WO2025024788A1

WO2025024788A1 - Stabilized glp-1 peptides and dual agonist peptides against glp-1r and gipr and methods of use thereof

Info

Publication number: WO2025024788A1
Application number: PCT/US2024/039809
Authority: WO
Inventors: Aaron SCHACHT; Arvind Kumar; Dharanesh Mahimapura GANGAIAH; Krishnakumar VASUDEVAN; Ali CAMARA
Original assignee: Biomedit LLC
Current assignee: Biomedit LLC
Priority date: 2023-07-27
Filing date: 2024-07-26
Publication date: 2025-01-30
Anticipated expiration: 2026-01-27

Abstract

The present application provides protease-resistant Glucagon-Like Peptide 1 (eGLP-1) peptides, and also provides protease-resistant dual agonist peptides against GLP-1R and GIPR, methods of making such peptides, as well as compositions comprising protease-resistant peptides and methods of treatment utilizing such peptides.

Description

Stabilized GLP-1 Peptides and Dual Agonist Peptides Against GLP-1R and GIPR and Methods of Use Thereof

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims priority from U.S. Provisional Patent Application Ser. No. 63/529,271, filed July 27, 2023, and U.S. Provisional Patent Application Ser. No. 63/540,427, filed September 26,

2023.

INCORPORATION OF SEQUENCE LISTING

[002] The sequence listing in ST.26 XML format entitled 2950-17_PCT_ST26, created on July 26,

2024, comprising 575,627 bytes, prepared according to 37 CFR 1.822 to 1.824, submitted July 26, 2024 with the filing of this application, is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[003] This invention relates to variant and stabilized forms of Glucagon-Like Peptide 1 (GLP-1), methods of use, and methods of production thereof, and also relates to dual agonists against GLP-1R and GIPR and their delivery via a live delivery platform, such as a genetically modified bacterium, to deliver therapeutic activity directly to the mucosa of an animal in need thereof.

BACKGROUND OF THE INVENTION

[004] Type 2 diabetes is a disease caused by high blood glucose due to insulin resistance and relative deficiency of insulin. Type 2 Diabetes Mellitus (T2DM) is most prevalent and characterized by a combination of interrelated metabolic disorder. Although various treatments such as tirzepatide, semaglutide, liraglutide, GLP1, GIP (7DTY_P), Medi7219, Exenatide (to name a few) are available for T2DM, still more improvements are needed to treat Felts catus and other species.

[005] Peptide therapeutics are progressively used in the treatment of T2DM. Their oral administration represents a significant advance in medicine but is challenged by gastrointestinal instability and ineffective uptake into the circulation. Exenatide, tirzepatide, semaglutide and liraglutide peptides are administered subcutaneously while Medi7219, and J229 are orally given. Drugs designed for T2DM either work as single agonist or dual or triple agonists. See Pechenov, et al., “Development of an orally delivered GLP-1 receptor agonist through peptide engineering and drug delivery to treat chronic disease,” Scientific Reports 1 1(1):22521 (2021).

[006] GLP-1 , a proglucagon-derived peptide produced by intestinal L cells, and GIP, produced by K cells, are used for treatment of type 2 diabetes. GLP-1 and GIP both stimulate insulin secretion and GLP- 1 slows down gastric emptying whereas GIP has no effect. Exenatide is a GLP-1 analogue originally found in the saliva of the Gila monster and has a 53% amino acid identity to GLP-1 (Bond, “Exenatide (Byetta) as a novel treatment option for type 2 diabetes mellitus,” Proceedings (Baylor University.

Medical Center) 19(3): 281-284 (2006). Liraglutide (4APD_ A) shares 97% sequence identity with GLP-1 and the addition of a C16 fatty acid side chain facilitates binding of the drug to circulating serum albumin (Garber, “Long-acting glucagon-like peptide 1 receptor agonists: a review of their efficacy and tolerability,” Diabetes Care 34(Suppl 2):S279-84 (2011). Furthermore, the replacement of alanine by glycine in position 8 (GLP-1-Gly8), as also utilized in Exenatide, significantly increases the insulinotropic effect (Lin, et al., “Oral Delivery of Pentameric Glucagon-Like Peptide- 1 by Recombinant Lactobacillus in Diabetic Rats," PloS One 1 l(9):e0162733 (2016)).

[0071 The GIPR and GLP-1R receptors each comprise an N-terminal extracellular domain (ECD), a central domain consisting of seven transmembrane a-helices, and a C-terminal, cytoplasmic domain that mediates intracellular signal transduction by physical association with a G protein. Tirzepatide agonist against GIPR and GLP-1R exhibit a similar peptide-receptor binding interface, whereas structural changes are observed at the extra cellular loop (ECLl, ECL3) and trans-membrane (TM1, TM3) regions. The N-terminal of tirzepatide overlaps well in both the receptors and interacts with the central transmembrane domain, causing a conformational rearrangement that activates the receptors. Residues from Tyr-l-Met-14 of the tirzepatide agonist make no contacts to the extracellular domain of the receptors. It is believed that hydrophobic interactions are involved in binding.

[008] The binding interface of GIP spans the a-helical C-terminal region of peptide comprising residues Asp-15-Lys-30 which interact with the extracellular domain of the receptors as shown in Figure 2. As shown in Figure 2, the C-terminal of GLP-1 agonist interacts with Extracellular domain whereas N- terminal of GLP-1 interacts with Transmembrane of the GLP-1R receptor. Figure taken from Cong, et al., “Molecular insights into ago-allosteric modulation of the human glucagon-like peptide- 1 receptor," Nature Communications, /2(l):3763 (2021).

[009] The helical peptide exposes hydrophobic residues toward the ECD, suggesting that binding is dominated by hydrophobic interactions. Structural studies of the GLP-1 peptide bound to the ECD confirm that the C-terminal a-helical region of GLP-1 is positioned within a binding cleft of the N- terminal of ECD. The hydrophobic faces of GLP-1 make the majority of interactions with the ECD and likely are the key contributors to ECD/peptide affinity. The ECD of the receptors folds down towards the TMD to stabilize the complex. The formation of polar network rearrangement shows that GLP-1 triggered GLP-1 R activation. Upon receptor activation, three layers of the polar network are reorganized as (i) central polar network, (ii) HETX motif polar network, (iii) TM2-6-7-helix 8 polar network (See Figure 2, Cong et al. (2021)). The ECD of the receptor recognizes and “catches” C-terminal part (residues 15-30) of the agonist after which the N-terminal part of the peptide moves and docks into the TMD. During this process, the well-conserved PLLG motif of the receptor in the middle of helix VI gets destabilized and changes conformation. The bottom of helix VI moves 10-20 A outwards, making an angle of 40 - 60* with helix VII and creates space in the intracellular part of the receptor for cytosolic protein complexes to bind.

[010] GLP-1 (7-37), GIP (1-42), Exenatide (1-39), tirzepatide (1-39), Semaglutide, Liraglutide, J211, Medi7219 (to name a few) are studied in detail. The N-terminal of the tirzepatide is more homologous to GIP peptide and the C-terminal is more like Exenatide (30-39aa). See Nauck and D’Alessio, “Tirzepatide, a dual GIP/GLP-1 receptor co-agonist for the treatment of type 2 diabetes with unmatched effectiveness regrading glycaemic control and body weight reduction,” Cardiovascular Diabetology, 2/(l):169 (2022) and Wang, “Designing a Dual GLP-1R/GTR Agonist from Tirzepatide: Comparing Residues Between Tirzepatide, GLP-1, and GIP,” Drug Design. Development and Therapy 16; 1547-1559 (2022). The main circulating GIP peptide is a GIP (1-42) variant, its N-terminal is truncated by DPP-4 resulting in GIP (3-42). GIP (1-42) is processed from the precursor protein proGIP by the prohormone convertase (PC), but also contains a PC2 cleavage motif, which cleaves at Gly31, Lys32 and Lys33 positions. After cleavage, two peptides GIP (l-30)-NH2 and GT (34-42) are generated. The C-terminal truncated GT (l-30)-NH2 peptide seems to have similar agonistic properties as full length GT (1-42) (Alana et al. “NMR structure of the glucose-dependent insulinotropic polypeptide fragment, GT(1- 30)amide,” Biochemical and Biophysical Research Communications, 325(\) (2004) and Gabe et al. “Molecular interactions of full-length and truncated GIP peptides with the GT receptor - A comprehensive review,” Peptides 725:170224 (2020). The last 12 residues of GT (1-42) are observed to be disordered and do not bind to the ECD, nor to residues close to the TMD, suggesting that these residues are of little importance for GTR binding and receptor activation. It is also observed that the presence of C-terminus affects the overall stability of the GIP peptide, and its presence improves the agonistic action of the N-terminally truncated GIP peptides, as compared to the same N-terminal truncations in the absence of the C terminus.

[Oil] GLP-1 is a 30 amino acid containing peptide secreted from intestinal L-cells in response to food intake. It exists in two equipotent forms: GLhP-1 (7-36)-NH2 and GLP-l(7-37). GLP-l(7-36) is more abundant. GLP-l(7-36) binds to and activates the GLP-1 receptor (GLP-1R) to exert its regulatory functions.

[012] Various modifications have been suggested in the art to engineer tirzepatide by substituting the non-natural AIB with a natural amino acid and to reduce proteolytic vulnerability. Tirzepatide is a GIP analogue and Wang et al., 2022 suggest that Gly can be a possible option for modification of AIB (Wang (2022)). However, this modification decreases binding affinity of the peptide for GLP-1 R while it increases binding to the GIPR receptor. Importantly, glycine is easily cleaved by the proteolytic enzyme elastase (Wang (2022)). [013] GLP-1 (7-37) has His at 7^th position and is crucial for GLP-1R receptor activation and to retain their insulinotropic activity. Replacement of His⁷ with Tip⁷ lowers the binding affinity and strongly lowers the activity of GLP-1 whereas the absence of Tyr* from the N-terminal of GIP dramatically decreases its activity. Tyr¹ and lie⁷ are key for activating GIPR receptor by GIP peptide. Incorporating Thr⁷ from GLP-1 into tirzepatide may lower its GIP activity. Tyr¹⁰ and lie¹² from GIP are used in MAR709 peptide and He¹² plays an important role in GIPR receptor activation. Tyr¹⁰ and He¹² are used in tirzepatide. Substitution of Tyr¹⁹ with Ala in GLP-1 decreases the binding affinity and activity of GLP-1 agonist. Aibl3 of tirzepatide seems to lower its GLP-1 activity without affecting its GIP activity (Wang (2022)).

[014] GLP-1 is an incretin hormone secreted by the L cells of the intestine that stimulates the secretion of insulin from the pancreas in a glucose-dependent manner. See, Chia and Egan, “Incretin-Based Therapies in Type 2 Diabetes Mellitus,” J Clin Endocrinol Metab. 93(10):3703-3716 (2008) and Muller et al., “Glucagon-like peptide 1 (GLP-1),” Mol Metab 30:72-130 (2019). Exogenously supplied GLP-1 analogues are used in the treatment of type 2 diabetes. It has been shown to play an important role in increasing the beta cell mass and has potent antidiabetic effects associated with weight loss. See, Baggio and Drucker, “Biology of incretins: GLP-1 and GIP,” Gastroenterology 132(6):2131-57 (2007).

[015] The amino acid sequence of GLP-1 (SEQ ID NO: 18) is highly conserved in mammals and has 100% identity in humans, mouse, rat, sheep, goat, cow, hamster, cat, and dogs, to name just a few (data not shown). GLP-1 is the endogenous ligand of GLP-1R, and it shows 53% identity with Exenatide (long- acting GLP-1 receptor agonists). See Bond, “Exenatide (Byetta) as a novel treatment option for type 2 diabetes mellitus,” Proceedings (Baylor University. Medical Center), 19(3):281-284 (2006). Circulating GLP-1 is rapidly inactivated by the serum protease dipeptidyl peptidase-4 (DPP-IV or DPP-4), which removes the two N-terminal amino acids. GLP-1 also has numerous sites of proteolytic lability to the digestive proteases like pepsin, trypsin, and chymotrypsin (Manandhar and Ahn, “Glucagon-like peptide- 1 (GLP-1) analogs: recent advances, new possibilities, and therapeutic implications,” Journal of Medicinal Chemistry, 58(3): 1020-1037 (2015). (See Figure 1).

[016] The inactive full-length form of GLP-1 (1-37) is processed into two active circulating forms, GLP-1 (7-37) and GLP-1 (7-36) amide, with the latter being the most abundant form found in blood. Both forms of GLP-1 have a very short half-life because they are sensitive to dipeptidyl peptidase-IV (DPP-IV) digestion in serum. GLP-1 also contains aromatic residues (Phel2, Tyrl9, Phe28, and Trp31) that are sensitive to chymotrypsin, pepsin and/or neprilysin. A summary of the known cleavage sites is presented in Figure 1. See Pechenov et al., “Development of an orally delivered GLP-1 receptor agonist through peptide engineering and drug delivery to treat chronic disease,” Sci Rep. 11(1 ):22521 (2021) PMID: 34795324. [0171 The half-life of GLP-1 has been reported as less than 2 minutes where concentrations return to baseline within 90 min after subcutaneous injection making it difficult to administer systemically. See, Kieffer et al., “Degradation of glucose-dependent insulinotropic polypeptide and truncated glucagon-like peptide 1 in vitro and in vivo by dipeptidyl peptidase IV,” Endocrinology 136(8):3585-96 (1995). Thus DPP-1V resistant GLP-1 analogs are needed for treatment of type 2 diabetes.

[018] Peptides have low toxicity and high specificity and are useful therapeutic agents in clinical applications (Bellmann-Sickert and Beck-Sickinger, “Peptide drugs to target G protein-coupled receptors,” Trends Pharmacol Sci 31(9):434-41 2010). Despite these advantages, therapeutic peptides are highly prone to proteolytic degradation during storage or when used for oral administration and generally require parenteral administration. See, McGregor, “Discovering and improving novel peptide therapeutics,” Curr Opin Pharmacol 8(5):616-9 (2008). For chronic diseases, such as diabetes, repeated injections are often needed that results in decreased patient compliance (Hamman and Steenekamp, 2011). Therefore, there is a great need for the development of improved GLP-1 peptides and less expensive and patient-friendly drug delivery methods that retain low toxicity and high specificity, are resistant to degradation and available for oral administration.

[019] There exists a number of patent publications directed to modified GLP-1 peptides. U.S. Patent Publication No. 2018/0162920, published June 14, 2018, to Revell and Bednarek (the ‘920 Publication), provides protease-resistant, lipidated GLP 1 analogs by the selective and strategic positioning of lipidation and alpha-functionalized amino acid substitution of amino acids in the peptide. More specifically, the ‘920 Publication discloses lipidation of certain lysine or cysteine residues. The engineered GLP-l peptides of the present application do not contain either lysine or cysteine. U.S. Patent No. 7,847,063, published December 7, 2010, to Sugita et al. provides recombinant GLP-1 derivatives. International Patent Publication No. 2020/023388, published January 30, 2020, to Alsina-Femandez et al., describes dosing regimens of GIP/GLP1 co-agonists. U.S. Patent No. 6,620,910, issued September 16, 2003, to Galas et al., provides modified GLP-1 peptides that include lysine and are susceptible to trypsin cleavage.

[020] The present applications provides for, and includes, GLP-1 derivatives having strong binding to the GLP-1 R receptor in humans, cat, and other mammals, and modified to provide substantial resistance to proteolysis from digestive enzymes such as DPP-IV, neprilysin, a-chymotrypsin, trypsin, elastase, or pepsin. The present application also reports the modification of tirzepatide sequences to conserve the dual agonistic properties, replacing the non-natural amino acids (e.g., alpha amino iso butyric acid) naturally occurring, codable amino acids, and making the dual agonist peptides resistant to gut peptidases. The present application provides for, and includes peptides combining the activity for glucagon-like peptide- 1 (GLP-1) with glucose-dependent insulinotropic polypeptide (GIP) to treat diabetes. Together GLP-1 and GIP stimulate insulin secretion and are responsible for the incretin effect. GLP-1R and GIPR are expressed on pancreatic p-cells and activation of these in the context of even modest elevations of blood glucose potently stimulate insulin secretion.

SUMMARY OF THE INVENTION

[0211 hi general, the present application relates to novel engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptides or pharmaceutically acceptable salts, solvates and/or other forms thereof., corresponding pharmaceutical compositions, methods and/or uses of the eGLP-1 polypeptides for treatment of metabolic diseases and/or related disorders. Further, in general, the present application relates to novel engineered dual agonist polypeptides or pharmaceutically acceptable salts, solvates and/or other forms thereof, corresponding pharmaceutical compositions, methods and/or uses of the dual agonist polypeptides for treatment of metabolic diseases and/or related disorders.

[022) The present application provides and includes, in one form thereof, engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of Formula I H G E G T S E S D V S Xn Xu X_l4 E G Q A A Q E X22 X₂₃ A X25 X₂₆ V D G X (I) (SEQ ID NO:1), wherein X12 = S or Q; X_l3 = S, Q, or Y; X_l4 - 1 or L; X22 = V, I, or F; X23 - V or I; X25 - V, I, or W; X₂₆ = I or V; and X₃₀ = R or S.

[023] More particularly, the application includes an engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of any one of SEQ ID NO:2 to Error! Reference source not found.. More particularly, the application includes an engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of Formula III (SEQ ID NO:382) or of any one of SEQ ID NO:372 to SEQ ID NO:380.

[024] In aspects, the present application includes engineered nucleic acid vectors encoding an engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of Formula I (Error! Reference source not found.). Also included are one or more vectors encoding one or more eGLP-1 peptides of Formula I (Error! Reference source not found.). Also included are concatemers of an engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of Formula I (SEQ ID NO:1). In aspects, the engineered nucleic acid vectors encode an engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of any one of Error! Reference source not found, to SEQ ID NO: 14, and combinations thereof. In aspects, the engineered nucleic acid vectors encode an engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of Formula III (SEQ ID NO:382) or of any one of SEQ ID NO:372 to SEQ ID NO:380.

[025] In aspects, the present application includes nucleic acid expression cassettes comprising a promoter for transcriptional expression, a nucleic acid sequence encoding a peptide comprising an eGLP- 1 of Formula I (SEQ ID NO: 1), and translation and transcription termination sequences. In aspects, the translation termination sequences comprise one of more in stop codons. In aspects, translation termination sequences comprise a terminal stop codon and one or more in-frame or out-of-frame stop codons 3* to the terminal stop codon. In aspects, the nucleic acid expression cassettes encode an engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of any one of Error! Reference source not found, to SEQ ID NO: 14, and combinations thereof. In aspects, the nucleic acid expression cassettes encode an engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of any one of Error! Reference source not found, to SEQ ID NO: 14, and combinations thereof. In aspects, the nucleic acid expression cassettes encode an engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of Formula III (SEQ ID NO:382) or of any one of SEQ ID NO:372 to SEQ ID NO:380, and combinations thereof.

[026] The present application further includes, and provides for, methods of making an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide having the amino acid sequence of Formula I (Error! Reference source not found.), the method comprising culturing a host cell transformed with an expression vector encoding an eGLP-1 polypeptide under conditions allowing expression of the eGLP-1 , and recovering the eGLP-1. Also included are methods of making an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide having the amino acid sequence of any one of Error! Reference source not found, to Error! Reference source not found., or combinations thereof, comprising culturing a host cell transformed with an expression vector encoding an eGLP-1 polypeptide under conditions allowing expression of the eGLP-1, and recovering the eGLP-1. Also included are methods of making an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide having the amino acid sequence of Formula III (SEQ ID NO:382) or of any one of SEQ ID NO:372 to SEQ ID NO:380, or combinations thereof, comprising culturing a host cell transformed with an expression vector encoding an eGLP-1 polypeptide under conditions allowing expression of the eGLP-1, and recovering the eGLP-1.

[027] In aspects, the present application provides host cells comprising polynucleotide sequences encoding an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide having the amino acid sequence of Formula I (SEQ ID NO:1). In aspects, host cells comprising polynucleotide sequences encoding an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide having the amino acid sequence of any one of SEQ ID NO: 2 to SEQ ID NO: 14, or combinations thereof. In aspects, host cells comprising polynucleotide sequences encoding an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide having the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO: 14, or combinations thereof. In other aspects, encoding an eGLP-1 polypeptide having the amino acid sequence of Formula III (SEQ ID NO:382) or of any one of SEQ ID NO:372 to SEQ ID NO:380, or combinations thereof.

[028] The present application further includes methods of treating or preventing a disease comprising administering to a subject in need of treatment thereof an effective amount of an engineered Glucagon- Like Peptide 1 (eGLP-1) polypeptide having the amino acid sequence of Formula 1 (SEQ ID NO:1) wherein the disease or disorder is selected from the group consisting of: lipodystrophy, dyslipidemia, hyperlipidemia, overweight, obesity, hypothalamic amenorrhea, Alzheimer’s disease, leptin deficiency, fatty liver disease, diabetes, type 1 diabetes, type II diabetes, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), metabolic syndrome X, metabolic dysfunction-associated steatohepatitis (MASH), and Huntington's Disease.

[029] The present application further includes methods of treating or preventing a disease or condition caused or characterized by hypoglycemia or impaired insulin release, comprising administering to a subject in need of treatment thereof an effective amount of an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide having the amino acid sequence of Formula I (Error! Reference source not found.). In aspects, the methods of treating or preventing a disease or condition caused or characterized by hypoglycemia or impaired insulin release, comprising administering to a subject in need of treatment thereof an effective amount of an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide having the amino acid sequence of any one of SEQ ID NO:2 to Error! Reference source not found., or combinations thereof. In aspects, the eGLP-1 polypeptide comprises the amino acid sequence of SEQ ID NO:3 to SEQ ID NO: 14. In aspects, the eGLP-1 polypeptide comprises or has the sequence of Formula III (SEQ ID NO.-382) or of any one of SEQ ID NO:372 to SEQ ID NO:380, or combinations thereof. [030] The present application further includes methods of treating or preventing a disease or condition caused or characterized by hypoglycemia or impaired insulin release, comprising administering to a subject in need of treatment thereof an effective amount of a host cell transformed to express an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide having the amino acid sequence of Formula I (SEQ ID NO: 1 ). Also included in aspects are methods of treating or preventing a disease or condition caused or characterized by hypoglycemia or impaired insulin release, comprising administering to a subject in need of treatment thereof an effective amount of a host cell transformed to express an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide having the amino acid sequence of SEQ ID NO:2 to SEQ ID NO: 14, and combinations thereof. In aspects, the eGLP-1 polypeptide comprises the amino acid sequence of SEQ ID NO:3 to SEQ ID NO: 14. In aspects, the eGLP-1 polypeptide comprises the amino acid sequence of Formula III (SEQ ID NO:382) or of any one of SEQ ID NO:372 to SEQ ID NO:380.

[031] The present application further includes, and provides for, administering a composition comprising an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide having the amino acid sequence of Formula I (SEQ ID NO: 1) for the treatment or prevention of a disease or condition caused or characterized by hypoglycemia or impaired insulin release. In aspects, the present application includes, and provides for administering a composition comprising an engineered Glucagon-Like Peptide 1 (eGLP- 1) polypeptide having the amino acid sequence of Formula I (SEQ ID NO:1) for glycemic control, promoting insulin production, reducing Ale, promoting B-cell mass, promoting weight loss, or reducing excess body weight. In addition, eGLP-1 polypeptides provided herein can be used for treatment of related disorders. In aspects, eGLP-1 polypeptides having the amino acid sequence of Error! Reference source not found, to SEQ ID NO: 14, or a combination thereof, are administered for glycemic control, promoting insulin production, reducing Ale, promoting B-cell mass, promoting weight loss, or reducing excess body weight, or combinations thereof. In aspects, eGLP-1 polypeptides having the amino acid sequence of Formula III (SEQ ID NO:382) or of any one of SEQ ID NO:372 to SEQ ID NO:380, or a combination thereof, are administered for glycemic control, promoting insulin production, reducing Ale, promoting B-cell mass, promoting weight loss, or reducing excess body weight, or combinations thereof. In an aspect, the composition is a cell expressing an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide having the amino acid sequence of Formula I (Error! Reference source not found.), a polypeptide having the amino acid sequence of SEQ ID NO:2 to SEQ ID NO: 14, or a polypeptide having the amino acid sequence of Formula III (SEQ ID NO:382) or of any of SEQ ID NO:372 to SEQ ID NO:380, or a combination thereof.

[032] The present application further includes, and provides for, administering a composition comprising an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide having the amino acid sequence of Formula I (SEQ ID NO: 1), a polypeptide having the amino acid sequence of SEQ ID NO:2 to SEQ ID NO: 14, or a polypeptide of Formula III (SEQ ID NO:382) or of any one of SEQ ID NO:372 to SEQ ID NO:380, or a combination thereof as a direct fed microbial (DFM) for the treatment or prevention of a condition caused or characterized by excess body weight, and the treatment of obesity, morbid obesity, obesity linked inflammation, obesity linked gallbladder disease, obesity induced sleep apnea, metabolic syndrome, pre-diabetes, insulin resistance, glucose intolerance, type 2 diabetes, type I diabetes, hypertension, atherogenic dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke or microvascular disease in a subject. In aspects, the composition comprising an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide, is a direct fed microbial (DFMs) composition comprising bacteria transformed with a nucleic acid encoding an engineered Glucagon-Like Peptide 1 (eGLP-1) a comprising the amino acid sequence of Formula I (SEQ ID NO:1), a polypeptide having the amino acid sequence of Error! Reference source not found, to Error!

Reference source not found., or of Formula III (SEQ ID NO:382) or of any one of SEQ ID NO:372 to SEQ ID NO:380, or a combination thereof. In aspects, the eGLP-1 polypeptide comprises the amino acid sequence of Error! Reference source not found, to Error! Reference source not found.. In one or more aspects, the composition comprising an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide, is an engineered composition comprising bacteria with a nucleic acid encoding engineered to secrete eGLP-1 comprising the amino acid sequence of Formula I (SEQ ID NO: 1), a polypeptide having the atnino acid sequence of SEQ ID NO:2 to SEQ ID NO: 14, or of Formula in (SEQ ID NO:382) or of any one of SEQ ID NO:372 to SEQ ID NO:380, or a combination thereof. In aspects, the host cell is engineered to secrete eGLP-1 by plasmid-based expression or by chromosomal integration.

[033] The present application provides and includes, in one form thereof, engineered polypeptides, particularly engineered dual agonist polypeptides, comprising the amino acid sequence of Formula II (SEQ ID NO: 111):

Y X₂ E G T Xe X₇ S D X10 S 1 X13 Xu D Xu I A Q X20 A X22 V Q X25 X₂₆1 A G G P S S G A P P (I) wherein X2 = V or K; X₆ =F, P, or S; X₇ = T, C, or E; Xio = Y, C, or E; X13 = A, S, Y, N, I, L, R, V, or K; Xu = L, K, H, or I; X₁₆ = K, R, H, or V; X20 = K, R, H, N; X22 = F, A, P; X25 = W, P, K, H, or I; and X26 = L or V.

[034] The present application further includes, and provides for, engineered polypeptides comprising the amino acid sequence of Formula II (SEQ ID NO:111) wherein X2 = V; Xs = P or S; X₇ = C or E; Xio = C or E; XI₃ = S; XM = L, H, or l; Xi₆ = H or V; X2o = H or N; X₂2 = A or P; X25 - P or I; and X₂6 = L or V.

[035] More particularly, the application includes dual agonist polypeptides comprising the amino acid sequence of any one of Error! Reference source not found, to SEQ ID NO: 147. In aspects, the application includes dual agonist polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO:116. In aspects, the application includes dual agonist polypeptides comprising the amino acid sequence of any one of Error! Reference source not found, to SEQ ID NO: 147. In aspects, the application includes dual agonist polypeptides comprising the amino acid sequence of Formula IV (SEQ ID NO:383) or of SEQ ID NO.381.

[036] The present application further includes, and provides for, an isolated polynucleotide encoding an engineered polypeptide comprising the amino acid sequence of sequence of Formula IT (SEQ ID NO: 111): wherein X2 = V or K; Xe =F, P, or S; X? = T, C, or E; Xio = Y, C, or E; X13 = A, S, Y, N, I, L, R, V, or K; Xu = L, K, H, or I; X_i6 = K, R, H, or V; X20 = K, R, H, N; X22 = F, A, P; X25 » W, P, K, H, or I; and X_M = L or V.

[037] The application includes isolated polynucleotide encoding dual agonist polypeptides comprising the amino acid sequence of any one of Error! Reference source not found, to SEQ ID NO: 147, of any one of SEQ ID NO: 113 to SEQ ID NO: 116, of any one of Error! Reference source not found, to SEQ ID NO:147, or of Formula IV (SEQ ID NO:383) or of SEQ ID NO:381.

[038] The present application further includes, and provides for, nucleic acid expression cassettes comprising one or more of: a nucleic acid sequence comprising a transcription promoter, a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of Formula II (SEQ ID NO: 111) wherein X₂ - V or K; Xs =F, P, or S; X₇ - T, C, or E; X_I0 = Y, C, or E; X₁₃ = A, S, Y, N, I, L, R, V, or K; X14 = L, K, H, or I; Xu = K, R, H, or V; Xzo = K, R, H, N; X22 = F, A, P; X25 - W, P, K, H, or I; and X26 = L or V; a nucleic acid sequence comprising a translation terminator; and a nucleic acid sequence comprising transcription terminator. In aspects the nucleic acid sequence encodes dual agonist polypeptides comprising the amino acid sequence of any one of Error! Reference source not found, to SEQ ID NO:147, of any one of SEQ ID NO: 113 to SEQ ID NO:116, of any one of Error! Reference source not found, to SEQ ID NO:147, or of Formula IV (SEQ ID NO:383) or of SEQ ID NO:381. [039] The present application further includes, and provides for, methods of making an engineered polypeptide comprising the amino acid sequence of Formula II (SEQ ID NO: 111) Y X2 E G T Xe X? S D X10 S I X13 XM D Xie I A Q X20 A X22 V Q X25 X26 I A G G P S S G A P P (I), wherein Xz = V or K; Xe =F, P, or S; X₇ = T, C, or E; X_w = Y, C, or E; X₁₃ = A, S, Y, N, I, L, R, V, or K; Xu = L, K, H, or I; Xu = K, R, H, or V; X20 = K, R, H, N; X22 = F, A, P; X25 = W, P, K, H, or I; and X₂₆ = L or V; the method comprising culturing a host cell transformed with an expression vector encoding an engineered polypeptide under conditions allowing expression of the engineered polypeptide, and recovering the engineered polypeptide.

[040] The present application further includes, and provides for, pharmaceutical compositions comprising an engineered polypeptide comprising the amino acid sequence of Formula IT (SEQ ID NO: 111) and a carrier.

[041] The present application further includes, and provides for, a pharmaceutical composition comprising a recombinant host cell comprising a polynucleotide encoding an engineered polypeptide of Formula II (SEQ ID NO: 1 11).

[042] The present application further includes, and provides for, host cells comprising the polynucleotide encoding an engineered polypeptide comprising the amino acid sequence of Formula II (SEQ ID NO:111): Y X₂ E G T X« X₇ S D X_I0 S I Xi₃ Xu D Xi₆ 1 A Q X2o A X22 V Q X25 X₂₆ 1 A G G P S S G A P P (I), wherein X₂ = V or K; Xe =F, P, or S; X₇ = T, C, or E; X_IO = Y, C, or E; X_I3 = A, S, Y, N, I, L, R, V, or K.; Xu = L, K, H, or I; Xu = K, R, PI, or V; X₂₀ = K, R, H, N; X22 = F, A, P; X25 = W, P, K» H, or I; and X26 = L or V.

[043] The present application further includes, and provides for, host cells comprising a vector comprising a polynucleotide encoding an engineered polypeptide comprising the amino acid sequence of Formula H (SEQ ID NO: 111): Y X2 E G T Xe X₇ S D X,o S I X_l3 X_I4 D X_I61 A Q X20 A X22 V Q X2J X₂₆ I A G G P S S G A P P (I), wherein X₂ = V or K; Xt =F, P, or S; X₇ - T, C, or E; X₁₀ = Y, C, or E; X_!3 = A, S, Y, N, I, L, R, V, or K; Xu = L, K, H, or I; Xu = K, R, H, or V; X20 = K, R, H, N; X22 - F, A, P; X25 = W, P, K, H, or I; and X₂₆ = L or V. [044] The present application further includes, and provides for, transformed cells comprising a nucleic acid expression cassette comprising one or more of: a nucleic acid sequence comprising a transcription promoter; a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of Formula JI (SEQ ID NO: 111): Y Xi E G T Xs X₇ S D Xio S I X.₃ Xu D Xi< I A Q X_a A X22 V Q X25 X26 I A G G P S S G A P P (I), wherein X₂ = V or K; Xs =F, P, or S; X₇ = T, C, or E; Xio = Y, C, or E; Xn = A, S, Y, N, I, L, R, V, or K; XM = L, K, H, or I; X.s » K, R, H, or V; X20 = K, R, H, N; X₂₂ = F, A, P; X_2$ = W, P, K, H, or I; and X₂s = L or V; a nucleic acid sequence comprising a translation terminator, and a nucleic acid sequence comprising transcription terminator.

[045] The present application further includes, and provides for, genetically engineered plants, or part thereof, comprising a recombinant nucleic acid encoding an engineered polypeptide of Formula II (SEQ ID NO:111) Y X₂ E G T Xs X₇ S D X10 S I X13 Xu D Xis I A Q X20 A X22 V Q X25 X₂s I A G G P S S G A P P (I), wherein X₂ = V or K; X₆ -F, P, or S; X₇ = T, C, or E; X_l0 = Y, C, or E; X_B - A, S, Y, N, I, L, R, V, or K; X_M = L, K, H, or I; X₎₆ = K, R, H, or V; X20 = K, R, H, N; Xy = F, A, P; Xy - W, P, K, H, or I; and X₂s = L or V.

[046] The present application further includes, and provides for, methods of treating or preventing a treatment of a condition caused or characterized by excess body weight, and the treatment of obesity, morbid obesity, obesity linked inflammation, obesity linked gallbladder disease, obesity induced sleep apnea, metabolic syndrome, pre-diabetes, insulin resistance, glucose intolerance, type 2 diabetes, type I diabetes, hypertension, atherogenic dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke or microvascular disease in a subject, comprising administering to a subject in need of treatment thereof an effective amount of the engineered polypeptides, the pharmaceutical compositions, or the cells provided herein.

[047] The present application further includes, and provides for, methods of treating or preventing a disease or condition caused or characterized by hypoglycemia or impaired insulin release, comprising administering to a subject in need of treatment thereof an effective amount of the engineered polypeptides, the pharmaceutical compositions, or the cells described herein.

