Stabilized GLP-1 peptides and dual agonist peptides against GLP-1r and GIPR and methods of use thereof

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. The application further provides variant albumin binding domain (ABD) sequences and peptides, compositions thereof, constructs and polypeptides including the variant ABD sequences, and methods utilizing such sequences and polypeptides.

Description

[0001] Atty. Docket No. 2950-42 P

[0002] Stabilized GLP-1 Peptides and Dual Agonist Peptides Against GLP-1R and GIPR and Methods of Use Thereof

[0003] INCORPORATION OF SEQUENCE LISTING

[0004]

[0001] A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference in its entirety. Said Sequence Listing has been submitted with the filing of this application as an electronic document encoded as XML in UTF-8 text. The electronic document, created on January 10, 2025, is entitled “2950-42P ST26.xml”, and is 636,742 bytes in size.

[0005] FIELD OF THE INVENTION

[0006]

[0002] 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. The application further relates to variant albumin binding domain (ABD) sequences and peptides, constructs and polypeptides including the variant ABD sequences, and methods utilizing the sequences and polypeptides.

[0007] BACKGROUND OF THE INVENTION

[0008]

[0003] 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 disorders. 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 Fells catus and other species.

[0009]

[0004] 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. Drags 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 delivety to treat chronic disease,” Scientific Reports 11(1 ):22521 (2021).

[0010]

[0005] 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.

[0011] Medical Center) 19(3):281 284 (2006). Liraglutide (4APD_A) shares 97% sequence identity with GLP-1 and the addition of a C 16 fatty acid side chain facilitates binding of the drug to circulating serum albumin Atty. Docket No. 2950-42 PCT

[0012] (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)).

[0013] [006} 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- 1 R exhibit a similar peptide-receptor binding interface, whereas structural changes are observed at the extra cellular loop (ECL1, 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.

[0014]

[0007] 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-1 R receptor. Figure taken from Cong, et al., “Molecular insights into ago-allosteric modulation of the human glucagon-like peptide- 1 receptor,” Nature Communications, 72(1): 763 (2021).

[0015]

[0008] 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-1R activation. Upon receptor activation, three layers of the polar network are reorganized as (i) central polar network, (ii) IIETX 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. Atty. Docket No, 2950-42 PCT

[0016]

[0009] 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, 27(1): 169 (2022) and Wang, “Designing a Dual GLP-1 R / GIPR Agonist from Tirzepatide: Comparing Residues Between Tirzepatide, GLP-1, and GIP,” Drug Design, Development and Therapy 76:1547-1559 (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 GIP (34-42) are generated. The C-terminal truncated GIP (l-30)-NH2 peptide seems to have similar agonistic properties as full length GIP (1-42) (Alana et al. “NMR structure of the glucose-dependent insulinotropic polypeptide fragment, GIP(1-30)amide,” Biochemical and Biophysical Research Communications, 325(1) (2004) and Gabe et ah “Molecular interactions of full-length and truncated GIP peptides with the GIP receptor - A comprehensive review,” Peptides 725:170224 (2020). The last 12 residues of GIP (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 GIPR binding and receptor activation. It is also observed that the presence of C-terminus affects the overall sta bility 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.

[0017]

[0010] 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-l (7~36)-NH2 and GLP- 1(7-37). GLP-1 (7-36) is more abundant. GLP-l(7-36) binds to and activates the GLP-1 receptor (GLP-1R) to exert its regulatory functions.

[0018] [Oil] 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- 1R while it increases binding to the GIPR receptor. Importantly, glycine is easily cleaved by the proteolytic enzyme elastase (Wang (2022)).

[0019]

[0012] GLP-1 (7-37) has His at 7thposition and is crucial for GLP-1R receptor activation and to retain their insulinotropic activity. Replacement of His7with Trp7lowers the binding affinity' and strongly lowers the activity of GLP-1 whereas the absence of Tyr1from the N-terminal of GIP dramatically Atty. Docket No. 2950-42 PCT

[0020] decreases its activity. Tyr1and He7are key for activating GIPR receptor by GIP peptide. Incorporating Thr7from GLP-1 into tirzepatide may lower its GIP activity. Tyr10and lie’12from GIP are used in MAR709 peptide and He12piays an important role in GIPR receptor activation. Tyr10and lie12are used in tirzepatide. Substitution of Tyr19with Ala in GLP-1 decreases the binding affinity and activity of GLP-1 agonist. Aib 13 of tirzepatide seems to lower its GLP- 1 activity without affecting its GIP activity (Wang (2022)).

[0021]

[0013] 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(T0):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 ceil 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).

[0022]

[0014] 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 I).

[0023]

[0015] 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 I. 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.

[0024]

[0016] 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 Atty. Docket No. 2950-42 PCT

[0025] peptide 1 in vitro and in vivo by dipeptidyl peptidase IV,” Endocrinology 136(8):3585-96 (1995). Thus DPP-IV resistant GLP-1 analogs are needed for treatment of type 2 diabetes.

[0026]

[0017] 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.

[0027]

[0018] 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-1 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 Calas et al., provides modified GLP-1 peptides that include lysine and are susceptible to trypsin cleavage.

[0028]

[0019] 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. Atty. Docket No. 2950-42 PCT

[0029] SUMMARY OF THE INVENTION

[0030]

[0020] 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 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.

[0031]

[0021] The present application provides and includes, in one form thereof, engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence selected from:

[0032] Formula V (SEQ ID NO.398):

[0033] Xi G E G T S E S D V S XI2X13X;4E X16Q A Xi9X20E X22X23A X25XMV D G X30(V), wherein Xi = H or Y; X,2= S, Q, A or M; Xi3= S, Q, Y, or M; Xu = I or L; Xie ~ G or M; Xi9= A or T; X20 = Q, V, or D; X22- V, I, F or H; X23= V or I; X25= V, I, or W; X26= I, V or L; and X30= R or S; Formula VI (SEQ ID NO:399):

[0034] Xi G E G T Xe T S DV S Xi2Xi3LE X16Q A X,9X2o E X221 A X25L V D G R (VI), wherein X, = H or Y; Xg = F or I; Xi2= S, A or M; X = Y or M; Xi6= G or M; X,9= A or T; X2o~ Q, V, or D; X22~ F or I I; and X23= I or W; or

[0035] Formula VII (SEQ ID NO:400):

[0036] Xi G E G T X6T S D V S X12X!3X14E Xi6Q A X19X20E X22X23A X23L V D G X30(VII), wherein Xi = H or Y; Xo F or I; Xt2= S, Q, A or M; X = S, Q, Y, or M; X14 = I or L; Xi6 G or M; X19 - A or T; X2e= Q, V, or D; X22= V, I, F or H; X23- V or I; X25= V, I, or W; and X50 - R or S.

[0037]

[0022] 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:387 to SEQ ID NO:389.

[0038]

[0023] 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 V (SEQ ID NO:398), Formula VI (SEQ ID NO:399), or Formula VII (SEQ ID NO:400). Also included are one or more vectors encoding one or more eGLP-1 peptides of Formula V (SEQ ID NO:398), Formula VI (SEQ ID NO:399), or Formula Vll (SEQ ID NO:400). Also included are concatemers of an engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of Formula V (SEQ ID NO:398), Formula VI (SEQ ID NO:399), or Formula VII (SEQ ID NO:400). In aspects, the engineered nucleic acid vectors encode an engineered Glucagon-Like Peptide I (eGLP-1) comprising the amino acid sequence of any one of SEQ ID NO:387 to SEQ ID NO:389, and combinations thereof. Atty. Docket No. 2950-42 PCT

[0039]

[0024] 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 V (SEQ ID NO:398), Formula VI (SEQ ID NO:399), or Formula VII (SEQ ID NO:400), 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-ffame stop codons 3’ to the terminal stop codon. In aspects, the nucleic acid expression cassetes encode an engineered Glucagon-Like Peptide I (eGLP-1) comprising the amino acid sequence of any one of SEQ ID NO:387 to SEQ ID NO:389, and combinations thereof.

[0040]

[0025] 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 V (SEQ ID NO:398), Formula VI (SEQ ID NO:399), or Formula VII (SEQ ID NO:400), 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 SEQ ID NO:387 to SEQ ID NO:389, 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.

[0041]

[0026] 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 V (SEQ ID NO:398), Formula VI (SEQ ID NO:399), or Formula VII (SEQ ID NO:400). 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:387 to SEQ ID NO:389, or combinations thereof.

[0042]

[0027] 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 V (SEQ ID NO:398), Formula VI (SEQ ID NO:399), or Formula VII (SEQ ID NO:400), 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 I diabetes, type II diabetes, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), metabolic syndrome X, metabolic dysfunction-associated steatohepatitis (MASH), Parkinson’s disease, and Huntington’s Disease. In aspects, the methods comprise administering an engineered Atty. Docket No. 2950-42 PCT

[0043] Glucagon-Like Peptide 1 (eGLP-1) polypeptide having the amino acid sequence of any one of SEQ ID NO. G87 to SEQ ID NO:389, or combinations thereof.

[0044]

[0028] 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 V (SEQ ID NO:398), Formula VI (SEQ ID NO:399), or Formula VII (SEQ ID NO:400). 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.387 to SEQ ID NO:389, or combinations thereof.

[0045]

[0029] 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 V (SEQ ID NO:398), Formula VI (SEQ ID NO:399), or Formula VII (SEQ ID NO:400). 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:387 to SEQ ID NO:389, and combinations thereof.

[0046]

[0030] 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 V (SEQ ID NO:398), Formula VI (SEQ ID NO:399), or Formula VII (SEQ ID NO.400) 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 V (SEQ ID NO:398), Formula VI (SEQ ID NO:399), or Formula VII (SEQ ID NO:400) 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 SEQ ID NO:387 to SEQ ID NO:389, or a combination thereof, are administered for glycemic control, promoting insulin production, reducing le, promoting 13-cell mass, promoting weight loss, or reducing excess body weight, or combinations thereof. In an aspect, Atty. Docket No. 2950-42 PCT

[0047] the composition is a cell expressing an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide having the amino acid sequence of Formula V (SEQ ID NO:398), Formula VI (SEQ ID NO:399), or Formula VII (SEQ ID NO:400), or a polypeptide having the amino acid sequence of SEQ ID NO:387 to SEQ ID NO:389, or a combination thereof.

[0048]

[0031] 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 V (SEQ ID NO:398), Formula VI (SEQ ID NO:399), or Formula VII (SEQ ID NO:400), or a polypeptide having the amino acid sequence of SEQ ID NO:387 to SEQ ID NO:389, 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, 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. 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 V (SEQ ID NO:398), Formula VI (SEQ ID NO:399), or Formula VII (SEQ ID NO:400), or a polypeptide having the amino acid sequence of SEQ ID NO:387 to SEQ ID NO:389, or a combination thereof. 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 V (SEQ ID NO:398), Formula VI (SEQ ID NO:399), or Formula VII (SEQ ID NO:400), or a polypeptide having the amino acid sequence of SEQ ID NO:387 to SEQ ID NO:389, or a combination thereof. In aspects, the host cell is engineered to secrete eGLP-1 by plasmid-based expression or by chromosomal integration.

[0049]

[0032] The present application provides and includes, in one form thereof, engineered polypeptides, particularly engineered dual agonist polypeptides, comprising the amino acid sequence of

[0050] Formula VIII (SEQ ID NO:401):

[0051] Y V E G T F T S D Y S I S L Xi5Xi61 A Q X2o A F X23Q W L I A G (VIII),

[0052] wherein Xir D or Q; Xis = R or Q; X2o= R or Q; and X23= V or M; or

[0053] Formula IX (SEQ ID NO:402):

[0054] Y X2E G T X6X7S D X10 S I X13XuXl5X161 A X!9X20 A X22X23Q X25X26I A G (IX), Atty. Docket No. 2950-42 PCT

[0055] wherein X?= V or K; X6= F, P or S: X? ~ T, C or E; Xio= Y, C or E; XB- A, S, Y, N, I, L, R, V or K; XM= L, K, H or I; X)5= D or Q; Xie = K, R, H, V or Q; X19- Q or V; X20= K, R, H, N or Q; X22= F, A or P; X23= V or M; X25= W, P, K, H or I; and X26- L or V.

[0056]

[0033] In aspects, an engineered dual agonist polypeptide of Formula VIII (SEQ ID NO:401 ) or Formula IX (SEQ ID NO:402) is provided, wherein the polypeptide further comprises the amino acids GPSSGAPP at its C-terminal end.

[0057]

[0034] More particularly, the application includes dual agonist polypeptides comprising the amino acid sequence of any one of SEQ ID NO:391 to SEQ ID NO:394.

[0058]

[0035] In aspects, an engineered dual agonist polypeptide of any one of SEQ ID NO:391 to SEQ ID NO:394 is provided, wherein the polypeptide further comprises the amino acids GPSSGAPP at its C-terminal end.

[0059]

[0036] In aspects, the present application includes engineered nucleic acid vectors encoding an engineered dual agonist polypeptide comprising the amino acid sequence of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402). Also included are one or more vectors encoding one or more dual agonist peptides of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402). Also included are concatemers of an engineered dual agonist polypeptide comprising the amino acid sequence of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402). In aspects, the engineered nucleic acid vectors encode an engineered dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO:39l to SEQ ID NO:394, and combinations thereof.

[0060]

[0037] In aspects, the present application includes nucleic acid expression cassettes comprising a promoter for transcriptional expression, a nucleic acid sequence encoding a peptide comprising a dual agonist polypeptide of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402), 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 dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO:391 to SEQ ID NO:394, and combinations thereof.

[0061]

[0038] The present application further includes, and provides for, methods of making an engineered dual agonist polypeptide having the amino acid sequence of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402), the method comprising culturing a host cell transformed with an expression vector encoding a dual agonist polypeptide under conditions allowing expression of the dual agonist polypeptide, and recovering the dual agonist polypeptide. Also included are methods of making an engineered dual agonist polypeptide having the amino acid sequence of any one of SEQ ID NO:391 to SEQ ID NO:394, or combinations thereof, comprising culturing a host cell transformed with an Atty. Docket No. 2950-42 PCT

[0062] expression vector encoding a dual agonist polypeptide under conditions allowing expression of the dual agonist polypeptide, and recovering the dual agonist polypeptide.

[0063]

[0039] In aspects, the present application provides host cells comprising polynucleotide sequences encoding an engineered dual agonist polypeptide having the amino acid sequence of Formula VIII (SEQ ID NO:401 ) or Formula IX (SEQ ID NO:402). In aspects, host cells comprising polynucleotide sequences encoding an engineered dual agonist polypeptide having the amino acid sequence of any one of SEQ ID NO:391 to SEQ ID NO:394, or combinations thereof.

[0064]

[0040] 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 dual agonist polypeptide having the amino acid sequence of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402), 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 I diabetes, type II diabetes, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), metabolic syndrome X, metabolic dysfunction- associated steatohepatitis (MASH), Parkinson’s disease, and Huntington's Disease. In aspects, the methods comprise administering an engineered dual agonist polypeptide having the amino acid sequence of any one of SEQ ID NO:391 to SEQ ID NO:394, or combinations thereof.

[0065]

[0041] 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 dual agonist polypeptide having the amino acid sequence of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402). 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 dual agonist polypeptide having the amino acid sequence of any one of SEQ ID NO:391 to SEQ ID NO:394, or combinations thereof.

[0066]

[0042] 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 cel! transformed to express an engineered dual agonist polypeptide having the amino acid sequence of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402). 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 dual agonist polypeptide having the amino acid sequence of SEQ ID NO:391 to SEQ ID NO:394, and combinations thereof. Atty. Docket No. 2950-42 PCT

[0067]

[0043] The present application further includes, and provides for, administering a composition comprising an engineered dual agonist polypeptide having the amino acid sequence of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402) 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 dual agonist polypeptide having the amino acid sequence of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402) for glycemic control, promoting insulin production, reducing Ale, 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. In aspects, dual agonist polypeptides having the amino acid sequence of SEQ ID NO:391 to SEQ ID NO:394, 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 dual agonist polypeptide having the amino acid sequence of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402), or a polypeptide having the amino acid sequence of SEQ IDNO.391 to SEQ ID NO:394, or a combination thereof.

[0068]

[0044] The present application further includes, and provides for, administering a composition comprising an engineered dual agonist polypeptide having the amino acid sequence of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402), or a polypeptide having the amino acid sequence of SEQ ID NO:391 to SEQ ID NO:394, 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 dual agonist polypeptide is a direct fed microbial (DFMs) composition comprising bacteria transformed with a nucleic acid encoding a dual agonist polypeptide comprising the amino acid sequence of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402), or a polypeptide having the amino acid sequence of SEQ ID NO:391 to SEQ ID NO:394, or a combination thereof In one or more aspects, the composition comprising an engineered dual agonist polypeptide is an engineered composition comprising bacteria with a nucleic acid encoding engineered to secrete dual agonist polypeptide comprising the amino acid sequence of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402), or a polypeptide having the amino acid sequence of SEQ ID NO:391 to SEQ ID NO:394, or a combination thereof. In aspects, the host cell is engineered to secrete dual agonist polypeptide by plasmid-based expression or by chromosomal integration. Atty. Docket No. 2950-42 PCT

[0069]

[0045] 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 VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402); 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 SEQ ID NO:391 to SEQ ID NO:394.

[0070]

[0046] The present application further includes, and provides for, nucleic acid expression cassetes 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 V (SEQ ID NO:398), Formula VI (SEQ ID NO:399), or Formula VII (SEQ ID NO:400); a nucleic acid sequence comprising a translation terminator; and a nucleic acid sequence comprising transcription terminator. In aspects the nucleic acid sequence encodes eGLP-1 polypeptides comprising the amino acid sequence of any one of SEQ ID NO:387 to SEQ ID NO:389.

[0071]

[0047] The present application further includes, and provides for, methods of making an engineered polypeptide comprising the amino acid sequence of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402); 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. In aspects, the method provided is for making an engineered polypeptide comprising the amino acid sequence of any one of SEQ ID NO:391 to SEQ ID NO:394.

[0072]

[0048] The present application further includes, and provides for, methods of making an engineered polypeptide comprising the amino acid sequence of Formula V (SEQ ID NO:398), Formula VI (SEQ ID NO:399) or Formula VII (SEQ ID NO:400); 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. In aspects, the method provided is for making an engineered polypeptide comprising the amino acid sequence of any one of SEQ ID NO:387 to SEQ ID NO:389.

[0073]

[0049] The present application further includes, and provides for, pharmaceutical compositions comprising an engineered polypeptide comprising the amino acid sequence of Formula VIII (SEQ ID NO:40l) or Formula IX (SEQ ID NO:402) and a carrier. In aspects, pharmaceutical compositions are provided comprising an engineered polypeptide comprising the amino acid sequence of any one or more of SEQ ID NO:391 to SEQ ID NO:394.

[0074]

[0050] The present application further includes, and provides for, pharmaceutical compositions comprising an engineered polypeptide comprising the amino acid sequence of Formula V (SEQ ID Atty. Docket No. 2950-42 PCT

[0075] NO:398), Formula VI (SEQ ID NO:399) or Formula VII (SEQ ID NO:400) and a carrier. In aspects, pharmaceutical compositions are provided comprising an engineered polypeptide comprising the amino acid sequence of any one or more of SEQ ID NO:387 to SEQ ID NO:389.

[0076] [051 J 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 VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402). In aspects, pharmaceutical compositions are provided comprising a recombinant host cell comprising a polynucleotide encoding an engineered polypeptide of any one or more of SEQ ID NO:391 to SEQ ID NO:394.

[0077]

[0052] 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 V (SEQ ID NO:398), Formula VI (SEQ ID NO:399) or Formula VII (SEQ ID NO:400). In aspects, pharmaceutical compositions are provided comprising a recombinant host cell comprising a polynucleotide encoding an engineered polypeptide of any one or more of SEQ ID NO:387 to SEQ ID NO: 89.

[0078]

[0053] The present application further includes, and provides for, host cells comprising the polynucleotide encoding an engineered polypeptide comprising the amino acid sequence of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402). 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 VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402).

[0079]

[0054] The present application further includes, and provides for, host cells comprising the polynucleotide encoding an engineered polypeptide comprising the amino acid sequence of Formula V (SEQ ID NO:398), Formula VI (SEQ ID NO:399) or Formula VII (SEQ ID NO:400). 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 V (SEQ ID NO:398), Formula VI (SEQ ID NO:399) or Formula VII (SEQ ID NO:400).

[0080]

[0055] 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 VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402); a nucleic acid sequence comprising a translation terminator; and a nucleic acid sequence comprising transcription terminator.

