more His residues, one or more Cys residues, the N-terminus and/or the C-terminus are PEGylated.

32. The peptide or the derivative thereof of any one of claims 27 to 31, wherein the one or more PEGylated amino acids comprise a Lys residue.

33. The peptide or the derivative thereof of claim 32, wherein the PEGylation is conducted via amine conjugation.

34. The peptide or the derivative thereof of any one of claims 27 to 33, wherein the one or more PEGylated amino acids comprise a Gin residue.

35. The peptide or the derivative thereof of claim 34, wherein the PEGylation is conducted via transglutaminase (TGase) mediated enzymatic conjugation.

36. The peptide or the derivative thereof of any one of claims 27 to 35, wherein the one or more PEGylated amino acids comprise a Cys residue.

37. The peptide or the derivative thereof of claim 36, wherein the PEGylation is conducted via thiol conjugation.

38. The peptide or the derivative thereof of any one of claims 1 to 37, wherein the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on glucagon-like peptide- 1 receptor (GLP-1 R), glucagon-like peptide-2 receptor (GLP-2R), gastric inhibitory peptide receptor (GIPR), and/or glucagon receptor (GCGR).

39. The peptide or the derivative thereof of any one of claims 1 to 38, wherein the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GLP-1 R and GCGR.

40. The peptide or the derivative thereof of any one of claims 1 to 38, wherein the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GLP-1 R and GIPR.

41. The peptide or the derivative thereof of any one of claims 1 to 38, wherein the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GLP-1 R and GLP-2R.

42. The peptide or the derivative thereof of any one of claims 1 to 38, wherein the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GIPR and GCGR.

43. The peptide or the derivative thereof of any one of claims 1 to 38, wherein the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GLP-2R and GIPR.

44. The peptide or the derivative thereof of any one of claims 1 to 38, wherein the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GLP-2R and GCGR.

45. The peptide or the derivative thereof of any one of claims 1 to 38, wherein the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GLP-1 R, GIPR and GCGR.

46. The peptide or the derivative thereof of any one of claims 1 to 38, wherein the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GLP-1 R, GLP-2R and GCGR.

47. The peptide or the derivative thereof of any one of claims 1 to 38, wherein the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GLP-1 R, GLP-2R and GIPR.

48. The peptide or the derivative thereof of any one of claims 1 to 38, wherein the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GLP-2R, GIPR and GCGR.

49. The peptide or the derivative thereof of any one of claims 1 to 38, wherein the peptide or the derivative thereof has independently an agonistic activity or an antagonistic activity on GLP-1 R, GLP-2R, GIPR and GCGR.

50. The peptide or the derivative thereof of any one of claims 1 to 49, wherein the peptide or the derivative thereof exhibits an EC50 value for GLP-1 R in the range of 100 pM to 1000 nM, as determined by a cell-based assay.

51. The peptide or the derivative thereof of any one of claims 1 to 50, wherein the peptide or the derivative thereof exhibits an EC50 value for GLP-1 R in the range of 2 nM to 100 nM, as determined by a cellbased assay, optionally wherein the peptide or the derivative thereof exhibits an I C50 value for GLP-1 R in the range of 10 pM to 10 pM, as determined by a cell-based assay.

52. The peptide or the derivative thereof of any one of claims 1 to 51 , wherein the peptide or the derivative thereof exhibits an EC50 value for GIPR in the range of 100 pM to 1000 nM, as determined by a cell-based assay.

53. The peptide or the derivative thereof of any one of claims 1 to 52, wherein the peptide or the derivative thereof exhibits an EC50 value for GIPR in the range of 100 pM to 2 nM, as determined by a cellbased assay.

54. The peptide or the derivative thereof of any one of claims 1 to 53, wherein the peptide or the derivative thereof exhibits an IC50 value for GIPR in the range of 10 pM to 10 pM, as determined by a cellbased assay.

55. The peptide or the derivative thereof of any one of claims 1 to 54, wherein the peptide or the derivative thereof exhibits an EC50 value for GCGR in the range of 100 pM to 1000 nM, as determined by a cell-based assay.

56. The peptide or the derivative thereof of any one of claims 1 to 55, wherein the peptide or the derivative thereof exhibits an EC50 value for GCGR in the range of 2 nM to 100 nM, as determined by a cellbased assay.