[048] The present application further includes, and provides for, a pharmaceutical composition comprising an engineered polypeptide comprising an amino acid sequence Formula II (SEQ ID NO: 111).

[049] The present application further includes, and provides for, pharmaceutical compositions comprising an engineered polypeptide comprising an amino acid sequence selected from any of SEQ ID NO: 112 to SEQ ID NO: 148. In aspects, the pharmaceutical composition includes dual agonist polypeptides comprising the amino acid sequence of any one of SEQ ID NO:113 to SEQ ID NO:116. [050] The present application further includes, and provides for, direct fed microbials (DFM) comprising bacteria transformed with a nucleic acid encoding an engineered polypeptide comprising the amino acid sequence of Formula II (SEQ ID NO: 111): Y Xz E G T X» X₇ S D Xto S I X_l3 X₁₄ D X₁₆1 A Q X20 A X22 V Q X25 X26 I A G G P S S G A P P (I), wherein X2 - V or K; X6 =F, P, or S; X₇ - T, C. or E; X10 = Y, C, or E; X_l3 = A, S, Y, N, I, L, R, V, or K; X₁₄ = L, K, H, or I; X,₆ = K, R, H, or V; X20 = K, R, H, N; X22 = F, A, P; X25 = W, P, K, H, or I; and Xze = L or V.

BRIEF DESCRIPTION OF THE DRAWINGS

[051] The present inventions are disclosed with reference to the accompanying drawings, wherein: [052] Figure 1 is a graphical representation of the proteolytic cleavage sites and enzymes in wild-type GLP-1 (Error! Reference source not found.).

[053] Figure 2 is a graphical representation of a comparison of the ECD conformation between the GLP-1 -bound to GLP-1 R. As shown, the C-terminal region of GLP-1 agonist interacts with extracellular domain whereas the N-terminal region of GLP-1 interacts with transmembrane of the GLP-1R receptor. From Cong et al., 2021, Figure 3a.

[054] Figure 3 is a graphical representation of the reorganization of the polar network of GLP-1 R by GLP-1 binding. In activated GLP-1 R receptor, three layers of the polar network are reorganized as (i) central polar network, (ii) HETX motif polar network, (iii) TM2-6-7-helix 8 polar network. From Cong et aL, 2021, Figure 4.

[055] Figure 4 is a graphical representation of the superimposed structures of reported peptides bound to the GLP-1R (A) and GIPR (B) receptors. (A) presents the superimposed GLP-1R receptors and various colours represent the different complexes with reported peptides namely, Tirzepatide (7FIM-red), GLP1 (6X18-pink, 6VCB-blue), Exendin-P5 (6B3J-green), truncated peptide agonist (5NX2-yellow), Exendin-4(7LLL-cyan), pro-glucagon (7LLY-white), peptide-20 (7VBH-grey), Non-acylated Tirzepatide (7VBI-brown), Taspoglutide (7KI1 -purple) and Semaglutide (7KI0-deep teal). (B) Reported peptides bound GIPR complexes and its structural superimposition where Non acylated Tirzepatide (7VAB-red), Tirzepatide (7RBT-bIue, 7FIY-grey), GIF (7RA3-white, 7DTY-cyan) and peptide-20 (7FIN-yellow).

[056] Figure 5 is a graphical representation oof Cryo-EM structures of hGLPl and HGIP bound to hGLP-lR. Cryo-EM structures of hGLPl and hGIP bound hGLP-lR, hGIPR in complex are represented in A and C, respectively the modelled hGLPl and hGIP bound hGLP-lR and hGIPR complexes are shown in B and D.

[057] Figure 6 is an alignment of cat and human GLP-1 R receptors. Boxes represent the binding pocket residues and differences in residues in cat and human binding pocket. Boxes represent the conserved binding pocket residues. Binding pocket residues P91, W92 L 145, Y146, and K198 in cGIPR (numbering based on SEQ ID NO; 221). [058] Figure 7 is a graphical representation of GLP-1 activity according to an aspect of the present specification.

[059] Figure 8 is a sequence alignment of reported and engineered GLP-1 analogs.

[060] Figure 9 presents the sequence of GLP-1 (Error! Reference source not found.) and GLP-1 -Gly8 (Error! Reference source not found.) modified to remove the DPP-4 protease cleavage site.

[061] Figure 10 is a cross species alignment of the GIPR receptor of human (SEQ ID NO:220) cat (SEQ ID NO:221), rat (SEQ ID NO: 222) and mouse (SEQ ID NO:223). Asterisks indicate conserved binding pocket residues.

[062] Figure 11 is a graphical representation of GLP-1 activity according to an aspect of the present specification. hGLP-1 peptide (green) docked against hGLP-lR receptor (yellow) and superimposed on the selected template 6X18 hGLP-1 (blue)/hGLP-l R(red).

[063] Figure 12 presents Tirzepatide peptide (green) docked against hGLP-1 R receptor (white) and superimposed on the selected template 7FIM Tirzepatide(blue)/hGLP-lR(red).

[064] Figure 13 presents a homology modelled cGIP structure superimposed on the hGIP Cryo-Em structure. Figure 13 A presents an alignment of the hGIP and cGIP sequences (row 1 and 2), related peptides in the art, and peptides of the present application. Figure 13 B presents a graphical representation presenting the single residue difference at position 18 (boxed in Figure 13 A). cGIP is shown in red and hGIP in blue. hGIP residue 18His is represented in yellow colour and cGIP 18 Arg in green.

[065] Figure 14: (A). cGLP-l(yellow) peptide was docked against cGLP-lR receptor(white) and superimposed on the template (6X18) selected for the hGLP-l(green)/hGLP-lR(blue) complex. (B). Different binding pose residues between human and cat were shown in cGLP-lR (magenta) and hGLP-lR (Cyan).

[066] Figure 15 (A) Residues (Leu393, Leu397, Glu396 and Thr400) around AIB2 of Tirzepatide against hGLP-lR and (B) residues (Leu375, Lys374, Ala359 and Glu355) around AIB2 against cGLP- 1R.

[067] Figure 16 Binding poses analysis of tirzepatide analogues after docking against cGLP-lR represent the difference in binding poses. (A,B) peptide-4 and 8 (cyan) mutation in tirzepatide(blue) shows that N-tenninal of the peptides were losing their helicity, while (C) peptide-6(cyan) is moving away from the binding region.

[068] Figure 17 is a graphical representation of GIP activity according to an aspect of the present specification.

[069] Figure 18 is a graph of the effects of the test materials (various peptides or L reuteri expressing peptides) on weekly body weight at Day 40. [070] Figure 19 depicts the effect of BEP009 on fat content in an EchoMRI test on Day 39 of a DIO study in mice. Vehicle is compared to liraglutide BEP009, LR3632 chassis, and the engineered strains BE105ENLR139 (IX GLP1 Cl.l BEP-009 sequence) and BE 105ENLR181 (Sx GLPl C1.1 BEP-009 sequence) were evaluated, with liraglutide as a control.

[071] Figure 20 depicts the effect of the GLP-1 peptides and constructs including BEP009-ABD on weekly body weight -- Day 82. Vehicle is compared to liraglutide, BEP009, LR3632 chassis, and the engineered strain BE105ENLR181 (5x GLP1 Cl.l BEP-009 sequence), with liraglutide as a control. [072] Figure 21 depicts the effect or test materials and peptides on weekly blood glucose in the DIO model up to Day 78. Vehicle is compared to liraglutide, BEP009, BEP-009-ABD fusion, LR3632 chassis, and the engineered strain BE105ENLR181 (5x GLP1 Cl.l BEP-009 sequence), with liraglutide as a control.

[073] Figure 22 depicts the effect on fasting glucose/AUC was evaluated in an OGTT test at Day 75. Vehicle is compared to liraglutide, BEP009, BEP-009-ABD fusion, LR3632 chassis, and the engineered strain BE105ENLR181 (5x GLP1 Cl.l BEP-009 sequence), with liraglutide as a control.

[074] Figure 23 depicts the effect on fat content was assessed in an EchoMRI at Day 81. Vehicle is compared to liraglutide, BEP009, BEP-009-ABD fusion, LR3632 chassis, and the engineered strain BE1O5ENLR181 (5x GLP1 Cl.l BEP-009 sequence), with liraglutide as a control.

[075] Figure 24 depicts the effect on lean mass in an EchoMRI at Day 81. Vehicle is compared to liraglutide, BEP009, BEP-009-ABD fusion, LR3632 chassis, and the engineered strain BE105ENLR181 (5x GLP1 Cl.l BEP-009 sequence), with liraglutide as a control. The change in lean mass (%) is graphed. [076] Figure 25 depicts the effect of the test materials and peptides on biweekly food intake (normalized to vehicle control) at Day 84. The % change in food intake (Day 43 - Day 48) is graphed.

[077] Figure 26 depicts a GLP- 1R cellular assay of various eGLP- 1 polypeptides and dual agonist polypeptides as well as a eGLP-1 - ABD fusion peptide peptides. GLP-1, tirzepatide and liraglutide were used as positive controls for GLP-1R activation.

[078] Corresponding reference characters indicate corresponding parts throughout the several views. The examples set out herein illustrate several embodiments of the invention but should not be construed as limiting the scope of the invention in any manner.

DETAILED DESCRIPTION

Deflmtions:

[079] As used herein the term “about’' refers to ± 10 %.

[080] The terms “comprises", “comprising”, “includes”, “including”, “having" and their conjugates mean “including but not limited to". It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of and/or "consisting essentially of* are also provided.

[081 ] The term “consisting of’ means “including and limited to”.

[082] The term “consisting essentially of’ means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[083] As used herein, the singular form “a”, “an" and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.

[084] Throughout this application, various embodiments of this disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[085] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between" a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[086] As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. [087] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[088] “Treatment” or “treat" or “treating" as used herein refers to an approach for obtaining beneficial or desired results. For purposes of the present application, beneficial or desired results include, but are not limited, to, alleviation of a symptom and/or diminishment of the extent of a symptom and/or preventing a worsening of a symptom associated with a disease or condition. In one aspect, “treatment” or “treating” includes one or more of the following: (a) inhibiting the disease or condition (e.g., decreasing one or more symptoms resulting from the disease or condition, and/or diminishing the extent of the disease or condition); (b) slowing or arresting the development of one or more symptoms associated with the disease or condition (e.g., stabilizing the disease or condition, delaying the worsening or progression of the disease or condition); and (c) relieving the disease or condition, e.g., causing the regression of clinical symptoms, ameliorating the disease state, delaying the progression of the disease, increasing the quality of life, and/or prolonging survival.

[089] “Therapeutically effective amount” or “effective amount” as used herein refers to an amount that is effective to elicit the desired biological or medical response, including the amount of a compound that, when administered to a subject for treating a disease, is sufficient to affect such treatment for the disease. The effective amount will vary depending on the compound, the disease, and its severity and the age, weight, etc., of the subject to be treated. The effective amount can include a range of amounts. As is understood in the art, an effective amount may be in one or more doses, i.e., a single dose or multiple doses may be required to achieve the desired treatment endpoint. An effective amount may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable or beneficial result may be or is achieved. Suitable doses of any co-administered compounds may optionally be lowered due to the combined action (e.g., additive or synergistic effects) of the compounds.

[090] “Fatty acid” as used herein is an unbranched alkanoic acid of at least six carbons, for example, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, or more carbons, in length. The fatty acid can contain 1, 2, 3, or more carboxylic acid groups. The fatty acid can include other functional groups, such as but not limited to, amides and phenyl rings. Exemplary fatty acids include hexanoic acid, octanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, 1,6- hexanedioic acid, 1,8-octanedioic acid, 1,10-decanedioic acid, 1,12-dodecanedioic acid, 1,14- tetradecanedioic acid, 1,16-hexadecanedioic acid, and 1,18-octadecanedioic acid.

[091] The term “subject” is meant any subject, particularly a mammalian subject, in need of treatment with a peptide or polypeptide provided herein. Mammalian subjects include, but are not limited to, humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, cows, apes, monkeys, orangutans, and chimpanzees, and so on. In one aspect, the subject is a human subject. In an aspect, the subject is a domesticated animal. In an aspect the subject is a cat or a dog.

[092] As described herein “alpha-methyl functionalized amino acids” refer to amino acids in which the first (alpha) carbon atom of the amino acid includes a methyl group (CH3) substituent bound to the alpha carbon. Alpha-methyl functionalized amino acids include any of the naturally occurring twenty amino acids that include such a functionalization. As described throughout, alpha-methyl functionalized amino acids can replace any native amino acid in a peptide. The term "native" amino acid refers to one of the standard 20 amino acids that exist in biologically generated proteins.

Peptides

[093] In general, the present application relates to novel engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptides or pharmaceutically acceptable salts, solvates and/or other forms thereof, corresponding pharmaceutical compositions, methods and/or uses of the eGLP-1 polypeptides for treatment of diabetes, obesity, and other metabolic disorders.

[094] In particular, the present application relates to a compound of Formula (I) or pharmaceutically acceptable salts, solvates and/or other forms thereof, corresponding pharmaceutical compositions, methods and/or uses for treatment of diabetes, obesity, and other metabolic disorders. More specifically, the present application relates to polypeptides having the amino acid sequences of SEQ ID NO:1 to SEQ ID NO:14. In aspects, the eGLP-1 polypeptide comprises the amino acid sequence of SEQ ID NO:3 to SEQ ID NO: 14. In aspects, the eGLP-1 polypeptide comprises the amino acid sequence of Formula III(SEQ ID NO:382) or of SEQ ID NO:372 to SEQ ID NO:380.

Table 1: Engineered GLP-1 sequences

095] Also included and provided for by the present applications are nucleic acid molecules encoding polypeptides having the amino acid sequences of Error! Reference source not found, to SEQ ID NO: 14, transformed cells having nucleic acid molecules encoding polypeptides having the amino acid sequences of SEQ ID NO:2 to SEQ ID NO: 14. [096] The present disclosure provides for, and includes, an engineered Glucagon-Like Peptide 1 (eGLP- 1) polypeptides comprising the amino acid sequence of Formula I (SEQ ID NO: 1)

H G E G T S E S D V S Xi₂ Xi3Xi4E G Q A A Q E X22 X23A X25 X ₂6V D G X3o (l) wherein X,₂ = S or Q, X|₃ = S, Q, or Y, X₎₄ = I or L, X22 = V, I, or F, X23 = V or 1, Xis = V, I, or W, X26 = I or V, and X30 = R or S. As used throughout the present application, the eGLP-1 polypeptides comprise a thirty (30) amino acid sequence where the subscripts refer to the amino acid in order from the amino terminus to the carboxy terminus. As provided herein and discussed in detail below, the polypeptides can be concatemerized to comprise two, three, four, five or more repeats of the core dGLP-1 sequence of Formula 1. Also included and provided for by the present specification are the addition of amino acids at either the amino or carboxy terminus. In aspects, the carboxy terminus comprises an amide. [097] In an aspect, the eGLP-1 polypeptide comprises the amino acid sequence of Formula I wherein XI₂ = Q, XB - S, Xi4 - I or L, X₂2 = V, I, or F, X23 - V or I, X25 = V, I, or W, X26 = I or V, and X3o = R (SEQ ID NO:1). In an aspect, the eGLP-1 polypeptide comprises the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO: 14. In aspects, the eGLP-1 polypeptide comprises the amino acid sequence of SEQ ID NO:3 to SEQ ID NO: 14. In aspects, the eGLP-1 polypeptide comprises the amino acid sequence of Formula III (SEQ ID NO:382) or of any one of SEQ ID NO:372 to SEQ ID NO:380 [098] As provided herein, the eGLP-1 polypeptides are substantially resistant to proteolytic degradation having been modified to eliminate protease recognition sites for the common proteolytic enzyme dipeptidyl peptidase 4 (DPP-IV or DPP-4, Gene 10:1803), neprilysin (membrane metalloendopeptidase (MME), Gene ID:4311, see also Gene ID:79258), the serine proteases a-chymotrypsin, trypsin, elastase, or acid proteases of the pepsin family.

[099] In general, the present application further relates to novel engineered dual agonist polypeptides or pharmaceutically acceptable salts, solvates and/or other forms thereof, corresponding pharmaceutical compositions, methods and/or uses of the dual agonist polypeptides for treatment of diabetes, obesity, and other metabolic disorders. As used herein, “dual agonist polypeptides” and “engineered polypeptides” are used interchangeably and refer to the polypeptides of Formula II (SEQ ID NO: 111). More specifically “dual agonist polypeptides" and “engineered polypeptides” are polypeptides of SEQ ID NO: 112 to SEQ ID NO: 147 or multimerized polypeptides thereof including SEQ ID NO: 164 to SEQ ID NO: 207. In aspects, the application includes dual agonist polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116 or multimerized polypeptides thereof including SEQ ID NO: 164 to SEQ ID NO: 207. Also included are dual agonist polypeptides comprising the amino acid sequence of Formula IV (SEQ ID NO:383) or of SEQ ID NO:381. Generally, the dual agonist polypeptides relate to tirzepatide (LY3298176, SEQ ID NO: 208), a GIF analogue and a unimolecular, bifunctional (GT and GLP-1 receptor) long-acting agonist Tirzepatide shares a maximum sequence identity of 95% to the dual agonist polypeptides of the present application.

[100 J In particular, the present application relates to a compound of Formula (I) or pharmaceutically acceptable salts, solvates and/or other forms thereof, corresponding pharmaceutical compositions, methods and/or uses for treatment of diabetes, obesity, and other metabolic disorders.

Y X2 E G T X5 X7 S D X10 S I X13 X14 D X16 I A Q X20 A X22 V Q W L I A G G P S S G A P P Formula (II) (SEQ ID NO: 111) wherein X₂ = V or K, Xe =F, P, or S, X₇ = T, C, or E, X10 = Y, C, or E, X13 = A, S, Y, N, I, L, R, V, or K, X14 = L, K, H, or I, Xi₆ = K, R, H, or V, X20 = K, R, H, N, X22 » F, A, P, X₂₅ = W, P, K, H, or I, and X26 = L or V.

[101] As used throughout the present application, the dual agonist polypeptides comprise a thirty (37) amino acid sequence where the subscripts refer to the amino acid in order from the amino terminus to the carboxy terminus. As provided herein and discussed in detail below, the polypeptides can be concatemerized to comprise two, three, four, five or more repeats of the core dual agonist polypeptide sequence of Formula II. Also included and provided for by the present specification are the addition of amino acids at either the amino or carboxy terminus. In aspects, the carboxy terminus comprises an amide.

[102] In aspects, the dual agonist polypeptides comprise a polypeptide having the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147. In aspects, the dual agonist polypeptides comprise a polypeptide having the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. The dual agonist polypeptides and their sequences of the present application are presented in Table 6. In other aspects, the dual agonist polypeptides comprise a polypeptide having the amino acid sequence of Formula IV (SEQ ID NO:383) or of SEQ ID NO:381.

Table 6: Dual Agonist Polypeptides

7FIM_V2/A13 YVEGTFTSDYSIALDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 112 7FIM_V2/S13 YVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 113 7FIM_V2/Y13 YVEGTFTSDYSIYLDKIAQKAFVQVVLIAGGPSSGAPP SEQ ID NO: 114 7FIM_V2/I13 YVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: U5 7FIM_ K_R YVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPS SEQ ID NO: 116 7FIMJ/2/Q13 YVEGTFTSDYSIQLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 117 7FIM.V2/L13 YVEGTFTSDYSILLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 118 7FIM_V2/R13 YVEGTFTSDYSIRLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 119 7FIM_K2/V13 YKEGTFTSDYSIVLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 120 7FIMJC2/K13 YKEGTFTSDYSIKLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 121 7FIM_R_H YVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPS SEQ ID NO: 122 Cl _ ‘ YVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPS SEQ ID NO: 123

Cl.l YVEGTPTSDCSISLDH I AQH APVQWLIAGGPSSGAPPPS SEQ ID NO: 124 C1.2 YVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPS SEQ ID NO: 125 C1.3 YVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO: 126

103] In aspects, the dual agonist polypeptide comprises a polypeptide of Formula (II), wherein Xz = V, X₆ --S, X₇ = C, X_l0 = C or E, X_l3 = S, X_M = I, X_l6 = V, X20 = H or N, X22 = A, X25 = I, and X26 = V.

[104] Also included are dual agonist polypeptides further comprising proline at the carboxy terminus (position 38). In a further aspect, the dual agonist polypeptides further comprise at the carboxy terminus two additional amino acids proline and serine (PS) at the carboxy terminus. The presence of the terminal proline and serine residues at the carboxy terminus is shared with other therapeutically active dual agonist polypeptides, tirzepatide (SEQ ID NO: 208), Exendin-4 (exenatide, SEQ ID NO: 212), Peptide_19 (SEQ ID NO: 210), and Peptide_20 (SEQ ID NO: 209) known in the art. In aspects, the dual agonist polypeptides comprise the amino acid sequence of any one of SEQ ID NO: 123 to SEQ ID NO: 147. In other aspects, the dual agonist polypeptides comprise a carboxy terminal amide.

[105] In aspects, the dual agonist polypeptide, pharmaceutically acceptable salts, solvates, or other forms, and corresponding pharmaceutical compositions comprises the amino acid sequence of any one of SEQ ID NO: 1 1 1 to SEQ ID NO: 147. In aspects, the dual agonist polypeptide, pharmaceutically acceptable salts, solvates, or other forms, and corresponding pharmaceutical compositions comprises the amino acid sequence of any one of SEQ ID NO: 1 13 to SEQ ID NO: 116. In certain aspects, the polypeptide, pharmaceutically acceptable salts, solvates, or other forms, and corresponding pharmaceutical compositions comprises an amino acid sequence of any one of SEQ ID NO: 145 to SEQ ID NO: 147. In other aspects, the polypeptide, pharmaceutically acceptable salts, solvates, or other forms, and corresponding pharmaceutical compositions comprises an amino acid sequence of any one of SEQ ID NO: 142 to SEQ ID NO: 144. In yet other aspects, the polypeptide, pharmaceutically acceptable salts, solvates, or other forms, and corresponding pharmaceutical compositions comprises an amino acid sequence of any one of SEQ ID NO: 137 to SEQ ID NO: 141 . In further aspects, the polypeptide, pharmaceutically acceptable salts, solvates, or other forms, and corresponding pharmaceutical compositions comprises an amino acid sequence of any one of SEQ ID NO: 127 to SEQ ID NO: 136. In aspects, the polypeptide, pharmaceutically acceptable salts, solvates, or other forms, and corresponding pharmaceutical compositions comprises an amino acid sequence of any one of SEQ ID NO: 123 to 127.

[106] As provided herein, in certain aspects the dual agonist polypeptides are resistant to proteolytic degradation having been modified to eliminate one or more of the protease recognition sites for the common proteolytic enzyme dipeptidyl peptidase 4 (DPP-IV or DPP-4, Gene ID: 1803), neprilysin (membrane metalloendopeptidase (MME), Gene ID:4311, see also Gene ID:79258), the serine proteases a-chymotrypsin, trypsin, elastase, or acid proteases of the pepsin family. In aspects, the protease resistant dual agonist polypeptides comprise the polypeptides presented in Table 10, Table 11, and Table 12. In other aspects, the dual agonist polypeptides comprise protease resistant polypeptides further modified to improve stability including the dual agonist polypeptides presented in Table 13.

[107] As used herein, the term “substantially resistant” means a peptide having a lower level of degradation compared to a wild-type, unmodified GLP-1 peptide (wt-GLP-1), or alternatively having a lower level of degradation compared to tirzepatide. In aspects, the eGLP-1 polypeptides maintain substantially the same receptor selectivity as the corresponding wt-GLP-1 polypeptide. In certain aspects, the eGLP-1 polypeptides provided herein exhibit increased receptor potency over the wt-GLP-1 polypeptide. The eGLP-1 polypeptides that are "substantially resistant" to proteolytic degradation can, for example, remain at least about 50% intact following exposure to an enzyme in conditions that the enzyme is generally active (e.g., suitable pH, temperature, other environmental conditions) for a defined period of time. The eGLP- 1 polypeptides provided herein can be substantially resistant to proteolytic degradation for a period of at least 4 hours, at least 8 hours, at least 12 hours, or at least 24 hours. In aspects, the dual agonist polypeptides maintain substantially the same receptor selectivity as the corresponding tirzepatide polypeptide. In certain aspects, the dual agonist polypeptides provided herein exhibit increased receptor potency over the tirzepatide polypeptide. The dual agonist polypeptides that are “substantially resistant" to proteolytic degradation can, for example, remain at least about 50% intact following exposure to an enzyme in conditions that the enzyme is generally active (e.g., suitable pH, temperature, other environmental conditions) for a defined period of time. The dual agonist polypeptides provided herein can be substantially resistant to proteolytic degradation for a period of at least 4 hours, at least 8 hours, at least 12 hours, or at least 24 hours. [1081 Iⁿ certain aspects, at least 60% of the eGLP-1 polypeptide remains intact following exposure to an enzyme in conditions that the enzyme is generally active for a defined period of time. In other aspects, the eGLP-1 polypeptide remains intact at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least about 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%, following exposure to an enzyme in conditions that the enzyme is generally active for a 24 hour period. In other aspects, the eGLP-1 polypeptides provided herein remain at least 60% intact for a period of at least 4 hours, at least 8 hours, at least 12 hours, or at least 24 hours.

[109] In certain aspects, at least 60% of the dual agonist polypeptide remains intact following exposure to an enzyme in conditions that the enzyme is generally active for a defined period of time. In other aspects, the dual agonist polypeptide remains intact at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least about 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%, following exposure to an enzyme in conditions that the enzyme is generally active for a 24 hour period. In other aspects, the dual agonist polypeptides provided herein remain at least 60% intact for a period of at least 4 hours, at least 8 hours, at least 12 hours, or at least 24 hours.

Multimers

[110] Multimerization of peptides has been shown to provide enhanced activity and better in vivo stability. See Sheard et al., “Peptide Multimerization as Leads for Therapeutic Development,” Biologies 2(1): 15-33 (2022). As reported by Lin et al., multimerized GLP-1 analogues can be expressed as either secreted or surface of bacteria. See Lin et al., “Oral Delivery of Pentameric Glucagon-Like Peptide-1 by Recombinant Lactobacillus in Diabetic Rats,” PLoS ONE I l(9):e0162733 (2016). Multimers including a terminal trypsin sensitive amino acid can be digested by intestinal trypsin to produce active, monomeric GLP-1 analogues. Id.

[111] The present application also provides for, and includes, multimers of the eGLP-1 polypeptides provided herein. Multimers include multiple copies of the eGLP-1 polypeptides up to pentamers (e.g., 5x copies). Midtimers of the eGLP-1 polypeptides of Table 2 are presented in Error! Reference source not found. ID NO:32 to SEQ ID NO:67. Also included are multimeric eGLP-1 polypeptides provided herein that are separated by linkers, such as those discussed below. Multimeric eGLP-1 polypeptides can be expressed in host cells using appropriate nucleic acid vectors using methods known in the art. In aspects, the multimerized eGLP-1 polypeptides can be purified from the host cells for therapeutic use. In other aspects, the multimerized eGLP-1 polypeptides can be provided as oral compositions of transgenic host cells expressing the multimers as described by Lin et al. Notably, the eGLP-1 polypeptides of SEQ ID NO:32 to SEQ ID NO:67 include a trypsin sensitive site at the carboxy terminus of each internal peptide of the multimer. Accordingly, it is believe that upon exposure to trypsin, the eGLP-1 polypeptide monomers of SEQ ID NO:3 to Error! Reference source not found, are released. Multimers of the eGLP-1 polypeptide of Formula III (SEQ ID NO:382) or of any of SEQ ID NO:372 to SEQ ID NO:380 are also contemplated and provided in aspects hereof.

[112] The present application also provides for, and includes, multimers of the dual agonist polypeptides provided herein. Multimers include multiple copies of the dual agonist polypeptides up to pentamers (e.g., 5x copies). Multimers of the dual agonist polypeptides of Table 6 are presented in SEQ ID NO: 148 to SEQ ID NO: 207. In aspects, the multimers of the dual agonist polypeptides of Table 6 are presented as SEQ ID NO: 164 to SEQ ID NO: 167. Also included are multimeric dual agonist polypeptides provided herein that are separated by linkers, such as those discussed below. Multimeric dual agonist polypeptides can be expressed in host cells using appropriate nucleic acid vectors using methods known in the art. In aspects, the multimerized dual agonist polypeptides can be purified from the host cells for therapeutic use. In other aspects, the multimerized dual agonist polypeptides can be provided as oral compositions of transgenic host cells expressing the multimers as described by Lin et al. Notably, the dual agonist polypeptides of SEQ ID NO: 148 to SEQ ID NO: 207 include a trypsin sensitive site at the carboxy terminus of each internal peptide. Accordingly, it is believed that upon exposure to trypsin, the dual agonist polypeptide monomers comprising the amino acid sequences of SEQ ID NO: 111 to SEQ ID NO: 147 are released.

Linkers end Fusions

[113] Certain eGLP-1 polypeptides provided herein are fusion proteins comprising an eGLP-1 polypeptide according to Formula I or the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO: 14, and the multimeric eGLP-1 polypeptides of SEQ ID NO:32 to SEQ ID NO:67 and one or more additional domains or amino acid sequences. In aspects, the additional domains or amino acid sequences include, but are not limited to one or more of a linker, an expressed peptide tag for purification, a hinge, or an Fc domain. Linkers, peptide tags, hinges, and Fc domains are well-known to those of ordinary skill in the art and can be incorporated into eGLP-1 polypeptides as described herein, and tested for potency, activity, and efficacy in treating hypoglycemic conditions, e.g., type-2 diabetes without undue experimentation according to the methods provided herein. As provided herein, the linkers, peptide tags, hinges, and Fc linkers can be added at either the N-terminal or C-terminal, or both, of the eGLP-1 polypeptides of the present application.

[114] In some aspects, the eGLP-1 polypeptide(s) may be linked directly or through a linker sequence, including one or more of the linkers or linker sequences described herein, to an additional domain or peptide sequence. In some aspects, the dual agonist polypeptide(s) may be linked directly or through a linker sequence, including one or more of the linkers or linker sequences described herein, to an additional domain or peptide sequence. In aspects the additional domain or peptide sequence may serve or have capability to stabilize, increase the half-life, protect e.g. from degradation or processing or clearance, or direct the engineered polypeptides to a particular location, cell type, tissue, organ or part of the body, such as through binding to a receptor or ligand. In aspects hereof the additional domain or peptide sequence may be an albumin binding domain. Exemplary peptide-ABD fusion polypeptides are provided, described and assessed in the examples herein. In addition or in another aspect, a transferrin receptor antibody or peptide may be linked in a fusion polypeptide. This addition aims to enhance bioavailability in the brain and can be linked including via the flexible linkers described herein. Transferrin receptors are also present in the gut, which mediates or facilitates the systemic translocation of the secreted peptide from the gut, including as described in Rue et al (Rue, L et al (2023) Pharmaceutics 15, 1748) and Meister et al (Meister SW (2020) Int J Mol Sci 21, 2999).

[115] Certain dual agonist polypeptides provided herein are fusion proteins comprising the dual agonist polypeptide according to Formula II (SEQ ID NO: 1 11) or the amino acid sequence of any one of SEQ ID NO: 112 to SEQ ID NO: 147 and one or more additional domains or amino acid sequences. In aspects, the fusion protein comprise the dual agonist polypeptide of SEQ ID NO: 1 13 to SEQ ID NO: 116 or the multimers SEQ ID NO: 164 to SEQ ID NO: 167. In aspects, the additional domains or amino acid sequences include, but are not limited to one or more of a linker, an expressed peptide tag for purification, a hinge, or an Fc domain. Linkers, peptide tags, hinges, and Fc domains are well-known to those of ordinary skill in the art and can be incorporated into the dual agonist polypeptides as described herein, and tested for potency, activity, and efficacy in treating hypoglycemic conditions, e.g., type-2 diabetes without undue experimentation according to the methods provided herein. As provided herein, the linkers, peptide tags, hinges, and Fc linkers can be added at either the N-terminal or C-terminal, or both, of the dual agonist polypeptides of the present application.

[116] Linkers used in various eGLP-1 polypeptides provided herein or used in various dual agonist polypeptides provided herein can facilitate formation of a desired structure. In some aspects, a polypeptide linker can comprise 1-50 amino acids, 1-25 amino acids, 25-50 amino acids, or 30-50 amino acids. Generally longer linkers correlate with higher activity (more flexible), but also decreased stability as the peptide becomes more exposed. Linkers can comprise, e.g., (Gly-Ser)_n, residues, where n is an integer of at least one, and up to, e.g., 4, 5, 6, 10, or more, optionally with some Glu or Lys residues dispersed through out to increase solubility. Alternatively, certain linkers do not comprise any Serine residues, e.g., where the linker is subject to O-linked glycosylation.

[117] Examples of suitable linkers include GS linkers. Since GS linkers do not share significant homology with known proteins, they are believed to be biologically inert and unlikely to have neomorphic effects or significant antigenicity. The length and amino acid sequence of a linker can be readily selected and optimized.

[118] In certain aspects, the linker comprises (GGGGS)n, wherein in 1, 2, 3, or 4. For example, certain specific linkers the amino acid sequence: G GGGGS GGGGS GGGGS GGGGSA (SEQ ID NO:69), A PPGGS GGGGS GGGGS GGGGSA (SEQ ID NO:70Error! Reference source not found.), GTGGGGS GGGGS GGGGS GGGGSA (SEQ ID NO:71), GGGGGS GGGGS GGGGS GGGGSA (SEQ ID NO:72Error! Reference source not found.), GGGGGSA (SEQ ID NO:73Error! Reference source not found.), GGGGGSGGGGSA (SEQ ID NO:74Error! Reference source not found.), GGGGGSGGGGS GGGGSA (SEQ ID NO:75), G KGGGS GGGGS GGGGS GGGGSA (SEQ ID NO:76), GGGGGS GGGGS GGGGS GGGGSA (SEQ ID NO:77), G GGGG GGGG GGGG GGGG A (SEQ ID NO:78) any combination thereof, any fragment thereof, or any variant thereof.

[119] In certain aspects, the linker comprises (GGSS)n (SEQ ID NO: 224), wherein in 1, 2, 3, 4, up to 10. For example, certain specific linkers the amino acid sequence: GGSS (SEQ ID NO: 224), GGSS GGSS (SEQ ID NO.-225), GGSS GGSS GGSS (SEQ ID NO:226), GGSS GGSS GGSS GGSS (SEQ ID NO:227), GGSS GGSS GGSS GGSS GGSS (SEQ ID NO:228), GGSS GGSS GGSS GGSS GGSS GGSS (SEQ ID NO:229), GGSS GGSS GGSS GGSS GGSS GGSS GGSS (SEQ ID NO:230), GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS (SEQ ID NO:231), GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS (SEQ ID NO:232), GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS (SEQ ID NO:233). As provided herein, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of SEQ ID NO: 127 to SEQ ID NO: 147.