[0081]

[0056] 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 VIII (SEQ ID NO:401 ) or Formula IX (SEQ ID NO:402). Atty. Docket No. 2950-42 PCT

[0082]

[0057] 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 V (SEQ ID NO.398), Formula VI (SEQ ID NO:399) or Formula VII (SEQ ID NO:400); a nucleic acid sequence comprising a translation terminator; and a nucleic acid sequence comprising transcription terminator.

[0083]

[0058] 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 V (SEQ ID NO:398), Formula VI (SEQ ID NO. G99) or Formula VII (SEQ ID NO:400).

[0084]

[0059] The present application further includes, and provides for, direct fed microbials (DFM) comprising bacteria transformed with a nucleic acid encoding an engineered dual agonist polypeptide comprising the amino acid sequence of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ I D NO:402) and / or encoding one or more engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of Formula V (SEQ ID NO:398), Formula VI (SEQ ID NO. G99) or Formula VII (SEQ ID NO:400). In aspects, the direct feed microbial (DFM) comprises bacteria transformed with a nucleic acid encoding one or more engineered dual agonist polypeptide selected from SEQ ID NO:391 to SEQ ID NO:394 and / or encoding one or more engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide selected from any of SEQ ID NO:387 to SEQ ID NO:389. In some aspects, said bacteria comprise a strain selected from the group consisting of Bacillus, Lactobacillus, Lactococcus, Enterococcus, Salmonella, and combinations thereof.

[0085]

[0060] The application further provides one or more variant albumin binding domain (ABD) sequence, wherein one or more trypsin site, or one or more Lys (K) or Arg (R), in the sequence LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NOG85) is substituted with an amino acid that is not susceptible to trypsin digestion. In an aspect, the variant albumin binding domain (ABD) sequence comprises the amino acid sequence selected from SEQ ID NO:395, SEQ ID NO:396 and SEQ ID NO:397. In some aspects, the variant albumin binding domain (ABD) sequence is covalently linked to one or more of a peptide therapeutic or one or more engineered polypeptide, particularly a peptide therapeutic or engineered polypeptide for use or application in type 2 diabetes mellitus (T2DM) or for treatment, control, or modulation of high blood glucose, metabolic disorders, excess body weight, or obesity. In aspects, the variant albumin binding domain (ABD) sequence is covalently linked to one or more of an engineered dual agonist or GLP-1 (eGLP-1) polypeptide. In embodiments, the variant albumin binding domain (ABD) sequence is covalently linked to one or more engineered dual agonist or GLP-1 (eGLP-1) polypeptide provided and described herein. Atty. Docket No. 2950-42 PCT

[0086]

[0061] The invention extends to variant albumin binding domain (ABD) based peptides, particularly wherein one or more of a peptide therapeutic or one or more engineered polypeptide, particularly a peptide therapeutic or engineered polypeptide for use or application in type 2 diabetes mellitus (T2DM) or for treatment, control, or modulation of high blood glucose, metabolic disorders, excess body weight, or obesity, is covalently Jinked to a variant albumin binding domain (ABD) sequence. In some aspects, the peptide therapeutic or engineered peptide covalently linked to a variant albumin binding domain (ABD) sequence described herein is selected from 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). In aspects, the invention extends to albumin binding domain based peptides, particularly wherein one or more of an engineered dual agonist or eGLP-1 polypeptide is covalently linked to an albumin binding domain (ABD) sequence. In aspects of this invention, the albumin binding domain (ABD) sequence comprises SEQ ID NO:384, SEQ ID NO:385 or SEQ ID NO:386 or comprises the variant albumin binding domain (ABD) sequence set out herein. In aspects, one or more of a peptide therapeutic or one or more engineered polypeptide, particularly a peptide therapeutic or engineered polypeptide for use or application in type 2 diabetes mellitus (T2DM) or for treatment, control, or modulation of high blood glucose, metabolic disorders, excess body weight, or obesity, is covalently linked to a variant albumin binding domain (ABD) sequence, wherein one or more trypsin site, or one or more Lys (K) or Arg (R), in the sequence LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO:385) is substituted with an amino acid that is not susceptible to trypsin digestion. In aspects, one or more of a peptide therapeutic or one or more engineered polypeptide is covalently linked to a variant albumin binding domain (ABD) sequence comprising the amino acid sequence selected from SEQ ID NO:395, SEQ ID NO:396 and SEQ ID NO:397.

[0087]

[0062] In aspects of the albumin binding domain (ABD) based peptides, the engineered dual agonist polypeptide sequence comprises the amino acid sequence of any of SEQ ID NO:391 to SEQ ID NO:394 or the eGLP-1 polypeptide sequence comprises the amino acid sequence of any of SEQ ID NO:387 to SEQ ID NO:389.

[0088]

[0063] The invention provides an engineered dual agonist or GLP-l (eGLP-1) polypeptide covalently linked to a variant albumin binding domain (ABD) sequence, wherein the engineered polypeptide sequence is selected from Formula I, Formula II, Formula III, Formula IV and the variant albumin binding domain (ABD) sequence is selected from SEQ ID NO:395, SEQ ID NO:396 and SEQ ID NO:397. In aspects thereof, engineered dual agonist or GLP-l (eGLP-1) polypeptide sequence is selected from SEQ IDNOs:3-14, SEQ ID NOs:372-380, SEQ ID NOs:l 12-147 and SEQ ID NOGS 1.

[0089]

[0064] Pharmaceutical compositions comprising the albumin binding domain based peptides and a carrier are provided. Atty. Docket No. 2950-42 PCT

[0090]

[0065] The invention includes an isolated polynucleotide encoding one or more variant albumin binding domain provided herein. In an aspect, the isolated polynucleotide encodes a variant albumin binding domain (ABD) sequence comprising the amino acid sequence selected from SEQ ID NO:395, SEQ ID NO:396 and SEQ ID NO:397. In an aspect, the isolated polynucleotide encodes a variant albumin binding domain (ABD) sequence comprising the amino acid sequence selected from SEQ ID NO:395, SEQ ID NO:396 and SEQ ID NO:397, and further encodes polypeptide comprising the engineered dual agonist polypeptide sequence comprising the amino acid sequence of any of SEQ ID NO:391 to SEQ ID NO:394 or the eGLP-1 polypeptide sequence comprising the amino acid sequence of any of SEQ ID NO:387 to SEQ IDNO:389. Host cells comprising the polynucleotide are also provided, including wherein said cell is a bacterial cell, plant cell, yeast cell, or algae cell.

[0091]

[0066] 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 1 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.

[0092]

[0067] 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.

[0093] BRIEF DESCRIPTION OF THE DRAWINGS

[0094]

[0068] The present inventions are disclosed with reference to the accompanying drawings, wherein:

[0095]

[0069] Figure 1 is a graphical representation of the proteolytic cleavage sites and enzymes in wild-type GLP-1 (SEQ ID NO: 18).

[0096]

[0070] 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 w hereas the N-terminal region of GLP-1 interacts with transmembrane of the GLP-1R receptor. From Cong et al., 2021, Figure 3a.

[0097]

[0071] 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) Atty. Docket No. 2950-42 PCT

[0098] central polar network, (ii) HETX motif polar network, (iii) TM2-6-7-helix 8 polar network. From Cong et al., 2021, Figure 4.

[0099]

[0072] Figure 4A and 4B is a graphical representation of the superimposed structures of reported peptides bound to the GLP-IR (A) and GIPR (B) receptors. (A) presents the superimposed GLP-IR 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-blue, 7FIY-grey), GIP (7RA3-white, 7DTY-cyan) and peptide-20 (7FIN-yellow).

[0100]

[0073] Figure 5A-5D is a graphical representation oof Cryo-EM structures of hGLPl and HGIP bound to hGLP-1 R. Cryo-EM structures of hGLPl and hGIP bound hGLP-1 R, hGIPR in complex are represented in A and C, respectively the modelled hGLPl and hGIP bound hGLP-1 R and hGIPR complexes are shown in B and D.

[0101]

[0074] Figure 6 is an alignment of cat and human GLP-IR 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 LI 45, Y146, and KI 98 in cGIPR (numbering based on SEQ ID NO: 221).

[0102]

[0075] Figure 7 is a graphical representation of GLP-1 activity according to an aspect of the present specification.

[0103]

[0076] Figure 8 is a sequence alignment of reported and engineered GLP-1 analogs.

[0104]

[0077] Figure 9A and 9B presents (A) the sequence of GLP- 1 (SEQ ID NO: 18) and GLP- 1 -Gly8 (SEQ ID NO: 19) modified to remove the DPP-4 protease cleavage site, and (B) depicts the structure of a 5XGLP-1 construct based on GLP-1-Gly8 or a trypsin stabilized peptide based from GLP-1-GLY8 with additional lysine (K) residue mutations.

[0105] (078] 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.

[0106]

[0079] 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-1 R receptor (yellow) and superimposed on the selected template 6X1 hGLP-1 (blue) / hGLP-lR(red).

[0107]

[0080] Figure 12 presents Tirzepatide peptide (green) docked against hGLP-lR receptor (white) and superimposed on the selected template 7FIM Tirzepatide(biue) / hGLP-lR(red). Atty. Docket No. 2950-42 PCT

[0108]

[0081] 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 1.3 A). cGIP is shown in red and hGIP in blue. hGIP residue 18His is represented in yellow colour and cGIP 18Arg in green.

[0109]

[0082] Figure 14A and 14B: (A). cGLP- 1 (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- 1 R (magenta) and hGLP- 1 R (Cyan).

[0110]

[0083] Figure 15A and 15B: (A) Residues (Leu393, Leu397, GIu396 and Thr400) around AIB2 of Tirzepatide against hGLP-lR and (B) residues (Leu375, Lys374, Ala359 and Glu355) around AIB2 against cGLP- 1 R.

[0111]

[0084] Figure 16A- 16C: Binding poses analysis of tirzepatide analogues after docking against cGLP- 1 R represent the difference in binding poses. (A, B) peptide-4 and 8 (cyan) mutation in tirzepatide(bkie) shows that N-terminal of the peptides w ere losing their helicity, while (C) peptide-6(cyan) is moving away from the binding region.

[0112]

[0085] Figure 17 is a graphical representation of GIP activity according to an aspect of the present specification.

[0113]

[0086] Figure I § is a graph of the effects of the test materials (various peptides or L reuteri expressing peptides) on weekly body weight at Day 40.

[0114]

[0087] 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 BE105ENLR181 (5x GLPl Cl.l BEP-009 sequence) were evaluated, with liraglutide as a control.

[0115]

[0088] Figure 20 depicts the effect of the GLP-1 peptides and constructs including BEP009-ABD on vreekly 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.

[0116]

[0089] 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. Atty. Docket No. 2950-42 PCT

[0117]

[0090] 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 (5xGLPl Cl.l BEP-009 sequence), with liraglutide as a control.

[0118]

[0091] 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 BE105ENLR181 (5x GLP1 Cl.l BEP-009 sequence), with liraglutide as a control.

[0119]

[0092] Figure 24 depicts the effect on lean mass in an EchoMRI at Day 1. 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.

[0120]

[0093] Figure 25 depicts the effect of the test materials and peptides on bi weekly food intake (normalized to vehicle control) at Day 84. The % change in food intake (Day 43 - Day 48) is graphed.

[0121]

[0094] Figure 26 depicts a GLP- 1 R 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.

[0122]

[0095] Figure 27 depicts a GLP- 1 R assay of various eGLP- 1 polypeptides and dual agonist polypeptides as well as variant albumin binding domain (ABD) peptides. GLP-1 and tirzepatide were used as positive controls for GLP- 1R activation.

[0123]

[0096] 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.

[0124] DETAILED DESCRIPTION

[0125] Definitions:

[0126]

[0097] As used herein the term “about” refers to ± 10 %.

[0127]

[0098] 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.

[0128]

[0099] The term “consisting of’ means “including and limited to”.

[0129]

[0100] 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. Atty. Docket No. 2950-42 PCT

[0130]

[0101] 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.

[0131]

[0102] 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.

[0132]

[0103] 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.

[0133]

[0104] 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.

[0134]

[0105] 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 li itations 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.

[0135]

[0106] “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 Atty. Docket No. 2950-42 PCT

[0136] 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.

[0137]

[0107] “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.

[0138]

[0108] “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.

[0139]

[0109] 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.

[0140]

[0110] 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. Atty. Docket No. 2950-42 PCT

[0141] Peptides

[0142] 111] 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.

[0143]

[0112] 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 NOG 82) or of SEQ ID NOG 72 to SEQ ID NO 80.

[0144] Table 1: Engineered GLP-1 sequences

[0145] Identifier Sequence SEQ ID NO

[0146] Formula 1 SEQ ID NO:1

[0147] Cl.l HGE GT FT S D VS S YLEGQAAQE F I AWLVDGR SEQ ID NO:2

[0148] C1.2 HGE GT S E S D VS S YLE GQAAQE F I AWL VDGR SEQ ID NO:3

[0149] C1.3 HGEGTS E S DVS S QLE GQAAQE F I AWL VDGR SEQ ID NO:4

[0150] Cl.4 HGE GT S E S DVS S Q I E GQAAQE FI AWLVDGR SEQ ID NO:5

[0151] C1.5 HGE GT S E S DVS QS I E GQAAQE F I AWLVDGR SEQ ID NO:6

[0152] C1.6 HGEGTSES DVSQSIEGQAAQEVIALWLVDGR SEQ ID NO:7

[0153] C1.7 HGE GT S E S DVS QS I EGQAAQE I VAWL VDGR SEQ ID NO:8

[0154] Cl.8 HGEGTS E S DVS QS I E GQAAQE I VA I VVDGR SEQ ID NO:9

[0155] C1.9 HGE GT S E S D V S Q S I EGQAAQE I VAV I VDGR SEQ ID NQ:10

[0156] C1.10 HGE GT S E S DVS QS I E GQAAQE VI AV I VDGR SEQ ID NO:11

[0157] Cl.8.1 HGE GT S E S DVS QS I EGQAAQE I VAIVVDGS SEQ ID NO:12

[0158] Cl.9.1 ™Z _ " " HCTGTSESDVSQSIEGQAAQE'lTOVIV^GS^ SEQTD N0i3..

[0159]

[0160] ci. io.1 SEQ ID NO:14

[0161] 113] Also included and provided for by the present applications are nucleic acid molecules encoding polypeptides having the amino acid sequences of SEQ ID NO:3 to SEQ ID NO: 14, transformed cells having nucleic acid molecules encoding polypeptides having the amino acid sequences of SEQ ID NO:3 to SEQ ID NO: 14.

[0162]

[0114] The present disclosure provides for, and includes, an engineered Glucagon-Like Peptide 1 (eGLP- 1) polypeptides comprising the amino acid sequence of Formula 1 (SEQ ID NO: 1)

[0163] H G E G T S E S D V S Xi2X!3X14E G Q A A Q E X22X23A X25X2«. V D G X3o (I)

[0164] wherein Xu = S or Q, XB::::S, Q, or V, Xu = I or L, X22= V, 1, or F, X23 = V or I, X25 = V, I, or W, X26 = 1 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 Atty. Docket No. 2950-42 PCT

[0165] 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 I. 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.

[0166]

[0115] In an aspect, the eGLP-1 polypeptide comprises the amino acid sequence of Formula I wherein X12 ~ Q, XB = S, X14 I or L, X22- V, I, or F, X23= V or I, X25 ~ V, I, or W, X26 = I or V, and X30 = 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 NOG82) or of any one of SEQ ID NO:372 to SEQ ID NOG80

[0167]

[0116] 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 ID: 1803), neprilysin (membrane metalloendopeptidase (MME), Gene ID:4 11, see also Gene ID:79258), the serine proteases a-chymotrypsin, trypsin, elastase, or acid proteases of the pepsin family.

[0168]

[0117] The present application further relates to additional novel engineered GLP-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. In some such aspects or embodiments, an engineered dual agonist polypeptide comprising the amino acid sequence selected from:

[0169] Formula V (SEQ ID NO:398):

[0170] Xi G E G T S E S D V S X12 X!3X14E X16Q A Xl9X20E X22X23A X25X26V D G X30(V), wherein Xi = H or Y; Xj2= S, Q, A or M; Xi?, = S, Q, Y, or M; Xj4= I or L; Xis = G or M; X19 ~ A or T; X20= Q, V, or D; X22- V, I, F or H; X23= V or I; X2S- V, I, or W; X26= I, V or L; and X30- R or S; Formula VI (SEQ ID NO:399):

[0171] Xi G E G T X6T S D V S X]2Xi3L E X16Q A X19 X20E X221 A X25L V D G R (VI),

[0172] wherein Xi = H or Y; Xa = F or I; X12 = S, A or M; X13 “ Y or M; Xn$= G or M; X19 = A or T; X»= Q, V, or D; X22F or H; and X2; = I or W; or

[0173] Formula VII (SEQ ID NO:400):

[0174] X) G E G T X6T S D V S X12X13Xl4E Xie Q A X19 X2o E X22X23A X25L V D G X30 (VII), wherein Xi = H or Y; Xf, = F or I; X12 = S, Q, A or M; X13 = S, Q, Y, or M; X,4= I or L; X^ G or M; X19~ A or T; X20= Q, V, or D; X22= V, I, F or H; X23= V or I; X25 = V, 1, or W: and X30= R or S.

[0175] In particular such aspects or embodiments, the engineered eGLP-1 polypeptide comprises the amino acid sequence of any one of SEQ ID NO:387 to SEQ ID NO:389. In aspects, the engineered polypeptide is Atty. Docket No. 2950-42 PCT

[0176] substantially resistant to proteolytic degradation. In aspects, the engineered polypeptide at least maintains substantially the same receptor potency as the corresponding wild-type, unmodified polypeptide. In aspects, the engineered polypeptide is substantially resistant to DPP-IV, neprilysin, a-chymotrypsin, trypsin, elastase, or pepsin, degradation.

[0177]

[0118] 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” is used interchangeably and refer to the polypeptides of Formula II (SEQ ID NO: 111), Formula VIII (SEQ ID NO:401) or Formula IX (SEQ I |D NO:402). 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. Particularly included are dual agonist polypeptides comprising the amino acid sequence of Formula VIII (SEQ ID NO:401 ) or Formula IX (SEQ ID NO:402) or polypeptides selected from SEQ ID NO:391 to SEQ ID NO:394. Generally, the dual agonist polypeptides relate to tirzepatide (LY3298176, SEQ ID NO: 208), a GIP analogue and a unimolecular, bifunctional (GIP and GLP-1 receptor) long- acting agonist.

[0178]

[0119] In particular, the present application relates to a compound of a dual agonist peptide of Formula II (SEQ ID NO: 111), Formula IV, Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402) 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.

[0179]

[0120] 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 VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402). 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.

[0180]

[0121] 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 Atty. Docket No. 2950-42 PCT

[0181] 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.

[0182]

[0122] In particular aspects, the dual agonist polypeptides comprise a polypeptide having the amino acid sequence of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402) or of SEQ ID NO:391 to SEQ ID NO:394.