57. The peptide or the derivative thereof of any one of claims 1 to 56, wherein the peptide or the derivative thereof exhibits an IC50 value for GCGR in the range of 10 pM to 10 pM, as determined by a cellbased assay.

58. The peptide or the derivative thereof of any one of claims 1 to 57, wherein the peptide or the derivative thereof exhibits: an EC50 value for GCGR in the range of 10 pM to 10 pM, as determined by a cell-based assay or an I C50 value for GCGR in the range of 10 pM to 10 pM, as determined by a cell-based assay.

59. A fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 60, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 60.

60. The fusion protein of claim 59, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 60.

61. A fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 61, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 61.

62. The fusion protein of claim 61, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 61.

63. A fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 62, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 62.

64. The fusion protein of claim 63, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 62.

65. A fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 63, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 63.

66. The fusion protein of claim 65, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 63.

67. A fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 64, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 64.

68. The fusion protein of claim 67, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 64.

69. A fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 65, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 65.

70. The fusion protein of claim 69, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 65.

71. A fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1 , GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 66, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 66.

72. The fusion protein of claim 71, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 66.

73. A fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 67, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 67.

74. The fusion protein of claim 73, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 67.

75. A fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 68, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 68.

76. The fusion protein of claim 75, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 68.

77. A fusion protein comprising a hinge CH2-CH3-Fc domain fused to a peptide or a derivative thereof having agonistic or antagonistic activity on the receptors of GLP-1, GLP-2, GIP, and/or glucagon, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 69, or a variant thereof having an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations, with respect to the amino acid sequence selected from SEQ ID NO: 69.

78. The fusion protein of claim 77, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 69.

79. The fusion protein of any one of claims 59 to 78, wherein the hinge CH2-CH3-Fc domain is derived from IgG, IgM, IgA, IgD, or IgE.

80. The fusion protein of claim 79, wherein the IgG is selected from IgG 1 , lgG2, 1 gG3, and I gG4.

81 . The fusion protein of any one of claims 59 to 80, wherein the hinge CH2-CH3-Fc domain is derived from lgG1.

82. The fusion protein of claim 81 , wherein the I gG 1 is human I gG 1 .

83. The fusion protein of claim 81 or claim 82, wherein the hinge CH2-CH3-Fc domain comprises an amino acid sequence that is at least about 95%, or at least about 97%, or at least about 97%, or at least about 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 4.

84. The fusion protein of any one of claims 59 to 80, wherein the hinge-CH2-CH3 Fc domain is derived from I gG4.

85. The fusion protein of claim 84, wherein the lgG4 is human I gG4.

86. The fusion protein of claim 84 or claim 85, wherein the hinge CH2-CH3-Fc domain comprises an amino acid sequence that is at least about 95%, or at least about 97%, or at least about 97%, or at least about 98% or at least 99% identical to the amino acid sequence SEQ ID NO: 1 , SEQ ID NO: 2, or SEQ ID NO: 3.

87. The fusion protein of any one of claims 59 to 86, wherein the hinge CH2-CH3-Fc domain is fused to the peptide or a derivative thereof via a linker.

88. The fusion protein of claim 84, wherein the linker has an amino acid sequence selected from SEQ ID NOs: 5 to 53.

89. An isolated polynucleotide encoding the peptide or the derivative thereof of any one of claims 1 to 23 or 39 to 58 or the fusion protein of any one of claims 59 to 88.

90. The isolated polynucleotide of claim 89, wherein the isolated polynucleotide is DNA.

91 . The isolated polynucleotide of claim 89, wherein the nucleic acid is RNA.

92. The isolated polynucleotide of claim 89, wherein the isolated polynucleotide is selected from mRNA, circular RNA (circRNA) and self-amplifying RNA (saRNA), optionally wherein the polynucleotide is modified.

93. The isolated polynucleotide of claim 92, wherein the mRNA a modified mRNA (mmRNA).

94. The isolated polynucleotide of claim 93, wherein the mmRNA comprises at least one nucleoside modification selected from pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio- pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5- taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4- thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1 - methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio- dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2- thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo- pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1 -methylpseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1 -methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy- cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy- 1-methyl- pseudoisocytidine, 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza- 2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2,6- diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy- adenine, inosine, 1 -methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6- thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7- methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1 -methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2- methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, and combinations thereof.