[120] In certain aspects, the linker comprises GSGGS (SEQ ID NO: 234), wherein in 1 , 2, 3, 4, up to 10. For example, certain specific linkers the amino acid sequence: GSGGS GSGGS (SEQ ID NO:235), GSGGS GSGGS GSGGS (SEQ ID NO:236), GSGGS GSGGS GSGGS GSGGS (SEQ ID NO:237), GSGGS GSGGS GSGGS GSGGS GSGGS (SEQ ID NO:238), GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS (SEQ ID NO:239), GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS (SEQ ID NO:240), GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS (SEQ ID NO:241), GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS (SEQ ID NO:242), or GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS (SEQ ID NO: 243). As provided herein, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of SEQ ID NO: 127 to 147.

[121] In certain aspects, the linker comprises GGGS (SEQ ID NO: 244), wherein in 1, 2, 3, 4, up to 10. For example, certain specific linkers the amino acid sequence: GGGS GGGS (SEQ ID NO:245), GGGS GGGS GGGS (SEQ ID NO:246), GGGS GGGS GGGS GGGS (SEQ ID NO:247), GGGS GGGS GGGS GGGS GGGS (SEQ ID NO:248), GGGS GGGS GGGS GGGS GGGS GGGS (SEQ ID NO:249), GGGS GGGS GGGS GGGS GGGS GGGS GGGS (SEQ ID NO:250), GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS (SEQ ID NO:251), GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS (SEQ ID NO:252), or GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS (SEQ ID NO:253). As provided herein, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of SEQ ID NO: 127 to SEQ ID NO: 147.

[122] In certain aspects, the linker comprises GGSG (SEQ ID NO: 254), wherein in 1, 2, 3, 4, up to 10. For example, certain specific linkers the amino acid sequence: GGSG GGSG (SEQ ID NO:255), GGSG GGSG GGSG (SEQ ID NO:256), GGSG GGSG GGSG GGSG (SEQ ID NO:257), GGSG GGSG GGSG GGSG GGSG (SEQ ID NO:258), GGSG GGSG GGSG GGSG GGSG GGSG (SEQ ID NO:259), GGSG GGSG GGSG GGSG GGSG GGSG GGSG (SEQ ID NO:260), GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG (SEQ ID NO:261), GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG (SEQ ID NO:262), or GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG (SEQ ID NO: 263). As provided herein, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of SEQ ID NO: 127 to 147.

[123] In certain aspects, the linker comprises GGSGG (SEQ ID NO: 264), wherein in 1, 2, 3, 4, up to 10. For example, certain specific linkers the amino acid sequence: GGSGG GGSGG (SEQ ID NO:265), GGSGG GGSGG GGSGG (SEQ ID NO:266), GGSGG GGSGG GGSGG GGSGG (SEQ ID NO:267), GGSGG GGSGG GGSGG GGSGG GGSGG (SEQ ID NO:268), GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG (SEQ ID NO:269), GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG (SEQ ID NO:270), GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG (SEQ ID NO:271), GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG (SEQ ID NO:272), or GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG (SEQ ID NO:273). As provided herein, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of SEQ ID NO: 127 to SEQ ID NO: 147.

[124] In certain aspects, the linker comprises GSGSG (SEQ ID NO: 274), wherein in 1, 2, 3, 4, up to 10. For example, certain specific linkers the amino acid sequence: GSGSG GSGSG (SEQ ID NO:275), GSGSG GSGSG GSGSG (SEQ ID NO:276), GSGSG GSGSG GSGSG GSGSG (SEQ ID NO:277), GSGSG GSGSG GSGSG GSGSG GSGSG (SEQ ID NO:278), GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG (SEQ ID NO.279), GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG (SEQ ID NO:280), GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG (SEQ ID NO:281), GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG (SEQ ID NO:282), or GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG (SEQ ID NO: 283). As provided herein, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of SEQ ID NO: 127 to SEQ ID NO: 147.

[125] In certain aspects, the linker comprises GSGGG (SEQ ID NO: 284), wherein in 1, 2, 3, 4, up to 10. For example, certain specific linkers the amino acid sequence: GSGGG GSGGG (SEQ ID NO:285), GSGGG GSGGG GSGGG (SEQ ID NO:286), GSGGG GSGGG GSGGG GSGGG (SEQ ID NO:287), GSGGG GSGGG GSGGG GSGGG GSGGG (SEQ ID NO:288), GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG (SEQ ID NO:289), GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG (SEQ ID NO:290), GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG (SEQ ID NO:291), GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG (SEQ ID NO.-292), or GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG (SEQ ID NO: 293). As provided herein, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116.

[126] In certain aspects, the linker comprises GGGSG (SEQ ID NO: 294), wherein in 1, 2, 3, 4, up to 10. For example, certain specific linkers the amino acid sequence: GGGSG GGGSG (SEQ ID NO:295), GGGSG GGGSG GGGSG (SEQ ID NO:296), GGGSG GGGSG GGGSG GGGSG (SEQ ID NO:297), GGGSG GGGSG GGGSG GGGSG GGGSG (SEQ ID NO:298), GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG (SEQ ID NO:299), GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG (SEQ ID N0:300), GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG (SEQ ID NO:301), GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG (SEQ ID NO:302), or GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG (SEQ ID NO: 303). As provided herein, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of SEQ ID NO: 127 to SEQ ID NO: 147.

[127] In certain aspects, the linker comprises GSSSG (SEQ ID NO: 304), wherein in 1, 2, 3, 4, up to 10. For example, certain specific linkers the amino acid sequence: GSSSG GSSSG (SEQ ID NO:305), GSSSG GSSSG GSSSG (SEQ ID NO:306), GSSSG GSSSG GSSSG GSSSG (SEQ ID NO:307), GSSSG GSSSG GSSSG GSSSG GSSSG (SEQ ID NO:308), GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG (SEQ ID NO:309), GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG (SEQ ID NO:310), GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG (SEQ ID NO:311), GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG (SEQ ID NO:312), or GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG (SEQ ID NO: 313). As provided herein, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of SEQ ID NO: 127 to SEQ ID NO: 147.

[128] In certain aspects, the linker comprises GSSSS (SEQ ID NO: 314), wherein in 1 , 2, 3, 4, up to 10. For example, certain specific linkers the amino acid sequence:, GSSSS GSSSS (SEQ ID NO:315), GSSSS GSSSS GSSSS (SEQ ID NO:316), GSSSS GSSSS GSSSS GSSSS (SEQ ID NO:317), GSSSS GSSSS GSSSS GSSSS GSSSS (SEQ ID NO:318), GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS (SEQ ID NO:319), GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS (SEQ ID NO:320), GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS (SEQ ID NO:321), GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS (SEQ ID NO:322), or GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS (SEQ ID NO: 323). As provided herein, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of SEQ ID NO: 127 to SEQ ID NO: 147.

[129] In certain aspects, the linker comprises GGGGS (SEQ ID NO: 324), wherein in 1, 2, 3, 4, up to 10. For example, certain specific linkers the amino acid sequence: GGGGS GGGGS (SEQ ID NO:325), GGGGS GGGGS GGGGS (SEQ ID NO:326), GGGGS GGGGS GGGGS GGGGS (SEQ ID NO:327), GGGGS GGGGS GGGGS GGGGS GGGGS (SEQ ID NO:328), GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS (SEQ ID NO:329), GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS (SEQ ID NO:330), GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS (SEQ ID NO:331), or GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 332). As provided herein, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of SEQ ID NO: 127 to SEQ ID NO: 147.

[130] In certain aspects, the linker comprises AAAGG (SEQ ID NO: 333), wherein in 1, 2, 3, 4, up to 10. For example, certain specific linkers the amino acid sequence:, AAAGG AAAGG (SEQ ID NO:334), AAAGG AAAGG AAAGG (SEQ ID NO:335), AAAGG AAAGG AAAGG AAAGG (SEQ ID NO:336), AAAGG AAAGG AAAGG AAAGG AAAGG (SEQ ID NO:337), AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG (SEQ ID NO:338), AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG (SEQ ID NO:339), AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG (SEQ ID NO:340), AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG (SEQ ID NO:341), or AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG (SEQ ID NO: 342). As provided herein, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116.

[131] In certain aspects, the linker comprises GGSAAAGG (SEQ ID NO: 343), wherein in 1, 2, 3, 4, up to 10. For example, certain specific linkers the amino acid sequence: GGSAAAGG GGSAAAGG (SEQ ID NO: 344), GGSAAAGG GGSAAAGG GGSAAAGG (SEQ ID NO:345), GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG (SEQ ID NO:346), GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG (SEQ ID NO:347), GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG (SEQ ID NO:348), GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG (SEQ ID NO:349), GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG (SEQ ID NO:350), GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG (SEQ ID NO:351), or GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG (SEQ ID NO: 352). As provided herein, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. In aspects, the linker can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of SEQ ID NO: 127 to SEQ ID NO: 147.

[132] In aspects, the eGLP-1 polypeptides or the dual agonist polypeptides can include peptide tags for detection, purification, or both. Suitable, non-limiting examples include Alfa-tag (SRLEEELRRRLTE, SEQ ID NO:79), Avi-tag (GLNDIFEAQKIEWHE, SEQ ID NO:80) , C-tag (EPEA, SEQ ID NO:81Error! Reference source not found.), Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL, SEQ ID NO:82), Dogtag (DIPATYEFTDGKHY1TNEPIPPK, SEQ ID NO:83), E-tag (GAPVPYPDPLEPR, SEQ ID NO:84), FLAG (DYKDDDDK, SEQ ID NO:85), G4T (EELLSKNYHLENEVARLKK, SEQ ID NO:86), HA (YPYDVPDYA, SEQ ID NO:87), His (HHHHHH, SEQ ID NO:88), Isopeptag (TDKDMTITFTNKKDAE, SEQ ID NO:89), Myc (EQKLISEEDL, SEQ ID NO:90), NE-Tag (TKENPRSNQEESYDDNES, SEQ ID NO:91), Poly Glutamate-tag (EEEEEEE, SEQ ID NO:92), Poly Arginine-tag (RRRRRRR, SEQ ID NO:93), RholD4-tag (TETSQVAPA, SEQ ID NO:94), SBP-tag (MDEKTTGWRGGHWEGLAGELEQLRARLEHHPQGQREP, SEQ ID NO:95), Sdytag (DPIYMIDNDKPIT, SEQ ID NO:96), SH3 (STVPVAPPRRRRG, SEQ ID NO:97), Snooptag (KLGDIEFIKVNK, SEQ ID NO:98), Softag 1 (SLAELLNAGLGGS, SEQ ID NO:99), Softag 3 (TQDPSRVG, SEQ ID NO: 100), Spot-tag (PDRVRAVSHWSS, SEQ ID NO: 101), Spytag (AHIVMVDAYKPTK, SEQ ID NO: 102), S-tag (KETAAAKFERQHMDS, SEQ ID NO: 103), Strep-tag (WSHPQFEK, SEQ ID NO: 104), T7tag (MASMTGGQQMG, SEQ ID NO: 105), TC-tag (EVHTNQDPLD, SEQ ID NO: 106), Ty-tag (CCPGCC, SEQ ID NO: 107), VSV-tag (YTDIEMNRLGK, SEQ ID NO: 108), Xpress-tag (DLYDDDDK, SEQ ID NO: 109) and HiBit (VSGWRLFKKIS, SEQ ID NO: 110). Nucleic acid sequences encoding eGLP-1 polypeptides or dual agonist polypeptides and peptide tags are fused in frame incorporate the peptide tag to the amino terminus or carboxy terminus of the eGLP-1 polypeptides or dual agonist polypeptides.

[133] As provided herein, the tag can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147. In aspects, the tag can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 114 to SEQ ID NO: 116. In aspects, the tag can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of SEQ ID NO: 127 to SEQ ID NO: 147. The tag can be joined via peptide bond to the amino or carboxy terminus of the amino acid sequence of any one of SEQ ID NO: 2 to SEQ ID NO: 14, or of any of SEQ ID NO:32 to SEQ ID NO:67, or any of Formula I (SEQ ID NO: 1) or Formula III (SEQ ID NO:382) or of SEQ ID NO: 372 to SEQ ID NO:380. In other aspects, the tag can be joined via peptide bond to a linker as provided above to the amino or carboxy terminus of the dual agonist polypeptides provided herein.

[134] Importantly, the effects of linker, peptide tag, hinge, and Fc linker addition can be assessed using the 3-D models of GLP-1R receptor structure molecular docking methods described below.

[135]

Modifications

[136] The present application further includes, and provides for, lipidated eGLP-1 polypeptides or lapidated dual agonist polypeptides. The lipidated eGLP-1 polypeptides or lipidated dual agonist polypeptides may exhibit enhanced properties, such as longer in vivo half-life, compared to the corresponding non-lipidated eGLP-1 polypeptides or lipidated dual agonist polypeptides.

[137] “Lipidation” refers to a process of covalently attaching one or more fatty acids or polyethylene glycol directly or indirectly eGLP-1 polypeptide or dual agonist polypeptide described herein. In aspects, the lipid moiety is covalently attached either through the amino- or the carboxy-terminus. In other aspects, a lipid moiety is covalently attached via an internal amino acid, for example arginine, glutamine, aspartate, glutamate, tyrosine, histidine, threonine, and serine.

[138] An eGLP-1 polypeptide or dual agonist polypeptide that has undergone lipidation is said to be lipidated. The process of covalent attachment can convert the carboxylic acid into another functional group, such as a secondary amide, or can occur at another functional group present on the fatty acid in order to retain the carboxylic acid present in the original fatty acid. The covalent attachment of the one or more fatty acids can be directly attached to an eGLP-1 polypeptide or dual agonist polypeptide provided herein, or indirectly attached through a divalent linker moiety between the one or more fatty acids and eGLP-1 polypeptide or dual agonist polypeptide. A divalent linker moiety can include one or more amino acids, a polyethylene glycol (PEG), or a combination thereof. A linker moiety containing a PEG can further exhibit other functional groups, such as an amide, as needed for covalent attachment. Linker moieties comprising one or more amino acids can be attached via the C-terminus, the N-terminus, the side chain, or any combination thereof.

[139] “Polyethylene glycol" or “PEG" is a polyether monovalent radical of general formula -(O-Clfa- CHz) n-OH, or divalent radical of formula -(O-CH2-CH2)n-O-, wherein n is an integer greater than 1. When followed by a number, the PEG indicates the number of repeated units in the moiety. For instance, PEG3 can correspond with a divalent radical of formula -(O-CFb-CH^-O-, while PEGS can correspond with a monovalent radical of formula -(O-CHj-CHzJg-OH.

[14®] PEGs are prepared by polymerization of ethylene oxide and are commercially available over a range of molecular weights from 300 Daltons (Da) to 10,000,000 Da. Lower molecular weight PEGs are generally available as pure oligomers, referred to as monodisperse, uniform, or discrete. These are used in certain aspects of the present application. In certain aspects, the PEG is PEG2, PEG3, PEG4, PEGS, PEG6, PEG7, PEGS, PEG9, PEG10, PEG11, PEGU, PEG18, or PEG24. In certain aspects, the PEG is PEG2, PEG6, or PEG24.

Polynucleotides

[141] The present specification provides for, and includes, nucleic acids encoding the eGLP-1 polypeptides of Formula I. In aspects, the nucleic acids encode an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 14. In aspects, the nucleic acids encode an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO: 14. In aspects, the nucleic acids encode an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO: 14, or of Formula III, or of SEQ ID NO: 382 or of any one of SEQ ID NO:372 to SEQ ID NO:380.

[142] The present specification provides for, and includes, nucleic acids encoding the dual agonist polypeptides of Formula II (SEQ ID NO: 111). In aspects, the nucleic acids encode a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147. In aspects, the nucleic acids encode a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. In aspects, the nucleic acids encode a dual agonist polypeptide comprising the amino acid sequence of any one of Formula IV, SEQ ID NO: 383, or of SEQ ID NO: 381.

[143] The terms “polynucleotide¹ or “nucleotide” as used herein are intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA).

[144] The term “nucleic acid” refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. When applied to a nucleic acid or polynucleotide, the term “isolated” refers to a nucleic acid molecule, DNA or RNA that has been removed from its native environment, for example, a recombinant polynucleotide encoding an eGLP-1 polypeptide or dual agonist polypeptide contained in a vector is considered isolated for the purposes of the present disclosure. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) from other polynucleotides in a solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present disclosure. Isolated polynucleotides or nucleic acids according to the present disclosure further include such molecules produced synthetically. In addition, a polynucleotide or a nucleic acid can include regulatory elements such as promoters, enhancers, ribosome binding sites, or transcription termination signals. [145J The term “vector” means a construct that is capable of delivering, and in some aspects, expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells. Also included and provided for by the present applications are eukaryotic viral vectors. Bacterial and eukaryotic viral vectors provide for simplified delivery of the eGLP-1 polypeptide or dual agonist polypeptide sequences to host cells.

[146] As used herein, the term “host cell” refers to a cell or a population of cells harboring or capable of harboring a recombinant nucleic acid encoding an eGLP-1 polypeptide or dual agonist polypeptide. Host cells can be prokaryotic cells (e.g., E. coli), or alternatively, the host cells can be eukaryotic, for example, fungal cells (e.g., yeast cells such as Saccharomyces cerivisiae, Pichia pastoris, or Schizosaccharomyces pombe), and various animal cells, such as insect cells (e.g., Sf-9) or mammalian cells (e.g., HEK293F, CHO, COS 7, NIH-3T3), and plant cells. See Chunfeng et al., “Expression of cholera toxin B- lumbrokinase fusion protein in Pichia pastoris— the use of transmucosal carriers in the delivery of therapeutic proteins to protect rats against thrombosis,” Appl. Biochem. Biotechnol. 169(2):636-50 (2013).

(147] The terms “composition” or “pharmaceutical composition” refer to compositions containing an eGLP-1 polypeptide or dual agonist polypeptide as provided herein, along with e.g., pharmaceutically acceptable carriers, excipients, or diluents for administration to a subject in need of treatment (e.g., a mammal subject being treated for a hypoglycemic condition, e.g., type-2 diabetes).

[148] The term “pharmaceutically acceptable” refers to compositions that are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and other mammals without excessive toxicity or other complications commensurate with a reasonable benefit/risk ratio.

(149] An “effective amount” is that amount of a polypeptide comprising an eGLP-1 polypeptide or dual agonist polypeptide provided herein, the administration of which to a subject, either in a single dose or as part of a series, is effective for treatment, e.g., treatment of type-2 diabetes. An amount is effective, for example, when its administration results in one or more of prevention or modulation of hyperglycemia, promotion of insulin synthesis, an increase in B-cell mass, weight loss or weight maintenance (e.g., prevention of weight gain), reduction in food intake, modulation of gastric acid secretion, or modulation of gastric emptying. This amount can be a fixed dose for all subjects being treated, or can vary depending upon the weight, health, and physical condition of the subject to be treated, the extent of glycemic control desired, the formulation of polypeptide, a professional assessment of the medical situation, and other relevant factors.

[150] The term “subject” is meant any subject, particularly a mammalian subject, in need of treatment with an eGLP-1 polypeptide or dual agonist polypeptide as provided herein. Mammalian subjects include, but are not limited to, humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, cows, apes, monkeys, orangutans, chimpanzees, and other non-human primates, and so on. In one aspect, the subject is a human subject. In another aspect, the subject is a cat.

[151] As used herein, a “subject in need thereof’ refers to an individual for whom it is desirable to treat, e.g., a subject diagnosed with a metabolic disease or disorder. In aspects, the subject has a hypoglycemic condition, or is a subject prone to contract a hypoglycemic condition, e.g., type-2 diabetes. Subjects in need of treatment include subjects having a metabolic disease or disorders which can be alleviated by control of food intake, weight loss, energy metabolism, plasma glucose levels, insulin levels, and/or insulin secretion, positive inotropic effects, reduction of catabolic effects, slowing of gastric emptying, obesity, diabetes and diabetes-related conditions, liver fat-associated inflammation and injury. Such conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, insulin-resistance and disorders thereof such as polycystic ovary syndrome, obesity, diabetes mellitus of any kind, including type 1, type 2, and gestational diabetes.

Methods of making

[152] This specification provides a method of making an eGLP-1 polypeptide or dual agonist polypeptide by any suitable method. For example, eGLP-1 polypeptides or dual agonist polypeptides provided herein can be produced recombinantly using a convenient vector/host cell combination as would be well known to the person of ordinary skill in the art. A variety of methods are available for recombinantly producing eGLP-1 polypeptides or dual agonist polypeptides. Generally, a polynucleotide sequence encoding the eGLP-1 polypeptide or dual agonist polypeptide is inserted into an appropriate expression vehicle, e.g., a vector that contains the necessary elements for the transcription and translation of the inserted coding sequence. The nucleic acid encoding eGLP-1 polypeptide or dual agonist polypeptide is inserted into the vector in proper reading frame. The expression vector is then transfected into a suitable host cell that will express the eGLP-1 polypeptide or dual agonist polypeptide. Suitable host cells include without limitation bacteria, yeast, or mammalian cells. A variety of commercially available host-expression vector systems can be utilized to express the eGLP-1 polypeptides or dual agonist polypeptides described herein.

[153] The recombinant expression of eGLP- 1 polypeptides or dual agonist polypeptides described herein can be accomplished through the construction of an expression vector containing a polynucleotide that encodes the polypeptides disclosed herein. Once a polynucleotide encoding an eGLP-1 polypeptide or dual agonist polypeptide has been obtained, the vector for the production of the polypeptide can be produced by recombinant DNA technology using techniques well known in the art.

[154] As will be recognized, the nucleic acid sequences encoding an eGLP-1 polypeptide or dual agonist polypeptide can vary due to codon degeneracy. Since amino acids can be coded by different codons, the same amino acid can be transferred to ribosomes by several different tRNAs. However, the use of synonymous codons is strongly biased in both the prokaryotic and eukaryotic systems, comprising both bias between codons recognized by the same transfer RNA and bias between groups of codons recognized by different synonymous tRNAs. See Bulmer, “Coevolution of codon usage and transfer RNA abundance,” Nature 325:728-730 (1987). Depending on the organism, a different nucleic acid encoding an eGLP-1 polypeptide as provided herein is envisioned and included in the present application.

[155] Methods of codon and expression optimization are known in art to optimize multiple parameters and factors including codon usage (e.g., Codon Adaptation Index [CAI], Effective Number of codons [ENc], Relative Synonymous Codon Usage [RSCU] and Synonymous Codon Usage Order [SCUO]), codon pair, tRNA usage (e.g., tRNA adaptation index [tAI]), GC-content, ribosome binding site (RBS), hidden stop codons, motif avoidance, restriction site removal, mRNA secondary structure of the genes (e.g., mRNA free energy) and hydropathy index optimization. See, for example. Sharp et al., “The Codon Adaptation Index — a measure of directional synonymous codon usage bias, and its potential applications,” Nucleic Acids Research, 15 (3), 1281-1295 (1987); U.S. Patent NO. 8,326,547, issued December 4, 2012, to Liu et al; and U.S. Patent Publication No. 2021/0366574, published November 25, 2021, to Fan. A number of online tools for codon optimization have been developed and examples include DNAWorks (available on the internet at helixweb(dot)nih(dot)gov/dnaw orks/), Jcat (available on the internet atwww(dot)jcat(dot)de/), Syntheticgenedesigner (available on the internet atuserpages(dot)umbc(dot)edu/~wugl/codon/sgd/), GeneDesign (available on the internet atgenedesign(dot)org/), Gene Designer2.0 (available on the internet atwww(dot)dna20(dot)com/resources/genedesigner), OPTIMIZER (available on the internet atgenomes(dot)urv(dot)es/OPTIMIZER) Visualgenedeveloper (available on the internet atwww(dot)visualgenedeveloper(dot)net/), Eugene (available on the internet atbioinformatics(dot)ua(dot)pt/eugene), mRNA Optimizer (available on the internet atbioinformatics(dot)ua(dot)pt/software/mRNA-optimiser), COOL (available on the internet atbioinfo(dot)bti(dot)a-star(dot)edu(dot)sg/COOL/) and D-Tailor (available on the internet atsourceforge(dot)net/projects/dtailor/).

[156] Once an eGLP-1 polypeptide or dual agonist polypeptide has been produced by recombinant expression, it can be purified by any method known in the art for purification of a protein, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In aspects, the eGLP-1 polypeptides or dual agonist polypeptides can be prepared with a peptide tag, either at the amino or carboxy terminus to enable purification, detection, or both. In certain aspects, the peptide tag includes a protease cleavage site to enable the proteolytic removal of the peptide tog, and the generation of the eGLP-1 polypeptide or dual agonist polypeptide.

[157] Alternatively, an eGLP-1 polypeptide or dual agonist polypeptide provided herein can be chemically synthesized by methods well known to those of ordinary skill in the art, e.g., by solid phase synthesis as described by Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154). Solid phase peptide synthesis can be accomplished, e.g., by using automated synthesizers, using standard reagents. The use of chemical synthesis methods to prepare an eGLP-1 polypeptide or dual agonist polypeptide allows for the incorporation of non-standard amino acids, including, but not limited L-amino acids and alpha-methyl amino acids.

Formulations and Compositions

[158] In certain aspects, eGLP-1 polypeptides or dual agonist polypeptides provided herein possess one or more criteria of acceptable solubility, ease in formulatability, plasma stability (e.g., resistance to proteolysis), and improved pharmacokinetic properties. In certain aspects, eGLP-1 polypeptide polypeptides or dual agonist polypeptides as disclosed are soluble in standard buffers over a broad pH range.

[159] In certain aspects, eGLP-1 polypeptides or dual agonist polypeptides as disclosed are acceptably stable against proteases in serum or plasma. Common degradation products of native GLP-1 (wild type or wtGLP-1) include DPP IV-, pepsin-, trypsin-, chymotrypsin-, neprilysin- and elastase-cleavage products. Cleavage products arise from the action of proteases present in plasma or in the digestive tract (for orally administered compositions). In contrast, the eGLP-1 polypeptides or dual agonist polypeptides provided herein are modified to eliminate or reduce proteolysis and thereby increase stability leading to higher sustained plasma levels.

[160] The present application further provides compositions, e.g., pharmaceutical compositions, that contain an effective amount of an eGLP-1 polypeptide or dual agonist polypeptide as provided herein, formulated for the treatment of metabolic diseases, e.g., obesity, diabetes. Also provided for, and included, are pharmaceutical compositions comprising a host cell transformed with a polynucleotide encoding an eGLP-1 polypeptide or dual agonist polypeptide as provided herein. In aspects, the host cells express a polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 14 or SEQ ID NO:32 to SEQ ID NO:67, or of SEQ ID NO:382 or SEQ ID NO:372 to SEQ ID NO:380. In aspects, the host cells express an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID N0:2 to SEQ ID NO: 14. In aspects, the host cells express eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO: 14. In aspects, the host cells express a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147 or SEQ ID NO: 148 or SEQ ID NO: 164 to SEQ ID NO: 207. In aspects, the host cells express a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. In aspects, the host cells express a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 383 or SEQ ID NO: 381.

[161] The pharmaceutical compositions provided herein can be formulated for particular routes of administration such as oral administration, parenteral administration, and rectal administration, etc. In addition, the pharmaceutical compositions of the present application can be made up in a solid form including capsules, tablets, pills, granules, powders, or suppositories, or in a liquid form including solutions, suspensions, or emulsions. The pharmaceutical compositions can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, and buffers etc.

[162] Compositions as provided herein can be formulated according to known methods. Suitable preparation methods are described, for example, in Remington's Pharmaceutical Sciences, 23rd Edition, A Adejare, ed., Elsevier - Saunders, Mosby, Churchill, Easton, Pa. (2021), which is incorporated herein by reference in its entirety. Compositions can be in a variety of forms, including, but not limited to an aqueous solution, an emulsion, a gel, a suspension, lyophilized form, or any other form known in the art. In addition, the composition can contain pharmaceutically acceptable additives including, for example, carriers, diluents, binders, stabilizers, and preservatives. Once formulated, compositions of the application can be administered directly to the subject. In some aspects, the formulated compositions are provided as dry compositions to be suspended or solubilized in a liquid carrier, typically an aqeous carrier, prior to administration.

[163] Carriers that can be used with compositions of the application are well known in the art, and include, without limitation, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, and polyamino acids such as poly L-lysine, poly L-glutamic acid, and the like. A variety of aqueous carriers can be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. Compositions can be sterilized by conventional, well known sterilization techniques, or can be sterile filtered. A resulting composition can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. Compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, Wetting agents and the like, for example, sodium acetate, Sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan mono laurate, triethanolamineoleate, etc.

[164] In aspects, the pharmaceutical compositions containing eGLP-1 polypeptides or dual agonist polypeptides or host cells engineered to express the eGLP-1 polypeptide or dual agonist polypeptide (the APIs) provided herein can be formulated as tablets and gelatin capsules comprising the API active ingredient together with a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbents, colorants, flavors and sweeteners. Tablets may be either film coated or enteric coated according to methods known in the art.

[165] Suitable compositions for oral administration include an effective amount of a compound of the application in the form of tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, chewables, or syrups or elixirs. Compositions intended for oral use are prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents, and preserving agents, in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with nontoxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients are, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, com starch, or alginic acid; binding agents, for example, starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets are uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. Formulations for oral use can be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.

Host Cells

[166] The present application further provides for, and includes, host cells transformed with polynucleotides encoding peptides having the amino acid sequence of Formula I. In an aspect, the polynucleotide is an engineered vector comprising polynucleotides encoding peptides having the amino acid sequence of Formula I. In aspects, the engineered vector encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NO:1 to SEQ ID NO: 14. In other aspects, the engineered vector encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In aspects, the engineered vector encodes an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO: 14. In aspects, the engineered vector encodes an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO: 14.

[167] The present application further provides for, and includes, host cells transformed with polynucleotides encoding peptides having the amino acid sequence of Formula II (SEQ ID NO: 111). In an aspect, the polynucleotide is an engineered vector comprising polynucleotides encoding peptides having the amino acid sequence of Formula II (SEQ ID NO: 111). In aspects, the engineered vector encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147. In aspects, the engineered vector encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116 or the multimerized polypeptides of SEQ ID NO: 164 to SEQ ID NO: 167. In other aspects, the engineered vector encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 148 to SEQ ID NO: 207.

[168] The expression vectors are prepared using standard methods. Generally, expression vectors include a promoter for transcriptional expression, a nucleic acid sequence encoding a peptide comprising an eGLP-1 of Formula I, and a transcriptional terminator. In aspects, the nucleic acid sequence encoding the eGLP-1 polypeptides, end with a stop codon for translation termination. Suitable amino acid sequences of Formula I include the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 14. In other aspects, the expression vectors encode a polypeptide comprising an amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In aspects, the expression vectors encode an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO: 14. In aspects, the expression vectors encode an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO: 14. In aspects, the polypeptides may further include one or sequences for secretion or membrane localization. In certain aspects, the eGLP-1 polypeptides may further comprise an expressed peptide tag for detection or purification.

[169] In other aspects, the engineered vector encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 145 to SEQ ID NO: 147. In aspects, the engineered vector encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116, or the multimerized polypeptides of SEQ ID NO:164 to SEQ ID NO: 167. In some aspects, the engineered vector encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 142 to SEQ ID NO: 144. In aspects, the engineered vector encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 137 to SEQ ID NO: 141. In aspects, the engineered vector encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 127 to SEQ ID NO: 136. In yet further aspects, the engineered vector encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 123 to SEQ ID NO: 127.

[170] The expression vectors are prepared using standard methods. Generally, expression vectors include a promoter for transcriptional expression, a nucleic acid sequence encoding a peptide comprising a dual agonist polypeptide of Formula II (SEQ ID NO: 111), and a transcriptional terminator. In aspects, the nucleic acid sequence encoding the dual agonist polypeptides, end with a stop codon for translation termination. Suitable amino acid sequences of Formula II include the amino acid sequences of SEQ ID NO: 1 12 to SEQ ID NO: 147. In aspects, suitable amino acid sequences of Formula II include the amino acid sequences of any one of SEQ ID NO: 113 to SEQ ID NO: 116. Other suitable amino acid sequences of Formula II include the amino acid sequences of any one of SEQ ID NO: 164 to SEQ ID NO: 167.

Other suitable amino acid sequences of Formula II include the amino acid sequences of SEQ ID NO: 148 to SEQ ID NO: 207. In aspects, the polypeptides may further include one or sequences for secretion or membrane localization. In certain aspects, the dual agonist polypeptides may further comprise an expressed peptide tag for detection or purification.

[171] The expression vectors described herein, are introduced into host cells using standard methods. Suitable cells include bacterial cells, plant cells, yeast cells, or algae cells. At least one copy of the recombinant nucleic acids is stably introduced into the host cell. In aspects two or more copies of the recombinant nucleic acids are introduced into the host cell.

[172] In aspects, the recombinant nucleic acids are integrated into the bacterial cell or other host cell to provide for stable expression of the desired eGLP-1 polypeptides or dual agonist polypeptides provided herein. In an aspect, the integration is into the host cell chromosome. In aspects, the recombinant nucleic acids integrate randomly into the host cell chromosome. In other aspects, the integration of the recombinant nucleic acids is targeted, for example through the use of transposase and appropriate targeting sequences.

[173] In aspects, the transformed host cell is a yeast cell. In an aspect, the yeast cell is a yeast cell of a strain of Pichia pas tor is. See Chunfeng et al. 2013. In other aspects, the transformed host cell is a bacterial cell of a bacterial genus selected from the group consisting of Bacillus, Lactobacillus, Lactococcus, Salmonella, Enterococcus. In an aspect, the Lactobacillus is Lactobacillus gasseri. See Lin et al., “Oral Delivery of Pentameric Glucagon-Like Peptide- 1 by Recombinant Lactobacillus in Diabetic Rats,” PloS One, 17(9);e0162733 (2011) and (2016 and Duan et al., Engineered commensal bacteria reprogram intestinal cells into glucose-responsive insulin-secreting cells for the treatment of diabetes,” Diabetes 64(5): 1794-803 (2015). In another aspect, the Lactobacillus is Lactobacillus plantarum. See Luo et al., “Antidiabetic effect of an engineered bacterium Lactobacillus plantarum-pMG36e -GLP-1 in monkey model,” Synth. Syst. Biotechnol. 6(4):272-282 (2021).

[174] Also included and provided by the present application, are transformed plant cells. In aspects, the recombinant nucleic acids are integrated into the genome of the plant cell. In other aspects, the recombinant nucleic acids are integrated into the chloroplast of a plant cell. As provided herein, the transformed plant cells may be regenerated into a plant or plant part, including seeds.