[0183] Table 6: Dual Agonist Polypeptides

[0184] j 7FIM_V2 / A13 YVEGTFTSDYSIALDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 112 j 7FIM_V2 / S13 YVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 113 j 7FIM_V2 / Y13 YVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 114 j 7FIMJ / 2 / I13 YVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 115 7FIMJC R YVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPS SEQ ID NO: 116 7FIM V2 / Q13 YVEGTFTSDYSIQLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 117 7FIM_V2 / L13 YVEGTFTSDYSILLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 118 7FIM_V2 / R13 YVEGTFTSDYSIRLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 119 7FIMJC2 / V13 YKEGTFTSDYSIVLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 120 7FIM_K2 / K13 YKEGTFTSDYSIKLDKIAQKAFVQWUAGGPSSGAPP SEQ ID NO: 121 7FIM R H YVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPS SEQ ID NO: 122 Cl YVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPS SEQ ID NO: 123 Cl.l YVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPS SEQ ID NO: 124 C1.2 YVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPS SEQ ID NO: 125 C1.3 YVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO: 126 C1.4 YVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO: 127 Cl.4.1.1 YVEGTPCSDCSISLDHIAQKAPVQPVIAGGPSSGAPPPS SEQ ID NO: 128 Cl.4.1.2 YVEGTPCSDCSISLDKIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO: 129 Cl.4.1.3 YVEGTPCSDCSISLDKIAQKAPVQPVIAGGPSSGAPPPS SEQ ID NO: 130 Cl.4.2.1 YVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO: 131 Cl.4.2.2 YVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPS SEQ ID NO: 132 Cl.4.2.3 YVEGTSCSDCSISLDHIAQHAAVQKVIAGGPSSGAPPPS SEQ ID NO: 133 Cl.4.2.4 YVEGTSCSDCSISKDHIAQHAAVQKVIAGGPSSGAPPPS SEQ ID NO: 134 Cl.4.2.5 YVEGTSCSDCSISKDKIAQHAAVQKVIAGGPSSGAPPPS SEQ ID NO: 135 Cl.4.2.6 YVEGTSCSDCSISKDKIAQKAAVQKVIAGGPSSGAPPPS SEQ ID NO: 136 Cl.4.1 YVEGTSCSDCSISHDKIAQKAAVQKVIAGGPSSGAPPPS SEQ ID NO: 137 Cl.4.2 YVEGTSCSDCS1SHDKIAQKAAVQHVIAGGPSSGAPPPS SEQ ID NO: 138 Cl.4.3 YVEGTSCSDCSISHDKIAQKAAVQIVIAGGPSSGAPPPS SEQ ID NO: 139 Cl.4.4 YVEGTSCSDCSISHDVIAQKAAVQIVIAGGPSSGAPPPS SEQ ID NO: 140 Cl.4.5 YVEGTSCSDCSISIDVIAQKAAVQIVIAGGPSSGAPPPS SEQ ID NO: 141 Cl.4.5.1 YVEGTSESDCSISIDVIAQKAAVQIVIAGGPSSGAPPPS SEQ ID NO: 142 Cl.4.5.2 YVEGTSCSDESISIDVIAQKAAVQIVIAGGPSSGAPPPS SEQ ID NO: 143 Cl.4.5.3 YVEGTSESDESISIDVIAQKAAVQIVIAGGPSSGAPPPS SEQ ID NO: 144 Cl.4.5.1.1 YVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO: 145 Cl.4.5.2.1 YVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO: 146

[0185]

[0186] Cl.4.5.3.1 YVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO: 147 Atty. Docket No. 2950-42 PCT

[0187]

[0123] In aspects, the dual agonist polypeptide comprises a polypeptide of Formula (II), wherein X2V, X6 — S, X7 ~ C, X]Q ~ C or E, X13=S, X14 — I, Xi6 ~ V, X20 ~ H or N, X22A, X25 ~ I, and X26 ~ V.

[0188]

[0124] 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 particular aspects, the dual agonist polypeptides comprise the amino acid sequence of any one of SEQ ID NO:391 to SEQ ID NO:394 In other aspects, the dual agonist polypeptides comprise a carboxy terminal amide.

[0189]

[0125] In aspects, the dual agonist polypeptide, pharmaceutically acceptable salts, solvates, or other forms, and corresponding pharmaceutical compositions comprises the a ino acid sequence of any one of SEQ IDNO:391 to SEQ ID NO:394.

[0190]

[0126] 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:43 I I, 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 1. In particular aspects, the protease resistant dual agonist polypeptides comprise the polypeptides presented in Table 22.

[0191]

[0127] The present application further relates to additional 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. In some such aspects or embodiments, an engineered dual agonist polypeptide comprises the amino acid sequence selected from:

[0192] Formula VIII (SEQ ID NO:401):

[0193] Y V E G

[0194]

[0195] T F T S D Y S I S L Xi5X16I A Q X20A F X23Q W L I A G (VIII),

[0196] wherein XIJ= D or Q; X;s ~ R or Q; X2o= R or Q; and X23= V or M; or

[0197] Formula IX (SEQ ID NO:402):

[0198] Y X2E G T X6X7S D X10 S I X13Xl4XJ5X161 A X19X20A X22 X23Q X25X261 A G (IX), Atty. Docket No. 2950-42 PCT

[0199] wherein X2= V or K; X6= F, P or S; X7 = T, C or E; Xio= Y, C or E; Xi3= A, S, Y, N, I, L, R, V or K; Xi4= L, K, II or I; X)5= D or Q; X]6= K, R, H. V or Q; X!9= Q or V; X20= K, R, H, N or Q; X23- F, A or P: X23= V or M; X25= W, P, K, II or I; and X26= L or V.

[0200]

[0128] In some such aspects or embodiments, such one or more polypeptide may further comprise the amino acids GPSSGAPP at its C-terminal end.

[0201]

[0129] Particular new engineered dual agonist polypeptides comprise the amino acid sequence of any one of SEQ ID NO:391 to SEQ ID NO:394. Any such dual agonist polypeptide(s) may further comprise the amino acids GPSSGAPP at its C-terminal end.

[0202]

[0130] In aspects, provided herein is an isolated polynucleotide encoding:

[0203] an engineered dual agonist polypeptide comprising the amino acid sequence of Formula VIII (SEQ ID NO:401) or of Formula IX (SEQ IDNO:402); and / or

[0204] an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide comprising the amino acid sequence of Formula V (SEQ ID NO:398), of Formula VI (SEQ ID NO:399), or of Formula VII (SEQ ID NO:400).

[0205]

[0131] In particular aspects, host cells comprising the isolated polynucleotide are provided.

[0206]

[0132] In aspects, provided herein is a pharmaceutical composition comprising:

[0207] an engineered dual agonist polypeptide comprising the a ino acid sequence of Formula VIII (SEQ ID NO:40l) or of Formula IX (SEQ ID NO:402); and / or

[0208] an engineered Glucagon-Like Peptide I (eGLP-1) polypeptide comprising the amino acid sequence of Formula V (SEQ ID NO:398), of Formula VI (SEQ ID NO:399), or of Formula VII (SEQ ID NO:400); and a carrier.

[0209]

[0133] Kits comprising the pharmaceutical compositions or the host cells are also provided and contemplated herein.

[0210]

[0134] Included herein 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 an engineered dual agonist polypeptide and / or an engineered Glucagon-Like Peptide 1 (eGLP-1) as described herein including the new additional aspect polypeptides.

[0211]

[0135] 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 dyslipide ia, 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 an engineered dual agonist polypeptide and / or an engineered Glucagon-Like Peptide 1 (eGLP-1) as described herein including the new additional aspect polypeptides are provided. In particular aspects Atty. Docket No. 2950-42 PCT

[0212] of the methods, the dual agonist polypeptide comprises the amino acid sequence of any one of SEQ ID N0G91 to SEQ ID NOG94 or of Formula VIII (SEQ ID NO:401) or of Formula IX (SEQ ID NO:402), or the engineered Glucagon-Like Peptide I (eGLP-1) comprises the amino acid sequence of any one of SEQ ID NO:387 to SEQ ID NO:389, or of Formula V (SEQ ID NO:398), of Formula VI (SEQ ID NO:399), or of Formula VII (SEQ ID N0:400).

[0213]

[0136] A direct fed microbial (DFM) is provided herein comprising bacteria transformed with a nucleic acid encoding one or more engineered dual agonist polypeptide of Formula VIII or Formula IX and / or encoding one or more engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of Formula V, Formula VI or Formula VII. The direct feed microbial may comprise bacteria transformed with a nucleic acid encoding one or more engineered dual agonist polypeptide selected from SEQ ID NOG91 to SEQ ID NOG94 and / or encoding one or more engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide selected from any of SEQ ID NO:387 to SEQ ID NO:389. In aspects, said bacteria may comprise a strain selected from the group consisting of Bacillus, Lactobacillus, Lactococcus, Enterococcus, Salmonella, and combinations thereof. In aspects, the bacteria may be a Lactobacillus reuteri or a Bacillus subtilis strain.

[0214]

[0137] The application further provides one or more variant albumin binding domain (ABD) sequence, wherein one or more trypsin site, or one or more Lys (K) or Arg (R), in the sequence LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NOG85) is substituted with an amino acid that is not susceptible to trypsin digestion. In an aspect, the variant albumin binding domain (ABD) sequence comprises the amino acid sequence selected from SEQ ID NO:395, SEQ ID NO:396 and SEQ ID NO:397.

[0215]

[0138] The invention extends albumin binding domain based peptides, particularly wherein one or more of the engineered dual agonist or eGLP-1 polypeptides is covalently linked to an albumin binding domain (ABD) sequence. In aspects of this invention, the albumin binding domain (ABD) sequence comprises SEQ ID NOG84, SEQ ID NOG85 or SEQ ID NOG86 or comprises the variant albumin binding domain (ABD) sequence of claim 27 or 28. In aspects of the albumin binding domain based peptides, the engineered dual agonist polypeptide sequence comprises the amino acid sequence of any of SEQ ID NOG91 to SEQ ID NOG94 or the eGLP-1 polypeptide sequence comprises the amino acid sequence of any of SEQ ID NO:387 to SEQ ID NO:389.

[0216]

[0139] The invention provides an engineered dual agonist or GLP-1 (eGLP-1) polypeptide covalently linked to a variant albumin binding domain (ABD) sequence, wherein the engineered polypeptide sequence is selected from Formula I, Formula II, Formula III, Formula IV and the variant albumin binding domain (ABD) sequence is selected from SEQ ID NOG95, SEQ ID NO.396 and SEQ ID Atty. Docket No. 2950-42 PCT

[0217] NO:397. In aspects thereof, engineered dual agonist or GLP-1 (eGLP-1) polypeptide sequence is selected from SEQ ID NOs:3-14, SEQ ID NOs:372-380, SEQ ID NOs:l 12-147 and SEQ ID NO:381.

[0218]

[0140] Pharmaceutical compositions comprising the albumin binding domain based peptides and a carrier are provided.

[0219]

[0141] The invention includes an isolated polynucleotide encoding one or more variant albumin binding domain provided herein. In an aspect, the isolated polynucleotide encodes a variant albumin binding domain (ABD) sequence comprising the amino acid sequence selected from SEQ ID NO:395, SEQ ID NO:396 and SEQ ID NO:397. In an aspect, the isolated polynucleotide encodes a variant albumin binding domain (ABD) sequence comprising the amino acid sequence selected from SEQ ID NO:395, SEQ ID NO:396 and SEQ ID NO:397, and further encodes polypeptide comprising the engineered dual agonist polypeptide sequence comprising the amino acid sequence of any of SEQ ID NO:3 1 to SEQ ID NO:394 or the eGLP-1 polypeptide sequence comprising the amino acid sequence of any of SEQ ID NO:387 to SEQ ID NO:389. Host cells comprising the polynucleotide are also provided, including wherein said cell is a bacterial cell, plant cell, yeast cell, or algae cell.

[0220]

[0142] 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 dual agonist polypeptide, or alternatively having a lower level of degradation compared to tirzepatide. In aspects, the polypeptides maintain substantially the same receptor selectivity as the corresponding wt polypeptide. In certain aspects, the eGLP- 1 polypeptides provided herein exhibit increased receptor potency over the wt-GLP- 1 polypeptide. In certain aspects, the dual agonist polypeptides provided herein exhibit increased receptor potency over the wt dual agonist polypeptide. The eGLP-1 or 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 engineered 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. Atty. Docket No. 2950-42 PCT

[0221]

[0143] 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.

[0222]

[0144] 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.

[0223] Multimers

[0224]

[0145] 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 11 (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.

[0225]

[0146] 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). Multimers of the eGLP-1 polypeptides of Table 2 are presented in SEQ 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. Atty. Docket No. 2950-42 PCT

[0226] Accordingly, it is believe that upon exposure to trypsin, the eGLP-1 polypeptide monomers of SEQ ID NO:6 to SEQ ID NO:14 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. Multimers of the eGLP-1 polypeptide of Formula V (SEQ ID NO:398), of Formula VI (SEQ ID NO:399), or of Formula VII (SEQ ID NO:400) or of any of SEQ ID NO:387 to SEQ ID NO:389 are also contemplated and provided in aspects hereof. Multimers including multiple copies of the eGLP-1 polypeptides up to pentamers (e.g., 5x copies) of Formula V (SEQ ID NO:398), of Formula VI (SEQ ID NO:399), or of Formula VII (SEQ ID NO:400), or of any of SEQ ID NO:387-389 are contemplated.

[0227]

[0147] 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). Thus, multimers including multiple copies of the dual agonist polypeptides up to pentamers (e.g., 5x copies) Formula VIII (SEQ ID NO:401) or of Formula IX (SEQ ID NO:402), or of SEQ ID NO:391-394 are contemplated. 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 interna! 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.

[0228] Linkers and Fusions

[0229]

[0148] 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. Certain eGLP-1 polypeptides provided herein are fusion proteins comprising an eGLP- 1 polypeptide according to Formula V (SEQ ID NO:398), of Formula VI (SEQ ID NO:399), or of Formula VII (SEQ ID NO:400) or the amino acid sequence of any one of SEQ ID NO:387 to SEQ ID NO:389, and multimeric eGLP-1 polypeptides thereof, 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 Atty. Docket No. 2950-42 PCT

[0230] 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.

[0231]

[0149] 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).

[0232]

[0150] Certain dual agonist polypeptides provided herein are fusion proteins comprising the dual agonist polypeptide according to Formula II (SEQ ID NO: 111) 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: 113 to SEQ ID NO: 116 or the multimers SEQ ID NO: 164 to SEQ ID NO: 167. Certain dual agonist polypeptides provided herein are fusion proteins comprising a dual agonist polypeptide according to Formula VIII (SEQ ID NO:401) or of Formula IX (SEQ ID NO:402) or the amino acid sequence of any one of SEQ ID NO:391 to SEQ ID NO:394, and multimeric eGLP-1 polypeptides thereof, 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 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 Atty. Docket No. 2950-42 PCT

[0233] 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.

[0234]

[0151] 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.

[0235]

[0152] 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.

[0236]

[0153] 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: 70), GTGGGGS GGGGS GGGGS GGGGSA (SEQ ID NO:71), GGGGGS GGGGS GGGGS GGGGSA (SEQ ID NO:72) GGGGGSA (SEQ ID NO:73), GGGGGSGGGGSA (SEQ ID NO:74) 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.

[0237]

[0154] 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 IDNO.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 Atty. Docket No. 2950-42 PCT

[0238] peptide bond to the amino or carboxy terminus of the amino acid sequence of SEQ ID NO: 127 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:387 to SEQ ID NO:389 or any one of SEQ ID NO:391 to SEQ ID NO:394.

[0239]

[0155] 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. 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:387 to SEQ ID NO:389 or any one of SEQ ID NO:391 to SEQ ID NO:394.

[0240]

[0156] 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. 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:387 to SEQ ID NO:389 or any one of SEQ ID NO:391 to SEQ ID NO:394. Atty. Docket No. 2950-42 PCT

[0241]

[0157] 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. 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:387 to SEQ ID NO:389 or any one of SEQ ID NO:391 to SEQ ID NO:394.

[0242]

[0158] 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. 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:387 to SEQ ID NO:389 or any one of SEQ ID NO:391 to SEQ ID NO:394.

[0243]

[0159] 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), Atty. Docket No. 2950-42 PCT

[0244] 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. 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:387 to SEQ ID NO:389 or any one of SEQ ID NO:391 to SEQ ID NO:394.

[0245]

[0160] 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. 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:387 to SEQ ID NOG 89 or any one of SEQ ID NO:391 to SEQ ID NO:394.

[0246]

[0161] In certain aspects, the linker comprises GGGSG (SEQ ID NO: 294), wherein n is 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 NO: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 Atty. Docket No. 2950-42 PCT

[0247] (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. 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:387 to SEQ ID NO:389 or any one of SEQ ID NO:391 to SEQ ID NO:394.

[0248]

[0162] 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. 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:387 to SEQ ID NO:389 or any one of SEQ ID NO:391 to SEQ ID NO:394.

[0249]

[0163] 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 Atty. Docket No. 2950-42 PCT

[0250] 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. 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:387 to SEQ ID NO:389 or any one of SEQ ID NO:391 to SEQ ID NO:394.

[0251]

[0164] 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. 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:387 to SEQ ID NO:389 or any one of SEQ ID NO:391 to SEQ ID NO:394.

[0252]

[0165] 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. 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:387 to SEQ ID NO:389 or any one of SEQ ID NO:391 to SEQ ID NO:394. Atty. Docket No. 2950-42 PCT

[0253]

[0166] 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. 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:387 to SEQ ID NO:389 or any one of SEQ ID NO:391 to SEQ ID NO:394.

[0254]

[0167] 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:81), Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL, SEQ ID NO: 82), Dogtag (DIPATYEFTDGKHYITNEPIPPK, 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 (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP, SEQ ID NO:95), Sdytag (DPIVMIDNDKPIT, 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 Atty. Docket No. 2950-42 PCT

[0255] (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 (Y TDIEMNRLGK, 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.

[0256]

[0168] 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 111 (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. 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:387 to SEQ ID NO:389, or of any of SEQ ID NO:391 to SEQ ID NO:394, or any of Formula VIII (SEQ ID NO:401), Formula IX (SEQ ID NO:402) and / or Formula V (SEQ ID NO:398), of Formula VI (SEQ ID NO:399), or of Formula VII (SEQ ID NO:400).

[0257]

[0169] Importantly, the effects of linker, peptide tag, hinge, and Fc linker addition can be assessed using the 3-D models of GLP-1 R receptor structure molecular docking methods described below.

[0258] Modifications

[0259]

[0170] The present application further includes, and provides for, lipidated eGLP-I 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.

[0260]

[0171] “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.

[0261]

[0172] 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 Atty. Docket No. 2950-42 PCT

[0262] 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 atachment 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 atached via the C -terminus, the N-terminus, the side chain, or any combination thereof.

[0263]

[0173] “Polyethylene glycol” or “PEG” is a polyether monovalent radical of general formula -(O-CH₂-CH₂)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-CH₂-CH₂)₃-O-, while PEG 8 can correspond with a monovalent radical of formula -(O-CH₂-CH₂)₈-OH.

[0264]

[0174] 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 w'eight 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, PEG5, PEG6, PEG7, PEG8, PEG9, PEG 10, PEG11, PEG 12, PEG 18, or PEG24. In certain aspects, the PEG is PEG2, PEG6, or PEG24.

[0265] Polynucleotides

[0266] 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:6 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. In aspects, the nucleic acids encode an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide comprising the amino acid sequence of Formula V (SEQ ID NO:398), of Formula VI (SEQ ID NO:399), or of Formula VII (SEQ ID NO:400), or of SEQ ID NO: 387 to SEQ ID NO: 389.

[0267]

[0175] 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 Atty. Docket No. 2950-42 PCT

[0268] 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. In aspects, the nucleic acids encode an engineered dual agonist polypeptide comprising the amino acid sequence of Formula VIII (SEQ ID NO:401) or of Formula IX (SEQ ID NO:402) or of SEQ ID NO:391 to SEQ ID NO: 394.

[0269]

[0176] The terms “polynucleotide' 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).

[0270]

[0177] 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.

[0271] 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.

[0272]

[0178] 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.

[0273]

[0179] 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 ceils 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 Schizosaccharomyc.es pombe), and various animal cells, such as insect cells (e.g., Sf-9) or mammalian cells (e.g., HEK293F, CHO, COS 7, N1H-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). Atty. Docket No. 2950-42 PCT

[0274]

[0180] 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).

[0275]

[0181] 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.

[0276]

[0182] 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 modu lation 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.

[0277]

[0183] 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.

[0278]

[0184] As used herein, a “subject in need thereof’ refers to an individual for w'hom 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. Atty. Docket No. 2950-42 PCT

[0279] Methods of making

[0280]

[0185] 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.

[0281]

[0186] 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.

[0282]

[0187] 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.

[0283]

[0188] 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 Atty. Docket No. 2950-42 PCT

[0284] 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 / OPTlMIZER) 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 / ).

[0285]

[0189] 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 ter inus 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 tag, and the generation of the eGLP-1 polypeptide or dual agonist polypeptide.

[0286]

[0190] 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.

[0287] Formulations and Compositions

[0288]

[0191] 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 Atty. Docket No. 2950-42 PCT

[0289] polypeptides or dual agonist polypeptides as disclosed are soluble in standard buffers over a broad pH range.

[0290]

[0192] 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.

[0291]

[0193] 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 NO: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. In aspects, the host cells express a dual agonist polypeptide comprising the amino acid sequence of Formula VIII (SEQ ID NO:401) or of Formula IX (SEQ ID NO:402), or an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide comprising the amino acid sequence of Formula V (SEQ ID NO:398), of Formula VI (SEQ ID NO:399), or of Formula VII (SEQ ID NO:400). In aspects, the host cells express a dual agonist polypeptide or eGLP- 1 polypeptide comprising one or more amino acid sequence selected from SEQ ID NO:391-394 or SEQ IDNO:387-389.

[0292]

[0194] 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 Atty. Docket No. 2950-42 PCT

[0293] 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, weting agents, emulsifiers, and buffers etc.