95. The isolated polynucleotide of claim 93 or claim 94, wherein the mmRNA further comprises a 5'-cap and/or a poly A tail.

96. An expression vector comprising the isolated polynucleotide of claim 89 or claim 90.

97. A expression vector of claim 96, wherein the expression vector is a mammalian expression vector.

98. A host cell comprising the isolated polynucleotide of any one of claim 89 or claim 90, or the expression vector of claim 96 or claim 97.

99. A pharmaceutical composition comprising the peptide or the derivative thereof of any one of claims 1 to 58 or the fusion protein of any one of claims 59 to 88, or the isolated polynucleotide of any one of claim 89 to 95, or the expression vector of claim 96 or claim 67, or the host cell of claim 98.

100. A pharmaceutical composition comprising the mmRNA of any one of claims 93 to 95, and a pharmaceutically acceptable carrier.

101. The pharmaceutical composition of claim 99 or claim 100, wherein the carrier is a lipidoid, a liposome, a lipoplex, a lipid nanoparticle, a polymeric nanoparticle, a peptide, a protein, a cell, a nanoparticle mimic, a nanotube, or a conjugate.

102. The pharmaceutical composition of claim 101 , wherein the pharmaceutical composition is formulated as a lipid nanoparticle (LNPs), a lipoplex, or a liposome.

103. The pharmaceutical composition of claim 102, wherein the pharmaceutical composition is formulated as a lipid nanoparticle (LNPs).

104. The pharmaceutical composition of claim 103, wherein the lipid nanoparticles comprise lipids selected from an ionizable lipid (e.g, an ionizable cationic lipid selected from DLin-DMA, DLin-K-DMA, DLin- KC2-DMA, DLin-MC3-DMA, 98N12-5, and C12-200); a structural lipid (e.g., distearoylphosphatidylcholine (DSPC)); cholesterol, and a polyethyleneglycol (PEG)-lipid (e.g, a PEG-diacylglycerol (DAG), a PEG- dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof, or a PEG- dilauryloxypropyl (C12, a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), or a PEG- distearyloxypropyl (C18)); 1 ,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); dioleoylphosphatidylethanolamine (DOPE); and the mmRNA.

105. The pharmaceutical composition of claim 104, wherein the lipid nanoparticles comprise (a) a cationic lipid comprising from 50 mol % to 85 mol % of the total lipid present in the particle; (b) a non-cationic lipid comprising from 13 mol % to 49.5 mol % of the total lipid present in the particle; and (c) a conjugated lipid that inhibits aggregation of particles comprising from 0.5 mol % to 2 mol % of the total lipid present in the particle.

106. The pharmaceutical composition of claim 104 or claim 105, wherein the lipid nanoparticles comprise a lipid selected from SM-102, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, and C12- 200; a cholesterol; and a PEG-lipid.

107. The pharmaceutical composition of any one of claims 104 to 106, wherein the pharmaceutical composition is formulated for parenteral administration.

108. The pharmaceutical composition of claim 107, wherein the pharmaceutical composition is formulated for intradermal, intramuscular, intraperitoneal, intraarticular, intravenous, subcutaneous, intraarterial or transdermal administration.

109. A method of treating or preventing hyperglycemia, diabetes, obesity or metabolic syndrome, nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), a cardiometabolic disease, or liver fibrosis or for reducing blood glucose, or for reducing fed and fasting blood glucose, or for reducing cardiovascular risk, or for decreasing body weight, decreasing food intake, decreasing blood glucose, decreasing liver adiposity, decreasing liver weight, decreasing subcutaneous white adipose tissue (sWAT), or for increasing glucose tolerance in a subject in need thereof, the method comprising administering to the subject the peptide or the derivative thereof of any one of claims 1 to 58 or the fusion protein of any one of claims 59 to 88, or the isolated polynucleotide of any one of claim 89 to 95, or the expression vector of claim 96 or claim 97, or the host cell of claim 98, or the pharmaceutical composition of claim 99 to 108.