[175] Direct fed microbials (DFMs), often also called probiotics, are microorganisms which colonize the gastrointestinal tract of an animal and provide some beneficial effect to that animal. In aspects, the microorganisms can be bacterial species, for example those from the genera Bacillus, Lactobacillus, Lactococcus, Salmonella, and Enterococcus. The microorganisms can also be yeast or molds in other aspects. The microorganisms can be provided to an animal orally or mucosally or, in the case of birds, provided to a fertilized egg, i.e. in ovo.

[176] In aspects of the present application, a DFM can be a host cell transformed with a polynucleotide sequence encoding an eGLP-1 polypeptide of Formula I. Suitable amino acid sequences of Formula I include the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 14. In other aspects, the polynucleotide sequences encode a polypeptide comprising an amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In aspects, the polynucleotide sequences encode an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO: 14. In aspects, the polynucleotide sequences encode an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO: 14.

[177] In aspects of the present application, a DFM can be a host cell transformed with a polynucleotide sequence encoding a dual agonist polypeptide of Formula II (SEQ ID NO: 111). DFMs are characterized as being generally safe (even denoted Generally Regarded as Safe (GRAS) and most are not naturally resistant to antibiotics. Probiotics and DFMs provide an attractive delivery method to provide therapeutic amounts of the dual agonist polypeptides provided herein.

[178] Suitable amino acid sequences of Formula II include the amino acid sequences of SEQ ID NO: 112 to SEQ ID NO: 147. In aspects, the polynucleotide sequences encode a dual agonist polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. In other aspects, the polynucleotide sequences encode a dual agonist polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 164 to SEQ ID NO: 167. In other aspects, the polynucleotide sequences encode a dual agonist polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 148 to SEQ ID NO: 207. In aspects, the polynucleotide sequences encode a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 123 to SEQ ID NO: 147. [179] As provided herein, the DFM host cells are engineered to express the eGLP-1 polypeptides on the bacterial surface or as a secreted form. DFMs are characterized as being generally safe (even denoted Generally Regarded as Safe (GRAS) and most are not naturally resistant to antibiotics. Probiotics and DFMs provide an attractive delivery method to provide therapeutic amounts of the eGLP-1 polypeptides provided herein.

DFMs as a delivery system

[180] Strains suitable as DFMs can provide an attractive and useful starting point for applications to produce eGLP-1 polypeptides or dual agonist polypeptides of the present application. In addition to being suitable for the production of eGLP-1 polypeptides or dual agonist polypeptides for purification, transformed DFMs are also suitable as live delivery systems for synthesis and delivery of the eGLP-1 polypeptides or dual agonist polypeptides described herein for use in therapy to treat metabolic disorders.

[181] Direct feed strains engineered (e.g., genetically modified) to express the eGLP-1 polypeptides or dual agonist polypeptides (engineered DFMs or eDFMs) have applicability as a delivery system which can constantly deliver useful therapeutic amounts of the eGLP-1 polypeptides or dual agonist polypeptides directly to the host. In aspects, the eDFM delivers therapeutic amounts of the eGLP-1 polypeptides or dual agonist polypeptides directly to the gastrointestinal tract. In certain aspects, the delivery system is a live recombinantly engineered DFM, such as a bacterium, which can reproduce in -- and even colonize in some instances - a host and directly deliver the eGLP-1 polypeptides or dual agonist polypeptides to the subject in need of treatment. Thus, eDFMs provide improved delivery platforms and systems, using suitable vectors and nucleic acid-based systems known in the art, for the rapid and effective expression of heterologous eGLP-1 polypeptides or dual agonist polypeptides of the present application. Suitable eGLP-1 polypeptides for an eDFM are polypeptides having the amino acid sequence of Formula I. In aspects, the eDFM expresses polypeptides comprising the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 14. In other aspects, the eDFMs express a polypeptide comprising an amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In aspects, the eDFM expresses an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO: 14. In aspects, the eDFM expresses an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NOG to SEQ ID NO: 14.

[182] Suitable dual agonist polypeptides for an eDFM are polypeptides having the amino acid sequence of Formula II (SEQ ID NO: 111). In aspects, the eDFM expresses polypeptides comprising the amino acid sequences of SEQ ID NO: 112 to SEQ ID NO: 147. In other aspects, the eDFM expresses a dual agonist polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. In other aspects, the eDFM expresses a dual agonist polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 164 to SEQ ID NO: 167. In other aspects, the eDFM expresses a dual agonist polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 148 to SEQ ID NO: 207. In aspects, the eDFM expresses a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 123 to SEQ ID NO: 147.

[183] As provided herein, the eDFM can express the desired polypeptides on the eDFM surface, or can be engineered to secret the eGLP-1 polypeptides or dual agonist polypeptides into the environment (e.g., the gastrointestinal tract).

[184] hi aspects, the DFM can be lyophilized. See Kwon et al., “Oral delivery of human biopharmaceuticals, autoantigens and vaccine antigens bioencapsulated in plant cells,” Adv Drug. Deliv. Rev. 65(6):782-99 (2013).

Host cells as a production system

[185] Recombinant protein production in microbial cells is an important aspect of the modem biotechnological industry. Intracellular expression of heterologous proteins in host cells is widely utilized and such proteins are isolated from a culture of producing host cells. The eGLP-1 polypeptides or dual agonist polypeptides of the present application can be expressed from plasmids transfected into bacterial cells or from encoding sequence(s) integrated in the host bacteria genome.

[186] In addition, recent achievements in secretory expression of recombinant proteins have encouraged both the scientific and industrial communities to apply and implement bacteria with a secretory ability for protein production. Using secretory-type host cells, synthesized eGLP-1 polypeptides or dual agonist polypeptides are secreted directly and accumulated in the extracellular medium. Secreted eGLP-1 polypeptides or dual agonist polypeptides provide cost-effective downstream purification processing. Further, secretion can permit production and isolation of target biomolecules and proteins without the need or requirement for lysing the host cells thereby simplifying the production and purification (if needed) processes. Also, secretory expression of eGLP-1 polypeptides or dual agonist polypeptides prevents accumulation of the desired eGLP-1 polypeptide or dual agonist polypeptide within host cells, which can limit cell growth and production, lead to cell toxicity and result in incorrect protein folding. See Mergulhao, et al. “Recombinant protein secretion in Escherichia coli,” Biotechnol Adv

23(3): 177-202 (2005); Song et al., “Improving Protein Production on the Level of Regulation of both Expression and Secretion Pathways in Bacillus subtilis," J Microbiol Biotechnol 25(7):963-77 (2015). Kits

[187] In some aspects, the present application provides for, and includes, pharmaceutical kits comprising an engineered Glucagon-Like Peptide 1 (eGLP-1) having the amino acid sequence of Formula I (e.g., the active pharmaceutical ingredient (API)). In other aspects, the kits comprise a recombinant host cell comprising a polynucleotide encoding an engineered Glucagon-Like Peptide 1 (eGLP-1) as the API. In aspects, in addition to the API, the pharmaceutical compositions further include an excipient selected from binders, coatings, colorings, disintegrants, flavors, glidants, lubricants, preservatives, sorbents, and carriers.

[188] In some aspects, the present application provides for, and includes, pharmaceutical kits comprising an engineered dual agonist polypeptide having the amino acid sequence of Formula II (SEQ ID NO: 111) (e.g., the active pharmaceutical ingredient (API)). In other aspects, the kits comprise a recombinant host cell comprising a polynucleotide encoding dual agonist polypeptide as the API. In aspects, in addition to the API, the pharmaceutical compositions further include an excipient selected from binders, coatings, colorings, disintegrants, flavors, glidants, lubricants, preservatives, sorbents, and carriers.

Methods of treatment

[189] eGLP-1 polypeptides or dual agonist polypeptides provided herein provide for one or more of prevention or modulation of hyperglycemia, promotion of insulin synthesis, inhibition of glucagon synthesis, an increase in B-cell mass, weight loss or weight maintenance (e.g., prevention of weight gain), reduction in food intake, modulation of gastric acid secretion, or modulation of gastric emptying.

[190] The present specification provides for, and includes, methods of treating a hypoglycemic condition, e.g., type-2 diabetes, comprising administering to a subject in need of treatment an eGLP-1 polypeptide as disclosed herein. Further provided is an eGLP-1 polypeptide for treatment of a hypoglycemic condition, e.g., type-2 diabetes. Further provided is use an eGLP-1 polypeptide as provided herein in the manufacture of a medicament for the treatment of a hypoglycemic condition, e.g., type-2 diabetes. In aspects, a method of treating a hypoglycemic condition, e.g., type-2 diabetes, comprising administering to a subject in need of treatment an eGLP-1 polypeptide or dual agonist polypeptide as disclosed herein, provides for reduced levels of hemoglobin A1C (HbAlc).

[191] The term “HbAlc” refers to the product of a non-enzymatic glycation of the hemoglobin B chain. The determination of HbAlc levels is well known to one skilled in the art. Tn monitoring the treatment of diabetes mellitus, the HbAlc value is of exceptional importance. As its production depends essentially on the blood sugar level and the life of the erythrocytes, the HbAlc serves as a “blood sugar memory” reflecting the average blood sugar levels of the preceding 4-6 weeks. Diabetic patients whose HbAlc value is consistently well adjusted by intensive diabetes treatment (i.e. <6.5% of the total hemoglobin in the sample), are significantly better protected against diabetic microangiopathy.

[192] The present application provides for, and includes methods of treatment comprising providing an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:1 to SEQ ID NO: 14, or a combination thereof, for preventing, slowing progression of, delaying, or treating a metabolic disorder. In other aspects, the method of treatment comprises providing a therapeutic amount of a polypeptide comprising an amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In other aspects, the method of treatment comprises providing a therapeutic amount of an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO: 14. In aspects, the method of treatment comprises providing a therapeutic amount of an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO: 14.

[193] The present application provides for, and includes methods of treatment comprising providing an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:1 to SEQ ID NO:14, or a combination thereof, for improving glycemic control and/or for reducing of fasting plasma glucose, of postprandial plasma glucose and/or of glycosylated hemoglobin HbAlc. In other aspects, the method of treatment comprises providing a therapeutic amount of a polypeptide comprising an amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In other aspects, the method of treatment comprises providing a therapeutic amount of an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO: 14. In aspects, the method of treatment comprises providing a therapeutic amount of an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO: 14.

[194] The present application provides for, and includes methods of treatment comprising providing an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO: I to SEQ ID NO: 14, or a combination thereof, for preventing, slowing, delaying or reversing progression from impaired glucose tolerance, impaired fasting blood glucose, insulin resistance and/or from metabolic syndrome to type 2 diabetes mellitus. In other aspects, the method of treatment comprises providing a therapeutic amount of a polypeptide comprising an amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In other aspects, the method of treatment comprises providing a therapeutic amount of an eGLP- 1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO: 14. In aspects, the method of treatment comprises providing a therapeutic amount of an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO: 14.

[195] The present application provides for, and includes methods of treatment comprising providing an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO. l to SEQ ID NO:14, or a combination thereof, for preventing, slowing progression of, delaying or treating of complications of diabetes mellitus selected from the group consisting of insulin resistance, glucose intolerance, elevated fasting blood glucose, prediabetes, type I diabetes, type II diabetes, gestational diabetes hypertension, dyslipidemia, or a combination thereof. In other aspects, the method of treatment comprises providing a therapeutic amount of a polypeptide comprising an amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In other aspects, the method of treatment comprises providing a therapeutic amount of an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO: 14 In aspects, the method of treatment comprises providing a therapeutic amount of an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO: 14. [1961 The present application provides for, and includes methods of treatment comprising providing an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:1 to SEQ ID NO: 14 or a combination thereof, for reducing body weight, body fat, or combinations thereof, or preventing an increase in body weight, body fat, or a combination of either, or facilitating a reduction in body weight, body fat, or combinations thereof. In other aspects, the method of treatment comprises providing a therapeutic amount of a polypeptide comprising an amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In other aspects, the method of treatment comprises providing a therapeutic amount of an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO: 14. In aspects, the method of treatment comprises providing a therapeutic amount of an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO: 14.

[197] The present application provides for, and includes methods of treatment comprising providing an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 14, or a combination thereof, for preventing or treating the degeneration of pancreatic beta cells and/or for improving and/or restoring or protecting the functionality of pancreatic beta cells and/or restoring the functionality of pancreatic insulin secretion.

[198] The present application provides for, and includes methods of treatment comprising providing an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO: I to SEQ ID NO: 14 or a combination thereof, for preventing, slowing, delaying or treating diseases or conditions attributed to an abnormal accumulation of liver or ectopic fat. In other aspects, the method of treatment comprises providing a therapeutic amount of a polypeptide comprising an amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In other aspects, the method of treatment comprises providing a therapeutic amount of an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO: 14. In aspects, the method of treatment comprises providing a therapeutic amount of an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO:14.

[199] The present application provides for, and includes methods of treatment comprising providing an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:1 to SEQ ID NO: 14, or a combination thereof, for maintaining or improving insulin sensitivity, or for treating or preventing hyperinsulinemia, insulin resistance, or both. In other aspects, the method of treatment comprises providing a therapeutic amount of a polypeptide comprising an amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67 In other aspects, the method of treatment comprises providing a therapeutic amount of an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO: 14. In aspects, the method of treatment comprises providing a therapeutic amount of an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO: 14. [2001 The present application provides for, and includes methods of treatment comprising providing an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:1 to SEQ ID NO: 14, or a combination thereof, for preventing, slowing progression of, delaying, or treating new onset diabetes after transplantation (NODAT) and/or post-transplant metabolic syndrome (PTMS). In other aspects, the method of treatment comprises providing a therapeutic amount of a polypeptide comprising an amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In other aspects, the method of treatment comprises providing a therapeutic amount of an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO: 14. Tn aspects, the method of treatment comprises providing a therapeutic amount of an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:3 to SEQ ED NO: 14.

[201] The present application provides for, and includes methods of treatment comprising providing an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:1 to SEQ ID NO: 14, or a combination thereof, for preventing, delaying, or reducing new-onset diabetes mellitus after transplantation (NODAT), PTMS associated complications or both, including micro- and macrovascular diseases and events, graft rejection, infection, and death. In other aspects, the method of treatment comprises providing a therapeutic amount of a polypeptide comprising an amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In other aspects, the method of treatment comprises providing a therapeutic amount of an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO: 14. In aspects, the method of treatment comprises providing a therapeutic amount of an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO: 14.

[202] The present application provides for, and includes methods of treatment comprising providing an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:1 to SEQ ID NO: 14, or a combination thereof, for treating hyperuricemia and hyperuricemia associated conditions. In other aspects, the method of treatment comprises providing a therapeutic amount of a polypeptide comprising an amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In other aspects, the method of treatment comprises providing a therapeutic amount of an eGLP-l polypeptide comprising the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO: 14. In aspects, the method of treatment comprises providing a therapeutic amount of an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO: 14.

[203] eGLP-1 polypeptides provided herein can be administered for glycemic control, promoting insulin production, promoting B-cell mass, promoting weight loss, or reducing excess body weight. In addition, eGLP-1 polypeptides provided herein can be used for treatment of related disorders. Examples of related disorders include without limitation: insulin resistance, glucose intolerance, prediabetes, increased fasting glucose, hypertension, dyslipidemia (or a combination of these metabolic risk factors), glucagonomas, cardiovascular diseases such as congestive heart failure, atherosclerosis, arteriosclerosis, coronary heart disease, or peripheral artery disease, stroke, respiratory dysfunction, or renal disease.

[204] The present application provides for, and includes methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147, or a combination thereof, for preventing, slowing progression of, delaying, or treating a metabolic disorder. In aspects, the methods provide for providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116 or the multimers of any one of SEQ ID NO: 164 to SEQ ID NO: 167. In aspects, the methods provide for providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 148 to SEQ ID NO: 207.

[205] The present application provides for, and includes methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147, or a combination thereof, for improving glycemic control and/or for reducing of fasting plasma glucose, of postprandial plasma glucose and/or of glycosylated hemoglobin HbAlc. In aspects, the methods for improving glycemic control include providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. In aspects, the methods for improving glycemic control include providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 127 to SEQ ID NO: 147, or combinations thereof. In aspects, the methods for improving glycemic control include providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 148 to SEQ ID NO: 207, or combinations thereof..

[206] The present application provides for, and includes methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147, or a combination thereof, for preventing, slowing, delaying or reversing progression from impaired glucose tolerance, impaired fasting blood glucose, insulin resistance and/or from metabolic syndrome to type 2 diabetes mellitus. In aspects, a method of preventing, slowing, delaying or reversing progression from impaired glucose tolerance, impaired fasting blood glucose, insulin resistance and/or from metabolic syndrome to type 2 diabetes mellitus treating by providing a peptide comprising the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. In an aspect, a method of preventing, slowing, delaying or reversing progression from impaired glucose tolerance, impaired fasting blood glucose, insulin resistance and/or from metabolic syndrome to type 2 diabetes mellitus treating by providing a peptide comprising the amino acid sequence of any one of SEQ ID NO: 127 to SEQ ID NO: 147, is provided. Also included are methods of providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 148 to SEQ ID NO:207.

[207] The present application provides for, and includes methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147, or a combination thereof, for preventing, slowing progression of, delaying or treating of a condition or disorder selected from the group consisting of complications of diabetes mellitus. In aspects, the present methods include providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. In aspects, the present methods include providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 127 to SEQ ID NO: 147. In further aspects, the present methods include providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 148 to SEQ ID NO: 207.

[208] The present application provides for, and includes methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147, or a combination thereof, for reducing body weight and/or body fat or preventing an increase in . body weight and/or body fat or facilitating a reduction in body weight and/or body fat. In aspects, methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116 or a combination thereof, for reducing body weight and/or body fat or preventing an increase in body weight and/or body fat or facilitating a reduction in body weight and/or body fat.

[209] Further provided are methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 148 to SEQ ID NO: 207 for reducing body weight and/or body fat or preventing an increase in body weight and/or body fat or facilitating a reduction in body weight and/or body fat. In aspects, a method for reducing body weight and/or body fat or preventing an increase in body weight and/or body fat or facilitating a reduction in body weight and/or body fat comprising providing a polypeptide having the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. This application further provides a method for reducing body weight and/or body fat or preventing an increase in body weight and/or body fat or facilitating a reduction in body weight and/or body fat comprising providing a polypeptide having the amino acid sequence of any one of SEQ ID NO: 127 to SEQ ID NO: 147.

[210] The present application provides for, and includes methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147, or a combination thereof, for preventing or treating the degeneration of pancreatic beta cells and/or for improving and/or restoring or protecting the functionality of pancreatic beta cells and/or restoring the functionality of pancreatic insulin secretion. In aspects, the dual agonist polypeptides comprise the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116, for the prevention or treatment the degeneration of pancreatic beta cells and/or for improving and/or restoring or protecting the functionality of pancreatic beta cells and/or restoring the functionality of pancreatic insulin secretion. In aspects, the dual agonist polypeptides comprise the amino acid sequence of any one of SEQ ID NO: 127 to SEQ ID NO: 147. In aspects, the application includes and provides providing a dual agonist polypeptide of any one of SEQ ID NO: 148 to SEQ ID NO: 207, for the prevention or treatment the degeneration of pancreatic beta cells and/or for improving and/or restoring or protecting the functionality of pancreatic beta cells and/or restoring the functionality of pancreatic insulin secretion.

[211] The present application provides for, and includes methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147, or a combination thereof, for preventing, slowing, delaying or treating diseases or conditions attributed to an abnormal accumulation of liver or ectopic fat. In aspects, the application includes dual agonist polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116, or a combination thereof, for preventing, slowing, delaying or treating diseases or conditions attributed to an abnormal accumulation of liver or ectopic fat. In aspects, the application includes dual agonist polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 164 to SEQ ID NO: 167, or a combination thereof, for preventing, slowing, delaying or treating diseases or conditions attributed to an abnormal accumulation of liver or ectopic fat. The present application provides for, and includes methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 127 to SEQ ID NO: 147, or a combination thereof, for preventing, slowing, delaying or treating diseases or conditions attributed to an abnormal accumulation of liver or ectopic fat. The present application provides for, and includes methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 148 to SEQ ID NO:207, or a combination thereof, for preventing, slowing, delaying or treating diseases or conditions attributed to an abnormal accumulation of liver or ectopic fat.

[212] The present application provides for, and includes methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147, or a combination thereof, for maintaining and/or improving the insulin sensitivity and/or for treating or preventing hyperinsulinemia and/or insulin resistance. In aspects, the application includes dual agonist polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116, or a combination thereof, for maintaining and/or improving the insulin sensitivity and/or for treating or preventing hyperinsulinemia and/or insulin resistance. In aspects, the application includes dual agonist polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 164 to SEQ ID NO: 167, or a combination thereof, for maintaining and/or improving the insulin sensitivity and/or for treating or preventing hyperinsulinemia and/or insulin resistance. In aspects, the polypeptides comprise the amino acid sequence of SEQ ID NO: 127 to SEQ ID NO: 147. The present application provides for, and includes methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 148 to SEQ ID NO: 207, or a combination thereof, for maintaining and/or improving the insulin sensitivity and/or for treating or preventing hyperinsulinemia and/or insulin resistance.

[213] The present application provides for, and includes methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147, or a combination thereof, for preventing, slowing progression of, delaying, or treating new onset diabetes after transplantation (NODAT) and/or post-transplant metabolic syndrome (PTMS). In aspects, the application inchides dual agonist polypeptides comprising the amino acid sequence of any one of SEQ ED NO: 113 to SEQ ID NO: 116, or a combination thereof, for preventing, slowing progression of, delaying, or treating new onset diabetes after transplantation (NODAT) and/or post-transplant metabolic syndrome (PTMS). In aspects, the application includes dual agonist polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 164 to SEQ ID NO: 167, or a combination thereof, for preventing, slowing progression of, delaying, or treating new onset diabetes after transplantation (NODAT) and/or post-transplant metabolic syndrome (PTMS). In aspects, the polypeptides comprise the amino acid sequence of SEQ ID NO: 127 to SEQ ID NO: 147. The present application provides for, and includes methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 148 to SEQ ID NO:207, or a combination thereof, for preventing, slowing progression of, delaying, or treating new onset diabetes after transplantation (NODAT) and/or post-transplant metabolic syndrome (PTMS).

[214] The present application provides for, and includes methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147, or a combination thereof, for preventing, delaying, or reducing NODAT and/or PTMS associated complications including micro- and macrovascular diseases and events, graft rejection, infection, and death. In aspects, the application includes dual agonist polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116, or a combination thereof, for preventing, delaying, or reducing NODAT and/or PTMS associated complications including micro- and macrovascular diseases and events, graft rejection, infection, and death. In aspects, the polypeptides comprise the amino acid sequence of any one of SEQ ID NO: 164 to SEQ ID NO: 167. In aspects, the polypeptides comprise the amino acid sequence of any one of SEQ ID NO: 127 to SEQ ID NO:147. The present application provides for, and includes methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 148 to SEQ ID NO:207, or a combination thereof, for preventing, delaying, or reducing NODAT and/or PTMS associated complications including micro- and macrovascular diseases and events, graft rejection, infection, and death.

[215] The present application provides for, and includes methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147, or a combination thereof, for treating hyperuricemia and hyperuricemia associated conditions. In aspects, the application includes dual agonist polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116, or a combination thereof, for treating hyperuricemia and hyperuricemia associated conditions. In aspects, the application includes dual agonist polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 164 to SEQ ID NO: 167, or a combination thereof, for treating hyperuricemia and hyperuricemia associated conditions. In aspects, the polypeptides comprise the amino acid sequence of SEQ ID NO: 127 to SEQ ID NO: 147. The present application provides for, and includes methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 148 to SEQ ID NO:207, or a combination thereof, for treating hyperuricemia and hyperuricemia associated conditions.

[216] The present application provides for, and includes methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147, or a combination thereof, for preventing, delaying, or reducing a neurodegenerative disease. In aspects, the application includes dual agonist polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116, or a combination thereof, for preventing, delaying, or reducing a neurodegenerative disease. In aspects, the application includes dual agonist polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 164 to SEQ ID NO: 167, or a combination thereof, for preventing, delaying, or reducing a neurodegenerative disease. In aspects, the polypeptides comprise the amino acid sequence of SEQ ID NO: 127 to SEQ ID NO: 147. The present application provides for, and includes methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 148 to SEQ ID NO:207, or a combination thereof, for preventing, delaying, or reducing a neurodegenerative disease (NDD). In aspects, methods of treating NDD comprise providing a polypeptide comprising the amino acid sequence of SEQ ID NO: 127 to SEQ ID NO: 147. In aspects, providing a dual agonist polypeptide for the treatment of NDD comprises providing a host cell expressing a polypeptide comprising SEQ ID NO: 111 to SEQ ID NO: 147. In aspects, providing a dual agonist polypeptide for the treatment of NDD comprises providing a host cell expressing a polypeptide comprising of any one of SEQ ID NO: 113 to SEQ ID NO: 116 or the multimers of SEQ ID NO: 164 to SEQ ID NO: 167. In certain aspects, the host cell expresses a polypeptide comprising SEQ ID NO: 127 to SEQ ID NO: 147. In aspects for NDD treatment, the host cell is a direct fed microbial expressing a polypeptide as provided herein. In aspects, the treatment of an NDD is a combination therapy for the treatment of a hypoglycemic condition.

[217] In aspects, the present application provides for the treatment, prevention, delay, or symptom reduction of Alzheimer’s disease (AD) comprising providing a therapeutic amount of a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147, or a combination thereof. In aspects, the treatment, prevention, delay, or symptom reduction of Alzheimer’s disease (AD) comprising providing a therapeutic amount of a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. In aspects, the treatment, prevention, delay, or symptom reduction of Alzheimer’s disease (AD) comprising providing a therapeutic amount of a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 164 to SEQ ID NO: 167.1n aspects, the AD is late-onset AD (also known as sporadic AD). In aspects, the present application provides for a method of reducing amyloid plaque burden, reducing tau phosphorylation, or a combination of both in subjects in need of treatment, prevention, delay, or symptom reduction of AD. In certain aspects, the methods of treatment of AD include providing a therapeutic amount of a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147, or a combination thereof for the prevention of loss of brain insulin receptors and synapses. In aspects, the methods of treatment of AD include providing a therapeutic amount of a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147, or a combination thereof for the prevention or reduction of cognitive impairments. In aspects, the methods of treatment of AD include providing a therapeutic amount of a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. In aspects, the methods of treatment of AD include providing a therapeutic amount of a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 164 to SEQ ID NO: 167. Also included in aspects of the present application, methods of treating AD comprise providing a polypeptide comprising the amino acid sequence of SEQ ID NO: 127 to SEQ ID NO: 147. In aspects, providing a dual agonist polypeptide comprises providing a host cell expressing a polypeptide comprising any one of SEQ ID NO: 111 to SEQ ID NO: 147. In aspects, providing a dual agonist polypeptide comprises providing a host cell expressing a polypeptide comprising any one of SEQ ID NO: 113 to SEQ ID NO: 116. In aspects, providing a dual agonist polypeptide comprises providing a host cell expressing a polypeptide comprising any one of SEQ ID NO: 164 to SEQ ID NO: 167. In certain aspects, the host cell expresses a polypeptide comprising SEQ ID NO: 127 to SEQ ID NO: 147. In aspects, the host cell is a direct fed microbial expressing a polypeptide as provided herein. In aspects, the treatment of an AD is a combination therapy for the treatment of a hypoglycemic condition. [2181 IB aspects, the present application provides for the treatment, prevention, delay, or symptom reduction of amylotrophic lateral sclerosis (ALS) comprising providing a therapeutic amount of a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147, or a combination thereof. In aspects, the treatment, prevention, delay, or symptom reduction of amylotrophic lateral sclerosis (ALS) comprising providing a therapeutic amount of a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. In aspects, the treatment, prevention, delay, or symptom reduction of amylotrophic lateral sclerosis (ALS) comprising providing a therapeutic amount of a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 164 to SEQ ID NO: 167. In aspects, methods of treating ALS comprise providing a polypeptide comprising the amino acid sequence of SEQ ID NO: 127 to SEQ ID NO: 147. In aspects, providing a dual agonist polypeptide for the treatment of ALS comprises providing a host cell expressing a polypeptide comprising SEQ ID NO: 111 to SEQ ID NO: 147. In certain aspects, the host cell expresses a polypeptide comprising SEQ ID NO: 127 to SEQ ID NO: 147. In aspects for ALS treatment, the host cell is a direct fed microbial expressing a polypeptide as provided herein. In aspects, the treatment of an ALS is a combination therapy for the treatment of a hypoglycemic condition.

[219] In aspects, the present application provides for the treatment, prevention, delay, or symptom reduction of Parkinson’s disease (PD) comprising providing a therapeutic amount of a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 11 1 to SEQ ID NO: 147, or a combination thereof. In aspects, the application includes dual agonist polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116, or a combination thereof, for the treatment, prevention, delay, or symptom reduction of Parkinson’s disease (PD) comprising providing a therapeutic amount of a dual agonist polypeptide. In aspects, methods of treating PD comprise providing a polypeptide comprising the amino acid sequence of SEQ ID NO: 164 to SEQ ID NO:167. In aspects, methods of treating PD comprise providing a polypeptide comprising the amino acid sequence of SEQ ID NO: 127 to SEQ ID NO: 147. In aspects, providing a dual agonist polypeptide for the treatment of PD comprises providing a host cell expressing a polypeptide comprising any one of SEQ ID NO: 111 to SEQ ID NO: 147. In aspects, providing a dual agonist polypeptide for the treatment of PD comprises providing a host cell expressing a polypeptide comprising any one of SEQ ID NO: 113 to SEQ ID NO: 116. In certain aspects, the host cell expresses a polypeptide comprising SEQ ID NO: 127 to SEQ ID NO: 147. In aspects for PD treatment, the host cell is a direct fed microbial expressing a polypeptide as provided herein. In aspects, the treatment of an PD is a combination therapy for the treatment of a hypoglycemic condition.

[220] Dual agonist polypeptides provided herein can be administered for glycemic control, promoting insulin production, promoting B-cell mass, promoting weight loss, or reducing excess body weight. In addition, dual agonist polypeptides provided herein can be used for treatment of related disorders. Examples of related disorders include without limitation: insulin resistance, glucose intolerance, prediabetes, increased fasting glucose, hypertension, dyslipidemia (or a combination of these metabolic risk factors), glucagonomas, cardiovascular diseases such as congestive heart failure, atherosclerosis, arteriosclerosis, coronary heart disease, or peripheral artery disease, stroke, respiratory dysfunction, or renal disease.

[221 ] As used herein, “treatment* is an approach for obtaining beneficial or desired clinical results. As provided herein, beneficial, or desired clinical results from the disclosed eGLP-1 polypeptides or dual agonist polypeptides include, without limitation, stabilized serum glucose and serum insulin levels, increased B-cell mass, or amelioration, palliation, stabilization, diminishment of weight gain. “Treatment" refers to both therapeutic treatment and prophylactic or preventative measures in certain aspects. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. By “treatment” is meant improved glycemic control in type-2 diabetes, and is not necessarily meant to imply complete cure of the relevant condition.

[2221 The route of administration of eGLP-1 polypeptides or dual agonist polypeptides provided herein can be, for example, oral, parenteral, by inhalation or topical. The term parenteral as used herein includes, e.g., intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. Another example of a form for administration is a solution for injection, in particular for intravenous or intraarterial injection or drip. EGLP-1 polypeptides or dual agonist polypeptides provided herein can be administered as a single dose or as multiple doses. In aspects, the eGLP-1 polypeptides or dual agonist polypeptides are configured for transdermal administration. In certain aspects, an eGLP-1 polypeptide or dual agonist polypeptide is administered orally or by subcutaneous injection.

[223] Parenteral formulations can be a single bolus dose, an infusion or a loading bolus dose followed with a maintenance dose. These compositions can be administered at specific fixed or variable intervals, e.g., once a day, or on an “as needed basis, e.g., based on patient-initiated blood glucose measurements. Dosage regimens also can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response).

[224] The amounts of eGLP-1 polypeptides or dual agonist polypeptides to be administered can be readily determined by one of ordinary skill in the art. Factors influencing the mode of administration and the respective amount of an eGLP-1 polypeptide or dual agonist polypeptide include, but are not limited to, the severity of the disease (e.g., the extent of obesity), the subject’s history, and the age, height, weight, health, and physical condition of the subject undergoing therapy. Similarly, the amount of an eGLP-1 polypeptide or dual agonist polypeptide to be administered will be dependent upon the mode of administration and whether the subject will undergo a single dose or multiple doses of this agent. In certain aspects, eGLP-1 polypeptides or dual agonist polypeptides provided herein can be administered once per day via injection.

[225] Also provided for, and included in the present methods of treatment are oral treatments of DFMs expressing an eGLP-1 polypeptide or dual agonist polypeptide as provided herein.

[226] The present eGLP-1 polypeptides or dual agonist polypeptides provide for, and include, methods of treatment of a condition caused or characterized by excess body weight, and the treatment of obesity, morbid obesity, obesity linked inflammation, obesity linked gallbladder disease, obesity induced sleep apnea, metabolic syndrome, pre-diabetes, insulin resistance, glucose intolerance, type 2 diabetes, type I diabetes, hypertension, atherogenic dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke or microvascular disease in a subject, by administering a compound of claim 1 to said subject, in an amount sufficient to treat a condition caused or characterized by excess body weight, and to treat obesity, morbid obesity, obesity linked inflammation, obesity linked gallbladder disease, obesity induced sleep apnea, metabolic syndrome, pre-diabetes, insulin resistance, glucose intolerance, type 2 diabetes, type I diabetes, hypertension, atherogenic dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke or microvascular disease. In other aspects, the method of treatment comprises providing a therapeutic amount of a polypeptide comprising an amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In other aspects, the method of treatment comprises providing a therapeutic amount of an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO: 14. In aspects, the method of treatment comprises providing a therapeutic amount of an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO: 14. The present application provides for a method of treatment comprising providing dual agonist polypeptides having the amino acid sequence of SEQ ID NO: 111 to SEQ ID NO: 147. In aspects, the application includes administering a dual agonist polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116, or a combination thereof. In aspects, the polypeptides comprise the amino acid sequence of SEQ ID NO: 127 to SEQ ID NO: 147. In other aspects, the dual agonist polypeptides comprise the amino acid sequence of any one of SEQ ID NO: 148 to SEQ ID NO:207.