[0294]

[0195] 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.

[0295]

[0196] 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.

[0296]

[0197] 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) Atty. Docket No. 2950-42 PCT

[0297] absorbents, colorants, flavors and sweeteners. Tablets may be either film coated or enteric coated according to methods known in the art.

[0298]

[0198] 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.

[0299] Host Cells

[0300]

[0199] 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.

[0301]

[0200] The present application further provides for, and includes, host cells transformed with polynucleotides encoding peptides having the amino acid sequence of Formula 11 (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 Atty. Docket No. 2950-42 PCT

[0302] 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.

[0303]

[0201] Engineered vectors are contemplated and provided herein encoding and host cells transformed with vectors encoding one or more of engineered polypeptides selected from a dual agonist polypeptide comprising the amino acid sequence of Formula VIII (SEQ ID NO:401) or of Formula IX (SEQ ID NO:402); and / or an engineered Glucagon-Like Peptide 1 (eGLP-i) polypeptide comprising the amino acid sequence of Formula V (SEQ ID NO:398), of Formula VI (SEQ ID NO:399), or of Formula VII (SEQ ID NO:400); or a polypeptide comprising the amino acid sequence of any of SEQ ID NOs:387-389 or of SEQ ID NOs:391-394.

[0304]

[0202] 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:6 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.

[0305]

[0203] 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. Atty. Docket No. 2950-42 PCT

[0306]

[0204] 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: 112 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.

[0307]

[0205] 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.

[0308]

[0206] 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.

[0309]

[0207] 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 pastor 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, Z7(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).

[0310]

[0208] 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 Atty. Docket No. 2950-42 PCT

[0311] 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.

[0312]

[0209] 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,

[0313]

[0210] 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.

[0314]

[0211] 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.

[0315]

[0212] In particular aspects, a direct fed microbial (DFM) comprising bacteria transformed with a nucleic acid encoding one or more engineered dual agonist polypeptide of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402) and / or encoding one or more engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of Formula V (SEQ ID NO:398), Formula VI (SEQ ID NO:399) or Formula VII (SEQ ID NO:400) is provided. The direct feed microbial (DFM) in aspects comprises bacteria transformed with a nucleic acid encoding one or more engineered dual agonist polypeptide selected from SEQ ID NO:391 to SEQ ID NO:394 and / or encoding one or more engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide selected from any of SEQ ID NO:387 to SEQ ID NO:389.

[0316]

[0213] Probiotics and DFMs provide an attractive delivery method to provide therapeutic amounts of the dual agonist polypeptides provided herein.

[0317]

[0214] 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, Atty. Docket No. 2950-42 PCT

[0318] 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.

[0319]

[0215] 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.

[0320] DFMs as a delivery system

[0321]

[0216] 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.

[0322]

[0217] 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 1. 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 NO:3 to SEQ ID NO: 14. Atty. Docket No. 2950-42 PCT

[0323]

[0218] 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.

[0324]

[0219] 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).

[0325]

[0220] In 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).

[0326] Host cells as a production system

[0327]

[0221] 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.

[0328]

[0222] 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

[0329] 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). Atty. Docket No. 2950-42 PCT

[0330] Kits

[0331]

[0223] 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.

[0332]

[0224] 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.

[0333] Methods of treatment

[0334]

[0225] 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.

[0335]

[0226] 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).

[0336]

[0227] 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. In 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 Atty. Docket No. 2950-42 PCT

[0337] 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.

[0338]

[0228] 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. 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 Formula V (SEQ ID NO:398), Formula VI (SEQ ID NO:399) or Formula VII (SEQ ID NO:400) or of SEQ ID NO:387 to SEQ ID NO:389.

[0339]

[0229] 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.. 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 Formula V (SEQ ID NO:398), Formula VI (SEQ ID NO:399) or Formula VII (SEQ ID NO:400) or of SEQ ID NO:387 to SEQ ID NOG 89.

[0340]

[0230] 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, 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- Atty. Docket No. 2950-42 PCT

[0341] 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-l polypeptide comprising the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO: 14. In aspects, the method of treatment comprises providing a therapeutic amount of an eGLP-l polypeptide comprising the amino acid sequence of any one of Formula V (SEQ ID NO:398), Formula VI (SEQ ID NO:399) or Formula VII (SEQ ID NO:400) or of SEQ ID NO:387 to SEQ ID NO:389.

[0342] [231 J 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 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-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-l polypeptide comprising the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO: 14. In aspects, the method of treatment comprises providing a therapeutic amount of an eGLP-l polypeptide comprising the amino acid sequence of any one of Formula V (SEQ ID NO:398), Formula VI (SEQ ID NO:399) or Formula VII (SEQ ID NO:400) or of SEQ ID NO:387 to SEQ ID NO:389.

[0343]

[0232] The present application provides for, and includes methods of treatment comprising providing an eGLP-l 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-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.

[0344]

[0233] The present application provides for, and includes methods of treatment comprising providing an eGLP-l 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 Ait}'. Docket No. 2950-42 PCT

[0345] improving and / or restoring or protecting the functionality of pancreatic beta cells and / or restoring the functionality of pancreatic insulin secretion.

[0346]

[0234] 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, 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:3 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. 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 Formula V (SEQ ID NO:398), Formula VI (SEQ ID NO:399) or Formula VII (SEQ ID NO:400) or of SEQ ID NO:387 to SEQ ID NO:389.

[0347]

[0235] 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:3 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 Formula V (SEQ ID NO:398), Formula VI (SEQ ID NO:399) or Formula VII (SEQ ID NO:400) or of SEQ ID NO:3 to SEQ ID NO: 14.

[0348]

[0236] 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:3 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. In aspects, the method of treatment comprises providing a therapeutic Atty. Docket No. 2950-42 PCT

[0349] amount of an eGLP-1 polypeptide comprising the amino acid sequence of any one of Formula V (SEQ ID NO:398), Formula VI (SEQ ID NO:399) or Formula VII (SEQ ID NO:400) or of SEQ ID NO:387 to SEQ ID NO:389.

[0350]

[0237] 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:3 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. 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 Formula V (SEQ ID NO:398), Formula VI (SEQ ID NO:399) or Formula VII (SEQ ID NO:400) or of SEQ ID NO:387 to SEQ ID NO:389.

[0351]

[0238] 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-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:3 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. 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 Formula V (SEQ ID NO.398), Formula VI (SEQ ID NO:399) or Formula VII (SEQ ID NO:400) or of SEQ ID NO:387 to SEQ ID NO:389.

[0352]

[0239] 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), Atty. Docket No. 2950-42 PCT

[0353] glucagonomas, cardiovascular diseases such as congestive heart failure, atherosclerosis, arteriosclerosis, coronary heart disease, or peripheral artery disease, stroke, respiratory dysfunction, or renal disease.

[0354]

[0240] 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. In aspects, the method of treatment comprises providing a therapeutic amount of a dual agonist polypeptide comprising the amino acid sequence of any one of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402) or of SEQ ID NO:391 to SEQ ID NO:397.

[0355]

[0241] 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. In aspects, the methods comprise providing a dual agonist polypeptide comprising the amino acid sequence of any one of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402) or of SEQ ID NO:391 to SEQ ID NO:397.

[0356]

[0242] 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 Atty. Docket No. 2950-42 PCT

[0357] 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. Provided are methods of providing a dual agonist polypeptide comprising the amino acid sequence of any one of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402) or of SEQ ID NO:391 to SEQ ID NO:397.

[0358]

[0243] 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. In aspects, the method of treatment comprises providing a therapeutic amount of a dual agonist polypeptide comprising the amino acid sequence of any one of Formula VIII (SEQ ID NO:401 ) or Formula IX (SEQ ID NO:402) or of SEQ ID NO:391 to SEQ ID NO:397.

[0359]

[0244] 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. In aspects, method of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402) or of SEQ ID NO:391 to SEQ ID NO:397 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.

[0360]

[0245] 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 Atty. Docket No. 2950-42 PCT

[0361] 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. In aspects, methods are provided comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402) or of SEQ ID NO:391 to SEQ ID NO:397.

[0362]

[0246] 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. In aspects, the application includes and provides providing a dual agonist polypeptide of any one of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402) or of SEQ ID NO:391 to SEQ ID NO:397, 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.

[0363]

[0247] 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: Atty. Docket No. 2950-42 PCT

[0364] 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 atributed to an abnormal accumulation of liver or ectopic fat. The present application provides for, and includes such methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402) or of SEQ ID NO:391 to SEQ ID NO:397.

[0365]

[0248] 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. The present application provides for, and includes such methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402) or of SEQ ID NO:391 to SEQ ID NO:397.

[0366]

[0249] 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, Atty. Docket No. 2950-42 PCT

[0367] 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 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). The present application provides for, and includes such methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402) or of SEQ ID NO:391 to SEQ ID NO:397.

[0368]

[0250] 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. The present application provides for, and includes such methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402) or of SEQ ID NO:391 to SEQ ID NO:397.

[0369]

[0251] 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 Atty. Docket No. 2950-42 PCT

[0370] 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. The present application provides for, and includes such methods of treatment comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402) or of SEQ ID NO:391 to SEQ ID NO:397.

[0371]

[0252] 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. Atty. Docket No. 2950-42 PCT

[0372]

[0253] 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. The present application provides for, and includes such methods of treatment comprising providing a dual agonist polypeptide comprising providing a host cell expressing a polypeptide comprising the amino acid sequence of any one of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402) or of SEQ ID NO:391 to SEQ ID NO:397. Atty. Docket No. 2950-42 PCT

[0373]

[0254] In 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.

[0374]

[0255] 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: 111 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. The present application provides for, and includes such methods comprising providing a dual agonist polypeptide comprising the amino acid sequence of any one of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402) or of SEQ ID NO:391 to SEQ ID NO:397. Atty. Docket No. 2950-42 PCT

[0375]

[0256] 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.

[0376]

[0257] 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-l 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.

[0377]

[0258] The route of administration of eGLP-l 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-l polypeptides or dual agonist polypeptides provided herein can be administered as a single dose or as multiple doses. In aspects, the eGLP-l polypeptides or dual agonist polypeptides are configured for transdermal administration. In certain aspects, an eGLP-l polypeptide or dual agonist polypeptide is administered orally or by subcutaneous injection.

[0378]

[0259] 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).

[0379]

[0260] The amounts of eGLP-l 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-l 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 Atty. Docket No. 2950-42 PCT

[0380] 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.

[0381]

[0261] 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.

[0382]

[0262] 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 1 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:3 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. 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 Formula V (SEQ ID NO:398), Formula VI (SEQ ID NO:399) or Formula VII (SEQ ID NO:400) or of SEQ ID NO:387 to SEQ ID NO:389. 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: 11 to SEQ ID NO: I f 6, 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. In aspects, the dual agonist polypeptides comprise the amino acid sequence of any one of Formula VIII (SEQ ID NO:401 ) or Formula IX (SEQ ID NO:402) or of SEQ ID NO:391 to SEQ ID NOG97. Atty. Docket No. 2950-42 PCT

[0383] [263 J 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. T he 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.

[0384] [264j 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.

[0385]

[0265] 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. In aspects, the medicaments comprise a therapeutic amount of an eGLP- 1 polypeptide comprising the amino acid sequence of any one of Formula V (SEQ ID NO:398), Formula VI (SEQ ID NO:399) or Formula VII (SEQ ID NO:400) or of SEQ ID NO:387 to SEQ ID NO:389.

[0386]

[0266] 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 Atty. Docket No. 2950-42 PCT

[0387] any one of SEQ ID NO: 113 to SEQ ID NO: 147. 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. In aspects, the dual agonist polypeptides comprise the amino acid sequence of any one of Formula VIII (SEQ ID NO:40l) or Formula IX (SEQ ID NO:402) or of SEQ ID NO:391 to SEQ ID NO:397.

[0388] Combination treatments

[0389]

[0267] In certain aspects an eGLP-1 polypeptide or dual agonist polypeptide as provided herein can be administered in combination with one or more additional therapies. In aspects, the eGLP-1 polypeptides or dual agonist polypeptides may be administered as part of a combination therapy with an agent for treatment of diabetes, obesity, dyslipidemia or hypertension.

[0390]

[0268] 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.

[0391]

[0269] SEQUENCES

[0392] SEQ IDNO:1 Formula I HGEGTSESDVSXXXEGQAAQEXXAXXVDGX

[0393] SEQ ID NO:2 C 1.1 HGEGTFTSD VS S YLEGQ AAQEFI A WL VDGR

[0394] SEQ ID NO:3 Cl.2 HGEGTSESDVSSYLEGQ QEFIAWLVDGR

[0395] SEQ IDNO:4 Cl.3 HGEGTSESDVSSQLEGQAAQEFIAWLVDGR

[0396] SEQ ID NO: 5 C1.4 HGEGTSESDVSSQIEGQAAQEFIAWLVDGR

[0397] SEQ IDNO:6 Cl.5 HGEGTSESDVSQSIEGQAAQEFIAWLVDGR

[0398] SEQ IDNO:7 Cl.6 HGEGTSESDV SQSIEGQAAQEVI WLVDGR

[0399] SEQ ID NO: 8 C1.7 HGEGTSESDVSQSIEGQAAQEIVAWLVDGR

[0400] SEQ ID NO:9 Cl.8 HGEGTSESDVSQSIEGQAAQEIVAIVVDGR

[0401] SEQ ID NO: 10 C1.9 HGEGTSESDVSQSIEGQAAQEIVAVIVDGR

[0402] SEQ ID NO: 11 Cl.10 HGEGTSESDVSQSIEGQAAQEVIAVIVDGR

[0403] SEQ ID NO: 12 Cl.8.1 HGEG TSESDVSQSIEGQAAQEIVAI VVDGS

[0404] SEQ ID NO: 13 Cl.9.1 HGEGTSESDVSQSIEGQAAQEIVAVIVDGS

[0405] SEQ ID NO: 14 Cl.10.1 HGEGTSESDVSQSIEGQAAQEVIAVIVDGS

[0406] SEQ ID NO: 15 hGLPIR MAGAPGPLRLAVLLLGMVGRAGPRPQGATVSLWETVQKWREYRRQCQRSLTE DPPPATDLFCNRTFDEYACWPDGEPGSFVNVSCPWYLPWASSVPQGHVYRFCTAEGLW Atty. Docket No. 2950-42 PCT

[0407] LQKDNSSLPWRDLSECEESKRGERSWGEEQLLFLYIIYTVAYALSFSALVIASAILLGFRH LHCTRNYIHLNLFASFILRALSVFIKDAALKWMYSTAAQQHQWDGLLSYQDSLSCRLVF LLMQYCVAANYYWLLVEGVYLYTLLAFSVFSEQW1FRLYVSIGWGVPLLFVVPWGIVKI LYEDEGCWTRNSNMNYWLIIRLPILFAIGVNFLIFVRVICIVVSKLKANLMCKTDIKCRLA KSTLTLIPLLGTIIEVIFAFVMDEHARGTLRFIKLFTELSFTSFQGLMVAILYCFVNNEVQLE FRKSWERWRLEHLHIQRDSSMKPLKCPTSSLSSGATAGSSMYTATCQASCS SEQ ID NO: 16 cGLPIR MAGAPSPLCLALLLLGAVGRAGPRPQGATVSLSETVQKWREYRHQCQRFLTEAP PPATGLFCNRTFDEYACWPDGLPGSFVNVSCPWYLPWASSVLQGHVYRFCTAEGLWLR QDNSSLPWRNLSECEESKRGERSSPEEQLLSFSIIYTVGYTLSFSALVIASAILLSFRHLHCT RNYIHLNLFASFILRALSVFIRDAVLKWMYSTAPQQHQWDGLLSYQDSLGCRLVFLLMQ YCVAANYYWLLVEGVYLYTLLAFSVFSEQRIFRLYLSIGWGVPLLFVIPWGIVKYLYEDE GCWTRNSNMNYWLIRLPILFAIGVNFLIFVRVICIVVSKLKANLMCKTDIKCRLAKSTLT LIPLLGTHEVVFAFVMDEHARGTLRFIKLFTELSFTSFQGLMVAILYCFVNNEVQMEFRRS WERWRLKHLHIQRDSSMKPLKCPTSSLTSGGTVGSSVYAASCQASCS SEQ ID NO: 17 Exendin-4 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS SEQ ID NO: 18 GLP-1 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR

[0408] SEQ ID NO: 19 GLP-1-Gly8 HGEGTFTSDVSSYLEGQAAKEFIAWLVKGR

[0409] SEQ ID NO:20 Trp 5xGLP-l HGEGTFTSDVSSYLEGQAAQEFIAWLVDGR

[0410] SEQ ID NO:21 Liraglutide HAEFTFTSDVSSYLEGQAAKEFIAWLVRGRG (hexadecanoyl group attached to lysine via a glutamic acid spacer)

[0411] SEQ ID NO:22 Semaglutide HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG (K20 is conjugated with a semiglutied pubchem id 90016781)

[0412] SEQ ID NO:23 J211 EUEGSFTSDVSSFLEGEAAAEFIAFVV^GG (K26 is alpha-K) SEQ ID NO:24 J229 K4EGS TSDVSSELEGEAAKEFIAFVV GG (K26 is lipidated) SEQ ID NO:25 Medi7219 HaEGSfTSDVsSKLEGEAAkEflAK VVEGG (lower case is alpha-methyl, US2018 / 0162920 SEQ ID 263)

[0413] SEQ ID NO:26 GLP1 A8G HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

[0414] SEQ ID NO:27 GLP1 K20Q HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

[0415] SEQ ID NO:28 GLP1 K28D HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

[0416] SEQ ID NO:29 Hid SE M AADIISTIGDLVKWIIDTVNKFKK.

[0417] SEQ ID NO:30 5xGLP 1 trp-ABDcon MHGEGTFTSDVSSYLEGQAAQEFIAWLVDGRHGEGTFTSDVSSYLEGQAAQEFI AWLVDGRHGEGTFTSDVSSYLEGQAAQEFIAWLVDGRHGEGTFTSDVSSYLEGQAAQE Atty. Docket No. 2950-42 PCT

[0418] FIAWLVDGRHGEGTFTSDVSSYLEGQAAQEFIAWLVDGRGAPVPYPDPLEPRGGGGSGG GGSGGGGSLKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA* SEQ ID NO:31 5xGLP1trp HGEGTFTSDVSSYLEGQAAQEFIAWLVDGRHGEGTFTSDVSSYLEGQAAQEFIA WLVDGRHGEGTFTSDVSSYLEGQAAQEFIAWLVDGRHGEGTFTSDVSSYLEGQAAQEFI AWLVDGRHGEGTFTSDVSSYLEGQAAQEFIAWLVDGRGAPVPYPDPLEPR SEQ IDNO:32 > C1.2-2x HGEGTSESDVSSYLEGQAAQEFIAWLVDGX HGEGTSESDVSSYLEGQAAQEFIAWLVDG (X - R or K)

[0419] SEQ IDNO:33 > C1.2-3x HGEGTSESDVSSYLEGQAAQEFIAWLVDGXHGEGTSESDVSSYLEGQAAQEFIA WLVDGXHGEGTSESDVSSYLEGQAAQEFIAWLVDG (X = R or K)

[0420] SEQ IDNO:34 > C1.2-4x HGEGTSESDVSSYLEGQAAQEFIAWLVDGXHGEGTSESDVSSYLEGQAAQEFIA WLVDGXHGEGTSESDVSSYLEGQAAQEFIAWLVDGXHGEGTSESDVSSYLEGQAAQEFI AWLVDG (X = R OR K)

[0421] SEQ ID NO:35 > C1.2-5x HGEGTSESDVSSYLEGQAAQEFIAWLVDGXHGEGTSESDVSSYLEGQAAQEFIA WLVDGXHGEGTSESDVSSYLEGQAAQEFIAWLVDGXHGEGTSESDVSSYLEGQAAQEFI AWLVDGXHGEGTSESDVSSYLEGQAAQEFIAWLVDG (X = R or K)

[0422] SEQ IDNO:36 > C1.3-2x HGEGTSESDVSSQLEGQAAQEFIAWLVDGX HGEGTSESDVSSQLEGQAAQEFIAWLVDG (X - R OR K)

[0423] SEQ IDNO:37 > C1.3-3x HGEGTSESDVSSQLEGQAAQEFIAWLVDGXHGEGTSESDVSSQLEGQAAQEFIA WLVDGXHGEGTSESDVSSQLEGQAAQEFIAWLVDG (X - R OR K)

[0424] SEQ IDNO:38 > C1.3-4x HGEGTSESDVSSQLEGQAAQEFIAWLVDGXHGEGTSESDVSSQLEGQAAQEFIA WLVDGXHGEGTSESDVSSQLEGQAAQEFIAWLVDGXHGEGTSESDVSSQLEGQAAQEFI AWLVDG (X = R OR K)

[0425] SEQ IDNO:39 > C1.3-5x HGEGTSESDVSSQLEGQAAQEFIAWLVDGXHGEGTSESDVSSQLEGQAAQEFIA WLVDGXHGEGTSESDVSSQLEGQAAQEFIAWLVDGXHGEGTSESDVSSQLEGQAAQEFI AWLVDGXHGEGTSESDVSSQLEGQAAQEFIAWLVDG (X - R OR K) Atty. Docket No. 2950-42 PCT

[0426] SEQ IDNO:40 > CL4-2x

[0427] HGEGTSESDVSSQIEGQAAQEFIA WLVDG XHGEGTSESDVSSQIEGQAAQEFIAW LVDG (X = R or K)

[0428] SEQ ID NO:41 > C1.4-3x

[0429] HGEGTSESDVSSQIEGQAAQEFIA WLVDGXHGEGTSESDVSSQIEGQAAQEFIAW LVDGXHGEGTSESDVSSQIEGQAAQEFIA WLVDG (X = R OR K)

[0430] SEQ IDNO:42 > C1.4-4x

[0431] HGEGTSESDVSSQIEGQAAQEFIA WLVDGXHGEGTSESDVSSQIEGQAAQEFIAW LVDGXHGEGTSESDVSSQIEGQAAQEFIA WLVDGXHGEGTSESDVSSQIEGQAAQEFIAW LVDG (X = R OR K)

[0432] SEQ ID NO:43 > C1.4-5x HGEGTSESDVSSQIEGQAAQEFIAWLVDGXHGEGTSESDVSSQIEGQAAQEFIAW LVDGXHGEGTSESDVSSQIEGQAAQEFIA WLVDGXHGEGTSESDVSSQIEGQAAQEFIAW LVDGXHGEGTSESDVSSQIEGQAAQEFIA WLVDG (X = R OR K)

[0433] SEQ IDNO:44 > C1.5-2x HGEGTSESDVSQSIEGQAAQEFIAWLVDGXHGEGTSESDVSQSIEGQAAQEFIAW LVDG (X = R or K)

[0434] SEQ IDNO:45 > C1.5-3x HGEGTSESDVSQSIEGQAAQEFIAWLVDGXHGEGTSESDVSQSIEGQAAQEFIAW LVDGXHGEGTSESDVSQSIEGQAAQEFIAWLVDG (X = R OR K)

[0435] SEQ IDNO:46 > C1.5-4x HGEGTSESDVSQSIEGQAAQEFIAWLVDGXHGEGTSESDVSQSIEGQAAQEFIAW LVDGXHGEGTSESDVSQSIEGQAAQEFIAWLVDGXHGEGTSESDVSQSIEGQAAQEFIAW LVDG (X = R OR K)

[0436] SEQ IDNO:47 > C1.5-5x HGEGTSESDVSQSIEGQAAQEFIAWLVDGXHGEGTSESDVSQSIEGQAAQEFIAW LVDGXHGEGTSESDVSQSIEGQAAQEFIAWLVDGXHGEGTSESDVSQSIEGQAAQEFIAW LVDGXHGEGTSESDVSQSIEGQAAQEFIAWLVDG (X = R OR K)

[0437] SEQ IDNO:48 > C1.6-2x HGEGTSESDVSQSIEGQAAQEVIAWLVDGXHGEGTSESDVSQSIEGQAAQEVIA WLVDG (X = R OR K)

[0438] SEQ IDNO:49 > C1.6-3x HGEGTSESDVSQSIEGQAAQEVIAWLVDGXHGEGTSESDVSQSIEGQAAQEVIA WLVDGXHGEGTSESDVSQSIEGQAAQEVIA WLVDG (X = R OR K) Atty. Docket No. 2950-42 PCT

[0439] SEQ IDNO:50 > C1.6-4x HGEGTSESDVSQSIEGQAAQEVIAWLVDGXHGEGTSESDVSQSIEGQAAQEVIA WLVDGXHGEGTSESDVSQSIEGQAAQEVIAWLVDGXHGEGTSESDVSQSIEGQAAQEVI AWLVDG (X = R OR K)

[0440] SEQ IDNO:51 > C1.6-5x HGEGTSESDVSQSIEGQAAQEVIAWLVDGXHGEGTSESDVSQSIEGQAAQEVIA WLVDGXHGEGTSESDVSQSIEGQAAQEVIAWLVDGXHGEGTSESDVSQSIEGQAAQEVI AWLVDGXHGEGTSESDVSQSIEGQAAQEVIAWLVDG (X = R OR K)

[0441] SEQ IDNO:52 > Cl.7-2x

[0442] HGEGTSESDVSQSIEGQAAQEIV AWL VDGXHGEGTSESDVSQSIEGQAAQ EIVA WLVDG (X = R OR K)

[0443] SEQ IDNO:53 > C1.7-3x

[0444] HGEGTSESDVSQSIEGQAAQEIVA WLVDGXHGEGTSESDVSQSIEGQAAQEIVA WLVDGXHGEGTSESDVSQSIEGQAAQEIVA WLVDG (X = R OR K)

[0445] SEQ IDNO:54 > C1.7-4x

[0446] HGEGTSESDVSQSIEGQAAQEIV AWL VDGXHGEGTSESDVSQSIEGQAAQEIVA WLVDGXHGEGTSESDVSQSIEGQAAQEIVAWLVDGXHGEGTSESDVSQSIEGQAAQEIV AWLVDG (X = R OR K)

[0447] SEQ IDNO:55 > Cl.7-5x

[0448] HGEGTSESDVSQSIEGQAAQEIVA WLVDGXHGEGTSESDVSQSIEGQAAQEIVA WLVDGXHGEGTSESDVSQSIEGQAAQEIVA WLVDGXHGEGTSESDVSQSIEGQAAQEIV AWLVDGXHGEGTSESDVSQSIEGQAAQEIVAWLVDG (X = R OR K)

[0449] SEQ IDNO:56 > C1.8-2x HGEGTSESDVSQSIEGQAAQEIVAIVVDGXHGEGTSESDVSQSIEGQAAQEIVAIV VDG (X = R OR K)

[0450] SEQ IDNO:57 > C1.8-3x HGEGTSESDVSQSIEGQAAQEIVAIVVDGXHGEGTSESDVSQSIEGQAAQEIVAIV VDGXHGEGTSESDVSQSIEGQAAQEIVAIVVDG (X = R OR K)

[0451] SEQ IDNO:58 > C1.8-4x HGEGTSESDVSQSIEGQAAQEIVAIVVDGXHGEGTSESDVSQSIEGQAAQEIVAIV VDGXHGEGTSESDVSQSIEGQAAQEIVAIVVDGXHGEGTSESDVSQSIEGQAAQEIVAIV VDG (X - R OR K)

[0452] SEQ IDNO:59 > C1.8-5x

[0453] HGEGTSESDVSQSIEGQAAQEIVAIVVDGXHGEGTSESDVSQSIEGQAAQEIVAIV Atty. Docket No. 2950-42 PCT

[0454] VDGXHGEGTSESDVSQSIEGQAAQEIVAIVVDGXHGEGTSESDVSQSIEGQAAQEIVAIV VDGXHGEGTSESDVSQSIEGQAAQEIVAIVVDG (X = ROR K)

[0455] SEQ ID NO:60 > C1.9-2x HGEGTSESDVSQSIEGQAAQEIVAVIVDGXHGEGTSESDVSQSIEGQAAQEIVAVI VDG (X = R or K)

[0456] SEQ ID NO:61 > C1.9-3x HGEGTSESDVSQSIEGQAAQEIVAVIVDGXHGEGTSESDVSQSIEGQAAQEIVAVI VDGXHGEGTSESDVSQSIEGQAAQEIVAVIVDG (X = R OR K)

[0457] SEQ ID NO: 62 > C1.9-4x

[0458] HGEGTSESDVSQSIEGQAAQEJVAVIVDGX1 IGEG TSESDVSQSIEGQAAQEIVAVI VDGXHGEGTSESDVSQS1EGQAAQEIVAVIVDGXHGEGTSESDVSQSIEGQAAQEIVAVI VDG (X = R OR K)

[0459] SEQ ID NO: 63 > C1.9-5x HGEGTSESDVSQSIEGQAAQEIVAVIVDGXHGEGTSESDVSQSIEGQAAQEIVAVI VDGXHGEGTSESDVSQSIEGQAAQEIVAVIVDGXHGEGTSESDVSQSIEGQAAQEIVAVI VDGXHGEGTSESDVSQSIEGQAAQEIVAVIVDG (X - R OR K)

[0460] SEQ IDNO:64 > C1.10-2x HGEGTSESDVSQSIEGQAAQEVIAVIVDGXHGEGTSESDVSQSIEGQAAQEVIAVI VDG (X = R OR K)

[0461] SEQ ID NO:65 > C1.10-3x HGEGTSESDVSQSIEGQAAQEVIAVIVDGXHGEGTSESDVSQSIEGQAAQEVIAVI VDGXHGEGTSESDVSQSIEGQAAQEVIAVIVDG (X = R OR K)

[0462] SEQ ID NO:66 > C1.10-4x HGEGTSESDVSQSIEGQAAQEVIAVIVDGXHGEGTSESDVSQSIEGQAAQEVIAVI VDGXHGEGTSESDVSQSIEGQAAQEVIAVIVDGXHGEGTSESDVSQSIEGQAAQEVIAVI VDG (X = R OR K)

[0463] SEQ IDNO:67 > C1.10-5x HGEGTSESDVSQSIEGQAAQEVIAVIVDGXHGEGTSESDVSQSIEGQAAQEVIAVI VDGXHGEGTSESDVSQSIEGQAAQEVIAVIVDGXHGEGTSESDVSQSIEGQAAQEVIAVI VDGXHGEGTSESDVSQSIEGQAAQEVIAVIVDG (X = R OR K)

[0464] SEQ ID NO: 68 E-tag GAPVPYPDPLEPR

[0465] SEQ ID NO:69 G GGGGS GGGGS GGGGS GGGGSA

[0466] SEQ ID NO: 70 A PPGGS GGGGS GGGGS GGGGSA

[0467] SEQ ID NO: 71 GTGGGGS GGGGS GGGGS GGGGSA Atty. Docket No. 2950-42 PCT

[0468] SEQ ID NO: 72 GGGGGS GGGGS GGGGS GGGGSA

[0469] SEQ ID NO: 73 GGGGGSA

[0470] SEQ ID NO: 74 GGGGGSGGGGSA

[0471] SEQ ID NO:75 GGGGGSGGGGS GGGGSA

[0472] SEQ IDNO:76 G KGGGS GGGGS GGGGS GGGGSA

[0473] SEQ ID NO:77 GGGGGS GGGGS GGGGS GGGGSA

[0474] SEQ ID NO:78 G GGGG GGGG GGGG GGGG A

[0475] SEQ ID NO:79 Alfa-tag SRLEEELRRRLTE

[0476] SEQ IDNO:80 Avi-tag GLNDIFEAQKIEWHE

[0477] SEQ ID NO:81 C-tag EPEA

[0478] SEQ ID NO: 82 Calmodulin-tag KRRWKKNFIAVSAANRFKKISSSGAL

[0479] SEQ IDNO:83 Dogtag DIPATYEFTDGKHYITNEPIPPK

[0480] SEQ IDNO:84 E-tag GAPVPYPDPLEPR

[0481] SEQ IDNO:85 FLAG DYKDDDDK

[0482] SEQ IDNO:86 G4T EELLSKNYHLENEVARLKK

[0483] SEQ ID NO:87 HA YPYDVPDYA

[0484] SEQ ID NO:88 His HHHHHH

[0485] SEQ ID NO:89 Isopeptag TDKDMTITFTNKKDAE

[0486] SEQ IDNO:90 Myc EQKLISEEDL

[0487] SEQ ID NO:91 NE-TagTKENPRSNQEESYDDNES

[0488] SEQ ID NO:92 Poly Glutamate-tag EEEEEEE

[0489] SEQ ID NO:93 Poly Arginine-tag RRRRRRR

[0490] SEQ IDNO:94 RholD4-tag TETSQVAPA

[0491] SEQ IDNO.95 SBP-tag MDEKITGWRGGHVVEGLAGELEQLRARLEHHPQGQREP SEQ ID NO:96 Sdytag DPIVMIDNDKPIT

[0492] SEQ ID NO:97 SH3 STVPVAPPRRRRG

[0493] SEQ ID NO:98 Snooptag KLGDIEFIKVNK

[0494] SEQ ID NO:99 Softag 1 SLAELLNAGLGGS

[0495] SEQ ID NO: 100 Softag 3 TQDPSRVG

[0496] SEQ ID NO: 101 Spot-tag PDRVRAVSHWSS

[0497] SEQ ID NO: 102 Spytag AHIVMVDAYKPIK

[0498] SEQ ID NO: 103 S-tag KETAAAKFERQHMDS

[0499] SEQ ID NO: 104 Strep-tag WSHPQFEK

[0500] SEQ ID NO: 105 T7tag MASMTGGQQMG Atty. Docket No. 2950-42 PCT

[0501] SEQ ID NO: 106 TC-tag EVHTNQDPLD

[0502] SEQ ID NO: 107 Ty-tag CCPGCC

[0503] SEQ ID NO: 108 VSV-tag YTDIEMNRLGK

[0504] SEQ ID NO: 109 Xpress-tag DLYDDDDK

[0505] SEQ ID NO: 110 HiBit VSGWRLFKKIS

[0506] SEQ IDNO:111 Formula II Y X2 E G T X6 X7 S D X10 S I X13 X14 D X16 I A Q X20 A X22 V Q X25 X26 I A G G P S S G A P P where X2 = V or K;; 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 1;; 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.

[0507] SEQ ID NO: 112 7FIM_V2 / A13 YVEGTFTSDYSIALDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 113 7FIM V2 / S 13 YVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 114 7FIM V2 / Y13 YVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 115 7FIM V2 / 113 YVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 116 7FIM_K_R YVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPS SEQ ID NO: 117 7FIM_V2 / Q 13 YVEGTFTSDYSIQLDKIAQKAFVQWLIAGGPSSGAPP SEQ IDNO:118 7FIM_V2 / L 13 YVEGTFTSDYSILLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 119 7FIM_V2 / R13

[0508] YVEGTFTSDYSIRLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 120 7FIM K2 / V13 YKEGTFTSDYSIVLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 121 7FIM K2 / K13 YKEGTFTSDYSIKLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 122 7FIM R H YVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPS SEQ ID NO: 123 Cl YVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPS

[0509] SEQ ID NO; 124 Cl.l YVEGTPTSDCSISLDHIAQIIAPVQWLIAGGPSSGAPPPS

[0510] SEQ ID NO: 125 C1.2 YVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPS

[0511] SEQ ID NO: 126 C1.3 YVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS

[0512] SEQ ID NO: 127 C1.4 YVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:128 Cl.4.1.1 YVEGTPCSDCSISLDHIAQKAPVQPVIAGGPSSGAPPPS SEQ ID NO: 129 Cl.4.1.2 YVEGTPCSDCSISLDKIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO: 130 Cl.4.1.3 YVEGTPCSDCSISLDKIAQKAPVQPVIAGGPSSGAPPPS SEQ ID NO: 131 Cl.4.2.1 YVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO: 132 Cl.4.2.2 YVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPS SEQ ID NO: 133 Cl.4.2.3 YVEGTSCSDCSISLDHIAQHAAVQKVIAGGPSSGAPPPS SEQ ID NO: 134 Ci.4.2.4 YVEGTSCSDCSISKDHIAQHAAVQKVIAGGPSSGAPPPS Atty. Docket No. 2950-42 PCT

[0513] SEQ IDNO:135 Cl.4.2.5 YVEGTSCSDCSISKDKIAQHAAVQKVIAGGPSSGAPPPS SEQ ID NO: 136 Cl.4.2.6 YVEGTSCSDCSISKDKIAQKAAVQKVIAGGPSSGAPPPS SEQ IDNO:137 CI.4.1 YVEGTSCSDCSISHDKIAQKAAVQKVIAGGPSSGAPPPS

[0514] SEQ ID NO: 138 Cl.4.2 YVEGTSCSDCSISHDKIAQKAAVQHVIAGGPSSGAPPPS SEQ ID O.139 Cl.4.3 YVEGTSCSDCSISHDKIAQKAAVQIVIAGGPSSGAPPPS SEQ IDNO:140 Cl.4.4 YVEGTSCSDCSISHDVIAQKAAVQIVIAGGPSSGAPPPS

[0515] SEQ ID NO: 141 Cl.4.5 YVEGTSCSDCSISIDVIAQKAAVQIVIAGGPSSGAPPPS

[0516] SEQ ID NO: 142 C 1.4.5.1 YVEGTSESDCSISIDVIAQKAAVQIVIAGGPSSGAPPPS SEQ ID NO: 143 Cl.4.5.2 YVEGTSCSDESISIDVIAQKAAVQIVIAGGPSSGAPPPS SEQ ID NO: 144 Cl.4.5.3 YVEGTSESDESISIDVIAQKAAVQIVIAGGPSSGAPPPS SEQ ID NO: 145 Cl.4.5.1.1 YVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO: 146 Cl.4.5.2.1 YVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO: 147 Cl.4.5.3.1 YVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO: 148 7FIM_V2 / S13_2x YVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSISLDKIA QKAFVQWLIAGGPSSGAPP SEQ ID NO: 149 7FIM_V2 / S 13_3x YVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSISLDKIA QKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 150 7FIM V2 / S 13_4x YVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSISLDKIA QKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPPXYV EGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 151 7FIM V2 / S 13__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 Atty. Docket No. 2950-42 PCT

[0517] SEQ ID NO: 154 7FIM V2 / Y 13_4x YVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIYLDKIA QKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPXYV EGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 155 7FIM_V2 / Y13_5x YVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIYLDKIA QKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPXYV EGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIYLDKIAQKAFVQ WLIAGGPSSGAPP SEQ IDNO:156 7FIM_V2 / I13_2x YVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQ KAFVQWLIAGGPSSGAPP SEQ IDNO:157 7FIM_V2 / I13_3x YVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQ KAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 158 7FIM_V2 / 113_4x YVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQ KAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPPXYVE GTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO: 159 7FIM_V2 / 113_5x YVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQ KAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPPXYVE GTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQKAFVQWLI AGGPSSGAPP SEQ ID NO: 160 7FIM K_R_2x YVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRI AQRAFVQWLIAGGPSSGAPPPS SEQ ID NO: 161 7FIM K_R _3x YVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRI AQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPP S SEQ ID NO: 162 7FIM K__R_4x

[0518] YVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRI Atty. Docket No. 2950-42 PCT

[0519] AQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPP SXYVEGTFTSDYSISLDR1AQRAFVQWLIAGGPSSGAPPPS

[0520] SEQ ID NO: 163 7FIM_K_R_5x YVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRI AQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPP SXYVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRIAQR AFVQWLIAGGPSSGAPPPS SEQ ID NO: 164 7FIM_V2 / S 13 / H 16 / H20_2x YVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHI AQIIAFVQWLIAGGPSSGAPPPS SEQ ID NO: 165 7FIM_V2 / S 13 / H 16 / H20_3x YVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHI AQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPP S SEQ ID NO: 166 7FIM_ V2 / SI3 / H16 / H20_4x YVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHI AQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPP SXYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPS SEQ ID NO: 167 7FIM_V2 / S 13 / H 16 / H20_5x YVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHI AQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPP SXYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHIAQH AFVQ WLIAGGPS SG APPPS SEQ ID NO: 168 Cl_2x YVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDHI AQHAPVQWLIAGGPSSGAPPPS SEQ ID NO:169 Cl_3x YVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDHI AQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPP S SEQ ID NO:170 Cl_4x YVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDH1 AQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPP SXYVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPS Atty. Docket No. 2950-42 PCT

[0521] SEQ ID NO:171 Cl 5x YVEGTPTSDYSISLDHIAQIIAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDHI AQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPP SXYVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDHIAQH APVQWLIAGGPSSGAPPPS SEQ ID NO:172 Cl.l_2x YVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHI AQHAPVQWLIAGGPSSGAPPPS SEQ ID NO:173 Cl.1_3x YVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHI AQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPP S SEQ ID NO:174 Cl.l_4x YVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHI AQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPP SXYVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPS

[0522] SEQ ID NO:175 Cl.l_5x YVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHI AQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPP SXYVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQH APVQWLIAGGPSSGAPPPS SEQ IDNO:176 C1.2__2x YVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHI AQHAPVQPLIAGGPSSGAPPPS SEQ ID NO:177 C1.2 3x YVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHI AQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPS SEQ ID NO:178 C1.2_4x YVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHI AQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQIIAPVQPLIAGGPSSGAPPPS XYVEGTPTSDCSISLDIIIAQHAPVQPLIAGGPSSGAPPPS SEQ ID NO:179 C1.2_5x YVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHI AQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPS Atty. Docket No. 2950-42 PCT

[0523] XYVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHA PVQPLIAGGPSSGAPPPS SEQ ID NO:180 C1.3 2x YVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHI AQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:181 Cl.3_3x YVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHI AQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:182 C1.3_4x YVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHI AQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS XYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:183 C1.3 5x YVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHI AQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS XYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHA PVQPVIAGGPSSGAPPPS SEQ ID NO:184 C1.4_2x YVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHI AQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:185 Cl.4_3x YVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHI AQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:186 Cl.4 4x YVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHI AQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS XYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:187 C1.4_5x YVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHI AQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS XYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHIAQHA PVQPVIAGGPSSGAPPPS Atty. Docket No. 2950-42 PCT

[0524] SEQ ID NO:188 C1.4.2.1_2x YVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHI AQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:189 Cl.4.2.1 3x YVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHI AQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:190 C1.4.2.1_4x YVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHI AQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS XYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS

[0525] SEQ ID NO:191 C1.4.2.1_5x YVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS XYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS 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 YVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHI AQHAAVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPP SXYVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQH AAVQPVIAGGPSSGAPPPS SEQ ID NO:196 Cl.4.5.1.1 _2x YVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIA QNAAVQIVIAGGPSSGAPPPS Atty. Docket No. 2950-42 PCT

[0526] SEQ ID NO:197 C1.4.5.1.1_3x YVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIA QNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO:198 C1.4.5.1.1_4x YVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIA QNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPSX YVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO:199 C1.4.5.1.1_5x YVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIA QNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPSX YVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIAQNAAV QIVIAGGPSSGAPPPS SEQ ID NO:200 C1.4.5.2.1_2x YVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESISIDVIA QNAAVQIVIAGGPSSGAPPPS SEQ ID NO:201 C1.4.5.2.1_3x YVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESISIDVIA QNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO:202 Cl.4.5.2.1 4x YVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESISIDVIA QNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESSISIDVIAQNAAVQIVIAGGPSSGAPPPSX YVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO:203 C1.4.5.2.1_5x YVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESISIDVIA QNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPSX YVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESSISIDVIAQNAAV QIVIAGGPSSGAPPPS SEQ ID NO:204 C1.4.5.3.1_2x YVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDESISIDVIA QNAAVQIVIAGGPSSGAPPPS SEQ ID NO:205 C1.4.5.3.1_3x YVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSEDESISIDVIA QNAAVQIVIAGGPSSGAPPPSXYVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPS Atty. Docket No. 2950-42 PCT

[0527] SEQ ID NO:206 C1.4.5.3.1_4x YVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDESISIDVIA QNAAVQIVIAGGPSSGAPPPSXYVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPSX YVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ IDNO:207 C1.4.5.3.1_5x YVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDESISIDVIA QNAAVQIVIAGGPSSGAPPPSXYVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPSX YVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDESISIDVIAQNAAV QIVIAGGPSSGAPPPS SEQ ID NO:208 Tirzepatide (7FIM)

[0528] YAEGTFTSDYSIALDKIAQKAFVQWLIAGGPSSGAPP

[0529] SEQ ID NO:209 Peptide-20 HXQGTFTSDKSKYLDERAAQDFVQWLLDGG PSSGAPPPS (X= AIB)

[0530] SEQ ID NO:210 Peptide_19 YXEGTFTSDYSIYLDKQAAXEFVNWLLAGGPSAPPPSK (X=AIB)

[0531] SEQ ID NO:211 Semaglutide HXEGTFTSDVSSYLEGQAAKEFIAWLVRGRG

[0532] SEQ ID NO:212 Exendin-4 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS SEQ ID NO:213 7DTY_P (GIP)

[0533] YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ SEQ ID NO:214 MEDI7219 HaEGSfTSDVsSKLEGEA AkEflAKVVEGG (lower case is alpha-methyl, US_2018 0162920_ A 1_263)

[0534] SEQ ID NO:215 J211 HaEGSfTSDVsSfLEGEAAkEflAfVVkGG (US_2018 0162920_A 1_2) SEQ ID NO:216 J229 HaEGSfTSDVsSFLEGEAAkEflAFVVKGG

[0535] (US-2018 0162920-A 1 _2)

[0536] SEQ ID NO:217 wtGLP-1 haegtftsdvssylegqaakefiawlvkgrg

[0537] SEQ ID NO:218 hGLPIR MAGAPGPLRLAVLLLGMVGRAGPRPQGATVSLWETVQKWREYRRQCQRSLTE DPPPATDLFCNRTFDEYACWPDGEPGSFVNVSCPWYLPWASSVPQGHVYRFCTAEGLW LQKDNSSLPWRDLSECEESKRGERSWGEEQLLFLYIIYTVAYALSFSALVIASAILLGFRH LHCTRNYIHLNLFASFILRALSVFIKDAALKWMYSTAAQQHQWDGLLSYQDSLSCRLVF LLMQYCVAANYYWLLVEGVYLYTLLAFSVFSEQWIFRLYVSIGWGVPLLFVVPWGIVKI LYEDEGCWTRNSNMNYWLIIRLPILFAIGVNFLIFVRVICIVVSKLKANLMCKTDIKCRLA KSRLTLIPLLGTHEVIFAFVMDEHARGTLRFIKLFTELSFTSFQGLMVAILYCFVNNEVQLE FRKSWERWRLEHLHIQRDSSMKPLKCPTSSLSSGATAGSSMYTATCQASCS Atty. Docket No. 2950-42 PCT

[0538] SEQ ID NO:219 cGLPIR magapsplclallllgavgragprpqgatvslsetvqkwreyrhqcqrflteapppatglfcnrtfdeyacwpdglpgsfvnv scpwylpwassvlqghvyrfctaeglwlrqdnsslpwrnlseceeskrgersspeeqllsfsiiytvgytlsfsalviasaillsfrhlhctrn yihlnlfasfilralsvfirdavlkwmystapqqhqwdgllsyqdslgcrlvfllmqycvaanyywllvegvylytllafsvfseqrifrlyl sigwgvpllfvipwgivkylyedegcwtrnsnmnywliirlpilfaigvnflifvrvicivvskikanlmcktdikcrlakstltlipllgthe vvfafvtndehargtlrfiklftelsftsfqgimvailycfvnnevqmefirswerwrlkhlhiqrdssmkplkcptssltsggtvgssvyaa scqascs

[0539] SEQ ID NO:220 hGIPR MTTSPILQLLLRLSLCGLLLQRAETGSKGQTAGELYQRWERYRRECQETLAAAEP PSGLACNGSFDMYVCWDYAAPNATARASCPWYLPWHHHVAAGFVLRQCGSDGQWGL WRDHTQCENPEKNEAFLDQRLILERLQVMYTVGYSLSLATLLLALLILSLFRRLHCTRNY IHINLFTSFMERAAAILSRDRLLPRPGPYEGDQALALWNQALAACRTAQIVTQYCVGAN YTWLEVEGVYLHSLLVLVGGSEEGHFRYYLLLGWGAPALFVIPWVIVRYLYENTQCWE RNEVKAIWWIIRTPILMTILINFLIFIRILGILLSKLRTRQMRCRDYRLRLARSTLTLVPLLG VHEVVFAPVTEEQARGALRFAKLGFEIFLSSFQGFLVSVLYCFINKEVQSEIRRGWHHCR LRRSLGEEQRQLPERAFRALPSGSGPGEVPTSRGLSSGTLPGPGNEASRELESYC SEQ ID NO:221 cGIPR MPNGPPWQLFEPLLWSWGPLLRRAETGSVGQTAGELYQRWERYRRECRETLEA VDPPAGLACNGSFDMYVCWDYAAPNATARASCPWYLPWHSHVATGFVERHCGSDGQ WGPWRDHSQCEDPEKNGAFQDQRLILERLQVMYTVGYSVSLATLLLALLILSFFRRLRC TRNYIHINLFTSFMLRAAAILTRDRLLPPPGPYPGDQAPVLWKPALAACRTAQIVTQYCV GANYTWLLVEGVYLHSLLVLVGGSEGGHFRCYVFLGWGAPALFVIPWVIVRYLYENTQ CWERNEVKAIWWIIRTPILVTISINFLIFIRILGILVSKLRTRQMRCPDYRLRLARSTLTLVP LLGVHEVVFAPVTEEQARGALRFAKLGFEIFLSSFQGFLVGVVYCFVNKEVQAEIRRCW HRCRLRHSLGEERRQPPERASRTPPTGSGPRPVATDRTPSLGALPGPGNEASRGLESHC SEQ ID NO:222 rGIPR MPLRLLLLLLWLWGLSLQRAETDSEGQTTGELYQRWERYGWECQNTLEATEPP SGLACNGSFDMYACWNYTAANTTARVSCPWYLPWYRQVAAGFVFRQCGSDGQWGSW RDHTQCENPEKNGAFQDQKLILERLQVVY'rVGYSLSLATLLLALLILSLFRRLHCTRNYI HMNLFTSFMLRAGAILTRDQLLPPLGPYTGNQTPTLWNQALAACRTAQILTQYCVGANY TWLLVEGVYLHFILLVVVRRSEKGHFRCYLLLGWGAPALFVIPWVIVRYLYENTQCWER NEVKAIWWIIRTPILITILINFLIFIRIGILVSKLRTRQMRCPDYRLRLARSTLTLMPLLGVH EVVFAPVTEEQAEGSLRFAKLAFEIFLSSFQGFLVSVLYCFINKEVQSEIRRLRLSLQEQCP RPHLGQAPRAVPLSSAPQEAAIRNALPSGMLHVPGDEVLESYC Atty. Docket No. 2950-42 PCT

[0540] SEQ ID NO:223 mGIPR MPLRLLLLLLWLWGLQWAETDSEGQTTTGELYQRWEHYGQECQKMLETTEPPS GLACNGSFDMYACWNYTAANTTARVSCPWYLPWFRQVSAGFVFRQCGSDGQWGSWR DHTQCENPEKNGAFQDQTLILERLQIMYTVGYSLSLTTLLLALLILSLFRRLHCTRNYIHM NLFTSFMLRAAAILTRDQLLPPLGPYTGDQAPTPWNQALAACRTAQIMTQYCVGANYT WLLVEGVYLHHLLVIVGRSEKGHFRCYLLLGWGAPALFVIPWVIVRYLRENTQCWERN EVKAIWWIIRTPILITILINFLIFIRIGILVSKLRTRQMRCPDYRLRLARSTLTLVPLLGVHE VVFAPVTEEQVEGSLRFAKLAFEIFLSSFQGFLVSVLYCFINKEVQSEIRQGWRHRRLRLS LQEQRPRPHQELAPRAVPLSSACREAAVGNALPSGMLHVPGDEVLESYC SEQ ID NO:224 GGSS

[0541] SEQ ID NO:225 GGSS GGSS

[0542] SEQ ID NO:226 GGSS GGSS GGSS

[0543] SEQ ID NO:227 GGSS GGSS GGSS GGSS

[0544] SEQ ID NO:228 GGSS GGSS GGSS GGSS GGSS

[0545] SEQ ID NO:229 GGSS GGSS GGSS GGSS GGSS GGSS

[0546] SEQ ID NO:230 GGSS GGSS GGSS GGSS GGSS GGSS GGSS

[0547] SEQ ID NO:231 GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS

[0548] SEQ ID NO:232 GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS

[0549] SEQ ID NO:233 GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS

[0550] SEQ ID NO:234 GSGGS

[0551] SEQ ID NO:235 GSGGS GSGGS

[0552] SEQ ID NO:236 GSGGS GSGGS GSGGS

[0553] SEQ ID NO:237 GSGGS GSGGS GSGGS GSGGS

[0554] SEQ ID NO:238 GSGGS GSGGS GSGGS GSGGS GSGGS

[0555] SEQ ID NO:239 GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS

[0556] SEQ ID NO:240 GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS

[0557] SEQ ID NO:241 GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS

[0558] SEQ ID NO:242 GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS SEQ ID NO:243 GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS SEQ ID NO:244 GGGS

[0559] SEQ ID NO:245 GGGS GGGS

[0560] SEQ ID NO:246 GGGS GGGS GGGS

[0561] SEQ ID NO:247 GGGS GGGS GGGS GGGS Atty. Docket No. 2950-42 PCT

[0562] SEQ ID NO:248 GGGS GGGS GGGS GGGS GGGS

[0563] SEQ ID NO:249 GGGS GGGS GGGS GGGS GGGS GGGS

[0564] SEQ ID NO:250 GGGS GGGS GGGS GGGS GGGS GGGS GGGS

[0565] SEQ ID NO:251 GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS

[0566] SEQ ID NO:252 GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS

[0567] SEQ ID NO:253 GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS SEQ ID NO:254 GGSG

[0568] SEQ ID NO:255 GGSG GGSG

[0569] SEQ ID NO:256 GGSG GGSG GGSG

[0570] SEQ ID NO:257 GGSG GGSG GGSG GGSG

[0571] SEQ ID NO:258 GGSG GGSG GGSG GGSG GGSG

[0572] SEQ ID NO:259 GGSG GGSG GGSG GGSG GGSG GGSG

[0573] SEQ ID NO:260 GGSG GGSG GGSG GGSG GGSG GGSG GGSG

[0574] SEQ ID NO:261 GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG

[0575] SEQ ID NO:262 GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG

[0576] SEQ ID NO:263 GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG SEQ ID NO:264 GGSGG

[0577] SEQ ID NO:265 GGSGG GGSGG

[0578] SEQ ID NO:266 GGSGG GGSGG GGSGG

[0579] SEQ ID NO:267 GGSGG GGSGG GGSGG GGSGG

[0580] SEQ ID NO:268 GGSGG GGSGG GGSGG GGSGG GGSGG

[0581] SEQ ID NO:269 GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG

[0582] SEQ ID NO:270 GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG

[0583] SEQ ID NO:271 GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG SEQ ID NO: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

[0584] SEQ ID NO:275 GSGSG GSGSG

[0585] SEQ ID NO:276 GSGSG GSGSG GSGSG

[0586] SEQ ID NO:277 GSGSG GSGSG GSGSG GSGSG

[0587] SEQ ID NO:278 GSGSG GSGSG GSGSG GSGSG GSGSG

[0588] SEQ ID NO:279 GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG Atty. Docket No. 2950-42 PCT

[0589] SEQ ID NO:280 GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG

[0590] SEQ ID NO:281 GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG

[0591] SEQ ID NO:282 GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG SEQ ID NO:283 GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG SEQ ID NO:284 GSGGG

[0592] SEQ ID NO:285 GSGGG GSGGG

[0593] SEQ ID NO:286 GSGGG GSGGG GSGGG

[0594] SEQ ID NO: 287 GSGGG GSGGG GSGGG GSGGG

[0595] SEQ ID NO:288 GSGGG GSGGG GSGGG GSGGG GSGGG

[0596] SEQ ID NO:289 GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG

[0597] SEQ ID NO:290 GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG

[0598] SEQ ID NO: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 SEQ ID NO:294 GGGSG

[0599] SEQ ID NO:295 GGGSG GGGSG

[0600] SEQ ID NO:296 GGGSG GGGSG GGGSG

[0601] SEQ ID NO:297 GGGSG GGGSG GGGSG GGGSG

[0602] SEQ ID NO:298 GGGSG GGGSG GGGSG GGGSG GGGSG

[0603] SEQ ID NO:299 GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG

[0604] SEQ ID NO:300 GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG SEQ ID NO:301 GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG SEQ ID NO:302 GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG SEQ ID NO:303 GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG SEQ ID NO:304 GSSSG

[0605] SEQ ID NO:305 GSSSG GSSSG

[0606] SEQ ID NO:306 GSSSG GSSSG GSSSG

[0607] SEQ ID NO:307 GSSSG GSSSG GSSSG GSSSG

[0608] SEQ ID NO:308 GSSSG GSSSG GSSSG GSSSG GSSSG Atty. Docket No. 2950-42 PCT

[0609] SEQ ID NO:309 GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG

[0610] SEQ IDNO.-310 GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG

[0611] SEQ ID NOG 11 GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG

[0612] SEQ ID NO:312 GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG SEQ ID NOG 13 GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG SEQ ID NOG 14 GSSSS

[0613] SEQ ID NO:315 GSSSS GSSSS

[0614] SEQ ID NO:316 GSSSS GSSSS GSSSS

[0615] SEQ ID NO:317 GSSSS GSSSS GSSSS GSSSS

[0616] SEQ ID NOG 18 GSSSS GSSSS GSSSS GSSSS GSSSS

[0617] SEQ ID NO:319 GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS

[0618] SEQ ID NOG20 GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS

[0619] SEQ ID NO:321 GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS

[0620] SEQ ID OG22 GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS SEQ ID NOG23 GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS SEQ ID NOG24 GGGGS

[0621] SEQ ID NOG25 GGGGS GGGGS

[0622] SEQ ID NOG26 GGGGS GGGGS GGGGS

[0623] SEQ ID NOG27 GGGGS GGGGS GGGGS GGGGS

[0624] SEQ IDNOG28 GGGGS GGGGS GGGGS GGGGS GGGGS

[0625] SEQ ID NOG29 GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS

[0626] SEQ IDNOG30 GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS

[0627] SEQ ID NOG 1 GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS SEQ ID NOG32 GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS SEQ ID NOG33 AAAGG

[0628] SEQ ID NOG34 AAAGG AAAGG

[0629] SEQ IDNOG35 AAAGG AAAGG AAAGG

[0630] SEQ ID NOG36 AAAGG AAAGG AAAGG AAAGG

[0631] SEQ ID NOG37 AAAGG AAAGG AAAGG AAAGG AAAGG

[0632] SEQ ID OG38 AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG

[0633] SEQ ID OG39 AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG

[0634] SEQ ID NOG40 AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG Atty. Docket No. 2950-42 PCT

[0635] SEQ ID NO:341 AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG SEQ ID NO:342 AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG SEQ IDNO:343 GGSAAAGG

[0636] SEQ ID NO:344 GGSAAAGG GGSAAAGG

[0637] SEQ ID NO:345 GGSAAAGG GGSAAAGG GGSAAAGG

[0638] SEQ ID NO:346 GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG

[0639] SEQ ID NO:347 GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG SEQ IDNO:348 GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG SEQ IDNO:349 GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG SEQ ID NO.-350 GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG SEQ ID NO:351 GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG SEQ ID NOG 52 GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG SEQ ID NOG 53 DPIVMIDNDKP1T Sdytag

[0640] SEQ ID NO:354 STVPVAPPRRRRG SH3

[0641] SEQ ID NO:355 KLGDIEFIKVNK Snooptag

[0642] SEQ ID NO:356 SLAELLNAGLGGS Softag 1

[0643] SEQ ID NO:357 TQDPSRVG Softag 3

[0644] SEQ ID NO:358 PDRVRAVSHWSS Spot-tag

[0645] SEQ ID NO:359 AHIVMVDAYKPTK Spytag

[0646] SEQ ID NO:360 KETAAAKFERQHMDS S-tag

[0647] SEQ ID NO:361 WSHPQFEK Strep-tag

[0648] SEQ ID NO:362 MASMTGGQQMG T7tag

[0649] SEQ ID NO:363 EVHTNQDPLD TC-tag

[0650] SEQ ID NO:364 CCPGCC Ty-tag

[0651] SEQ ID NO:365 YTD1EMNRLGK VSV-tag

[0652] SEQ ID NO:366 DLYDDDDK Xpress-tag

[0653] SEQ ID NO:367 VSGWRLFKKIS HiBit Atty. Docket No. 2950-42 PCT

[0654] SEQ ID NO:368 YAEGTFISDYSIAMDKIRQQDFVNWLLAQKGKKNDWK

[0655]

[0270] Embodiments of engineered Glucagon-Like Peptide 1:

[0656] 1. An engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of Formula I (SEQ ID NO: I):

[0657] H G E G T S E S D V S X12XI3X14E G Q A A Q E X22X23A X2JX26V D G X30(I) wherein Xj2= S or Q; Xj2~ S, Q, or Y; X14 = I or L; X22= V, I, or F; X23= V or I; X2s = V, I, or W; X26- I or V; and X30= R or S

[0658] or of Formula III (SEQ ID NO: 382):

[0659] Xi G E G T S E S D V S XI2XI3XI4E X]6Q A X19X20E X22X23A X25X26V D G X30(III), wherein Xi = H or Y; Xi? = S, Q, A or M; X» = S, Q, Y, or M; Xu = I or L; Xie = G or M; Xi? = 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 X3o - R or S.