[227] The eGLP-1 polypeptide or dual agonist polypeptide compounds described find use in preventing weight gain or promoting weight loss. As used herein, preventing means inhibiting or reducing weight gain when compared to the absence of treatment, and is not necessarily meant to imply complete cessation of weight gain. The peptides may cause a decrease in food intake and/or increased energy expenditure, resulting in the observed effect on body weight. In aspects, independently of their effect on body weight, the eGLP-1 polypeptides or dual agonist polypeptides provided herein have a beneficial effect on circulating glucose levels, glucose tolerance, and/or on circulating cholesterol levels, being capable of lowering circulating LDL levels and increasing HDL/LDL ratio. Thus the eGLP-1 polypeptides or dual agonist polypeptides provided herein can be used for direct or indirect therapy of any condition caused or characterized by excess body weight, such as the treatment and/or prevention of obesity, morbid obesity, obesity linked inflammation, obesity linked gallbladder disease, obesity induced sleep apnea.

[228] The present application provides for, and includes, the use of eGLP-1 polypeptides or dual agonist polypeptides for the treatment of pre-diabetes, insulin resistance, glucose intolerance, type 2 diabetes, type I diabetes, hypertension or atherogenic dyslipidemia (or a combination of two or more of these metabolic risk factors), atherosclerosis, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke and microvascular disease. In aspects, the effects in these conditions may be as a result of or associated with their effect on body weight or may be independent thereof. In aspects, the eGLP-1 polypeptides or dual agonist polypeptides are for use in the treatment of obesity.

[229] The eGLP-1 polypeptides or dual agonist polypeptides provided herein are compounds suitable for use in a method of medical treatment, particularly for use in a method of treatment of a condition as described above. The eGLP-1 polypeptides or dual agonist polypeptides provided herein are useful for the preparation of medicaments for the treatment of a condition as described above. In other aspects, the medicaments comprise a therapeutic amount of a polypeptide comprising an amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In other aspects, the medicaments comprise a therapeutic amount of an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO: 14. In aspects, the medicaments comprise a therapeutic amount of an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO: 14.

[230] The present application provides for a method of treatment comprising providing dual agonist polypeptides having the amino acid sequence of SEQ ID NO: 111 to SEQ ID NO: 147 In aspects, the method of treatment comprises providing dual agonist polypeptides having the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 147. hi aspects, the polypeptides comprise the amino acid sequence of SEQ ID NO: 127 to SEQ ID NO: 147. In other aspects, the dual agonist polypeptides comprise the amino acid sequence of any one of SEQ ID NO: 148 to SEQ ID NO:207.

Combination treatments

[231] In certain aspects an eGLP-1 polypeptide as provided herein can be administered in combination with one or more additional therapies. In aspects, the eGLP-1 polypeptides may be administered as part of a combination therapy with an agent for treatment of diabetes, obesity, dyslipidemia or hypertension.

[232] The additional therapy can include one or more existing standard therapies type-2 diabetes or other hypoglycemic condition, or new therapies. In certain aspects, the one or more additional therapies can include, without limitation, blood sugar monitoring, diet modifications, exercise, insulin, a thiazolidinedione, a sulfonylurea, an incretin, metformin, a glyburide, a dipeptidyl peptidase 4 inhibitor, a bile acid sequestrant, or any combination thereof

[233] SEQUENCES

SEQ ID NO: 16 cGLPIR

LIPLLGTHEWFAFVMDEHARGTLRHKLFTELSFTSFQGLMVAILYCFVNNEVQMEFRRS

WERWRLKHLHIQRDSSMKPLKCPTSSLTSGGTVGSSVYAASCQASCS

SEQ ID NO: 17 Exendin-4 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS

SEQ ID NO: 18 GLP-1 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR

SEQ ID NO: 19 GLP-1-Gly8 HGEGTFTSDVSSYLEGQAAKEFIAWLVKGR

SEQ ID NO:20 Trp 5xGLP-l HGEGTFTSDVSSYLEGQAAQEFIAWLVDGR

SEQ ID NO:21 Liraglutide HAEFTFTSDVSSYLEGQAAKEFIAWLVRGRG

(hexadecanoyl group attached to lysine via a glutamic acid spacer)

SEQ ID NO:22 Semaglutide HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG (K20 is conjugated with a semiglutied pubchem id 90016781)

SEQ ID NO:23 J211 H4EGSFTSDV,?SFI.EGEAAXEMAFWXGG (K26 is alpha-K) SEQ ID NO:24 J229 H^EGSTH'SDVSS^EGEAAKEFlA^WTraG (K26 is lipidated) SEQ ID NO:25 Medi7219 HaEGSfTSDVsSKLEGEAAkEflAKWEGG (lower case is alpha-methyl, US2018/0162920 SEQ ID 263)

SEQ ID NO:26 GLP1 ASG HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

SEQ ID NO:27 GLP1_K2OQ HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

SEQ ID NO:28 GLP1JK28D HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

SEQ ID NO:29 Hid SEMAADnSTIGDLVKWUDTVNKFKK

SEQ ID NO:30 SxGLP 1 trp-ABDcon

MHGEGTFTSDVSSYLEGQAAQEFIAWLVDGRHGEGTFTSDVSSYLEGQAAQEFI AWLVDGRHGEGTFTSDVSSYLEGQAAQEFIAWLVDGRHGEGTFTSDVSSYLEGQAAQE FIAWLVDGRHGEGTFTSDVSSYLEGQAAQEFIAWLVDGRGAPVPYPDPLEPRGGGGSGG GGSGGGGSLKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDETLKA*

SEQ ID NO:31 SxGLPltrp

HGEGTFTSDVSSYLEGQAAQEFIAWLVDGRHGEGTFTSDVSSYLEGQAAQEFIA WLVDGRHGEGTFTSDVSSYLEGQAAQEFIAWLVDGRHGEGTFTSDVSSYLEGQAAQEFT AWLVDGRHGEGTFTSDVSSYLEGQAAQEFIAWLVDGRGAPVPYPDPLEPR

SEQ ID NO:32 >C1.2-2x HGEGTSESDVSSYLEGQAAQEF1AWLVDGX

HGEGTSESDVSSYLEGQAAQEFIAWI.VDG (X = R or K)

SEQ ID NO:33 >C1.2-3x

HGEGTSESDVSSYLEGQAAQEFIAWLVDGXHGEGTSESDVSSYLEGQAAQEFIA WLVDGXHGEGTSESDVSSYLEGQAAQEFIAWLVDG (X = R or K)

SEQ ID NO:34 >C1.2-4x

HGEGTSESDVSSYLEGQAAQEFIAWLVDGXHGEGTSESDVSSYLEGQAAQEFIA WLVDGXHGEGTSESDVSSYLEGQAAQEFIAWLVDGXHGEGTSESDVSSYLEGQAAQEFI AWLVDG (X = R OR K)

SEQ ID NO:35 >C1.2-5x

HGEGTSESDVSSYLEGQAAQEFIAWLVDGXHGEGTSESDVSSYLEGQAAQEFIA

WLVDGXHGEGTSESDVSSYLEGQAAQEFIAWLVDGXHGEGTSESDVSSYLEGQAAQEFI AWLVDGXHGEGTSESDVSSYLEGQAAQEFIAWLVDG (X = R or K)

SEQ ID NO:36 X?1.3-2x HGEGTSESDVSSQLEGQAAQEFIAWLVDGX HGEGTSESDVSSQLEGQAAQEF1AWLVDG (X = R OR K)

SEQ ID NO:37 >C1.3-3x

HGEGTSESDVSSQLEGQAAQEFIAWLVDGXHGEGTSESDVSSQLEGQAAQEFIA

WLVDGXHGEGTSESDVSSQLEGQAAQEFIAWLVDG (X = R OR K)

SEQ ID NO:38 >C1.3-4x

HGEGTSESDVSSQLEGQAAQEFIAWLVDGXHGEGTSESDVSSQLEGQAAQEFIA

WLVDGXHGEGTSESDVSSQLEGQAAQEFIAWLVDGXHGEGTSESDVSSQLEGQAAQEFI AWLVDG (X = R OR K)

SEQ 1D NO:39 X?1.3-5x

HGEGTSESDVSSQLEGQAAQEFIAWI.VDGXHGEGTSESDVSSQLEGQAAQEFIA

WLVDGXHGEGTSESDVSSQLEGQAAQEFIAWLVDGXHGEGTSESDVSSQLEGQAAQEFI AWLVDGXHGEGTSESDVSSQLEGQAAQEFIAWLVDG (X = R OR K)

SEQ ID NO:40 >CL4-2x

HGEGTSESDVSSQ1EGQAAQEFIAWLVDGXHGEGTSESDVSSQIEGQAAQEFIAW LVDG (X = R orK)

SEQ ID NO:41 >C1.4-3x

HGEGTSESDVSSQIEGQAAQEFIAWLVDGXHGEGTSESDVSSQIEGQAAQEFIAW LVDGXHGEGTSESDVSSQIEGQAAQEFIAWLVDG (X - R OR K)

SEQ ID NO142 >C1.4-4x

HGEGTSESDVSSQIEGQAAQEFIAWLVDGXHGEGTSESDVSSQIEGQAAQEFIAW

LVDGXHGEGTSESDVSSQIEGQAAQEFIAWLVDGXHGEGTSESDVSSQ1EGQAAQEFIAW LVDG (X = R ORK)

SEQ ID NO:43 >C1.4-5x

HGEGTSESDVSSQIEGQAAQEFIAWLVDGXHGEGTSESDVSSQIEGQAAQEFIAW LVDGXHGEGTSESDVSSQIEGQAAQEFIAWLVDGXHGEGTSESDVSSQIEGQAAQEFIAW LVDGXHGEGTSESDVSSQIEGQAAQEFIAWLVDG (X = R OR K) SEQ JD NO:44 >C1.5-2x

HGEGTSESDVSQSIEGQAAQEFIAWLVDGXHGEGTSESDVSQS1EGQAAQEFIAW LVDG (X = R orK)

SEQ ID NO:45 >C1.5-3x

HGEGTSESDVSQSIEGQAAQEFIAWLVDGXHGEGTSESDVSQSIEGQAAQEFIAW LVDGXHGEGTSESDVSQSIEGQAAQEFIAWLVDG (X = R OR K)

SEQ ID NO:46 >C1.5-4x

HGEGTSESDVSQSIEGQAAQEFIAWLVDGXHGEGTSESDVSQSIEGQAAQEFIAW LVDGXHGEGTSESDVSQSIEGQAAQEFIAWLVDGXHGEGTSESDVSQSIEGQAAQEFIAW LVDG (X = RORK)

SEQ ID NO:47 >C1.5-5x

HGEGTSESDVSQSIEGQAAQEFIAWLVDGXHGEGTSESDVSQSIEGQAAQEFIAW LVDGXHGEGTSESDVSQSIEGQAAQEFIAWLVDGXHGEGTSESDVSQSIEGQAAQEFIAW LVDGXHGEGTSESDVSQSIEGQAAQEFIAWLVDG (X = R OR K)

SEQ ID NO:48 >C1.6-2x

HGEGTSESDVSQSIEGQAAQEVIAWLVDGXHGEGTSESDVSQSIEGQAAQEVIA

WLVDG (X = R OR K)

SEQ ID NO:49 >C1.6-3x

HGEGTSESDVSQSIEGQAAQEVIAWLVDGXHGEGTSESDVSQSIEGQAAQEV1A WLVDGXHGEGTSESDVSQSIEGQAAQEV1AWLVDG (X = R OR K)

SEQ 1D NO:50 >C1.6-4x HGEGTSESDVSQSIEGQAAQEVLAWLVDGXHGEGTSESDVSQSIEGQAAQEVIA WLVDGXHGEGTSESDVSQSIEGQAAQEVIAWLVDGXHGEGTSESDVSQSIEGQAAQEVI AWLVDG (X = ROR K)

SEQ ID NO:51 >C1.6-5x HGEGTSESDVSQSIEGQAAQEVIAWLVDGXHGEGTSESDVSQSIEGQAAQEVIA WLVDGXHGEGTSESDVSQSIEGQAAQEVIAWLVDGXHGEGTSESDVSQSIEGQAAQEVI AWLVDGXHGEGTSESDVSQSIEGQAAQEVIAWLVDG (X = R OR K)

SEQ ID NO:52 >C1.7-2x

HGEGTSESDVSQSIEGQAAQEIVAWI.VDGXHGEGTSESDVSQSIEGQAAQEIVA

WLVDG (X - RORK)

SEQ ID NO:53 >CL7-3x

HGEGTSESDVSQSIEGQAAQEIVAWLVDGXHGEGTSESDVSQSIEGQAAQEIVA WLVDGXHGEGTSESDVSQS1EGQAAQEIVAWLVDG (X = R OR K) SEQ ID NO:54 >C1.7-4x

HGEGTSESDVSQSIEGQAAQEIVAWLVDGXHGEGTSESDVSQSIEGQAAQEIVA WLVDGXHGEGTSESDVSQSIEGQAAQEIVAWLVDGXHGEGTSESDVSQSIEGQAAQEIV AWLVDG (X = R OR K)

SEQ ID NO:55 >C1.7-5x

HGEGTSESDVSQSIEGQAAQEIVAWLVDGXHGEGTSESDVSQSIEGQAAQEIVA WLVDGXHGEGTSESDVSQSIEGQAAQEIVAWLVDGXHGEGTSESDVSQSIEGQAAQErV AWLVDGXHGEGTSESDVSQSIEGQAAQEIVAWLVDG (X - R OR K)

SEQ ID NO:56 >CL8-2x

HGEGTSESDVSQSIEGQAAQEIVAIVVDGXHGEGTSESDVSQSIEGQAAQEIVAIV VDG (X = RORK)

SEQ ID NO:57 >C1.8-3x

HGEGTSESDVSQSIEGQAAQEIVAIVVDGXHGEGTSESDVSQSIEGQAAQEIVAIV VDGXHGEGTSESDVSQSIEGQAAQEIYAIWDG (X - R OR K)

SEQ ID NO:58 >C1.84x

HGEGTSESDVSQSIEGQAAQE1VAIWDGXHGEGTSESDVSQSIEGQAAQEIVAIV VDGXHGEGTSESDVSQSIEGQAAQE1VAIWDGXHGEGTSESDVSQS1EGQAAQETVAIV VDG (X- RORK)

SEQ ID NO:59 >C1.8-5x

HGEGTSESDVSQSIEGQAAQEIVAIWDGXHGEGTSESDVSQSDEGQAAQEIVAIV VDGXHGEGTSESDVSQSffiGQAAQEIVAIWDGXHGEGTSESDVSQSIEGQAAQEIVAIV VDGXHGEGTSESDVSQSIEGQAAQEIVAIWDG (X = R OR K)

SEQ IDNO:60 >C1.9-2x

HGEGTSESDVSQSIEGQAAQEIVAVIVDGXHGEGTSESDVSQSIEGQAAQEIVAV1 VDG (X = R or K)

SEQ ID NO:61 >C1.9-3x

HGEGTSESDVSQS1EGQAAQEIVAVIVDGXHGEGTSESDVSQSIEGQAAQEIVAVI VDGXHGEGTSESDVSQSIEGQAAQEIVAVIVDG (X = R OR K)

SEQ ID NO:62 >C1.9-4x

HGEGTSESDVSQSIEGQAAQEIVAVIVDGXHGEGTSESDVSQSIEGQAAQEIVAVI VDGXHGEGTSESDVSQSIEGQAAQEIVAVIVDGXHGEGTSESDVSQSIEGQAAQEIVAVI VDG (X = RORK)

SEQ ID NO:63 >CL9-5x

HGEGTSESDVSQS1EGQAAQEIVAVIVDGXHGEGTSESDVSQSIEGQAAQEIVAVI

SEQ£DNO:64 >C1.10-2x

VDG(X = RORK)

SEQIDNO:65 >C1.10-3x

HGEGTSESDVSQSIEGQAAQEVIAVIVDGXHGEGTSESDVSQSIEGQAAQEVIAVI VDGXHGEGTSESDVSQSIEGQAAQEVIAVIVDG (X = R OR K)

SEQIDNO:66 >C1.10-4x

HGEGTSESDVSQS1EGQAAQEVIAVIVDGXHGEGTSESDVSQSIEGQAAQEVIAVI VDGXHGEGTSESDVSQSIEGQAAQEVIAVrVDGXHGEGTSESDVSQSIEGQAAQEVIAVI VDG(X = RORK)

SEQIDNO:67 >C1.10-5x

SEQIDNO:68 E-tag GAPVPYPDPLEPR SEQIDNO:69 G GGGGS GGGGS GGGGS GGGGSA SEQIDNO:70 A PPGGS GGGGS GGGGS GGGGSA SEQIDNO:71 GTGGGGS GGGGS GGGGS GGGGSA SEQIDNO:72 GGGGGS GGGGS GGGGS GGGGSA SEQIDNO:73 GGGGGSA SEQIDNO:74 GGGGGSGGGGSA SEQIDNO:75 GGGGGSGGGGS GGGGSA SEQIDNO:76 G KGGGS GGGGS GGGGS GGGGSA SEQIDNO:77 GGGGGS GGGGS GGGGS GGGGSA SEQ1DNO:78 G GGGG GGGG GGGG GGGG A SEQIDNO:79 Alfa-tag SRLEEELRRRLTE SEQIDNO:80 Avi-tag GLNDIFEAQKIEWHE SEQIDNO:81 C-tag EPEA SEQIDNO:82 Calmodulin-tag KRRWKKNFIAVSAANRFKKISSSGAL SEQIDNO:83 Dogtag DIPATYEFTDGKHYITNEPIPPK SEQ ID NO:84 E-tag GAPVPYPDPLEPR SEQ ID NO:85 FLAG DYKDDDDK

SEQIDNO:111 FomulaJI Y X2 E GTX6 X7 S DX10 S 1X13 X14D X161 AQX20 A X22VQX25 X26IAGGPS SGAPPwhereX2-VorK;;X6=F, P, or S;; X7 = T, C, or E;; X10 = Y, C, or E;; X13 = A, S, Y, N, I, L, R, V, or K;; X14 = L, K, H, or I;; X16 = K, R, H, or V;; X20 = K, R, H, N;; X22 = F, A, P;; X25 = W, P, K, H, or I;; X26 = L or V. SEQIDN0:112 7FIM_V2/A13 YVEGTFTSDYSIALDKIAQKAFVQWLIAGGPSSGAPP

SEQIDN0:113 7FIM_V2/S 13 YVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPP

SEQIDN0:114 7FIM_V2/Y13 YVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPP

SEQIDN0.115 7FIM_V2/113 YVEGTFTSDYS11LDKIAQKAFVQWLIAGGPSSGAPP

SEQ1DNO:116 7FIMJC.R YVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPS

SEQ1DNO:117 7FIM_V2/Q13 YVEGTFTSDYSIQLDKIAQKAFVQWLIAGGPSSGAPP

SEQIDN0:118 7FIM_V2/L13 YVEGTFTSDYSILLDKIAQKAFVQWLIAGGPSSGAPP

SEQIDN0:119 7FIM V2/R13

YVEGTFTSDYSIRLDKIAQKAFVQWLIAGGPSSGAPP

SEQIDNO:120 7FIM_K2/V13 YKEGTFrSDYSIVLDKIAQKAFVQWLIAGGPSSGAPP SEQIDN0:121 7FIMJC2/K13 YKEGTFTSDYSIKLDKIAQKAFVQWLIAGGPSSGAPP SEQIDNO:122 7FIM R H YVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPS SEQIDNO:123 Cl YVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPS SEQ1DNO.124 Cl.l YVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPS SEQIDNO:125 C1.2 YVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPS SEQIDNO:126 C1.3 YVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQIDNO:127 C1.4 YVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQIDNO:128 Cl.4.1.1 YVEGTPCSDCSISLDHIAQKAPVQPVIAGGPSSGAPPPS SEQIDNO:129 Cl.4.1.2 YVEGTPCSDCSISLDKIAQHAPVQPVIAGGPSSGAPPPS SEQIDNO:130 Cl.4.1.3 YVEGTPCSDCSISLDKIAQKAPVQPVIAGGPSSGAPPPS SEQIDN0:131 Cl.4.2.1 YVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQIDNO:132 Cl.4.2.2 YVEGTSCSDCSISLDH1AQHAAVQPVIAGGPSSGAPPPS SEQ1DNO:133 Cl.4.2.3 YVEGTSCSDCSISLDHIAQHAAVQKVIAGGPSSGAPPPS SEQIDNO:134 Cl.4.2.4 YVEGTSCSDCSISKDHIAQHAAVQKVIAGGPSSGAPPPS SEQIDNO:135 Cl.4.2.5 YVEGTSCSDCSISKDKIAQHAAVQKVIAGGPSSGAPPPS SEQIDNO:136 Cl.4.2.6 YVEGTSCSDCS1SKDKIAQKAAVQKVIAGGPSSGAPPPS SEQIDNO:137 C 1.4.1 YVEGTSCSDCS1SHDKIAQKAAVQKVIAGGPSSGAPPPS SEQIDNO:138 C 1.4.2 YVEGTSCSDCS1SHDKIAQKAAVQHVIAGGPSSGAPPPS SEQIDNO:139 Cl.4.3 YVEGTSCSDCSISHDKIAQKAAVQIVIAGGPSSGAPPPS SEQIDNO:140 C 1.4.4 YVEGTSCSDCSISHDVIAQKAAVQIVIAGGPSSGAPPPS SEQIDNO:141 C 1.4.5 YVEGTSCSDCSISIDVIAQKAAVQIVIAGGPSSGAPPPS SEQIDNO:142 Cl.4.5.1 YVEGTSESDCSISIDVIAQKAAVQIVIAGGPSSGAPPPS SEQIDNO:143 Cl.4.5.2 YVEGTSCSDESISIDVIAQKAAVQIVIAGGPSSGAPPPS SEQIDNO:144 Cl.4.5.3 YVEGTSESDESISIDVIAQKAAVQIVIAGGPSSGAPPPS SEQ ID NO: 145 Cl.4.5.1.1 YVEGTSESDCSISIDVIAQNAAVQIV1AGGPSSGAPPPS

SEQ ID NO: 146 CI.4.5.2.1 YVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPS

SEQ ID NO: 147 Cl.4.5.3.1 YVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPS

SEQ ID NO: 148 7FIM_V2/S13_2x YVEGTFTSDYSISLDKIAQKAFVQWL1AGGPSSGAPPXYVEGTFTSDYSISLDKIA QKAFVQWLIAGGPSSGAPP

SEQ ID NO: 149 7FIM_V2/S 13_3x

YVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSISLDKIA QKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO:150 7FIM_V2/S13_4x

YVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSISLDKIA QKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPPXYV EGTFTSDYSISLDK1AQKAFVQWUAGGPSSGAPP SEQ ID NO: 151 7FIM_V2/S13_5x

YVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSISLDKIA QKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPPXYV EGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSISLDKIAQKAFVQW LIAGGPSSGAPP

SEQ ID NO: 152 7FIM_V2/Y13_2x

YVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIYLDKIA QKAFVQWLIAGGPSSGAPP

SEQ ID NO: 153 7FIM_V2/Y13_3x

YVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIYLDKIA QKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 154 7FIM_V2/Y13_4x

YVEGTFTSDYS1YLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIYLDKIA QKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIYLDKIAQKAFVQWL1AGGPSSGAPPXYV EGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPP

SEQ ID NO: 155 7FIM_V2/Y13_5x

YVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIYLDKIA QKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPXYV EGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIYLDKIAQKAFVQ WLIAGGPSSGAPP SEQ ID NO: 156 7FIM_V2/I13_2x

YVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQ KAFVQWLIAGGPSSGAPP SEQ ID NO.157 7FIM_V2/I13_3x

YVEGTFTSDYSHLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQ KAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 158 7FIM_V2/I13_4x

YVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQ KAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPPXYVE GTFTSDYS11LDKIAQKAFVQWLIAGGPSSGAPP

SEQ ID NO: 159 7FIM_V2/I13_5x

YVEGTFTSDYSIILDK1AQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQ KAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPPXYVE GTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQKAFVQWLI AGGPSSGAPP

SEQ ID NO: 160 7FIM_K ,R_2x

YVEGTFTSDYSISLDR1AQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRI AQRAFVQWLIAGGPSSGAPPPS

SEQ ID NO: 161 7FIM_K_R_3x

YVEGTFTSDYSISLDR1AQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRI AQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPP S

SEQ ID NO: 162 7FIM_K_R_4x

YVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDR1 AQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPP SXYVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPS

SEQ ID NO: 163 7FIM_K_R_5x

YVEGTFTSDYSISLDR1AQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRI AQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPP SXYVEGTFTSDYS1SLDRIAQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRIAQR AFVQWLIAGGPSSGAPPPS

SEQ ID NO:164 7FIM_V2/S13/H16/H20_2x

YVEGTFTSDYS1SLDHIAQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHI AQHAFVQWLIAGGPSSGAPPPS SEQ ID NO:165 7FIM_V2/S13/H16/H20_3x

YVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHI AQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPP S

SEQ ID NO:166 7FIM_V2/S13/H16/H20_4x

YVEGTFTSDYSISLDHIAQHAFVQWIJAGGPSSGAPPPSXYVEGTFTSDYSISLDHI AQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPP SXYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPS

SEQ ID NO: 167 7FIM_V2/S 13/H16/H20_5x

YVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHI AQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPP SXYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHIAQH APVQWLIAGGPSSGAPPPS

SEQ ID NO: 168 Cl_2x

YVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDHI AQHAPVQWLIAGGPSSGAPPPS

SEQ ID NO: 169 C1 3x

YVEGTPTSDYSISLDHIAQHAPVQWLTAGGPSSGAPPPSXYVEGTPTSDYSISLDHI AQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPP S

SEQ IDNO:170 Cl_4x

YVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDHI AQHAPVQWLTAGGPSSGAPPPSXYVEGTPTSDYSISLDH1AQHAPVQWLIAGGPSSGAPPP SXYVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPS

SEQ ID N0.171 Cl_5x

YVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDHI AQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPP SXYVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDHIAQH APVQWLIAGGPSSGAPPPS

SEQ ID NO: 172 Cl.l_2x

YVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHI AQHAPVQWLIAGGPSSGAPPPS

SEQ ID NO: 173 Cl.l_3x

YVEGTPTSDCSISLDH1AQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDH1 AQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPP S

SEQ ID NO:174 Cl.l_4x

YVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHI AQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPP SXYVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPS

SEQ IDNO:175 Cl.l_5x

YVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHI AQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDH1AQHAPVQWLIAGGPSSGAPPP SXYVEGTPTSDCS1SLDHIAQHAPVQWL1AGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQH APVQWLIAGGPSSGAPPPS

SEQ ID NO:176 C1.2_2x

YVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHI AQHAPVQPLIAGGPSSGAPPPS

SEQ ID NO:177 C1.2_3x

YVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHI AQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPS SEQ ID NO:178 C1.2_4x

YVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHI AQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPS XYVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPS

SEQ ID NO:179 C1.2_5x

YVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHI AQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPS XYVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHA PVQPLIAGGPSSGAPPPS

SEQ ID NO:180 C1.3_2x

YVEGTPTSDCSISLDH1AQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHI AQHAPVQPVIAGGPSSGAPPPS

SEQ ID NO:181 C1.3_3x

YVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHI AQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:182 C1.3_4x

YVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDin AQHAPVQPV1AGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS XYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS

SEQ IDNO:183 C1.3_5x

YVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHI AQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS XYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHA PVQPVIAGGPSSGAPPPS

SEQ IDNO:184 CL4_2x

YVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHI AQHAPVQPV1AGGPSSGAPPPS

SEQ IDNO:185 C1.4_3x

YVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHI AQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ IDNO:186 C1.4_4x

YVEGTPCSDCSISLDHIAQHAPVQPVTAGGPSSGAPPPSXYVEGTPCSDCSISLDHI AQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS XYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS

SEQ IDNO:187 C1.4_5x

YVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHI AQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS XYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHIAQHA PVQPVIAGGPSSGAPPPS

SEQ ID NO:188 C1.4.2.1_2x

YVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHI AQHAPVQPVIAGGPSSGAPPPS

SEQ 1D NO:189 C1.4.2.1_3x

YVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHI AQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO: 190 Cl .4.2. l_4x

YVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHI AQHAPVQPV1AGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS XYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS

SEQ ID NO:191 C1.4.2.1_5x

YVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHI AQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS XYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHA PVQPVIAGGPSSGAPPPS

SEQ ID NO: 192 C1.4.2.2_2x

YVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHI AQHAAVQPVIAGGPSSGAPPPS

SEQ ID NO: 193 C1.4.2.2_3x

YVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHI AQHAAVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPP S

SEQ ID NO:194 C1.4.2.2_4x

YVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHI AQHAAVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPP SXYVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPS SEQ ID NO: 195 C1.4.2.2_5x

YVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDH1 AQHAAVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPP SXYVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQH AAVQPVIAGGPSSGAPPPS

SEQ ID NO:196 C1.4.5.1.1_2x

YVEGTSESDCS1SIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIA QNAAVQIVIAGGPSSGAPPPS

SEQ ID NO:197 C1.4.5.1.I_3x

YVEGTSESDCSISIDVIAQNAAVQTVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIA QNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSIS1DVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO:198 C1.4.5.1.1_4x

YVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIA QNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIAQNAAVQ1VIAGGPSSGAPPPSX YVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPS

SEQ ID NO:199 C1.4.5.1.1_5x

YVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIA QNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPSX YVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIAQNAAV QIVIAGGPSSGAPPPS SEQ ID N0:200 C1.4.5.2.1_2x

YVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESISIDVIA QNAAVQJVIAGGPSSGAPPPS

SEQ ID NO:201 Cl.4.5.2. l_3x

YVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESISIDV1A QNAAVQ1VIAGGPSSGAPPPSXYVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO:202 Cl.4.5.2. l_4x

YVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESISIDVIA QNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPSX YVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO:203 C1.4.5.2.1_5x

YVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESISIDVIA QNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPSX YVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESISIDVIAQNAAV QIVIAGGPSSGAPPPS

SEQ ID NO:204 C1.4.5.3.1_2x

YVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDESISIDVIA QNAAVQIVIAGGPSSGAPPPS

SEQ ID NO:205 C1.4.5.3.1_3x

YVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDESISIDVIA QNAAVQIVIAGGPSSGAPPPSXYVEGTSESDES1SIDVIAQNAAVQ1VIAGGPSSGAPPPS SEQ ID NO:206 Cl.4.5.3. l_4x

YVEGTSESDESISroVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDESISIDVTA QNAAVQIVIAGGPSSGAPPPSXYVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPSX YVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPS

SEQ ID NO:207 C1.4.5.3.1_5x

YVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDESISIDVIA QNAAVQIVIAGGPSSGAPPPSXYVEGTSESDESTSIDVIAQNAAVQIVIAGGPSSGAPPPSX YVEGTSESDESISIDVIAQNAAVQIV1AGGPSSGAPPPSXYVEGTSESDESISIDVIAQNAAV QIVIAGGPSSGAPPPS

SEQ ID NO.-208 Tirzepatide (7FIM)

YAEGTFTSDYSIALDKIAQKAFVQWLIAGGPSSGAPP

SEQ ID NO:209 PPeeppttiiddee__2200 HXQGTFTSDKSKYLDERAAQDFVQWLLDGG PSSGAPPPS (X= AIB) SEQ ID NO:210 Peptide_19 YXEGTFTSDYSIYLDKQAAXEFVNWLLAGGPSAPPPSK (X=AIB) SEQ ID N0:211 Semaglutide HXEGTFTSDVSSYLEGQAAKEFTAWLVRGRG SEQ ID NO:212 Exendin-4 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS SEQ ID NO:213 7DTY P (GIP)

YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ

SEQ ID NO:214 MMEEDDII77221199 HaEGSfTSDVsSKLEGEAAkEflAKWEGG (lower case is alpha-methyl, US_2018_0162920_Al_263)

SEQ ID NO:215 JJ221111 HaEGSfTSDVsSfLEGEAAkEflAfWkGG (US_2018_0162920_Al_2) SEQ ID NO:216 JJ222299 HaEGSfTSDVsSFLEGEAAkEflAFWKGG

(US_2018_0162920_A1_2)

SEQ ID NO:217 wtGLP-1 haegtftsdvssylegqaakefiawlvkgrg SEQ ID NO:218 hGLPIR

SEQ ID NO:219 cGLPIR magapsplclallllgavgragprpqgatvslsetvqkwreyrhqcqrflteapppatglfcnrtfdeyacwpdglpgsfvnv scpwylpwassvlqghvyrfctaeglwlrqdnsslpwmlseceeskrgersspeeqllsfsiiytvgytlsfsalviasaillsfrhlhctm yihlnlfasfilralsvfirdavlkwmystapqqhqwdgllsyqdslgcrlvfllmqycvaanyywllvegvylytllafsvfseqrifrlyl sigwgvpllfvipwgivkylyedegcwtrnsnmnywliirlpilfaigvnflifvrvicivvsklkanhncktdikcrlakstltlipllgthe wfafvmdehargtlrfiklftelsftsfqglmvailycfvnnevqmefrrswerwrlkhlhiqrdssmkplkcptssltsggtvgssvyaa scqascs

SEQ ID NO:220 hGIPR

RNEVKAIWWIIRTPILMTILINFLIFIRILGILLSKLRTRQMRCRDYRLRLARSTLTLVPLLG VHEWFAPVTEEQARGALRFAKLGFEIFLSSFQGFLVSVLYCFINKEVQSEIRRGWHHCR LRRSLGEEQRQLPERAFRALPSGSGPGEVPTSRGLSSGTLPGPGNEASRELESYC SEQ ID NO:221 cGIPR MPNGPPWQLFLPLLWSWGPLLRRAETGSVGQTAGELYQRWERYRRECRETLEA VDPPAGLACNGSFDMYVCWDYAAPNATARASCPWYLPWHSHVATGFVLRHCGSDGQ WGPWRDHSQCEDPEKNGAFQDQRLILERLQVMYTVGYSVSLATLLLALLILSFFRRLRC TRNYIHINLFTSFMLRAAAILTRDRLLPPPGPYPGDQAPVLWKPALAACRTAQIVTQYCV GANYTWLLVEGVYLHSLLVLVGGSEGGHFRCYVFLGWGAPALFVIPWVIVRYLYENTQ CWERNEVKAIWWIIRTPILVTISINFLIFIRILGILVSKLRTRQMRCPDYRLRLARSTLTLVP LLGVHEWFAPVTEEQARGALRFAKLGFEIFLSSFQGFLVGWYCFVNKEVQAEIRRCW HRCRLRHSLGEERRQPPERASRTPPTGSGPRPVATDRTPSLGALPGPGNEASRGLESHC SEQ ID NO:222 rGIPR MPLRLLLLLLWLWGLSLQRAETDSEGQTTGELYQRWERYGWECQNTLEATEPP SGLACNGSFDMYACWNYTAANTTARVSCPWYLPWYRQVAAGFVFRQCGSDGQWGSW RDHTQCENPEKNGAFQDQKLILERLQVVYTVGYSLSLATLLLALLILSLFRRLHCTRNYI HMNLFTSFMLRAGAILTRDQLLPPLGPYTGNQTPTLWNQALAACRTAQILTQYCVGANY TWLLVEGVYLHHLLWVRRSEKGHFRCYLLLGWGAPALFVIPWVTVRYLYENTQCWER NEVKAIWWIIRTPILITILINFLIFIRILGILVSKLRTRQMRCPDYRLRLARSTLTLMPLLGVH EWFAPVTEEQAEGSLRFAKLAFEIFLSSFQGFLVSVLYCFINKEVQSE1RRLRLSLQEQCP RPHLGQAPRAVPLSSAPQEAAIRNALPSGMLHVPGDEVLESYC SEQ ID NO:223 mGIPR MPLRLLLLLLWLWGLQWAETDSEGQTTTGELYQRWEHYGQECQKMLETTEPPS GLACNGSFDMYACWNYTAANTTARVSCPWYLPWFRQVSAGFVFRQCGSDGQWGSWR DHTQCENPEKNGAFQDQTLILERLQIMYTVGYSLSLTTLLLALLILSLFRRLHCTRJNYIHM NLFTSFMLRAAAILTRDQLLPPLGPYTGDQAPTPWNQALAACRTAQIMTQYCVGANYT WLLVEGVYLHHLLVIVGRSEKGHFRCYLLLGWGAPALFVIPWVrVRYLRENTQCWERN EVKAIWWIIRTPILITILINFLIFIRILGILVSKLRl'RQMRCPDYRLRLARSTLTLVPLLGVHE WFAPVTEEQVEGSLRFAKLAFEIFLSSFQGFLVSVLYCFINKEVQSEIRQGWRHRRLRLS LQEQRPRPHQELAPRAVPLSSACREAAVGNALPSGMLHVPGDEVLESYC SEQ ID NO:224 GGSS SEQ ID NO:225 GGSS GGSS SEQ ID NO:226 GGSS GGSS GGSS SEQ ID NO:227 GGSS GGSS GGSS GGSS SEQIDNO:228 GGSS GGSS GGSS GGSS GGSS SEQIDNO:229 GGSS GGSS GGSS GGSS GGSS GGSS SEQIDNO:230 GGSS GGSS GGSS GGSS GGSS GGSS GGSS SEQIDNO:231 GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS SEQIDNO:232 GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS SEQIDNO:233 GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS SEQIDNO:234 GSGGS SEQIDNO:235 GSGGS GSGGS SEQIDNO:236 GSGGS GSGGS GSGGS SEQ ID NO:237 GSGGS GSGGS GSGGS GSGGS SEQIDNO:238 GSGGS GSGGS GSGGS GSGGS GSGGS SEQIDNO:239 GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS SEQIDNO:240 GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS SEQIDNO:241 GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS SEQIDNO:242 GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS SEQIDNO:243 GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS

GSGGS

SEQIDNO:244 GGGS SEQIDNO:245 GGGSGGGS SEQIDNO:246 GGGS GGGS GGGS SEQIDNO:247 GGGS GGGS GGGS GGGS SEQIDNO:248 GGGS GGGS GGGS GGGS GGGS SEQIDNO:249 GGGS GGGS GGGS GGGS GGGS GGGS SEQTDNO:250 GGGS GGGS GGGS GGGS GGGS GGGS GGGS SEQIDNO:251 GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS SEQIDNO:252 GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS SEQ ID NO:253 GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS SEQIDNO:254 GGSG SEQIDNO:255 GGSGGGSG SEQIDNO:256 GGSG GGSG GGSG SEQIDNO:257 GGSG GGSG GGSG GGSG SEQIDNO:258 GGSG GGSG GGSG GGSG GGSG SEQ ID NO:259 GGSG GGSG GGSG GGSG GGSG GGSG SEQ ID NO:260 GGSG GGSG GGSG GGSG GGSG GGSG GGSG SEQIDNO:261 GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG SEQIDNO:262 GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG SEQ ID NO:263 GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG SEQ ID NO:264 GGSGG SEQ ID NO:265 GGSGG GGSGG

SEQ ID NO:266 GGSGG GGSGG GGSGG SEQIDNO:267 GGSGG GGSGG GGSGG GGSGG SEQIDNO:268 GGSGG GGSGG GGSGG GGSGG GGSGG SEQIDNO:269 GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG SEQIDNO:270 GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG SEQIDNO:271 GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG SEQIDNO:272 GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG

SEQ ID NO:273 GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG

SEQ ID NO:274 GSGSG SEQ ID NO:275 GSGSG GSGSG SEQIDNO:276 GSGSGGSGSGGSGSG SEQIDNO.-277 GSGSG GSGSG GSGSG GSGSG SEQIDNO:278 GSGSG GSGSG GSGSG GSGSG GSGSG SEQ ID NO:279 GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG SEQ ID NO:280 GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG SEQ ID NO:281 GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG SEQ ID NO:282 GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG SEQIDNO:283 GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG

SEQ ID NO:284 GSGGG SEQ ID NO:285 GSGGG GSGGG SEQ ID NO:286 GSGGG GSGGG GSGGG SEQ ID NO:287 GSGGG GSGGG GSGGG GSGGG SEQ ID NO:288 GSGGG GSGGG GSGGG GSGGG GSGGG SEQIDNO:289 GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG SEQ ID NO:290 GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG SEQIDNO:291 GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG SEQ ID NO:292 GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG

SEQ ID NO:293 GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG

SEQIDNO:294 GGGSG

SEQIDNO:295 GGGSG GGGSG

SEQIDNO:296 GGGSG GGGSG GGGSG

SEQIDNO:297 GGGSG GGGSG GGGSG GGGSG

SEQIDNO:298 GGGSG GGGSG GGGSG GGGSG GGGSG

SEQ ID NO:299 GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG

SEQ ID N0:300 GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG

SEQIDNO:301 GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG

SEQIDNO:302 GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG

GGGSG

SEQIDNO:303 GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG

GGGSG GGGSG

SEQIDNO:304 GSSSG

SEQIDNO:305 GSSSG GSSSG

SEQIDNO:306 GSSSG GSSSG GSSSG

SEQIDNO:307 GSSSG GSSSG GSSSG GSSSG

SEQIDNO:308 GSSSG GSSSG GSSSG GSSSG GSSSG

SEQIDNO:309 GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG

SEQIDNO:310 GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG

SEQIDN0:311 GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG

SEQIDNO:312 GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG

SEQIDNO:313 GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG

GSSSG

SEQIDNO:314 GSSSS

SEQIDNO:315 GSSSS GSSSS

SEQIDNO:316 GSSSS GSSSS GSSSS

SEQIDNO.-317 GSSSS GSSSS GSSSS GSSSS

SEQIDNO:318 GSSSS GSSSS GSSSS GSSSS GSSSS

SEQIDNO:319 GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS SEQ ID NO:320 GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS

SEQIDNO:350 GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG

GGSAAAGG GGSAAAGG GGSAAAGG

SEQIDNO:35I GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG

GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG

SEQ ID NO:352 GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG

GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG

SEQIDNO:353 DPIYMTDNDKP1T Sdytag SEQIDNO:354 STVPVAPPRRRRG SI-13 SEQ ID NO:355 KLGD1EFIKVNK Snooptag SEQIDNO:356 SLAELLNAGLGGS Softag 1 SEQIDNO:357 TQDPSRVG Softag 3 SEQIDNO:358 PDRVRAVSHWSS Spot-tag SEQIDNO:359 AHIVMVDAYKPTK Spytag SEQ ID NO:360 KETAAAKFERQHMDS S-tag SEQ ID NO:361 WSHPQFEK Strep-tag SEQIDNO:362 MASMTGGQQMG T7tag SEQIDNO:363 EVHTNQDPLD TC-tag SEQIDNO:364 CCPGCC Ty-tag SEQIDNO:365 YTDIEMNRLGK VSV-tag SEQIDNO:366 DLYDDDDK Xpress-tag SEQ ID NO:367 VSGWRLFKKIS HiBit SEQ ID NO:368 YAEGTFISDYSIAMDKIRQQDFVNWLLAQKGKKNDWK

[234] Embodiments of engineered Glucagon-Like Peptide 1 :

I . An engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of Formula 1 (SEQ ID NO: 1):

HGEGTSESDVSXI₂XBXI4EGQAAQEX22X23AX₂5 X26VDGX3O(I) wherein X12 = S or Q; X12 = S, Q, or Y; Xi4 = lor L; X22 = V, I, or F; X23 " V or 1; X₂$ = V, I, or W ; X26 = I or V; and X₃₀ - R or S or of Formula III (SEQ ID NO: 382):

Xi G E G T S E S D V S XUXBXME X_lt Q A X_l9 X20 E X22X23 A X25X26 V D G X30 (III), wherein Xj = H or Y; X12 = S, Q, A or M; XB ~ S, Q, Y, or M; X14 = 1 or L; Xu = G or M; X19 A or T; X20 = Q, V, or D; X22 = V, I, or F; X23 “ V or I; X25 « V, 1, or W; X26 = I or V; and X_M == R or S. 2. The eGLP-1 of embodiment 1 , wherein Xu = Q; Xia = S; Xu = I or L; X22 = V, I, or F; X23 = V or I; X25 = V, I, or W; X26 = I or V; and X» = R.

3. The eGLP-1 of embodiment 1 comprising the amino acid sequence of any one of SEQ ID NO:1 to SEQ ID NO: 14, or of SEQ ID NO:372 to SEQ ID NO:380.

4. The eGLP- 1 polypeptide of embodiment 1 , wherein the polypeptide comprises a multimer of two, three, four, or five copies of an eGLP-1 polypeptide of Formula I or of Formula III

5. The eGLP-1 polypeptide of embodiment 4, wherein the polypeptide comprises an amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67.

6. The eGLP-1 of any one embodiments 1 to 5, wherein the eGLP-1 comprises a C-terminal amide;

7. The eGLP-1 of any one embodiments 1 to 6, wherein the eGLP-1 comprises one or more alphamethyl amino acids.

8. The eGLP-1 of any one embodiments 1 to 7, wherein the eGLP-1 further comprises a lipid moiety covalently bonded to the amino terminus, carboxy terminus, or an amino acid of Formula I.

9. The eGLP-1 of any one embodiments 1 to 8, wherein the eGLP-1 is substantially resistant to proteolytic degradation.

10. The eGLP- 1 of embodiment 9, wherein the eGLP- 1 is substantially resistant to DPP-IV, neprilysin, a- chymohypsin, trypsin, elastase, or pepsin, degradation.

11. The eGLP-1 of embodiment 9, wherein the eGLP-1 is substantially resistant to DPP-TV and neprilysin degradation.

12. The eGLP- 1 of embodiment 9, wherein the eGLP-1 is substantially resistant to a-chymotrypsin, trypsin, elastase, or pepsin, degradation.

13. The eGLP- 1 of any one of embodiments 1 to 13, wherein the eGLP-1 at least maintains substantially the same receptor potency as the corresponding wild-type, unmodified GLP-1 .

14. The eGLP-1 of any one of embodiments 1 to 14, wherein the eGLP-1 at least maintains substantially the same receptor selectivity as the corresponding wild-type, unmodified GLP-1. 15. The eGLP-1 of any one of embodiments 1 to 14, wherein the eGLP-1 exhibits increased receptor potency over the corresponding wild-type, unmodified GLP-1.

Polynucleotides

16. A polynucleotide encoding an engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of Formula I (SEQ ID NO: 1)

H G E G T S E S D V S XIZXBXUE G Q A A Q E X22X23 A X25X26 V D G X30 (D wherein X12 - S or Q; Xu ” S, Q, or Y; Xu - 1 or L; X22 ^=: V, I, or F; X23 = V or I; X23 == V, I, or W; X?s = I or V; and X30 - R or S, or of Formula III (SEQ ID NO: 382):

Xt G E G T S E S D V S X12X13X14E Xi_S Q A X19 X20 E X22X23 A X25X24 V D G X30 (III), wherein X> = H or Y; X12 - S, Q, A or M; X13 = S, Q, Y, or M; Xu = I or L; Xu” G or M; X19 = A or T; X2o= Q, V, or D; X22 = V, I, or F; X23 = V or I; X25 V, L or W; X_I6 = I or V; and X30 == R or S.

17. The polynucleotide of embodiment 16, wherein said polynucleotide is isolated.

18. An engineered vector comprising a polynucleotide encoding an engineered Glucagon-Like Peptide 1 (eGLP-1 ) comprising the amino acid sequence of Formula 1

H G E G T S E S D V S Xi₂X!3Xt₄E G Q A A Q E X22X23 A X25X26V D G X30 (1) wherein X.2 = S or Q; X12 - S, Q, or Y; Xu = I or L; X22 = V, I, or F; X23 = V or I; X25 = V, I, or W; X₂₆ = I or V; and X30 = Ror S, or of Formula III (SEQ ID NO: 382):

Xi G E G T S E S D V S X.ZXB XUE X_l6 Q A X_l9 X₂₀ E X22X23 A X25X26 V D G X30 (III), wherein Xi = H or Y; Xu ~ S, Q, A or M; Xn = S, Q, Y, or M; Xu = I or L; Xu= G or M; X19 - A or T; X20 = Q, V, or D; X22 » V, 1, or F; X_2} = V or I; X25 “ V, 1, or W; X₂₆ = 1 or V; and X30 - R or S.

19. A nucleic acid expression cassette comprising one or more of: a nucleic acid sequence comprising a transcription promoter; a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of Formula

I:

H G E G T S E S D V S X,₂XI₃XM E G Q A A Q E X22X23 A X25X26 V D G X30 (I) wherein X12 =" S or Q; X12 = S, Q, or Y; Xu - 1 or L; X22 = V, I, or F; X23 - V or I; X25 = V, I, or W; X26 - 1 or V; and X30 - R or S, or of Formula HI (SEQ ID NO: 382): Xi G E G T S E S D V S XUXIJXME X_t« Q A X_!9 X₂₀ E X22X23 A X25X26 V D G X30 (HI), wherein Xi = H or Y; Xu == S, Q, A or M; Xu =- S, Q, Y, or M; Xu = I or L; Xie ~ G or M;

Xi₉ = A or T; X_M- Q, V, or D; X22 - V, 1, or F; X₂₎ = V or I; X25 = V, I, or W; Xz₆ « 1 or V; and X30 = R or S; a nucleic acid sequence comprising a translation terminator; and a nucleic acid sequence comprising transcription terminator.

20. The nucleic acid expression cassette of embodiment 19, wherein said polypeptide further comprises one or more in-frame amino acid sequences comprising a secretion signal sequence, peptide tag, or combination thereof.

21. A method of making an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide comprising the amino acid sequence of Formula I,

H G E G T S E S D V S XuXuXuE G Q A A Q E X22X23 A X2sX26 V D G X30 (D wherein Xu = S or Q; Xu ~ S, Q, or Y; Xu - 1 or L; X22 = V, I, or F; X23 ~ V or I; X25 - V, 1, or W; Xzt = I or V; and X30 = R or S, or of Formula HI (SEQ ID NO: 382):

Xi G E G T S E S D V S X12 X13X14E XI₆ Q A X₁₉ X20 E X22X23 A X25X26 V D G X30 (HI), wherein Xi = H or Y; Xu = S, Q, A or M; X13 - S, Q, Y, or M; Xu = I or L; Xu= G or M; X,₉ = A or T; X20 = Q, V, or D; X₂₂ = V, I, or F; X23 = V or 1; X₂₅ = V, I, or W; Xze = I or V; and X30 = R or S; said method comprising culturing a host cell transformed with an expression vector encoding said eGLP-1 polypeptide under conditions allowing expression of said eGLP-1 polypeptide, and recovering said eGLP-1 polypeptide.

22. A pharmaceutical composition comprising an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide comprising the amino acid sequence of Formula 1

H G E G T S E S D V S Xi₂X₁₃Xi4E G Q A A Q E X_?2X23 A XI₅X₂₅V D G X3O (I) wherein Xu = S or Q; Xu =• S, Q, or Y; Xu = I or L; X22 ” V, I, or F; X23 = V or I; X25 = V, I, or W; X₂₆ = I or V; and XM = R or S; or of Formula III (SEQ ID NO: 382):

X; G E G T S E S D V S X_i2 Xu X_!4 E Xi₆ Q A X_l9 X20 E X22 X23 A X25 X 26 V D G X₃₀ (HI), wherein Xi = H or Y; Xu = S, Q, A or M; X13 = S, Q, Y, or M; Xu = I or L; Xu = G or M; X19 = A or T; X20 = Q, V, or D; X22 = V, I, or F; X23 - V or I; X25 = V, I, or W; X26 = I or V; and X30 = R or S; and a carrier. 23. A pharmaceutical composition comprising a recombinant host cell comprising a polynucleotide encoding an engineered Glucagon-Like Peptide 1 (eGLP-1).

24. The pharmaceutical composition of embodiment 23, further comprising a carrier.

25. A host cell comprising a polynucleotide encoding an engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of Formula 1

HGEGTSESDVSXi2X₁₃Xi4EGQAAQEX22X2₃AX₂₅ X26VDGX₃o(I) wherein X12 ™ S or Q; X12 - S, Q, or Y; Xu = I or L; X22 - V, I, or F; X23 = V or I; X25 ~ V, I, or W; X26 - I or V; and X_M = R or S, or of Formula III (SEQ ID NO: 382):

X, G E G T S E S D V S X12X13 X₁₄E Xu Q A X₁₉ Xzo F. X22X23 A X25X26 V D G X_w (Ill), wherein Xi = H or Y; X12 - S, Q, A or M; X_i3 = S, Q, Y, or M; X_!4 = I or L; X,₆ = G or M; Xi₉ » A or T; X₂₀ = Q, V, or D; X22 = V, 1, or F; X23 = V or I; X25 = V, I, or W; X26 = I or V; and XM = Ror S.

26. A host cell comprising a vector comprising a polynucleotide encoding an engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of Formula I

HGEGTSESDVSX_I2X_I3XUEGQAAQEX22 X23 AX25X26VDGX3O(I) wherein X_I2 = S or Q; Xi₂ - S, Q, or Y; X_l4 = I or L; Xn - V, I, or F; X₂₃ = V or I; X25 = V, I, or W; X₂₆ = I or V; and X_M = R or S, or of Formula III (SEQ ID NO: 382):

Xi G E G T S E S D V S XI₂X_!3XI₄E Xu Q A X_l9 X20 E X22 X2₃ A X25X26 V D G X_M (III), wherein Xi = H or Y; X12 - S, Q, A or M; Xn = S, Q, Y, or M; X_H = I or L; X_i6 = G or M; X19 « Aor T; X2o = Q, V, or D; X22 = V, I, or F; X₂₃ = Vor I; X25 = V J, or W; X26 - 1 or V; and X30 = Ror S.

27. A transformed cell comprising a nucleic acid expression cassette comprising one or more of: a nucleic acid sequence comprising a transcription promoter; a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of Formula I:

H GEGT S E S D VSXi2XnXi₄E GQ A AQ EX₂2 X23 AX25X26 VDG X₃o (I) wherein X12 “ S or Q; X12 = S, Q, or Y; Xu = I or L; X22 = V, I, or F; Xz₃ -≡ V or I; X25 = V, I, or W; X₂₆ = I or V; and XJO - R or S, or of Formula III (SEQ ID NO: 382): XiGEGTSESDVS XnXisXi₄E X₁₆ Q A X_l9 X20 E X22 X₂₃ A X25X26 V D G X₃₀ (III), wherein X> = H or Y; Xu = S, Q, A or M; Xu = S, Q, Y, or M; Xu = I or L; Xu = G or M;

Xi₉ = A or T; X₂₀- Q, V, or D; X22 = V, I, or F; Xia = V or I; XM = V, I, or W; X2₆ = I or V; and X30 = R or S; a nucleic acid sequence comprising a translation terminator; and a nucleic acid sequence comprising transcription terminator.

28. The transformed cell of embodiment 27, wherein said cell is a bacterial cell, plant cell, yeast cell, or algae cell.

29. The transformed cell of embodiment 26 or 28, wherein said cell comprises two or more copies of said recombinant nucleic acid.

30. The transformed cell of any one of embodiments 28 or 28, wherein said bacterial cell comprises an integration of said nucleic acid expression cassette into the bacterial chromosome.

31. The transformed cell of embodiment 30, wherein said integration in the bacterial chromosome is an integration into a transposase locus.

32. The transformed cell of embodiment 28, wherein said cell is a yeast cell.

33. The transformed cell of embodiment 32, wherein said yeast cell is a cell of a strain of Pichia pastoris.

34. The transformed cell of embodiment 28, wherein said cell is a bacterial cell of a bacterial genus selected from the group consisting of Bacillus, Lactobacillus, Salmonella, Lactococcus, Enterococcus.

35. The transformed cell of embodiment 28, wherein said transformed cell is a plant cell.

36. The transformed cell of embodiment 36, wherein said recombinant nucleic acid is integrated into the chloroplast of said plant cell.

37. The transformed cell of embodiment 36, wherein said recombinant nucleic acid is integrated into the genome of said plant cell.

38. A genetically engineered plant, or part thereof, comprising a recombinant nucleic acid encoding an engineered Glucagon-Like Peptide 1 (eGLP-1) according to embodiment 1. 39. The genetically engineered plant, or part thereof, of embodiment 38, wherein said part thereof is a plant seed.

40. The genetically engineered plant, or part thereof, of embodiment 38, wherein said recombinant nucleic acid is integrated into the chloroplast of said plant, or part thereof.

41. The genetically engineered plant, or part thereof, of embodiment 38, wherein said recombinant nucleic acid is integrated into the genome of said plant cell.

42. A kit comprising the pharmaceutical composition of any one of embodiments 22 to 41,

43. The kit of embodiment 42, comprising the host cell of embodiment 25 or 26, the transformed cell of any one of embodiments 27 to 37, or the genetically engineered plant, or part thereof, of any one of embodiments 38 to 41, and a carrier.

44. A method of treating or preventing a treatment of a condition caused or characterized by excess body weight, and the treatment of obesity, morbid obesity, obesity linked inflammation, obesity linked gallbladder disease, obesity induced sleep apnea, metabolic syndrome, pre-diabetes, insulin resistance, glucose intolerance, type 2 diabetes, type I diabetes, hypertension, atherogenic dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke or microvascular disease in a subject, comprising administering to a subject in need of treatment thereof an effective amount of the eGLP-1 of any one of embodiments 1 to 15, the pharmaceutical composition of embodiment 22, or the host cell of embodiment 25.

45. A method of treating or preventing a disease or condition caused or characterized by hypoglycemia or impaired insulin release, comprising administering to a subject in need of treatment thereof an effective amount of the eGLP-1 of any one of embodiments 1 to 15, the pharmaceutical composition of embodiment 22, or the host cell of embodiment 25.

46. The method of embodiments 44 or 45, wherein the disease or condition is diabetes or obesity.

47. The method of embodiment 46, wherein the disease or condition is type-2 diabetes.

48. The method of any one of embodiments 44 to 47, wherein the administration further improves glycemic control, provides body weight control, improves P-cell function and mass, reduces the rate of gastric acid secretion and gastric emptying, or any combination thereof. 49. The method of any one of embodiments 44 to 48, wherein the eGLP-1 or the pharmaceutical composition is administered orally, by injection, or transdermally.

50. The method of any one of embodiments 44 to 48, wherein the host cell is administered orally.

51. The method of any one of embodiments 44 to 47, wherein the eGLP-1 or a pharmaceutical composition thereof is administered orally.

52. The method of embodiment 49, wherein the injection is administered subcutaneously or intravenously.

53. The method of any one of embodiments 44 to 52, wherein the eGLP-1 or the pharmaceutical composition is administered once per day.

54. The method of any one of embodiments 44 to 53, further comprising administering one or more additional therapies.

55. The method of embodiment 54, wherein said additional therapy comprises blood sugar monitoring, diet modifications, exercise, insulin, a thiazolidinedione, a sulfonylurea, an incretin, metformin, a glyburide, a dipeptidyl peptidase 4 inhibitor, a bile acid sequestrant, or any combination thereof.

56. The method of any one of embodiments 44 to 55, wherein the subject is a human, a non-human primate, a cat, or a dog.

57. The method of any one of embodiments 44 to 56, wherein the subject is a cat.

58. A pharmaceutical composition comprising an engineered Glucagon-Like Peptide 1 of Formula I: H G E G T S E S D V S XHXB XUE G Q A A Q E X22X23 A X_2JX26V D G XJO (I) wherein Xu = S or Q; Xu = S, Q, or Y; XU = I or L; X22 = V, I, or F; X23 = V or I; X25 = V, I, or W; X26 = I or V; and X30 = R or S, or of Formula HI (SEQ ID NO: 382): Xi G E G T S E S D V S XUXBXUE XII Q AX19 X20 E X22X23 A X25X26 V D G X» (HI), wherein Xi = H or Y; Xu = S, Q, A or M; X13 = S, Q, Y, or M; Xu = I or L; Xu = G or M; X19 = A or T; X?.o= Q, V, or D; X22 = V, I, or F; X23 = V or I; X25 = V, I, or W; X26 = I or V; and X30 = R or S.

59. A pharmaceutical composition comprising an engineered Glucagon-Like Peptide 1 (eGLP-1) comprising an amino acid sequence selected from any of SEQ ID NO: 1 to SEQ ID NO: 14. 60. The pharmaceutical composition of embodiments 58 or 59, wherein said composition is a lyophilized composition of transformed plant cells or transformed bacterial cells.

61. A direct fed microbial (DFM) comprising bacteria transformed with a nucleic acid encoding an engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of Formula I: H G E G T S E S D V S XuXBXuE G Q A A Q E XzzXziA XziXzfiV D G Xw a) wherein Xu = S or Q; Xu = S, Q, or Y; Xu = I or L; Xu = V, I, or F; X23 = V or I; Xs = V, I, or W; Xz6 = I orV; and XM = R or S, or of Formula III (SEQ ID NO: 382):

Xi G E GT S E S D V S XuXuXuE X,_s Q A X9 X20 E X22X23 A X₅X₂₆V D G X30 (HI), wherein Xi = H or Y; Xu = S, Q, A or M; Xu = S, Q, Y, or M; Xu = I or L; Xu = G or M; X19 = A or T; X20- Q, V, or D; Xu - V, 1, orF; X23 = V or I; Xu = V, I, or W; X₂₆ = I or V; and X30 = R or S.

62. The direct fed microbial (DFM) of embodiment 61, wherein said eGLP-1 polypeptide comprising an amino acid sequence selected from any of SEQ ID NO: 1 to SEQ ID NO:14 or SEQ ID NO:32 to SEQ ID NO:67 or of SEQ ID NO: 372 to SEQ ID NO:380.

63. The direct fed microbial (DFM) of embodiment 61, wherein said eGLP-1 polypeptide comprising an amino acid sequence selected from any of SEQ ID NOs:2 to 14.

64. The direct fed microbial (DFM) of embodiment 61, wherein said eGLP-1 polypeptide comprising an amino acid sequence selected from any of SEQ ID NOs:3 to 14.

65. The DFM of one of embodiments 61 to 64, wherein said bacteria comprise a strain selected from the group consisting of Bacillus, Lactobacillus, Lactococcus, Salmonella, Enterococcus, and combinations thereof.

66. The DFM of embodiment 65, wherein said transformed bacteria is a strain of Bacillus subtilis.

67. The DFM of embodiment 66, wherein said transformed bacterial of Bacillus subtilis and said DFM further comprises one or more B. amyloliquefaciens strains.

68. The DFM of embodiment 66, wherein said transformed Bacillus subtilis bacterial strain and said DFM further comprises one or more anaerobic cellulolytic bacteria. 69. The DFM of embodiment 68, wherein said one or more anaerobic cellulolytic bacteria is a member of the phylum Bacillota.

70. The DFM of embodiment 69, wherein said member of the phylum Bacillota is a bacterial strain of Ruminococcus flavefaciens.

71. The DFM of embodiment 68, wherein said one or more anaerobic cellulolytic bacteria is a member of the genus Bacteroides.

72. The DFM of embodiment 71, wherein said member of the genus Bacteroides is Bacteroides fragilis.

73. The DFM of embodiment 63, wherein said bacterial strain is a strain of Lactobacillus.

74. The DFM of embodiment 73, wherein said strain of Lactobacillus is a strain of Lactobacillus gasseri.

75. The DFM of embodiment 73, wherein said strain of Lactobacillus is a strain of Lactobacillus plantarum.

76. The DFM of any one of embodiments 61 to 75, wherein said DFM is lyophilized.

[235] Embodiments of engineered dual agonist polypeptides:

Embodiment 1. An engineered polypeptide comprising the amino acid sequence of Formula II (SEQ ID NO:111):

Y Xz E G T X< X₇ S D Xio S I XB X_l4 D Xie I A Q Xzo A X22 V Q XM X₂< I A G G P S S G A P P (I) wherein

or of Formula IV (SEQ ID NO: 383): Y X2 E G T Xe X? S D Xio S I X13 X,₄ D X₁₆1 A X19 X20 A X22 V Q X25 X26 1 A G G P S S G A P P (IV), wherein X₂ - V or K; X₆ -F, P, or S; X₇ = T, C, or E; X_l0 = Y, C, or E; X,₃ = A, S, Y, N, I, L, R, V, or K; Xu = L, K, H, or I; X_l6 - K, R, H, orV; XJ₉ = Q orV; X₂₀= K, R, H, N; X₂₂ = F, A, P; X25 - W, P, K, H, or I; and X26 = L or V.

Embodiment 2. The engineered polypeptide of embodiment 1 , wherein

X₂ = V; X_e = P or S; X₇ = C or E; X_i0 = C or E; X13 = S; Xu = L, H, or l; X,₆ = H or V; X2o = H or N; X22 = A or P; X25 = P or I; and X₂« = L or V.

Embodiment 3. The engineered polypeptide of embodiment 1, wherein

X₂ = V; Xe =S; X? = C; X10 = C orE; X_n = S; Xu ⁵³1; X_t6 = V; X20 = H or N; X22 = A; X₂₅ = I; andX₂6 = V.

Embodiment 4. The engineered polypeptide of embodiment 1, further comprising a proline at position 38. Embodiment 5. The engineered polypeptide of embodiment 4, further comprising a serine at position 39. Embodiment 6. The engineered polypeptide of embodiment 1, comprising the amino acid sequence of any one of SEQ ID NO:111 to SEQ ID NO: 147 or of SEQ ID NO: 381.

Embodiment 7. The engineered polypeptide of embodiment 6, wherein the polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116.

Embodiment 8. The engineered polypeptide of embodiment 6, wherein the polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 142 to SEQ ID NO: 144.

Embodiment 9. The engineered polypeptide of embodiment 6, wherein the polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 137 to SEQ ID NO: 141.

Embodiment 10. The engineered polypeptide of embodiment 6, wherein the polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 127 to SEQ ID NO: 136.

Embodiment 11. The engineered polypeptide of embodiment 6, wherein the polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 123 to SEQ ID NO: 127.

Embodiment 12. The engineered polypeptide of any one of embodiments 1 to 11 , wherein the engineered polypeptide comprises a C-terminal amide.

Embodiment 13. The engineered polypeptide of any one of embodiments 1 to 11, wherein the polypeptide comprises a multimer of two, three, four, or five copies of an engineered polypeptide of SEQ ID NO:111 to SEQ ID NO:122 or SEQ ID NO:123 to SEQ ID NO:147 or SEQ ID NO: 381.

Embodiment 14. The engineered polypeptide of embodiment 13, wherein the polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 148 to SEQ ID NO:207.

Embodiment 15. The engineered polypeptide of any one of embodiments 1 to 11 , wherein the engineered polypeptide comprises one or more alpha-methyl amino acids. Embodiment 16. The engineered polypeptide of any one of embodiments 1 to 11, wherein the engineered polypeptide further comprises a lipid moiety covalently bonded to the amino terminus, carboxy terminus, or an amino acid of Formula II (SEQ ID NO: 111) or of Formula IV (SEQ ID NO:383). Embodiment 17. The engineered polypeptide of any one of embodiments 1 to 11, wherein the engineered polypeptide is substantially resistant to proteolytic degradation.

Embodiment 18. The engineered polypeptide of embodiment 17, wherein the engineered polypeptide is substantially resistant to DPP-IV, neprilysin, a-chymotrypsin, trypsin, elastase, or pepsin, degradation.

Embodiment 19. The engineered polypeptide of any one of embodiments 1 to 16, wherein the engineered polypeptide at least maintains substantially the same receptor potency as the corresponding wild-type, unmodified polypeptide.

Embodiment 20. The engineered polypeptide of any one of embodiments 1 to 16, wherein the engineered polypeptide at least maintains substantially the same receptor potency as tirzepatide.

Embodiment 21. The engineered polypeptide of any one of embodiments 1 to 16, wherein the engineered polypeptide at least maintains substantially the same receptor selectivity as the corresponding wild-type, unmodified polypeptide.

Embodiment 22. The engineered polypeptide of any one of embodiments 1 to 16, wherein the engineered polypeptide exhibits increased receptor potency over the corresponding wild-type, unmodified polypeptide.

Embodiment 23. An isolated polynucleotide encoding an engineered polypeptide comprising the amino acid sequence of sequence of Formula II (SEQ ID NO:111): wherein X₂ = V or K; X₆ =F, P, or S; X₇ = T, C, or E; X_I0 = Y, C, or E; Xu = A, S, Y, N, I, L, R, V, or K; Xu = L, K, H, or I; X₁₆ = K, R, H, or V; X₂₀ = K, R, H, N; Xn = F, A, P; X25 = W, P, K, H, or I; and X₂₆ = L or V, or of Formula IV (SEQ ID NO: 383):

Y X₂ E G T X6 X₇ S D Xio S I Xu Xu D X_l<i I A Xi₉ X₂o A X₂₂ V Q X₂₅ X₂6 l A G G P S S G A P P (IV), wherein X₂ = V or K; Xe =F, P, or S; X₇ = T, C, or E; Xio - Y, C, or E; Xu = A, S, Y, N, I, L, R, V, or K; Xu = L, K, H, or I; Xu = K, R, H, or V; X19 = Q or V; X₂₀= K, R, H, N; X22 = F, A, P; X25 = W, P, K, H, or I; and XM = L or V.