[0660] 2. The eGLP-l of embodiment 1, wherein X12 = Q; X13 = S; Xu = I or L; X22- V, L or F; X23= V or I;

[0661] X?5 = V, I, or W; X26 = I or V; and X50 = R.

[0662] 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.

[0663] 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

[0664] 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.

[0665] 6. The eGLP-1 o f 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.

[0666] 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.

[0667] . The eGLP-1 of any one embodiments 1 to 8, wherein the eGLP-1 is substantially resistant to proteolytic degradation.

[0668] 10. The eGLP-1 of embodiment 9, wherein the eGLP-1 is substantially resistant to DPP-IV, neprilysin, a- chymotrypsin, trypsin, elastase, or pepsin, degradation.

[0669] 11. The eGLP-1 of embodiment 9, wherein the eGLP-1 is substantially resistant to DPP-IV and neprilysin degradation.

[0670] 12. The eGLP-1 of embodiment 9, wherein the eGLP-1 is substantially resistant to a-chymotrypsin, trypsin, elastase, or pepsin, degradation. Atty. Docket No. 2950-42 PCT

[0671] 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.

[0672] 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.

[0673] 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.

[0674] Polynucleotides

[0675] 16. A polynucleotide encoding an engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of Formula I (SEQ ID NO: 1)

[0676] H G E G T S E S D V S Xt2Xi3X14E G Q A A Q E X22X23A X25X26V D G X30(I)

[0677] wherein Xj2= S or Q; Xi? = S, Q, or Y; Xu - I or L: X22= V, I, or F; X23= V or I; X23= V, I, or W; X2r, = I or V; and X3o = R or S,

[0678] or of Formula III (SEQ ID NO: 382):

[0679] Xi G E G T S E S D V S Xii X13Xu E X16Q A X19X20E X22X23A X25X26V D G X30(III), wherein Xi = H or Y; Xi2= S, Q, A or M; Xi3= S, Q, Y, or M; Xu = I or L; X = G or M; Xi? = 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.

[0680] 17. The polynucleotide of embodiment 16, wherein said polynucleotide is isolated.

[0681] 18. An engineered vector comprising a polynucleotide encoding an engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of Formula I

[0682] H G E G T S E S D V S X12X!3XuE G Q A A Q E X? X23A X25X26V D G X30(I)

[0683] wherein Xj2= S or Q; Xi = S, Q, or Y; Xu - I or L; X2= V, I, or F; X3= V or I; X23= V, I, or W; X26 = I or V; and X3o = R or S,

[0684] or of Formula III (SEQ ID NO: 382):

[0685] Xi G E G T S E S D V S X12X]3X)4E Xi6Q A X19X20E X22X23A X25X26V D G X30(III), wherein Xi = H or Y; Xi2= S, Q, A or M; X|3= S, Q, Y, or M; Xu = I or L; Xis= G or M; Xi? = A or T: X2o= Q, V, or D; X22= V, I, or F; X23= V or I; X?s = V, I, or W; X26 = I or V; and X3o = R or S.

[0686] 19. A nucleic acid expression cassette comprising one or more of:

[0687] a nucleic acid sequence comprising a transcription promoter;

[0688] a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of Formula I:

[0689] H G E G T S E S D V S X.2X!3Xi4E G Q A A Q E X22X23A X25X26V D G X30(I)

[0690] wherein Xi2= S or Q; Xi? ~ S, Q, or Y; X = J or L; X22= V, I, or F; X3= V or I; X2s - N, I, or W; X2s = I or V; and X3o = R or S, Atty. Docket No. 2950-42 PCT

[0691] or of Formula III (SEQ ID NO: 382):

[0692] Xi GE G TS E S D V S X!2X!3X)4E Xi6Q A X!9X20E X22X23A X25X26V D G X30(III), wherein X, = H or Y; X|2= S, Q, A or M; X13 ~ S, Q, Y, or M; X14 = I or L; X = G or M;

[0693] Xi? - 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 X30 = R or S;

[0694] a nucleic acid sequence comprising a translation terminator; and

[0695] a nucleic acid sequence comprising transcription terminator.

[0696] 20. The nucleic acid expression cassete 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.

[0697] 21. A method of making an engineered Glucagon-Like Peptide 1 (eGLP-l) polypeptide comprising the amino acid sequence of Formula I,

[0698] H G E G T S E S D V S XI2X!3XJ4E G Q A A Q E X22X23A X25X26V D G X30(I)

[0699] wherein X

[0700]

[0701] 12 = S or Q; X12 ~ S, Q, or Y: Xn = I or L; X22= V, I, or F; X23= V or I; X23= V, I, or W; = I or V; and X3Q = R or S,

[0702] or of Formula III (SEQ ID NO: 382):

[0703] X1G E G T S E S D V S Xi2X!3X14E Xi6Q A X19 X20E X22X23A X25X26V D G X30 (HI), wherein Xi = H or Y; X(2::= S, Q, A or M; X13 = S, Q, Y, or M; XM = 1 or L; XM = G or M; X19 ~ A or T; X2o = Q,

[0704]

[0705] V, or D; X22= V, I, or F; X23= V or I; X23= V, I, or W; X26 = I or V; and X30 = R or S;

[0706] said method comprising culturing a host cell transformed with an expression vector encoding said eGLP-l polypeptide under conditions allowing expression of said eGLP-l polypeptide, and recovering said eGLP-l polypeptide.

[0707] 22. A pharmaceutical composition comprising an engineered Glucagon-Like Peptide 1 (eGLP-l) polypeptide comprising the amino acid sequence of Formula I

[0708] H G EG T S E S D V S Xu X)3X14E G Q A A Q E X22X23A X25X26V D G X30 (I)

[0709] wherein Xj2= S or Q; Xi2= S, Q, or Y; X« = I or L; X22= V, I. or F; X23= V or I; X23= V, I, or W; XM = I or V; and X30 = R or S;

[0710] or of Formula III (SEQ ID NO: 382):

[0711] Xi G EG T S E S D V S X12Xt3X!4E Xf6Q A X19 X20E X22X23A X25X26V D G X30(III), wherein Xi = FI or Y; Xj2= S, Q, A or M; Xi3= S, Q, Y, or M; X;4= I or L: X = G or M; X19 = A or T; X2o = Q, V, or D; X22” V, I, or F; X23= V or I; X2s = V, 1, or W; XM:::I or V; and X30 = R or S;

[0712] and a carrier. Atty. Docket No. 2950-42 PCT

[0713] A pharmaceutical composition comprising a recombinant host cell comprising a polynucleotide encoding an engineered Glucagon-Like Peptide 1 (eGLP-l).

[0714] The pharmaceutical composition of embodiment 23, further comprising a carrier.

[0715] A host cell comprising a polynucleotide encoding an engineered Glucagon-Like Peptide 1 (eGLP-l) comprising the amino acid sequence of Formula I

[0716] H G E G T S E S D V S Xi2XI3Xu E G Q A A Q E X22X23A X2SX26V D G X30(I)

[0717] wherein Xn = S or Q; Xi2= S, Q, or Y; Xu = I or L; X22= V, I, or F; X23= V or I; X23= V, I, or W; X26= I or V; and X30- R or S,

[0718] or of Formula III (SEQ ID NO: 382):

[0719] Xi G E G T S E S D V S Xj2Xi3Xj4 E Xt6 Q A X19 X2o E X22X23A X23X26 V D G X30 (III), wherein

[0720]

[0721] Xi = H or Y; Xn = S, Q, A or M; Xi3= S, Q, Y, or M; X14 = 1 or L; XM- G or M; Xt9 = 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.

[0722] A host cell comprising a vector comprising a polynucleotide encoding an engineered Glucagon-Like Peptide 1 (eGLP-l) comprising the amino acid sequence of Formula I

[0723] H G E G T S E S D V S X!2X)3Xu E G Q A A Q E X22X23A X25X26V D G X30 (1) wherein Xi2= S or Q; Xi2= S, Q, or Y; X14 = 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,

[0724] or of Formula III (SEQ ID NO: 382):

[0725] X) G E GT S E S D V S XiZX13X14E Xi6Q A X19 X20E X22X23A X25X26V D G X30(III), wherein Xi = H or Y; Xt2= S, Q, A or M; Xu = S, Q, Y, or M; X14 = I or L; Xu = G or M; X19 - A or T; X

[0726]

[0727] 20= Q, V, or D; X22= V, I, or F; X23= V or I; X25== V, I, or W; X26= I or V; and X30= Ror S.

[0728] 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:

[0729] H G E G T S E S D V S X12 X]3X!4E G Q A A Q E X22X23A X25X26V D G X30(I) wherein Xi? = S or Q; Xi2= S, Q, or Y; X14 = I or L; X22= V, I, or F; X23= V or I; X23= V, I, or W; X26 = I or V; and X3o ~ R or S,

[0730] or of Formula III (SEQ ID NO: 382):

[0731] X. G E G T S E S D V S X12X!3X14E X16Q A Xi9X20E X22X23A X25X26V D G X30(III), wherein Xi = H or Y; Xi2= S, Q, A or M; Xl3= S, Q, Y, or M; Xt4= I or L; X = G or M;

[0732] X1 = A or T; X20= Q, V, or D: X22= V, 1, or F; X23= V or I; X2S- V, I, or W; X26= I or V; and X30 = R or S; Atty. Docket No. 2950-42 PCT

[0733] a nucleic acid sequence comprising a translation terminator; and

[0734] a nucleic acid sequence comprising transcription terminator.

[0735] The transformed cell of embodiment 27, wherein said cell is a bacterial cell, plant cell, yeast cell, or algae cell.

[0736] The transformed cell of embodiment 26 or 28, wherein said cell comprises two or more copies of said recombinant nucleic acid.

[0737] 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.

[0738] The transformed cell of embodiment 30, wherein said integration in the bacterial chromosome is an integration into a transposase locus.

[0739] The transformed cell of embodiment 28, wherein said cell is a yeast ceil.

[0740] The transformed cell of embodiment 32, wherein said yeast cell is a cell of a strain of Pichia pastoris, 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.

[0741] The transformed cell of embodiment 28, wherein said transformed cell is a plant cell.

[0742] The transformed cell of embodiment 36, wherein said recombinant nucleic acid is integrated into the chloroplast of said plant cell.

[0743] The transformed cell of embodiment 36, wherein said recombinant nucleic acid is integrated into the genome of said plant cell.

[0744] 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.

[0745] The genetically engineered plant, or part thereof, of embodiment 38, wherein said part thereof is a plant seed.

[0746] 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.

[0747] The genetically engineered plant, or part thereof, of embodiment 38, wherein said recombinant nucleic acid is integrated into the genome of said plant cell.

[0748] A kit comprising the pharmaceutical composition of any one of embodiments 22 to 41.

[0749] 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.

[0750] 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 Atty. Docket No. 2950-42 PCT

[0751] 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 I to 15, the pharmaceutical composition of embodiment 22, or the host cell of embodiment 25.

[0752] A method of treating or preventing a disease or condition caused or characterized by hypoglyce ia 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.

[0753] The method of embodiments 44 or 45, wherein the disease or condition is diabetes or obesity.

[0754] The method of embodiment 46, wherein the disease or condition is type-2 diabetes.

[0755] The method of any one of embodiments 44 to 47, wherein the administration further improves glycemic control, provides body weight control, improves 0-cell function and mass, reduces the rate of gastric acid secretion and gastric emptying, or any combination thereof.

[0756] 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.

[0757] The method of any one of embodiments 44 to 48, wherein the host cell is administered orally.

[0758] The method of any one of embodiments 44 to 47, wherein the eGLP-1 or a pharmaceutical composition thereof is administered orally.

[0759] The method of embodiment 49, wherein the injection is administered subcutaneously or intravenously.

[0760] The method of any one of embodiments 44 to 52, wherein the eGLP-1 or the pharmaceutical composition is administered once per day.

[0761] The method of any one of embodiments 44 to 53, further comprising administering one or more additional therapies.

[0762] 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. 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.

[0763] The method of any one of embodiments 44 to 56, wherein the subject is a cat.

[0764] A pharmaceutical composition comprising an engineered Glucagon-Like Peptide 1 of Formula I:

[0765] H G E G T S E S D V S X^MiE G Q A A Q E X22X23A X25X26V D G X30(I) Atty. Docket No. 2950-42 PCT

[0766] wherein X!2= S or Q; Xi2= S, Q, or Y; X14= I or L; X22= V, I, or F; X23- V or I: X25= V, L or W; X26 = 1 or V; and X30 R or S,

[0767] or of Formula III (SEQ ID NO: 382):

[0768] Xi G E G T S E S D V S X12 X!3X14E X16Q A X!9X20E X22 X23A X25X26 V D G X30 (HI), wherein Xi = H or Y; X!2= S, Q, A or M; X13 = S, Q, Y, or M; X14- I or L: X16= G or M; X!9- A or T; X20= Q, V, or D; X22= V, L or F; X23= V or I; X25= V, L or W; X26= I or V; and X30 = R or S.

[0769] 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.

[0770] The pharmaceutical composition of embodiments 58 or 59, wherein said composition is a lyophilized composition of transformed plant cells or transformed bacterial cells.

[0771] 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:

[0772] H G E G T S E S D V S X12X!3X,4E G Q A A Q E X22X23A X25X26V D G X30 (I) wherein X|2= S or Q: X12 = S, Q, or Y; X14 = I or L; X22~ V, I, or F; X23= V or I; X25 = V, I, or W; XM = I or V; and X30 R or S,

[0773] or of Formula III (SEQ ID NO: 382):

[0774] X, G E G T S E S D V S Xi2X!3X14E X16Q A X!9X20E X22X23A X25X26V D G X30 (HI), wherein X, = H or Y; Xj2= S, Q, A or M: X13 = S, Q, Y, or M; X|4- I or L; X,6 = G or M; X;9= A or T; X2Q= 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.

[0775] 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.

[0776] 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.

[0777] 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.

[0778] 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.

[0779] The DFM of embodiment 65, wherein said transformed bacteria is a strain of Bacillus subtilis.

[0780] The DFM of embodiment 66, wherein said transformed bacterial of Bacillus subtilis and said DFM further comprises one or more B. amyloliquefaciens strains. Atty. Docket No. 2950-42 PCT

[0781] 68. The DFM of embodiment 66, wherein said transformed Bacillus sublilis bacterial strain and said DFM further comprises one or more anaerobic cellulolytic bacteria.

[0782] 69. The DFM of embodiment 68, wherein said one or more anaerobic cellulolytic bacteria is a member of the phylum Bacillota.

[0783] 70. The DFM of embodiment 69, wherein said member of the phylum Bacillota is a bacterial strain of Ruminococcusflavefaciens.

[0784] 71. The DFM of embodiment 68, wherein said one or more anaerobic cellulolytic bacteria is a member of the genus Bacteroides.

[0785] 72. The DFM of embodiment 71, wherein said member of the genus Bacteroides is Bacteroides fragilis.

[0786] 73. The DFM of embodiment 63, wherein said bacterial strain is a strain of Lactobacillus.

[0787] 74. The DFM of embodiment 73, wherein said strain of Lactobacillus is a strain of Lactobacillus gasseri.

[0788] 75. The DFM of embodiment 73, wherein said strain of Lactobacillus is a strain of Lactobacillus planlarum.

[0789] 76. The DFM of any one of embodiments 61 to 75, wherein said DFM is lyophilized.

[0790] (271] Embodiments of engineered dual agonist polypeptides:

[0791] Embodiment 1. An engineered polypeptide comprising the amino acid sequence of Formula II (SEQ ID NO: 111):

[0792] Y X2E G T X« X7S D Xio S I XBX!4D X]61 A Q X20A X22V Q X25X261 A G G P S S G A P P (I) wherein

[0793] X2= V or K; [tirzepatide = A]

[0794] Xe =F, P, or S; [tirzepatide = F]

[0795] X7= T, C, or E; [tirzepatide = T]

[0796] Xio = Y, C, or E; [tirzepatide = Y]

[0797] XB- A, S, Y, N, I, L. R, V, or K; [tirzepatide = A]

[0798] X14 = L, K, H, or I; [tirzepatide ~ L]

[0799] Xie = K, R, H, or V; [tirzepatide = K]

[0800] X2o = K, R, H, N; [tirzepatide = K]

[0801] X22~ F, A, P; [tirzepatide = F]

[0802] X2s = W, P, K, H, or I; and [tirzepatide = W]

[0803] X26 = L or V,

[0804] or of Formula IV (SEQ ID NO: 383): Y X2E G T X6 X7S D Xio

[0805]

[0806] S I X!3XI4D XI61 A Xl9X20 A X22V Q X25 X26I A G G P S S G A P P (IV), Atty. Docket No. 2950-42 PCT

[0807] 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, 1, L, R, V, or K; XI4= L, K, H, or I; X]6- K, R, H, or V; X,9= Q or V; X20- K, R, H, N; X22= F, A, P; X25- W, P, K, H, or I; and X26 L or V.

[0808] Embodiment 2. The engineered polypeptide of embodiment 1, wherein

[0809] X2= V; X6= P or S; X7= C or E: Xio = C or E; X]3- S: X!4= L, H, or I; X)6= H or V; X20 = H or N; X22 ~ A or P; X25 = P or I; and X26 - L or V.

[0810] Embodiment 3. The engineered polypeptide of embodiment 1, wherein

[0811] X2= V; X6=S; X7= C: X10= C or E; Xu = S; X!4= I; Xi6= V; X20= H orN; X22= A; X25- I; andX?6 “ V.

[0812] 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.

[0813] 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.

[0814] 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.

[0815] Embodiment 9. The engineered polypeptide of embodiment 6, wherein the polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 1.37 to SEQ ID NO: 141.

[0816] 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.

[0817] 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.

[0818] Embodiment 12. The engineered polypeptide of any one of embodiments 1 to 11, wherein the engineered polypeptide comprises a C-terminal amide.

[0819] 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.

[0820] 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.

[0821] 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. Atty. Docket No. 2950-42 PCT

[0822] 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.

[0823] 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.

[0824] 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.

[0825] 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.

[0826] 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.

[0827] 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.

[0828] Embodiment 23. An isolated polynucleotide encoding an engineered polypeptide comprising the amino acid sequence of sequence of Formula II (SEQ ID NO: 111):

[0829] wherein X2- V or K; X6=F, P, or S; X7= T, C, or E; Xio = Y, C, or E; X,3= A, S, Y, N, I, L, R, V, or K; X!4= 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,

[0830] or of Formula IV (SEQ ID NO: 383):

[0831] Y X2E G T XsX7S D Xi0S I X13X)4D X161 A Xi9X2o A X22 V Q X25 X2s 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; Xio = Y, C, or E; Xi3= A, S, Y, N, I, L, R, V, or K; X14= L, K, H, or I: X16= K, R, H, or V; Xi9- Q or V; X20= K, R, H, N; X22 = F, A, P; X25- W, P, K, H, or I; and X26= L or V.

[0832] Embodiment 24. An engineered vector comprising the polynucleotide of embodiment 23.

[0833] Embodiment 25. A nucleic acid expression cassette comprising one or more of:

[0834] a nucleic acid sequence comprising a transcription promoter;

[0835] a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of Formula II (SEQ ID NO: 111): Atty. Docket No. 2950-42 PCT

[0836] wherein X2= V or K; Xs =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; X!4= 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,

[0837] or of Formula IV (SEQ ID NO: 383):

[0838] Y X2E G T X6X7S D X10 S I X13 XM D X161 A Xl9X20 A X22V Q X25X26l A G G P S S G AP P (IV), wherein X2= V or K; Xs =F, P, or S; X? = T, C, or E; X10::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, II, or V; X!9= Q or V; X20- K, R, H, N; X22= F, A, P; X25= W, P, K, H, or I; and X26= L or V;

[0839] a nucleic acid sequence comprising a translation terminator; and

[0840] a nucleic acid sequence comprising transcription terminator.

[0841] 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.