Embodiment 24. An engineered vector comprising the polynucleotide of embodiment 23. Embodiment 25. A nucleic acid expression cassette comprising one or more of: a nucleic acid sequence comprising a transcription promoter; a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of Formula II (SEQ ID NO: 111): wherein X2 = V or K; X» =F, P, or S; X? = T, C, or E; Xw = Y, C, or E; Xu = A, S, Y, N, I, L, R, V, or K; X_l4 = L, K, H, or I; Xw - K, R, H, or V; X₂₀ - K, R, H, N; X22 = F, A, P; X25 = W, P, K, H, or I; and X26 = L or V, or of Formula IV (SEQ ID NO: 383):

YXZEGTXSXTSDXWSIXU X,₄D XW I AX19X20 AX22 VQ X₂₅ X₂₆1 A G GP S S G A PP (IV), wherein X₂ = V or K; Xs =F, P, or S; X₇ = T, C, or E; Xw = Y, C, or E; X₎₃ = A, S, Y, N, I, L, R, V, or K; X[₄ = L, K, H, or I; Xw = K, R, H, or V; X,₉ = Q or V; X2o= K, R, H, N; X22 = F, A, P; XJS = W, P, K, H, or I; and X2s = L or V; a nucleic acid sequence comprising a translation terminator; and a nucleic acid sequence comprising transcription terminator.

Embodiment 26. The nucleic acid expression cassette of embodiment 25, wherein said polypeptide further comprises one or more in-frame amino acid sequences comprising a secretion signal sequence, peptide tag, or combination thereof.

Embodiment 27. A method of making an engineered polypeptide comprising the amino acid sequence of Formula 11 (SEQ ID NO: 111)

Y X₂ E GT X4X7 S D Xw S IXu Xi₄ D Xie I A QX20 A X22 V Q Xzs X261 AG GP S S G A P P (I), wherein X₂ = V or K; X« =F, P, or S; X₇ = T, C, or E; Xw = Y, C, or E; X13 = A, S, Y, N, I, L, R, V, or K; X_l4 = L, K, H, or I; Xw = K, R, H, or V; X20 = K, R, H, N; X_tt = F, A, P; X₂₅ = W, P, K, H, or I; and X₂₆ "= L or V, or of Formula IV (SEQ ID NO: 383):

YX₂EGTX_sX₇SDXwSIXi3 Xi₄DXwIAXwX₂oAX₂₂VQX_2JX₂6lAGGPSSGAPP(IV), wherein X2 = V or K; X₆ =F, P, or S; X₇ = T, C, or E; Xw = Y, C, or E; X13 = A, S, Y, N, I, L, R, V, orK; X₁₄ = L, K, H, or I; Xw = K, R, H, or V; X₁₉ = Qor V; X_M= K, R, H, N; X22 = F, A, P; X25 = W, P, K, H, or I; and X₂₆ = L or V; said method comprising culturing a host cell transformed with an expression vector encoding an engineered polypeptide under conditions allowing expression of the engineered polypeptide, and recovering the engineered polypeptide.

Embodiment 28. A pharmaceutical composition comprising an engineered polypeptide comprising the amino acid sequence of Formula II (SEQ ID NO: 111) or Formula TV (SEQ ID NO:383) and a carrier. Embodiment 29. A pharmaceutical composition comprising a recombinant host cell comprising a polynucleotide encoding an engineered polypeptide of Formula II (SEQ ID NO: 111 ) or of Formula IV (SEQ ID NO: 383).

Embodiment 30. The pharmaceutical composition of embodiment 29, further comprising a carrier. Embodiment 31. A host cell comprising the polynucleotide encoding an engineered polypeptide comprising the amino acid sequence of Formula II (SEQ ID NO: 111) YX₂EGTX₆X7SDXIOSIX_U XI₄D Xu I AQ X20 AX22 V Q X₂< X₂₄1 AG GP S S G AP P (I), wherein X₂ - V or K; X₄ =F, P, or S; X₇ == T, C, or E; X_l0 = Y, C, or E; X₁₃ - A, S, Y, N, I, L, R, V, orK; XM = L, K, H, or I; X_]6 - K, R, H, or V; XM = K, R, H, N; X22 = F, A, P; X₂₅ - W, P, K, H, or I; and X₂₄ = L or V, or of Formula IV (SEQ ID NO: 383): YX₂EGTX₆X₇SDXioSIX_l3 Xu D X>₆1 AXI₉ XM AX₂₂ VQ X25 X₂₄1 A G G P S S G APP(IV), wherein X₂ = V or K; X₄ =F, P, or S; X₇ - T, C, or E; X_l0 = Y, C, or E; X_n = A, S, Y, N, I, L, R, V, or K; Xu = L, K, H, or I; X_]6 = K, R, H, or V; X_l9 = QorV; X20- K, R, H, N; X_M = F, A, P; X_2$ = W, P, K, H, or I; and X₂₆ = L or V.

Embodiment 32. A host cell comprising a vector comprising a polynucleotide encoding an engineered polypeptide comprising the amino acid sequence of Formula IT (SEQ ID NO: 111): YX₂EGTX₆X₇SDX|₀STXBXI4DXI₆IAQX₂CAX₂₂VQX_2SX₂₆IAGGPSSGAPP(I), wherein X₂ = V or K; Xe -F, P, or S; X₇ - T, C, or E; X10 - Y, C, or E; X_B = A, S, Y, N, I, L, R, V, or K; X14 = L, K, H, or I; X_l4 = K,R, H, or V; X20 “ K, R, H, N; X₂₂ = F, A, P; Xy - W, P, K, H, or I; and X₂₄ = L or V, or of Formula IV (SEQ ID NO: 383):

YX2EGTX6X₇SDXioSIX_l3 XUDXI₄IAXI₉X₂₀AX₂₂VQX₂₃ X₂₄ IAGGPSSG APP(IV), wherein X? = V or K; Xe -F, P, or S; X₇ = T, C, or E; X10 = Y, C, or E; X_l3 - A, S, Y, N, 1, L, R, V, or K; Xu - L, K, H, or I; X_l4 = K, R, H, dr V; X_l9 = Q or V; X₂₀- K, R, H, N; X₂₂ = F, A, P; Xy - W, P, K, H, or I; and X₂₆ = L or V.

Embodiment 33. A transformed cell comprising a nucleic acid expression cassette comprising one or more of: a nucleic acid sequence comprising a transcription promoter; a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of Formula II (SEQ ID NO:111): YX₂EGTX₆X₇SDX_loSIXi₃Xi4DX₁6lAQX₂oAX₂₂VQX_2SX₂6lAGGPSSGAPP(I), wherein X₂ = V or K; X₆ =F, P, or S; X₇ - T, C, or E; X,_o = Y, C, or E; X₁₃ - A, S, Y, N, I, L, R, V, or K; X,₄ = L, K, H, or I; X₎₆ = K, R, H, or V; XM = K, R, H, N; X₂₂ = F, A, P; X25 = W, P, K, H, or I; and X« - L or V, or of Formula IV (SEQ ID NO: 383):

YX2EGTX6X₇SDX_I0SIXi₃ X₁₄DX_l6lAX_l9X2oAX₂₂VQX₂₅ X₂61AGGPSSGAPP(IV), wherein X2 - V or K; X« -F, P, or S; X₇ - T, C, or E; X10 = Y, C, or E; X_l3 = A, S, Y, N, I, L, R, V, or K; Xu ⁼ L, K, H, or I; Xu = K, R, H, or V; X_]9 - Q or V; X₂₍r- K, R, H, N; X₂₂ = F, A, P; X2_$ - W, P, K, H, or I; andX₂₆ = L or V; a nucleic acid sequence comprising a translation terminator; and a nucleic acid sequence comprising transcription terminator.

Embodiment 34. The transformed cell of embodiment 33, wherein said cell is a bacterial cell, plant cell, yeast cell, or algae cell.

Embodiment 35. The transformed cell of embodiment 32 or 33, wherein said cell comprises two or more copies of said recombinant nucleic acid.

Embodiment 36. The transformed cell of any one of embodiments 32 to 35, wherein said cell comprises an integration in the bacterial chromosome.

Embodiment 37. The transformed cell of embodiment 36, wherein said integration in the bacterial chromosome is an integration into a transposase locus.

Embodiment 38. The transformed cell of any one of embodiments 32 to 35, wherein said cell is a yeast cell.

Embodiment 39. The transformed cell of embodiment 38, wherein said yeast cell is a cell of a strain of Pichia pastoris.

Embodiment 40. The transformed cell of any one of embodiments 32 to 35, wherein said cell is a bacterial cell of a bacterial genus selected from the group consisting of Bacillus, Lactobacillus, Lactococcus, Enterococcus.

Embodiment 41. The transformed cell of any one of embodiments 32 to 35, wherein said transformed cell is a plant cell.

Embodiment 42. The transformed cell of embodiment 41, wherein said recombinant nucleic acid is integrated into the chloroplast of said plant cell.

Embodiment 43. The transformed cell of embodiment 41, wherein said recombinant nucleic acid is integrated into the genome of said plant cell.

Embodiment 44. A genetically engineered plant, or part thereof, comprising a recombinant nucleic acid encoding an engineered polypeptide of Formula II (SEQ ID NO: 111)

Y X2 E G T Xs Xr 8 D Xio S I XB Xu D Xie 1 A Q Xzo A X22 V Q X25 X24 1 A G G P S S G A P P (I), wherein X2 = V or K; Xe =F, P, or S; X7 = T, C, or E; X10 = Y, C, or E; Xu = A, S, Y, N, I, L, R, V, or K; X14 = L, K, H, or I; X₁₆ = K, R, H, or V; Xro = K, R, H, N; Xz2 = F, A, P; X2$ = W, P, K, H, or I; and X₂e = L or V, or of Formula IV (SEQ ID NO: 383):

Y X₂ E G T X₆ X₇ S D X_!0 S I Xi3 Xu D Xi₆ I A Xi9 X20 A X22 V Q X₂₅ X26 l A G G P S S G A P P (IV), wherein X2 = V or K; Xe =F, P, or S; X? - T, C, or E; Xie = Y, C, or E; X13 - A, S, Y, N, I, L, R, V, or K; Xu = L, K, H, or I; X_l6 = K, R, H, or V; X₁₉ = Q or V; X2o= K, R, H, N; X22 = F, A, P; X_2$ = W, P, K, H, or I; and Xzt = L or V. Embodiment 45. The genetically engineered plant, or part thereof, of embodiment 44, wherein said part thereof is a plant seed.

Embodiment 46. The genetically engineered plant, or part thereof, of embodiment 44 or 45, wherein said recombinant nucleic acid is integrated into the chloroplast of said plant, or part thereof.

Embodiment 47. lire genetically engineered plant, or part thereof, of embodiment 46, wherein said recombinant nucleic acid is integrated into the genome of said plant cell.

Embodiment 48. A kit comprising the pharmaceutical composition of embodiment 28 or embodiment 29. Embodiment 49. The kit of embodiment 48, comprising the host cell of embodiment 31 and a carrier.

Methods

Embodiment 50. A method of treating or preventing a treatment of a condition caused or characterized by excess body weight, and the treatment of obesity, morbid obesity, obesity linked inflammation, obesity linked gallbladder disease, obesity induced sleep apnea, metabolic syndrome, prediabetes, insulin resistance, glucose intolerance, type 2 diabetes, type I diabetes, hypertension, atherogenic dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke or microvascular disease in a subject, comprising administering to a subject in need of treatment thereof an effective amount of the engineered polypeptide of any one of embodiments 1 to 22, the pharmaceutical compositions of embodiment 28 to 30, or cells of embodiments 31 to 47.

Embodiment 51. A method of treating or preventing a disease or condition caused or characterized by hypoglycemia or impaired insulin release, comprising administering to a subject in need of treatment thereof an effective amount of the engineered polypeptide of any one of embodiments 1 to 22, the pharmaceutical compositions of embodiment 28 to 30, or cells of embodiments 31 to 47.

Embodiment 52. The method of embodiment 50, wherein the disease or condition is diabetes or obesity.

Embodiment 53. The method of embodiment 52, wherein the disease or condition is type-2 diabetes.

Embodiment 54. The method of any one of embodiments 50 to 53, wherein the administration further improves glycemic control, provides body weight control, improves P-cell function and mass, reduces the rate of gastric acid secretion and gastric emptying, or any combination thereof.

Embodiment 55. The method of any one of embodiments 50 to 54, wherein the engineered polypeptide or the pharmaceutical composition is administered orally, by injection, or transdermally. Embodiment 56. The method of any one of embodiments 50 to 54, wherein the host cell is administered orally. Embodiment 57. The method of embodiment 50, wherein the engineered polypeptide or a pharmaceutical composition thereof is administered orally.

Embodiment 58. The method of embodiment 55, wherein the injection is administered subcutaneously or intravenously.

Embodiment 59. The method of any one of embodiments 50 to 58, wherein the engineered polypeptide or the pharmaceutical composition is administered once per day.

Embodiment 60. The method of any one of embodiments 50 to 59, further comprising administering one or more additional therapies.

Embodiment 61. The method of embodiment 60, wherein the additional therapy comprises blood sugar monitoring, diet modifications, exercise, insulin, a thiazolidinedione, a sulfonylurea, an incretin, metformin, a glyburide, a dipeptidyl peptidase 4 inhibitor, a bile acid sequestrant, or any combination thereof.

Embodiment 62. The method of any one of embodiments 50 to 61 , wherein the subject is a human, a non-human primate, a cat, or a dog.

Embodiment 63. A pharmaceutical composition comprising an engineered polypeptide comprising an amino acid sequence Formula II (SEQ ID NO:111).

Embodiment 64. A pharmaceutical composition comprising an engineered polypeptide comprising an amino acid sequence selected from any of SEQ ID NO: 112 to SEQ ID NO: 147 or of SEQ ID NO: 381. Embodiment 65. The pharmaceutical composition of embodiment 64, wherein said composition is a lyophilized composition of transformed plant cells or transformed bacterial cells.

Embodiment 66. A direct fed microbial (DFM) comprising bacteria transformed with a nucleic acid encoding an engineered polypeptide comprising the amino acid sequence of Formula II (SEQ ID NO: 111):

Y XZ E G T XS XT S D XIO S I XB X_H D X_I6 I A Q Xa A X22 V Q X25 X* I A G G P S S G A P P (I), wherein Xz = V or K; Xe =F, P, or S; X₇ = T, C, or E; X₁₀ = Y, C, or E; X_n = A, S, Y, N, I, L. R, V, or K; Xu - L, K, H, or I; Xi6 = K, R, H, or V; X_M = K, R, H, N; Xz2 = F, A, P; Xzs = W, P, K, H, or 1; and X₂« = L or V, or of Formula IV (SEQ ID NO: 383):

Y X₂ E G T Xs X₇ S D X10 S I X13 Xu D Xi» I A X19 X20 A X22 V Q X25 X₂« I A G G P S S G A P P (IV), wherein X₂ = V or K; X₆ =F, P, or S; X₇ = T, C, or E; Xw = Y, C, or E; XB = A, S, Y, N, I, L, R,

V, or K; Xu = L, K, H, or I; Xis = K, R, H, or V; X₁₉ = Q or V; Xa= K, R, H, N; X22 = F, A, P; X25 = W, P, K, H, or I; and X₂₆ = L or V. Embodiment 67. The direct fed microbial (DFM) of embodiment 66, wherein said engineered polypeptide comprising an amino acid sequence selected from any of SEQ ID NO: 112 to SEQ ID NO: 17 or SEQ ID NO: 148 to SEQ ID NO:207, or of SEQ ID NO: 381.

Embodiment 68. The DFM of embodiment 66, wherein said bacteria comprise a strain selected from the group consisting of Bacillus, Lactobacillus, Lactococcus, Enterococcus, and combinations thereof.

Embodiment 69. The DFM of embodiment 68, wherein said transformed bacteria is a strain of

Bacillus subtilis.

Embodiment 70. The DFM of embodiment 68, wherein said transformed bacterial of Bacillus subtilis and said DFM further comprises one or more B. amyloliquefaciens strains.

Embodiment 71. The DFM of embodiment 68, wherein said transformed bacterial of Bacillus subtilis and said DFM further comprises one or more anaerobic cellulolytic bacteria.

Embodiment 72. The DFM of embodiment 68, wherein said one or more anaerobic cellulolytic bacteria is a member of the phylum Bacillota.

Embodiment 73. The DFM of embodiment 72, wherein said member of the phylum Bacillota is a bacterial strain of Ruminococcus flavefaciens.

Embodiment 74. The DFM of embodiment 68, wherein said one or more anaerobic cellulolytic bacteria is a member of the genus Bacteroides.

Embodiment 75. The DFM of embodiment 74, wherein said member of the genus Bacteroides is

Bacteroides fragilis.

Embodiment 76. The DFM of embodiment 68, wherein said bacterial strain is a strain of

Lactobacillus.

Embodiment 77. The DFM of embodiment 76, wherein said strain of Lactobacillus is a strain of

Lactobacillus gasseri.

Embodiment 78. The DFM of embodiment 76, wherein said strain of Lactobacillus is a strain of Lactobacillus plantarum.

Embodiment 79. The DFM of any one of embodiments 67 to 78, wherein said DFM is lyophilized.

[236] While the present disclosure has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof to adapt to particular situations without departing from the scope of the present disclosure. Therefore, it is intended that the present disclosure not be limited to the particular embodiments disclosed as the best mode contemplated for carrying out the present disclosure, but that the present disclosure will include all embodiments falling within the scope and spirit of the appended claims. EXAMPLES

EXAMPLE 1

Homology Modeling

[237] 3-D complexed crystal structures of human GLP-1R (hGLP-lR) in PDB database (Berman et al., “The Protein Data Bank," Nucleic Acids Research, 28(1), 235-242 (2000)) are obtained from the Protein Data Bank (PDB) database. There were forty-eight peptides bound hGLP-lR receptor complexes available in PDB database. 11 out of 48 complex structures are selected for superimposition of receptor bound complexes as shown in Table 2.

Table 2: Selected pdb structures for superimposition and template selection

[238] Superimposition shows that all the 11 complex models align well and show 1.659A rmsd with each other as represented in Figure 4.

[239] The hGLP-lR-hGLPl bound complex structure (RCSB PDB - 6X18) are selected based on factors such as full-length structure, complexed with peptide, resolution as well as year of publication. The hGLP-lR/hGLPl bound complex model is designed to standardize & benchmark the parameters for modeling cat receptors. The resulting superimposed models are illustrated in Figure 4.

[240] The cGLP- 1 R structure is modeled based on PDBid: 6X 18 based on the high similarity between human & cat GLP-1R receptors. The benchmarked parameters from the human complexes (e.g., hGLP- IR/hGLP) are utilized to design the complexed cat receptor structure (cGLP-lR/cGLP) model.

[241] Models are validated by checking the stereochemical properties in a Ramachandran plot and sequence alignment is performed to check the similarity between human and cat receptors and their respective peptides. [2421 Both cat and human GLP-1R receptors show 92% identity. Further analysis after sequence alignment reveals that the binding pocket residues are largely conserved (Figure 6). Differences between human and cat are observed at three residues (Leul44-Phe, Tyrl45-Ser and Lysl97-Arg). Two residues, (Pro90, Trp91 of ECD) are not present in cat.

Benchmarking Docking parameters

[243] Molecular docking of the hGLP-1 and hGIP peptides is performed against the hGLP-lR receptor. Binding pose analysis of the hGLP-lR/hGLP-1 complexe showed that the peptides are binding on the same pose with overall rmsd of 0.030A and 0.795A respectively. The majority of the sidechain orientations, inter-spatial distances and interactions are preserved in both the complexes.

[244] The N-terminal of the GLP-1 peptide shows peptide-protein interactions with trans-membrane residues as His7-Q234(TM3), Ala8-L388/L384(TM7), Glu9-L388/R190(TM2)/Y152(TMl), GlylO- N300, Thrl l-R380(TM7), Phel2-L388(TM7)/L141/L144(TM1), Thrl3-K197(TM2), Serl4- N300(ECL2), Aspl5-R380, Vall6-L201 and Serl7-T298/R299(ECL2)/Y205(TM2) [for clarity peptide residues are mentioned in 3-letter code] (Zhao et al., 2022) while the C-terminus forms a peptide-protein interaction network with extra cellular domain (ECD) of hGLP-1 R receptor shown in Figure 2.

Sequence comparison between GLP-1 endogenous ligands in vertebrates

[245] A complex model for the GLP- 1 R receptors are built and the endogenous ligands are docked. Model building shows that cGLP-lR/cGLPl co-model has recognizable similarity with hGLP-1 R/hGLPl templates. Binding poses of cat receptors after superimposition over their respective template are analyzed. cGLP-lR and hGLP-lR co-models in complex with GLP-1 align well with side chain orientations preserved (data not shown).

Designing GLP-1 analogues using Ligand-based approach

[246] Exenatide has a decreased sensitivity to DDP-4, resulting a longer half-life. Liraglutide shares 97% sequence identity with GLP-1. It has an additional Cl 6 fatty acid side chain. It increases duration of action to 24 hr and enabling once-daily injection of the peptide. See Garber, “Long-acting glucagon-like peptide 1 receptor agonists: a review of their efficacy and tolerability,” Diabetes Care, 34(Suppl 2):S279- 84 (2011).

[247] In GLP I [Gly⁸] analogue, Ala to Gly change significantly improves resistance against proteolytic inactivation by DPP-4. Lin et al., “Oral Delivery of Pentameric Glucagon-Like Peptide- 1 by Recombinant Lactobacillus in Diabetic Rats,” PloS One, 1 /(9);e0162733 (2011). This modification is also observed in Exenatide. Here, we have considered GLP-1 -Gly⁸ sequence for preparing GLP-1 analogues and tried to make it resistant from proteolytic enzymes. The 26* and 34* positions of GLP-1 contain Lys residues, which are important proteolytic cleavage sites for Trypsin. Literature survey suggested that replacement of Lys to Gin at 26* position and Lys to Asp at 34* position significantly improves resistance against Trypsin (Figure 9). [248] Further, we have incorporated multiple modifications by analyzing proteolytic cleavage sites of different peptides. See Figure 1. For design purposes, both subcutaneous and orally given peptides are analyzed and to engineer peptides which are resistant from multiple proteolytic cleavage enzymes.

[249] Peptides are designed by mutating specific residues. Further, energy minimization and molecular docking followed by conformational sampling is performed for each peptide. Engineered peptides changes, their docking energies, and cleavage sites residues are mentioned in sequential manner in Table 3. In depth analysis of each peptide, shows that peptides are binding in the same interacting pocket, maintaining their helicity, and showing better affinity for both the receptors.

Table 3: GLP-1 engineered analogues, their modifications, cleavage sites, binding energies for against hGLP-lR and cGLP-lR

[250] Peptides C1.8, C1.9 and CLIO are selected for farther engineering. To make them trypsin resistant at 36^th position, an Arg³⁶ to Ser³⁶ change is incorporated. Newly designed peptides C.1.8.1 , C.1.9.1 and C.1.10.1, are trypsin resistant, and their energy scores are also comparable (Table 4).

Table 4: Designed GLP-1 analogues, their modifications, cleavage sites, binding energies to hGLP-lR and cGLP-lR

GLP1 activity assay

[251] Cloned human GLP-1 receptor-expressing ChemiBrite cells are made by stable transfection of HEK293 cells with ChemiBrite clytin and the GLP-1 receptor and a promiscuous G protein to couple the receptor to the calcium signaling pathway. The cells are stability-tested cells and are ready for luminescent analysis of agonists, antagonists and modulators at the GLP-1 receptor.

[252] Representative data for activation of GLP-1 receptor stably expressed in HEK293 cells, induced by GLP-l(7-36) using a luminescent calcium flux assay are presented in Figure 7. GLP-lR-expressing HEK293 cells are loaded with 10 pM Coelenterazine-h for 3 hours at room temperature and calcium flux in response to the indicated ligand is determined on a FLIPR Tetra with ICCD camera in 96-well format with a final concentration of 0.5% DMSO. Luminescence signal obtained in this experiment is 260,000 RLU (Relative Light Units) as measured by AUG (are under curve) for 80 seconds post agonist addition using the manufacturer’s protocol. Similarly, parental cells (Cat. No. HTSHEK-2L) are tested to determine the specificity of the resulting signal.

[253] Peptides of the present specification are tested for GLP-1 activity in vitro and in vivo.

Efficacy evaluation of engineered GLP-1 peptides in mouse and rat models of Type 2 and Type 1 Diabetes

[254] GLP-1 peptides are evaluated for efficacy in mouse and rat models of Type 2 and Type 1 diabetes administered as peptides or as Direct Fed Microbials (DFMs) incorporating expression vectors for the GLP-1 peptides of the present specification (“therapeutic peptides and host cells”). In vivo activity is evaluated by either injection (subcutaneous or intravenous) or by oral administration. Alternatively, host cells from Bacillus, Lactobacillus, Lactococcus, Salmonella, and Enterococcus species are transformed and selected for expression of GLP-1 using traditional methods for use in oral administration as DFMs.

[255] The therapeutic peptide and host cell candidates delivering GLP1 are evaluated for efficacy in mice and rats using different models of type 2 and type 1 diabetes, including diet induced obesity (DIO), ob'/ob' and db7db" models of type 2 diabetes. See, for example on the internet at www(dot)jax(dot)org/news-and-insights/jax-blog/2015/july/choosing-among-type-ii-diabetes-mouse- models). Obesity is one of the greatest risk factors linked to diabetes in humans, cats, and other species. Similar to humans, some mouse strains become obese when fed high-fat or so-called “Western” diets. Among mouse strains, C57BL/6 is among the most sensitive to this diet-induced obesity (DIO) and these mice develop severe obesity, increased glucose intolerance, moderate insulin resistance and elevated glucose levels. Therefore, DIO is a popular model for studying pre-diabetes and diabetes-related metabolic syndrome in humans. Similarly, mice homozygous for the obese (Lep⁰⁴) and the diabetes (Lept'^tb) mutations are the earliest diabetic mouse models characterized, and remain a popular choice for diabetes researchers. Leptin is a hormone that regulates appetite, and mutations in either the leptin gene (Lep) or its receptor (Lepr) induce unregulated feeding (hyperphagia), resulting in subsequent obesity and frank diabetes.

[256] An example study design for evaluating our engineered candidates for efficacy in a DIO model of type 2 diabetes is shown below in Table 5. Test samples are delivered either directly by injection or orally by gavage of engineered candidates or host cells expressing engineered candidates (e.g., DFM). In summary, the study contains 3 or 4 groups with 12 DIO mice (Jackson Labs) fed with high fat diet (HFD) for 16 weeks in each group. Group 1 is a negative control with sham dosing. Group 2 is intraperitoneally treated with Liraglutide daily for 12 weeks. For DFM treatments, Group 3 is orally gavaged with the unmodified host strain at 1 x 10’CFUs/mice/day for 12 weeks in 0.2-0.4 ml volume as a negative control. Group 4 is the test group and is provided the engineered peptides either as injected peptide compositions (subcutaneous or intravenously), as oral formulations, or as an orally gavaged DFM comprising a transformed host cell expressing the engineered peptides. DFMs provided by oral are transformed to deliver GLPI at 1 x 10⁹CFUs/mice/day for 12 weeks in 02-0.4 ml volume. For all mice, the following parameters are measured:

Table 5: Study design for evaluating engineered candidates in a DIO model of type 2 diabetes.

EXAMPLE 2: Homology Modeling

[257] 3-D complexed crystal structures of human GLP-1R (hGLP-lR) & GIPR (hGIPR) in PDB database (Berman, et al., “The Protein Data Bank,” Nucleic Acids Research 28(1):235— 242 (2000)) are obtained from the Protein Data Bank (PDB) database. There were forty-eight peptides bound hGLP-lR receptor complexes available in PDB database. 11 out of 48 complex structures are selected for superimposition of receptor bound complexes as shown in Table 7. Table 7: Selected pdb structures for superimposition and template selection

[258] Superimposition shows that all the 11 complex models align well and show 1.659A rmsd with each other as represented in Figure 4A. Similarly, nine peptide bound hGIPR complex structures were available in PDB (Table 7) and all 9 bound complex structures showed 0.995A rmsd with each other as shown in Figure 4B.

[259] The hGLP-lR-hGLPl bound complex structure (RCSB PDB - 6X18) and the hGlPR- hGTP bound complex structure (RCSB PDB - 7DTY) are selected based on factors such as full- length structure, complex with peptide, resolution, as well as year of publication. The structures are used as templates for designing the cGIPR complex. The hGLP-lR/hGLPl and hGIPR/hGIP bound complex models are designed to standardize & benchmark the parameters for modeling cat receptors. The resulting models are illustrated in Figure 5.

[260] The cGLP- 1 R structure is modeled based on PDBid: 6X18 and the cGIPR structure is modeled based on PDB Ids: 7DTY & 7RA3 based on the high similarity between human & cat GLP-1R & GIPR receptors. The benchmarked parameters from the human complexes (e.g., hGLP-lR/hGLP, hGIPR/hGIP) are utilized to design the complexed cat receptor structure (cGLP-lR/cGLP, cGIPR/GIP) model.

[261] Models are validated by checking the stereochemical properties in a Ramachandran plot, and sequence alignment is performed to check the similarity between human and cat receptors and their respective peptides.

[262] Both cat and human GLP-1R receptors show 92% identity. Further analysis after sequence alignment reveals that the binding pocket residues are largely conserved (Figure 6). Differences between human and cat are observed at three residues (Leul44-Phe, Tyrl45-Ser and Lysl97-Arg). Two residues, (Pro90, Trp91 of ECD) are not present in cat (Figure 6).

[263] Similarly, cGIPR contains 84.65% identity with hGIPR and cross species analysis shows that binding pocket residues are conserved in human, rat, mouse, and cat (Figure 10). Six binding pocket residues (67Met, Tyr68, Tyr87, Leu88, Pro89 and Trp90) are not available in cat because the cat sequence is shorter in length (Figure 10). EXAMPLE 3: Benchmarking Docking parameters

[264] Molecular docking of the hGLP-1 and hGIP peptides is performed against hGLP- 1 R and hGIPR receptors, respectively. Binding pose analysis of hGLP-1 R/hGLP-1 and hGIPR/hGIP complexes showed that the peptides were binding on the same pose with overall rmsd of 0.030A and 0.795A respectively. The majority of the sidechain orientations, inter-spatial distances and interactions are preserved in both the complexes.

[265] N-terminal of the GLP-1 peptide shows peptide-protein interactions with trans-membrane residues as His7-Q234(TM3), Ala8-L388/L384(TM7), Ghi9-L388/R190(TM2)/Y152(TMl), Glyl0-N300, Thrll-R380(TM7), Phel2-L388(TM7)/L141/L144(TM1), Thrl3-K197(TM2), Serl4-N300(ECL2), Aspl5-R380, Vall6-L201 and Serl7-T298/R299(ECL2)/Y205(TM2) (for clarity peptide residues are mentioned in 3-letter code] (Zhao et al., 2022) while the C-terminus forms a peptide-protein interaction network with extra cellular domain (ECD) of hGLP-1 R receptor shown in Figure 11.

[266] Docking of tirzepatide-hGLPIR shows that the peptide is binding in the same region as it binds in the reported hGLP-1 R receptor with RMSD 1.205 A. During peptide-protein interaction analysis, it is observed that N-terminal of tirzepatide is interacting with Tyrl-Q234, AIB2-L388, Glu3-Y152/R190, Phe6-Y148/L388/L141/L144, Thr7-T298, Ser8-N300, Asp9-R380, TyrlO- Y145/L141/L201, Seri 1-Y205/T298, AIB13-L141 [for clarity peptide residues are mentioned in 3-letter code] residues of GLP-1 R (as shown in Figure 12) and the C-terminal interacts with the ECD of the receptor.

[267] Similar studies are performed on hGIPR receptor by molecular docking of hGIP and tirzepatide. Tirzepatide is a dual agonist for GLP-1R and GIPR. Docking of tirzepatide showed interactions Tyrl-Q234/W296, AIB2-L374/1378, Glu3-R183/Y145, Thr5-R300, Phe6- Y141/L134/L137/ L374/L378, Thr7-R190, Ser8-N290, Asp9-R370, Tyr10-Q138/R196, Serl L E288, AIB13-R131 (Zhao et al., 2022).

EXAMPLE 4: Sequence comparison between cat & human endogenous ligands (GIP and GLP-1)

[268] cGIP contains 97% sequence identity with hGIP. The single residue difference is observed at position 18 (His-Arg) (Figure 13). Further, the 2QKH peptide structure selected as a template for cGIP peptide modelling shows a 0.982 A deviation from hGIP. However, GLP-1 has no difference between cat and human sequences. While comparing with tirzepatide, GIP has 8 position differences in both species whereas GLP-1 has 13 position differences between them. [269] Complex models for cGLP-lR and cGIPR receptors are built and their respective endogenous ligands as well as tirzepatide peptides are docked. Model building shows that cGLP- IR/cGLPl co-model has recognizable similarity with hGLP-lR/hGLPl templates. Binding poses of cat receptors after superimposition over their respective template are analyzed. cGLP- 1R and hGLP-lR co-models in complex with GLP-1 are aligning well, side chain orientations are preserved. There are three residue differences in the binding pocket of both the species as shown in Figure 14.

EXAMPLE 5: Docking Tirzepatide in cat receptors

[270] Tirzepatide is docked against cGLP-1 R and cGIPR receptors and confirms that it is also bound in the same interacting pocket as in human. Inter-spatial distance and interactions are preserved. After superimposition of tirzepatide bound hGLP-lR and hGIPR complexes, 0.846A and 1.588A rmsd is observed.

EXAMPLE 6: Modification of Tirzepatide

Substitution of non-natural AIB amino add

[271] The 2^nd and 13^th residues of tirzepatide are modified from non-natural (AIB) to natural amino acids, starting with an alignment of the reported peptides. Tirzepatide, Peptide-20, Peptide- 19, Semaglutide and Exendin-4 are docked against the hGLP-lR and cGLP-lR receptors and their binding energies analyzed (reported in Table 8).

Table 8: Docking binding energies to hGLP-lR and cGLP-lR against reported peptides

[272] The peptide with the maximal calculated binding affinity against hGLP- 1 R is Peptide-20 while Exendin-4 shows the maximal calculated binding affinity against cGLP-lR.

[273] Residues around the AIB are analyzed for both the human and cat receptor GLP-1R. In the hGLP-lR, the Leu³⁹³, Leu³⁹⁷, Glu³⁹⁶, Thr⁴⁰⁰ residues are observed around AIB² (peptide residue as shown in Figure 15(A)) and Leu³⁷⁵, Lys³⁷⁴, Ala³⁵⁹, Glu³⁵⁵ residues are observed around AIB² in cGLP-lR receptor (Figure 15 (B)).

[274] Tirzepatide-related peptides are prepared by substituting non-natural to natural amino acids and to protect proteolytic vulnerability. Tirzepatide is GIP analogue and the literature suggested that Gly can be a possible option for modification of AIB (Wang, 2022). However, this modification decreases binding affinity of the peptides for GLP-1R while increasing binding for the GIPR receptor. Replacement of AIB with Gly also introduces a cleavage site for the proteolytic enzyme elastase (Wang, 2022).

[275] GLP-1 (7-37) has His at 7^th position and is believed to be crucial for GLP-1R receptor activation and to retain insulinotropic activity. Replacement of His⁷ with Tip⁷ lowers the binding affinity and strongly lowers the activity of GLP-1 whereas the absence of Tyr¹ from the N-terminal of GIP dramatically decreases its activity. Tyr* and Re⁷ are key for activating GIPR receptor by GIP peptide. Accordingly, it is predicted that incorporating Thr⁷ from GLP-1 into tirzepatide may lower its GIP activity. Tyr¹⁰ and He¹² from GIP are used in the MAR709 peptide and lie¹² plays an important role in GIPR receptor activation. Tyr¹⁰ and lie¹² are used in tirzepatide. Substitution of Tyr¹⁹ with Ala in GLP-1 decreases the binding affinity and activity of GLP-1 agonist. Aib¹³ of tirzepatide is reported to lower its GLP-1 activity without affecting its GIP activity (Wang, 2022).

[276] A ligand based approach is applied for modification of AIB into natural amino acids in tirzepatide. After analysis of different peptide sequences using the docking proposed peptides to the receptor models as described above, amino acid combinations are mutated in tirzepatide peptide sequence (see Table 9). Conformational sampling and molecular docking is performed for each modified peptide for both the hGLP-lR and cGLP-lR receptors. [277 J Docking scores of modified peptides and top ranked poses are visually inspected as shown in Table 9. Peptide with changes as in peptides 2 and 3 bind in the same pose as tirzepatide and both the peptides show better binding affinities for hGLP-lR and cGLP-lR. In contrast, analogues with changes as in peptides 4 and 8 lose their helicity (Figure 16 (A, B)) at N-terminal of peptide against cGLP-lR but showing better interactions with hGLP-lR. Peptide 6 is not binding in the binding pocket well and showing outward movement from the receptor (Figure 16 C). Similarly, peptides 5 and 7 lose helicity and do not bind in the same pose as tirzepatide. Peptide 9 and 10 modifications are susceptible to the proteolytic enzyme Trypsin. Table 9: Modifications in Tirzepatide at 2nd and 13th positions

found.

Modification of DPP-4 and trypsin protease sensitive sites

[278] To protect the modified tirzepatides against both serum and gastrointestinal proteases, the tirzepatide sequence is further modified by substituting in natural amino acids at predicted vulnerable sites in multiple phases, considering each proteolytic enzyme one by one. Proteolytic enzyme cleavage sites are illustrated in Figure 1.

[279] Peptide 3 presented in Table 9, is selected for further modifications and after each modification cleavage sites analysis is performed. The subsequent changes lead to the evolution of Table 9 peptides that show resistance against DPP-4 and Trypsin, but remain sensitive to chymotrypsin cleavage at positions 1, 14, 16, and 20 (see Table 10).

Table 10: Tirzepatide modifications in Phase-I

[280] In Phase-2, after the modifications as shown in Table 10, peptides show resistance against DPP-4, however trypsin (site 16, 20) and chymotrypsin cleavage (site 1, 14) sites are still present. Molecular docking studies are performed against hGLP-lR and cGLP-lR reveals that the modified peptides are interacting in the same binding pocket, but their helicity is disrupted (see Table 11).

Table 1 kTirzepatide modifications in Phase-2 and their docking scores against hGLP-lR and cGLP-lR

[281] To prevent the uncoiling in the designed peptides, additional substitutions are introduced into the peptides of Table 11. These peptides maintain their helicity.

Table 12: Peptide modifications to remove protease sensitive sites

[282] Further modifications are introduced to peptide Cl.4.26 of Table 12 to increase peptide stability (see Table 13).

Table 13: Modified peptides with improved stability

[283] The peptides are evaluated for binding by calculating the binding energies to the human and cat receptors and presented in Table 14. As provided herein, peptides showed resistance against the different proteolytic enzyme cleavage sites and have binding energies that are closer to that of tirzepatide.

Table 14: Docking scores to hGLP-lR and cGLP-lR

[284] Tirzepatide peptides are conjugated to a C20 fatty diacid moiety via a linker to Lys20 (avoiding cleavage by Trypsin and facilitate binding to serum albumin) making them difficult to express and deliver via a bacterial system, for example via Lactobacillus. To provide for deliverability via a Lactobacillus system, Lys²⁰ is mutated to Asn²⁰, leading to Phase-6 series (see Table 15).

Table 15: Tirzepatide modifications in Phase-6 and their docking scores against hGLP-lR and cGLP-lR.

[285] Peptides C.1.4.5.1.1, Cl.4.5.2.1 and Cl.4.5.3.1 are engineered as part of Phase-6 (see Table 15). The engineered peptides have resistance against DPP-4, chymotrypsin, and trypsin and binding energies comparable to tirzepatide.

EXAMPLE 7: Dual Agonist Activity Assay

GIP-1

[286] Cloned human GLP-1 receptor-expressing ChemiBrite cells are made by stable transfection of HEK293 cells with ChemiBrite clytin and the GLP-1 receptor and a promiscuous G protein to couple the receptor to the calcium signaling pathway. The cells are stability-tested cells and are ready for luminescent analysis of agonists, antagonists and modulators at the GLP-1 receptor. Similar methods for the in vitro measurement of GLP-1 activity are known in the art.

[287] Representative data for activation of GLP-1 receptor stably expressed in HEK293 cells, induced by GLP-1 (7-36) using a luminescent calcium flux assay are presented In Figure 24. GLP-1 R-expressing HEK293 cells are loaded with 10 pM Coelenterazine-h for 3 hours at room temperature and calcium flux in response to the indicated ligand is determined on a FLIPR Tetra with ICCD camera in 96-well format with a final concentration of 0.5% DMSO. Luminescence signal obtained in this experiment is 260,000 RLU (Relative Light Units) as measured by AUG (are under curve) for 80 seconds post agonist addition using the manufacturer’s protocol. Similarly, parental cells (Cat. No. HTSHEK-2L) are tested to determine the specificity of the resulting signal.

[288] Peptides of the present specification are tested for GLP-1 activity in vitro and in vivo.

GIPR Activity

[289] Cloned human GIP receptor-expressing CHO-Kl/GIP/Gal5 cells are made by stable transfection of CHO cells with human GIPR receptor and a promiscuous G protein to couple the receptor to the calcium signaling pathway. The cells are stability-tested cells and are ready for luminescent analysis of agonists of GIPR. Similar methods for the in vitro measurement of GLP-1 activity are known in the art.

[290] Representative data for activation of GIPR receptor stably expressed in CHO cells, induced by GIP using a luminescent calcium flux assay are presented in Figure 17. The cells are loaded with Calcium-4 prior to being stimulated with agonist GIP. The intracellular calcium change is normalized and measured by FLIPR. The relative fluorescent units (RFU) are normalized and plotted against the log of the cumulative doses of GIP (Mean ± SD, n = 2). The ECso of GIP on the cells of this representative example is 0.12 pM.

[291] The GIPR activity of dual agonist polypeptides are tested for activity using the luminescent assay and the results are presented in Table 16. Six replicates are each tested from a concentration of 1x1 O’⁹ M (nM) to 1000 nM and the average RLU and activity assessed and presented at a concentration at 1 pM.

Table 16: GIPR activity as measured by luminescence assay.

Efficacy evaluation of engineered dual agonist polypeptides in mouse and rat models of Type 2 and Type 1 Diabetes

[292] Dual agonist polypeptides are evaluated for efficacy in mouse and rat models of Type 2 and Type 1 diabetes administered as peptides or as Direct Fed Microbials (DFMs) incorporating expression vectors for the Dual agonist polypeptides of the present specification (“therapeutic peptides and host cells”). In vivo activity is evaluated by either injection (subcutaneous or intravenous) or by oral administration. Alternatively, host cells from Bacillus, Lactobacillus, Lactococcus, Salmonella, and Enterococcus species are transformed and selected for expression of Dual agonist polypeptides using traditional methods for use in oral administration as DFMs.

[293] The therapeutic peptide and host cell candidates delivering Dual agonist polypeptides are evaluated for efficacy in mice and rats using different models of type 2 and type 1 diabetes, including diet induced obesity (DIO), ob7ob" and db /db" models of type 2 diabetes. See, for example on the internet at www(dot)jax(dot)org/news-and-insights/jax-blog/2015/july/choosing- among-type-ii-diabetes-mouse-models). Obesity is one of the greatest risk factors linked to diabetes in humans, cats, and other species. Similar to humans, some mouse strains become obese when fed high-fat or so-called “Western” diets. Among mouse strains, C57BL/6 is among the most sensitive to this diet-induced obesity (DIO) and these mice develop severe obesity, increased glucose intolerance, moderate insulin resistance and elevated glucose levels. Therefore, DIO is a popular model for studying pre-diabetes and diabetes-related metabolic syndrome in humans. Similarly, mice homozygous for the obese (Zep°⁶) and the diabetes (Lepr^ltb) mutations are the earliest diabetic mouse models characterized, and remain a popular choice for diabetes researchers. Leptin is a hormone that regulates appetite, and mutations in either the leptin gene (Lep) or its receptor (Lepr) induce unregulated feeding (hyperphagia), resulting in subsequent obesity and frank diabetes.

(294] An example study design for evaluating the engineered candidates for efficacy in a DIO model of type 2 diabetes is shown below in Table 17. Test samples are delivered either directly by injection or orally by gavage of engineered candidates or host cells expressing engineered candidates (e.g., DFM). In summary, the study contains 3 or 4 groups with 12 DIO mice (Jackson Labs) fed with high fat diet (HFD) for 16 weeks in each group. Group 1 is a negative control with sham dosing. Group 2 is intraperitoneally treated with tirzepatide daily for 12 weeks. For DFM treatments, Group 3 is orally gavaged with the unmodified host strain at 1 x 10' CFUs/mice/day for 12 weeks in 0.2-0.4 ml volume as a negative control. Group 4 is the test group and is provided the engineered peptides either as injected peptide compositions (subcutaneous or intravenously), as oral formulations, or as an orally gavaged DFM comprising a transformed host cell expressing the engineered peptides. DFMs provided by oral are transformed to deliver GLP1 at 1 x 10⁹CFUs/mice/day for 12 weeks in 0.2-0.4 ml volume. For all mice, the following parameters are measured:

8. Body weight, food intake weekly

9. Body composition week 0, 4, and 8

10. OGTT, week 6

11. Non-fasting blood glucose weekly

12. Blood (serum) samples (5 aliquots for each) for insulin, adipokines, clinical chemistry, liver function, serum lipids - week 2, 4, 6, and 8

13. Fecal samples every week for microbiome profiling

14. Necropsy at the end - intestine, pancreas, liver, adipose tissue, muscle in RNALater for qPCR and separate samples for histopathology/ immunohistochemistry

Table 17: Study design for evaluating engineered candidates in a DIO model of type 2 diabetes.

EXAMPLE 8

Design and Generation of Fusion Peptides - Albumin Binding Domain

[295] Glucagon-like peptide- 1 (GLP-1) is considered to be a promising peptide for the treatment of type 2 diabetes mellitus (T2DM), however, the extremely short half-life of GLP-1 limits its clinical application. Albumin-binding domain (ABD) with high affinity for human serum albumin (HSA) is a suitable scaffold for half-life extension of therapeutic peptides and proteins. A previous report has shown that the albumin binding domain extends the half-life of native glucagon-like peptide- 1 (Tan H et al (2021) Eur J Pharmacol 890:173650; doi.org/10.1016/j.ejphar.2020.173650). Engineered GLP-1 polypeptides and also dual agonist polypeptides provided and described herein were fused to ABD sequence.

[296] The naturally occurring ABD is a small, three-helical protein domain (Nilvebrant and Hober (2013) Comp Struct Biotechnol J 6, 1-8). G148-ABD3 (GA3), expressed by streptococcal strain 0148, is one of the best characterized domains and has been developed widely to extend the half-lives of therapeutic peptides or proteins (Nilvebrant and Hober (2013) Comp Struct Biotechnol J 6, 1-8; Stork et al. (2007) Prot Eng Des Sei 20, 569-576; Gapizov et al. (2019) Biotechnol Appl Biochem 66, 617-625). Inspired by the promising application of GA3 in half-life extension, many variants have been designed to further improve the pharmacodynamic and pharmacokinetic profiles of fusion partners. ABD035, selected by phage display technology, showed an apparent affinity of 50- 50 fM for HSA, corresponding to several orders of magnitude of improvement compared with wild-type GA3 (1.2 nM) (Jonsson et al. (2008) Prot Eng Des Sei 21 , 515-527). ABDCon, with an affinity of 75 pM for HSA, is a variant designed by a consensus sequence design method (Jacobs et al. (2015) Prot Eng Des Sei 28, 385-393).

[297] The sequences of the GA3, AB DO 35 and ABDCon albumin binding domain (ABD) sequences are aligned and provided below (gaps in the aligned sequences are shown as amino acid differences are shown in bold):

GA3 LAEAKVLANRELDKYGV- SDYYKNLINNAKTVEGVKALIDEILAALP (SEQ ID NO:384)

ABD035 LAEAKVLANRELDKYGV- SDFYKRLINKAKTVEGVBALKLHILAALP (SEQ ID NO:385)

ABDCon LKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA- - (SEQ ID NO:386)

[298] ABD-fusions of GLP-1 polypeptide and of dual agonist polypeptide were generated whereby fusions comprising Glp-1 -peptide-/-Linker-/-ABD and Glp-l-peptide-/-Linker-/-ABD constructs were generated. The peptides were linked to exemplary ABD sequence ABD035 (SEQ ID NO:385). The ABD fusion constructs generated and the base peptide sequences are provided below (the ABD sequence is shown in bold).

[299] Various

EXAMPLE 9

Efficacy Evaluation of Engineered L. reuteri strains and Glp-1 peptide (BEP009 and BEP009-ABD) in DIO mice

[300] GLP-1 peptides and GLP-1 peptide-ABD fusions were evaluated for efficacy in a mouse model of Type 2 and Type 1 diabetes administered as peptides or as Direct Fed Microbtals (DFMs) incorporating expression vectors for the GLP-1 peptides of the present specification (“therapeutic peptides and host cells”). In particular, a diet induced obesity (DIO) model was utilized. In vivo activity was evaluated by intraperitoneal injection.

[301] Objective: Evaluate the efficacy of engineered L. reuteri 3632 delivering one copy of peptide GLP-1 Cl.l (SEQ ID NO: 2) (designatedlX BEP009) andL reuteri 3632 delivering five copies of peptide GLP-1 Cl.l (SEQ ID NO: 2) (designated 5X BEP009) in a diet-induced obesity (DIO) mouse model. Liraglutide was used as a positive control to validate the DIO model. Peptide GLP-1 Cl .l (SEQ ID NO: 2) (designated BEP009) administered via intraperitoneal injection served as an injectable peptide control, while L. reuteri 3632 functioned as a chassis control.

[302] The study design is shown in the below Table 18:

Table 18

Liraglutide: A short-acting GLP-1 agonist used for the treatment of type 2 diabetes and obesity. BEP009: A trypsin- and DPPIV-resistant version of GLP-1.

BEI05ENLR139: L. reuteri 3632 strain with single copy integration of the BEP009 (IX BEP009) expression cassette.

BE 105ENLR181 : L reuteri 3632 strain with integration of five copies of BEP009 (5X BEP009), linked by trypsin-susceptible sites.

[303] Among mouse strains, C57BL/6 is among the most sensitive to this diet-induced obesity (DIO) and these mice develop severe obesity, increased glucose intolerance, moderate insulin resistance and elevated glucose levels. Therefore, DIO is a popular model for studying pre-diabetes and diabetes-related metabolic syndrome in humans.

[304] C57BL/6J mice were used at 6 weeks of age and 0-week obese. The mice were fed a high fat diet (HFD). Body weight was assessed every week. Resting glucose was assessed twice a week. Food intake was evaluated every 2 weeks. Evaluations and aspects of the clinical mouse study included the following. In a first part of the study OGTT was done on Day 33, EchoMRI on Day 39. Study Adaptations then included administration of BEP009-ABD to Group 5 Day 48 onwards. Further, Dose escalation of BEP009 and BEP009-ABD was included as a study adaptation from Day 48 onwards. Fecal pellets were assessed for colonization Day 48 onwards (to assess L reuteri colonization). In a further part of the study, OGTT was done on Day 75 and EchoMRI on Day 81. Administration of the test articles was stopped on Day 85. The study was ended on Day 99 and OGTT and EchoMRI were evaluated on Day 99.

[305] The effects of the test materials on weekly body weight at Day 40 is depicted in Figure 18. Compared to vehicle control, liraglutide consistently reduced body weight. Compared to vehicle control, BEP009 injected i/p had a slight effect on body weight from day 19 onwards. The chassis L. reuteri 3632 and the engineered strains BE105ENLR139 (IX GLP1) and BE105ENLR181 (5x GLP1) had minimal to no effect on body weight in this study.

[306] The effect of BEP009 on fat content in an EchoMRI test on Day 39 was evaluated. The results are depicted in Figure 19. As expected, compared to vehicle control, liraglutide reduced body fat content in an EchoMRI test on day 39. Compared to vehicle control, BEP009 had a slight effect on body fat content in an EchoMRI test on day 39. Compared to vehicle control, the chassis LR3632 and the engineered strains BE105ENLR139 and BE105ENLR181 also showed no effect on body fat content in an EchoMRI test on day 39.

[307] This initial phase of the study showed that the DIO model is functioning as expected. Control peptide liraglutide showed significant reduction in weekly body weight and also showed significant reduction in resting and fasting blood glucose and AUC in an OGTT. BEP-009 showed a minor reduction in weekly body weight and no significant effect on blood glucose. BEP009 may be rapidly cleared by renal processes, preventing it from reaching therapeutic concentrations. This observation supports the hypothesis that BEP009 needs to maintain consistently higher concentrations in circulation to access the hypothalamus and induce weight loss.

[308] Based on the initial results the study was adapted and changes instilled on day 48. Group 3: Starting on day 48, the dose of BEP009 was increased to 2X for the first 3 weeks and 4X for the last 2 weeks. Group 5: Based on BEP009's minimal impact on weekly body weights and its lack of effect on the AUC or fasting blood glucose levels in the OGTT test on day 33, which suggests that BEP009 might be rapidly cleared by renal processes, we hypothesized that adding ABD could extend its half-life. This extension would potentially allow BEP009 to achieve therapeutic concentrations and thus affect body weight and glucose levels. To evaluate this hypothesis, we stopped administration of BE1O5ENLR139 (IX GLP1) on Day 47 and started administration of BEP009-ABD (the eGLP-l-ABD fusion construct) daily at 2X dose from Day 48 onwards for the first 3 weeks and 4X for the last 2 weeks. We also started collecting fecal pellets on a weekly basis to understand the colonization dynamics of engineered strains. Group 1, 2, 4 & 6: No major changes.

[309] The results of evaluating the effect of the GLP-1 peptides and constructs including BEP009-ABD on weekly body weight - Day 82 were determined and are shown in Figure 20. As expected, compared to vehicle control, daily administration of liraglutide effectively reduced high fat diet (HFD)-induced weight gain. Compared to vehicle control, daily administration of BEP009 showed marginal reduction in HFD- induced weight gain. Compared to vehicle control, daily administration of BEP009-ABD effectively reduced HFD-induced weight gain and this effect was comparable to liraglutide. Compared to vehicle control, administration of the chassis resulted in slightly higher body weight gains compared to vehicle control. Compared to vehicle control, the engineered strain had no significant effect on HFD-induced body weight gain.

[310] Up to Day 78, the effect on weekly blood glucose was determined. The results are shown in Figure 21. As expected, compared to vehicle control, liraglutide showed trend towards reduced blood glucose throughout the study although the effect was prominent after day 50. Compared to vehicle control, BEP009-ABD showed marked reduction in blood glucose immediately after its administration on day 48 and this effect persisted throughout the rest of the study period.

[311] At Day 75, the effect on fasting glucose/ AUG was evaluated in an OGTT test. Results are shown in Figure 22. As expected, compared to vehicle control, liraglutide reduced fasting blood glucose and AUC significantly in OGTT test on day 75. Compared to vehicle control, BEP009 injected i/p showed marginal effect on AUC and fasting blood glucose in OGTT test on day 75. Compared to vehicle control, BEP009-ABD reduced fasting blood glucose and AUC in an OGTT test on day 75 and this reduction was either slightly better than or comparable to liraglutide. Compared to vehicle control, the chassis L reuteri 3632 and the engineered strain BE105ENLR181 (5x GLP1) showed slightly increased fasting glucose and AUC in OGTT test on day 75.

[312] At Day 81, the effect on fat content was assessed in an EchoMRI. The results are depicted in Figure 23. As expected, compared to vehicle control, liraglutide reduced fat content in EchoMRI test on day 81. Compared to vehicle control, BEP009 injected i/p showed marginal reduction in fat content in EchoMRI on day 81 . Compared to vehicle control, BEP009-ABD reduced fat content in an EchoMRI test on day 81 and this reduction was slightly better than BEP009. Compared to vehicle control, the chassis L reuteri 3632 and the engineered strain BE105ENLR181 (5x GLP1) showed no reduction in fat content.

[313] Also at Day 81 the effect on lean mass in an EchoMRI was evaluated and the results are shown in Figure 24. As expected, compared to vehicle control, liraglutide reduced lean body mass in EchoMRI test on day 81. Compared to vehicle control, BEP009 injected i/p slightly increased the lean mass in EchoMRI on day 81. Compared to vehicle control, BEP009-ABD reduced lean mass in an EchoMRI test on day 81 and this reduction was comparable to liraglutide. Compared to vehicle control, the chassis L. reuteri 3632 and the engineered strain BE105ENLR181 (5x GLP1) showed slight increase in lean mass compared to vehicle control.

[314] The effect of the test materials and peptides on biweekly food intake (normalized to vehicle control) - Day 84 was determined and results shown in Figure 25. As expected, compared to vehicle control, daily administration of liraglutide effectively reduced cumulative food intake (total g/animal/day).Compared to vehicle control, daily administration of BEP009 showed reduction in cumulative food intake (total g/animal/day). Compared to vehicle control, daily administration of BEP009-ABD also effectively reduced cumulative food intake (total g/animal) and this effect was comparable to liraglutide but less than BEP009. Administration of the chassis strain had no effect on cumulative food intake compared to vehicle control. Administration of the engineered strains resulted in slightly higher cumulative food intake compared to vehicle control.

[315] In conclusion from this study, liraglutide showed significant reduction in all the relevant parameters assessed. BEP-009 peptide alone showed marginal reduction in weekly body weight, in fat and lean mass and in fasting blood glucose. The peptide-ABD fusion BEP-009-ABD demonstrated significant reduction in weekly body weight, in bi-weekly food intake, in fat and lean mass and in resting and fasting blood glucose. Addition of ABD is able to achieve optimal systemic concentrations to result in optimal efficacy. BEP009-ABD had a more pronounced effect on glucose than body weight, suggesting higher doses may be needed for weight reduction. While its glucose impact matched or exceeded liraglutide, its effect on weight was less. This might be due to inefficient crossing or back diffusion from the BBB to systemic circulation, hindering brain concentration. However, the similar or superior glucose activity compared to liraglutide indicates that BEP009-ABD's moderate weight impact isn’t due to reduced potency.

[316] Group treated with BE105ENLR181 showed a modest trend towards affecting body weight and glucose levels compared to the chassis. Groups treated with L. reuteri 3632, BE105ENLR139 & BE105ENLR181 exhibited a trend towards slightly higher body weights, food intake, resting and fasting blood glucose levels, and AUG in GTT tests. L reuteri is a probiotic and has been shown to improve body weight and other parameters on its own in animals.

[317] STUDY 2

[318] Another DIO mice study was conducted to specifically confirm the earlier study results and also to further assess the ABD fusion peptide, including at various dose levels. Included in the study was investigating the impact of various doses of BEP009-ABD on body weight, adipose tissue, lean mass, and glucose levels. Also, assess changes in body weight, adipose tissue, lean mass, and glucose levels after discontinuing BEP009-ABD administration. The study design is shown below in Table 19. Study design:

[319] In this DIO study, C57BL/6J mice, 16-weeks of age 10-week obese were fed a HFD diet Body weight was assessed every day and resting blood glucose was determined twice a week. Assays were EchoMRI on Day 14, Fasting blood glucose on Day 15, IPGTT on Day 15. Then in the study, stop administering test articles from Day 15 onwards. The study concluded on Day 29. EchoMRI on Day 28, Fasting blood glucose on Day 29, IPGTT on Day 29.

[320] The key learnings and conclusions from this study include:

[321] Study Adaptation for Groups 2.3 & 4

[322] Stop administering test articles on Day 15 to all groups to understand the potential half-life of BEP009-ABD. After stopping test article administration for two weeks, the following data were collected: Body weight daily, resting blood glucose weekly, echoMRI on Day 28 and IPGTT on Day 29.

[323] Key Learnings

[324] Body weight: Compared to the vehicle control group, Group 2, which received liraglutide, showed a steady weight gain after discontinuing peptide administration. However, their weight gain did not reach the level observed in the vehicle control group. Similarly, Group 3, treated with a low dose of BEP009-ABD, experienced a slight weight gain after stopping peptide administration, which was comparable to the vehicle control group. In contrast, Group 4, which received a high dose of BEP009- ABD, also exhibited steady weight gain following the cessation of peptide administration, but this gain did not match the level of the vehicle control group.

[325] Lean & adipose mass: Two weeks after discontinuing peptide administration, the effects of liraglutide, BEP009-ABD (low dose), and BEP009-ABD (high dose) on lean mass were restored to levels comparable to the vehicle control. However, while BEP009-ABD (low dose) also restored adipose mass io levels similar to the vehicle control, the effects of liraglutide and BEP009-ABD (high dose) on adipose mass were not fully restored.

[326| Glucose levels: Resting blood glucose, fasting blood glucose, and the area under the curve (AUG) in the intraperitoneal glucose tolerance test (IPGTT) were all restored to levels comparable to the vehicle control two weeks after stopping peptide administration.

[327] Conclusions

[328] The significant rebound weight gain, increased glucose levels and restoration of lean and adipose mass observed in Group 2 after stopping liraglutide is expected due to its short half-life of 12-24 hours. Similarly, the rebound weight gain, increased glucose levels and restoration of lean and adipose mass in Groups 3 (low dose) and 4 (high dose) following the cessation of BEP009-ABD is anticipated because albumin has a short half-life of approximately 24-36 hours in mice.

EXAMPLE 10

[329] Additional variant GLP-1 and dual agonist peptides were generated. Initial evaluation of the peptides was undertaken to assess their activity for GLP-1 R activation. The peptides are shown below in Table 20. Amino acid changes and variants of the BEP-009 (Cl.l) Glp-1 polypeptide sequence (SEQ ID NO:2) are shown in bold. Amino acid changes and variants of the BEP-018 (7FIM_K_R) dual agonist polypeptide sequence (SEQ ID NO:116) are shown in bold.

Table 20 - Variants of GLP-1 and dual agonist peptides

[330] The above peptides as well as engineered eGLP-1 - ABD fusion peptide described above (denoted BEP-105-009-ABD) (SEQ ID NO:370) were evaluated in a GLP-1R cellular assay. In the standard protocol, lOpM native coelenterazine was added and the mixtures incubated for 3 hours at 30°C. About 1 pM of agonists were added in each instance. The results are depicted in Figure 26. GLP-1, tirzepatide and liraglutide were used as positive controls for GLP-1 R activation. All of the eGLP-1, dual agonist and the ABD fusion peptides were active for GLP-1R activation.

[331] Based on and including the above Glp-1 polypeptides, a further consensus sequence for engineered GLP-1 polypeptide is provided herein as Formula DI (SEQ ID NO: 382):

Xi G E G T S E S D V S Xu Xi j XM E Xi« Q A X19 X20 E X22 X« A XM X 26 V D G XJO (III), wherein Xi = H or Y; Xi2 = S, Q, A or M; Xu = S, Q, Y, orM; Xi< = I or L; Xie = G or M; X19 = A or T; X2O= Q, V, orD; X22 = V, I, or F; X23 = V or I; X25 = V, I, or W; X26 = I or V; and X3o = R or S.

[332] Based on and including the above dual agonist polypeptides, a further consensus sequence for engineered dual agonist polypeptide is provided herein as Formula IV (SEQ ID NO: 383): Y X2 E G T X6 X₇ S D Xio S I Xi3 X_l4 D Xie I A X,₉ X20 A X22 V Q X2S X26 I A G G P S S G A P P (IV), wherein X2 = V or K; Xe =F, P, or S; X₇ - T, C, or E; Xio = Y, C, or E; X₁₃ = A, S, Y, N, I, L, R, V, or K; XM = L, K, H, or I; X_l6 = K, R, H, or V; X19 = Q or V; X₂o= K, R, H, N; X22 = F, A, P; X25 = W, P, K, H, or I; and X26 = L or V.

[333] While the inventions have been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof to adapt to particular situations without departing from the scope of the invention. Therefore, it is intended that the inventions not be limited to the particular embodiments disclosed as the best mode contemplated for carrying out this invention, but that the inventions will include all embodiments frilling within the scope and spirit of the appended claims.

Claims

Claims:

1. An engineered dual agonist polypeptide comprising the amino acid sequence of Formula II

(SEQ ID NO: 111):

YX2EGTX6X7SDX10SIX13X14DX16IAQX20AX22VQX25X26IAGGPSSGAPP

(I) wherein

or of Formula IV (SEQ ID NO: 383):

YX2EGTX6X7SDX10SIX13 Xi4DX₁₆IAXi9X2oAX22VQX25 X2₆ lAGGPSSGAPP(IV), wherein X2 = V or K; Xe =F, P,orS;X₇ = T, C,orE; X₁₀ = Y, C.orE; X|₃ = A,S, Y,N, I, L, R, V, or K;

X_l4 = L, K, H, or I; X₁₆ = K, R, H, or V; X19 = Q or V; X2o= K, R, H, N; X22 = F, A, P; X25 = W, P, K, H, or I; and X26 - L or V.

2. The engineered dual agonist polypeptide of claim 1 , wherein

X2 = V;X6=PorS;X₇ = CorE; Xio = CorE; Xi₃ = S; Xu^L, H,orl; Xi₆ = HorV; X20 = H or N; X22 = A or P; X25 = P or I; and X26 = L or V.

3. The engineered dual agonist polypeptide of claim 1 , comprising the amino acid sequence of any one of SEQ ID NO: 112 to SEQ ID NO: 147 or of SEQ ID NO: 381.

4. The engineered dual agonist polypeptide of claim 1 , comprising the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116.

5. An engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide comprising the amino acid sequence of Formula I:

HGEGTSESDVSX₁iXi3Xi4EGQAAQEX22 X23AX25X26VDGX₃o(l) wherein

or of Formula III (SEQ ID NO: 382):

Xi G E G T S E S D V S XUXIJXME XI« Q A Xu X₂o E X22X23 A X25X26 V D G X30 (III), wherein Xi = H or Y; X_l2 = S, Q, A or M; X_L1 == S, Q, Y, or M; X₁₄ = I or L; Xu = G or M; Xu = A or T; Xro = Q, V, or D; X22“ V, I, or F; X23 - V or 1; X₂5 = V, I, or W; X26 = 1 or V; andXjo ~ R or S.

6. The engineered polypeptide of claim 5 comprising the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO: 14 or of SEQ ID NO: 372 to SEQ ID NO: 380.

7. The engineered polypeptide of claim 1 or claim 5, wherein the engineered polypeptide is substantially resistant to proteolytic degradation.

8. The engineered polypeptide of claim 1 or claim 5, wherein the engineered polypeptide at least maintains substantially the same receptor potency as the corresponding wild-type, unmodified polypeptide.

9. The engineered polypeptide of claim 1 or claim 5, wherein the engineered polypeptide is substantially resistant to DPP-IV, neprilysin, a-chymotrypsin, trypsin, elastase, or pepsin, degradation.

10. An isolated polynucleotide encoding: an engineered dual agonist polypeptide comprising the amino acid sequence of Formula II (SEQ ID NO: 111) or of Formula IV (SEQ ID NO:383); and/or an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide comprising the amino acid sequence of Formula I (SEQ ID NO:1) or of Formula III (SEQ ID NO:382).

11. A pharmaceutical composition comprising: an engineered dual agonist polypeptide comprising the amino acid sequence of Formula II (SEQ ID NO: 111) or of Formula IV (SEQ ID NO:383); and/or an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide comprising the amino acid sequence of Formula I (SEQ ID NO: 1) or of Formula m (SEQ ID NO:382); and a carrier.

12. A host ceil comprising the polynucleotide of claim 10.

13. The host cell of claim 12, wherein said cell is a bacterial cell, plant cell, yeast cell, or algae cell.

14. A kit comprising the pharmaceutical composition of claim 11 or the host cell of claim 12, and a carrier.

15. A method of treating or preventing a disease or condition caused or characterized by hypoglycemia or impaired insulin release, comprising administering to a subject in need of treatment thereof an effective amount of an engineered dual agonist polypeptide of claim 1 and/or an engineered Glucagon-Like Peptide 1 (eGLP-1) of claim 5, a pharmaceutical composition of claim 11, or a host cell of claim 12.

16. A method of treating or preventing a treatment of a condition caused or characterized by excess body weight, and the treatment of obesity, morbid obesity, obesity linked inflammation, obesity linked gallbladder disease, obesity induced sleep apnea, metabolic syndrome, prediabetes, insulin resistance, glucose intolerance, type 2 diabetes, type I diabetes, hypertension, atherogenic dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke or microvascular disease in a subject, comprising administering to a subject in need of treatment thereof an effective amount of the engineered dual agonist polypeptide of claim 1 and/or an engineered Glucagon-Like Peptide 1 (eGLP-1) of claim 5, a pharmaceutical composition of claim 11 , or a host cell of claim 12.

17. The method of claim 15 or 16, wherein the disease or condition is diabetes or obesity.

18. The method of claim 15 or 16, wherein the dual agonist polypeptide, the eGLP-1 polypeptide or the pharmaceutical composition is administered orally, by injection, or transdermally.

19. The method of claim 15 or 16, wherein the subject is human, cat, dog, or non-human primate.

20. A pharmaceutical composition comprising an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide comprising an amino acid sequence selected from any of any one of SEQ ID NO: 112 to SEQ ID NO: 147 or of SEQ ID NO: 381

21. A pharmaceutical composition comprising an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide comprising an amino acid sequence selected from any of SEQ ID NO: 1 to SEQ ID NO: 14 or SEQ ID NO:32 to SEQ ID NO: 67. l ' l. A direct fed microbial (DFM) comprising bacteria transformed with a nucleic acid encoding an engineered dual agonist polypeptide of Formula II or Formula IV and/or encoding an engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of Formula I or Formula IIL

23. The DFM of claim 22, wherein said bacteria comprise a strain selected from the group consisting of Bacillus, Lactobacillus, Lactococcus, Enterococcus, Salmonella, and combinations thereof.