[0842] Embodiment 27. A method of making an engineered polypeptide comprising the amino acid sequence of Formula II (SEQ ID NO: 111)

[0843] Y X2E G T X6X?S D Xl0S I X!3XM D X)61 A Q X» A X22 V Q X25X261 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.o = Y, C, or E; Xl3= A, S, Y, N, I, L, R, V, or K; Xw= L, K, H, or I; Xi« = 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,

[0844] or of Formula IV (SEQ ID NO: 383):

[0845] Y X2E G T X6X7S D X10 S I X13 X!4D Xis I A X19 X20 A X22V Q X25X261 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; Xi3= A, S, Y, N, I, L, R, V, or K; XI4- L, K, H, or I; Xi6= K, R, H, or V; X19 = Q or V; X20= K, R, H, N; X22 = F, A, P; X2; ~ W, P, K, H, or I; and X% ~ L or V;

[0846] 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.

[0847] Embodiment 28. A pharmaceutical composition comprising an engineered polypeptide comprising the amino acid sequence of Formula II (SEQ ID NO: 111 ) or Formula IV (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).

[0848] Embodiment 30. The pharmaceutical composition of embodiment 29, further comprising a carrier. Atty. Docket No, 2950-42 PCT

[0849] Embodiment 31. A host cell comprising the polynucleotide encoding an engineered polypeptide comprising the amino acid sequence of Formula II (SEQ ID NO: 111)

[0850] Y X2E G T X6X7 S D X)0S I Xi3Xt4D X161 A Q X2o A X22V Q X25X261 A G G P S S G A P P (I), wherein X2= V or K; X« =F, P, or S; X? = T, C, or E; Xio = Y, C, or E; Xu = A, S, Y, N, I, L, R, V, or K; XM= 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,

[0851] or of Formula IV (SEQ ID NO: 383):

[0852] Y X2E G T X6X7S D X10S I X13X!4D X161 A X!9X20A X22V Q X25X26I A G G P S S G A P P (IV), wherein X2= V or K; Xs =F, P, or S; X? = T, C, or E; Xio = Y, C, or E; XB = A, S, Y, N, I, L, R, V, or K; X14= L, K, H, or I; Xt6= K, R, H, or V; X!9= Q or V; X20= K, R, H, N; X22= F, A, P; X25= W, P, K, H, or 1; and X26- L or V.

[0853] Embodiment 32. A host cell comprising a vector comprising a polynucleotide encoding an engineered polypeptide comprising the amino acid sequence of Formula II (SEQ ID NO:111):

[0854] Y X2E G T Xs X7S D X!0S I Xi3X14D X161 A Q X20A X22V Q X25X261 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; Xf0= Y, C, or E; XB = A, S, Y, N, I, L, R, V, or K; X]4= 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 X2« ~ L or V,

[0855] or of Formula IV (SEQ ID NO: 383):

[0856] Y X2E G T Xs X7S D X1BS I Xi3XwD X161 A X)9X20A X22V Q X25X2SI 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; X10= Y, C, or E; XB = A, S, Y, N, 1, L, R, V, or K; Xu - L, K, H, or I; X16= K, R, H, or V; Xt9= Q or V; X20= K, R, H, N; X22= F, A, P; X25= W, P, K, H, or I; and X26- L or V.

[0857] Embodiment 33. A transformed cell comprising a nucleic acid expression cassete 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):

[0858] Y X2E G T X6X7S D X!0S I X X)4D XJ6I A Q X20A X22V Q X25X261 A G G P S S G A P P (I), wherein X2= V or K; Xe:::F, P, or S; X? = T, C, or E; Xio - Y, C, or E; Xi 3 = A, S, Y, N, I, L, R, V, or K; X14= L, K, H, or I; Xt6= K, R, H, or V; X20= K, R, H, N; X22= F, A, P; X25= W, P, K, H, or I; and XM - L or V,

[0859] or of Formula IV (SEQ ID NO: 383):

[0860] Y X2E G T X6X7S D XwS I X13Xt4D Xi61 A XI9X20A X22V Q X2s X261 A G G P S S G A P P (IV), wherein X2= V or K; Xo =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; Xt= L, K, H, or I; Xi6- K, R, H, or V; X)9= Q or V; X20= K, R, H, N; X22= F, A, P; X25 = W, P, K, H, or I; and X26 = L or V; Atty. Docket No. 2950-42 PCT

[0861] a nucleic acid sequence comprising a translation terminator; and

[0862] a nucleic acid sequence comprising transcription terminator.

[0863] Embodiment 34. The transformed cell of embodiment 33, wherein said cell is a bacterial cell, plant cell, yeast cell, or algae cell.

[0864] Embodiment 35. The transformed cell of embodiment 32 or 33, wherein said cell comprises two or more copies of said recombinant nucleic acid.

[0865] Embodiment 36. The transformed cell of any one of embodiments 32 to 35, wherein said cell comprises an integration in the bacterial chromosome.

[0866] Embodiment 37. The transformed cell of embodiment 36, wherein said integration in the bacterial chromosome is an integration into a transposase locus.

[0867] Embodiment 38. The transformed cell of any one of embodiments 32 to 35, wherein said cell is a yeast cell.

[0868] Embodiment 39. The transformed cell of embodiment 38, wherein said yeast cell is a cell of a strain of Pichia pastor is.

[0869] 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.

[0870] Embodiment 41. The transformed cell of any one of embodiments 32 to 35, wherein said transformed cell is a plant cell.

[0871] Embodiment 42. The transformed cell of embodiment 41, wherein said recombinant nucleic acid is integrated into the chloroplast of said plant cell.

[0872] Embodiment 43. The transformed cell of embodiment 41, wherein said recombinant nucleic acid is integrated into the genome of said plant cell.

[0873] 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)

[0874] Y X2E G T X6X7S D Xi0S I X13X!4D Xi61 A Q X20A X22V Q X25X261 A G G P S S G A P P (I), wherein X2= V or K; Xg ~F, P, or S; X? = T, C, or E; Xjo = 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; X22- F, A, P; X25= W, P, K, H, or I; and XM = L or V,

[0875] or of Formula IV (SEQ ID NO: 383): Y X2E G T X6

[0876]

[0877] X7S D Xio S I X13X14D X161 A Xi, X20A X22V Q X25X2SI 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; X10= Y, C, or E; Xl3= A, S, Y, N, 1, L, R, V, or K; Xu = L, K, H, or I; Xu - K, R, H, or V; X!9= Q or V; X20= K, R, H, N; X22= F, A, P; X25= W, P, K, H, or I; and X26= L or V. Atty. Docket No. 2950-42 PCT

[0878] Embodiment 45. The genetically engineered plant, or part thereof, of embodiment 44, wherein said part thereof is a plant seed.

[0879] 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. The genetically engineered plant, or part thereof, of embodiment 46, wherein said recombinant nucleic acid is integrated into the genome of said plant cell.

[0880] Embodiment 48. A kit comprising the pharmaceutical composition of embodiment 28 or embodiment 29.

[0881] Embodiment 49, The kit of embodiment 48, comprising the host cell of embodiment 31 and a carrier.

[0882] Methods

[0883] 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, pre¬ diabetes, insulin resistance, glucose intolerance, type 2 diabetes, type 1 diabetes, hypertension, atherogenic dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke or mierovascular 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.

[0884] 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.

[0885] Embodiment 52. The method of embodiment 50, wherein the disease or condition is diabetes or obesity.

[0886] Embodiment 53. The method of embodiment 52, wherein the disease or condition is type-2 diabetes.

[0887] 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.

[0888] 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. Atty. Docket No. 2950-42 PCT

[0889] Embodiment 57. The method of embodiment 50, wherein the engineered polypeptide or a pharmaceutical composition thereof is administered orally.

[0890] Embodiment 58. The method of embodiment 55, wherein the injection is administered subcutaneously or intravenously.

[0891] 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.

[0892] Embodiment 60. The method of any one of embodiments 50 to 59, further comprising administering one or more additional therapies.

[0893] 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 giyburide, a dipeptidyl peptidase 4 inhibitor, a bile acid sequestrant, or any combination thereof.

[0894] 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.

[0895] Embodiment 63. A pharmaceutical composition comprising an engineered polypeptide comprising an amino acid sequence Formula II (SEQ ID NO: 111).

[0896] 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.

[0897] 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):

[0898] Y X2E G T Xg X7S D Xio S I X13X14D X161 A Q X2QA X22V Q X25X261 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; Xl3- 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 X2s “ L or V,

[0899] or of Formula IV (SEQ ID NO: 383):

[0900] Y X2E G T X6X7S D Xio S I XnXu D X]6I A Xi9X20A X22V Q X25X261 A G G P S S G A P P (IV), wherein X

[0901]

[0902] 2= V or K; =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, II, or I; Xis = 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. Atty. Docket No. 2950-42 PCT

[0903] 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.

[0904] 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.

[0905] Embodiment 69. The DFM of embodiment 68, wherein said transformed bacteria is a strain of Bacillus subtilis.

[0906] 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.

[0907] 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.

[0908] Embodiment 72. The DFM of embodiment 68, wherein said one or more anaerobic cellulolytic bacteria is a member of the phylum Bacillota.

[0909] Embodiment 73. The DFM of embodiment 72, wherein said member of the phylum Bacillota is a bacterial strain of Ruminococcus flavefaciens.

[0910] Embodiment 74. The DFM of embodiment 68, wherein said one or more anaerobic cellulolytic bacteria is a member of the genus Bacteroides.

[0911] Embodiment 75. The DFM of embodiment 74, wherein said member of the genus Bacteroides is Bacteroides fragilis.

[0912] Embodiment 76. The DFM of embodiment 68, wherein said bacterial strain is a strain of Lactobacillus.

[0913] Embodiment 77. The DFM of embodiment 76, wherein said strain of Lactobacillus is a strain of Lactobacillus gasseri.

[0914] Embodiment 78. The DFM of embodiment 76, wherein said strain of Lactobacillus is a strain of Lactobacillus plantarum.

[0915] Embodiment 79. The DFM of any one of embodiments 67 to 78, wherein said DFM is lyophilized.

[0916]

[0272] Further Additional Embodiments and Variant Peptides

[0917] Embodiment 80. An engineered dual agonist polypeptide comprising the amino acid sequence selected from:

[0918] Formula VIII (SEQ ID NO:401):

[0919] Y V E G T F T S D Y S I S L Xi5Xi61 A Q X2o A F X23Q W L I A G (VIII),

[0920] wherein Xis= D or Q; Xis = R or Q; X2o= R or Q; and X23= V or M; or

[0921] Formula IX (SEQ ID NO:402): Atty. Docket No. 2950-42 PCT

[0922] Y X2E G T X6X7S D Xw S I Xi3X!4Xi5Xl61 A X19X20A X22X23Q X23X26I A G (IX), wherein X2- V or K; X6= F, P or S; X7~ T, C or E; Xio= Y, C or E; Xt3- A, S, Y, N, I, L, R, V or K; Xi4= L, K, H or I; Xi 5= D or Q; X = K, R, H, V or Q; Xi9= Q or V: X2o= K, R, H, N or Q; X22^ F, A or P; X23= V or M; X23= W, P, K, H or 1; and X26= L or V.

[0923] Embodiment 81. The engineered dual agonist polypeptide of embodiment 80, wherein the polypeptide further comprises the amino acids GPSSGAPP at its C -terminal end.

[0924] Embodiment 82. The engineered dual agonist polypeptide of embodiment 80, comprising the amino acid sequence of any one of SEQ ID NO:391 to SEQ ID NO:394.

[0925] Embodiment 83. The engineered dual agonist polypeptide of embodiment 82, wherein the polypeptide further comprises the amino acids GPSSGAPP at its C-terminai end.

[0926] Embodiment 84. An engineered Glucagon-Like Peptide 1 (eGLP-l) polypeptide comprising the amino acid sequence selected from:

[0927] Formula V (SEQ ID NO:398):

[0928] Xi G E G T S E S D V S X12 X;3Xu E Xi6 Q A X19 X2o E X22X23A X23X26 V D G X3o (V), wherein X, = H or Y; X,2= S, Q, A or M; X13 ~ S, Q, Y, or M; X14 = I or L; Xi6 = G or M; X19 = A or T; X20- Q, V, or D; X22= V, I, F or H; X23= V or I; X25= V, I, or W; X26= I, V or L; and X30 = R or S; Formula VI (SEQ ID NO:399):

[0929] Xi G E G T Xi T S D V S XI2X[3L E X16Q A X!9X20E X221 A X23L V D G R (VI),

[0930] wherein Xi = H or Y; X6= F or I Xi2= S, A or M; X!3= Y or M; Xi6= G or M; X)9= A or T; X2B= Q, V, or D; X22= F or H; and X25 = I or W; or

[0931] Formula VII (SEQ ID NG:400):

[0932] X, G E G TX^ T S D V S Xr X13Xl4E Xl6Q A X)9X20 E X22X23A X25 L V D G X30(VII), wherein Xi = H or Y; Xe = F or I; X;2~ 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, F or H; X23= V or I; X25- V, I, or W; and X30= R or S.

[0933] Embodiment 85. The engineered polypeptide of embodiment 84, comprising the amino acid sequence of any one of SEQ ID NO.387 to SEQ ID NO:389.

[0934] Embodiment 86. The engineered polypeptide of embodiment 80 or embodiment 84, wherein the engineered polypeptide is substantially resistant to proteolytic degradation.

[0935] Embodiment 87. The engineered polypeptide of embodiment 80 or embodiment 84, wherein the engineered polypeptide at least maintains substantially the same receptor potency as the corresponding wild-type, unmodified polypeptide.

[0936] Embodiment 88. The engineered polypeptide of embodiment 80 or embodiment §4, wherein the engineered polypeptide is substantially resistant to DPP-IV, neprilysin, a-chymotrypsin, trypsin, elastase, or pepsin, degradation. Atty. Docket No. 2950-42 PCT

[0937] Embodiment 89. An isolated polynucleotide encoding:

[0938] an engineered dual agonist polypeptide comprising the amino acid sequence of Formula VIII (SEQ ID NO:401) or of Formula IX (SEQ ID NO:402); and / or

[0939] an engineered Glucagon-Like Peptide 1 (eGLP-1)...

Claims

1. Atty. Docket No. 2950-42 PCT2.Claims:

1. A variant albumin binding domain (ABD) sequence, wherein one or more trypsin site, or one or more Lys (K) or Arg (R), in the sequence LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO:385) is substituted with an amino acid that is not susceptible to trypsin digestion.

2. The variant albumin binding domain (ABD) sequence of claim 1, comprising the amino acid sequence selected from SEQ ID NO.395, SEQ ID NO.-396 and SEQ ID NO:397.

3. The variant albumin binding domain (ABD) sequence of claim 1 or 2, covalently linked to a peptide therapeutic or engineered polypeptide for use or application in type 2 diabetes mellitus (T2DM) or for treatment, control, or modulation of high blood glucose, metabolic disorders, excess body weight, or obesity.

4. The variant albumin binding domain (ABD) sequence of claim 3, wherein the ABD sequence is covalently linked to an engineered dual agonist polypeptide or Glucagon-Like Peptide 1 (eGLP-1) polypeptide.

5. An engineered dual agonist polypeptide comprising the amino acid sequence selected from: Formula VIII (SEQ ID NO:401):8.Y V E G T F T S D Y S I S L X15X16I A Q X20A F X23Q W L I A G (VIII),9.wherein X15= D or Q; X16= R or Q; X20= R or Q; and X23= V or M; or10.Formula IX (SEQ ID NO:402):11.Y X2E G T X6X- S D Xio S I X13X14X)5X161 A X!9X20 A X23X23Q X25X26I A G (IX), wherein X2= V or K; 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; X15= D or Q; X16= K, R, H, V or Q; X19= Q or V; X20= K, R, H, N or Q; X22= F, A or P; X23= V or M; X25= W, P, K, H or I; and X26= L or V.

6. The engineered dual agonist polypeptide of claim 5, wherein the polypeptide further comprises the amino acids GPSSGAPP at its C-terminal end.Atty. Docket No. 2950-42 PCT7. The engineered dual agonist polypeptide of claim 5, comprising the amino acid sequence of any of SEQ ID NO:391 to SEQ ID NO:394.

8. The engineered dual agonist polypeptide of claim 7, wherein the polypeptide further comprises the amino acids GPSSGAPP at its C-terminal end.

9. An engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide comprising the amino acid sequence selected from:16.Formula V (SEQ ID NO:398):17.Xi G E G T S E S D V S Xl2X13Xi4E X16Q A Xi9X20E X22X23A X25X26V D G X3o (V), wherein Xi = H or Y; X12 = S, Q, A or M; Xu ~ S, Q, Y, or M; X14 = I or L; Xi6” G or M; Xu = A or T; X20= Q, V, or D; X22= V, I, F or H; X23= V or I; X25= V, I, or W; X26= I, V or L; and X30~ R or S; Formula VI (SEQ ID NO:399):18.X1G E G T X6T S D V S X12X13L E X16Q A X19X20E X22I A X25L V D G R (VI),19.wherein X1= H or Y; X6= F or I; X12= S, A or M; X13= Y or M; X16= G or M; X19= A or T; X20= Q, V, or D; X22= F or H; and X25= I or W; or20.Formula VII (SEQ ID NO:400):21.X1G E G T X6T S D V S X12X13X14E X16Q A X19X20E X22X23A X25L V D G X30(VII), wherein X1= H or Y; X6= F or I; X12= S, Q, A or M; X13= S, Q, Y, or M; X14= I or L; X16= G or M; X19= A or T; X20= Q, V, or D; X22= V, I, F or H; X23= V or I; X25= V, I, or W; and X30= R or S.

10. The engineered polypeptide of claim 9, comprising the amino acid sequence of any one of SEQ ID NO:87 to SEQ ID NO:389.

11. The engineered polypeptide of claim 5 or claim 9, wherein the engineered polypeptide is substantially resistant to proteolytic degradation.

12. The engineered polypeptide of claim 5 or claim 9, wherein the engineered polypeptide at least maintains substantially the same receptor potency as the corresponding wild-type, unmodified polypeptide.

13. The engineered polypeptide of claim 5 or claim 9, wherein the engineered polypeptide is substantially resistant to DPP-IV, neprilysin, a-chymotrypsin, trypsin, elastase, or pepsin, degradation,14. An isolated polynucleotide encoding:Atty. Docket No. 2950-42 PCT27.an engineered dual agonist polypeptide comprising the amino acid sequence of Formula VIII (SEQ ID NO:401) or of Formula IX (SEQ ID NO:402); and / or28.an engineered Glucagon-Like Peptide 1 (eGLP-1) polypeptide comprising the amino acid sequence of Formula V (SEQ ID NO:398), of Formula VI (SEQ ID NO:399), or of Formula VII (SEQ ID NO:400).

15. A pharmaceutical composition comprising:30.an engineered dual agonist polypeptide comprising the amino acid sequence of Formula VIII (SEQ ID NO:401) or of Formula IX (SEQ ID NO:402); and / or31.an engineered Glucagon-Like Peptide 1 (eGLP- 1 ) polypeptide comprising the amino acid sequence of Formula V (SEQ ID NO:398), of Formula VI (SEQ ID NO:399), or of Formula VII (SEQ ID NO:400); and a carrier.

16. A host cell comprising the polynucleotide of claim 14,17. The host cell of claim 16, wherein said cell is a bacterial cell, plant cell, yeast cell, or algae cell.

18. A kit comprising the pharmaceutical composition of claim 15 or the host cell of claim 16, and a carrier.

19. 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 5 and / or an engineered Glucagon-Like Peptide 1 (eGLP-1) of claim 9, a pharmaceutical composition of claim 15, or a host cell of claim 16.

20. The method of claim 19, wherein the dual agonist polypeptide comprises the amino acid sequence of any one of SEQ ID NO:391 to SEQ ID NO:394, or wherein the engineered Glucagon-Like Peptide 1 (eGLP-1) comprises the amino acid sequence of any one of SEQ ID NO:387 to SEQ ID NO:389.

21. 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 engineered dual agonist polypeptide of claim 5 and / or an engineered Glucagon-Like Peptide 1 (eGLP-1) of claim 9, a pharmaceutical composition of claim 15, or a host cell of claim 16.Atty. Docket No. 2950-42 PCT22. The method of claim 21, wherein the dual agonist polypeptide comprises the amino acid sequence of any one of SEQ ID NO:391 to SEQ ID NO:394, or wherein the engineered Glucagon-Like Peptide I (eGLP-1) comprises the amino acid sequence of any one of SEQ ID NO:387 to SEQ ID NO:389.

23. The method of claim 19, 20, 21 or 22, wherein the disease or condition is diabetes or obesity.

24. The method of claim 19, 20, 21 or 22, wherein the dual agonist polypeptide, the eGLP-1 polypeptide or the pharmaceutical composition is administered orally, by injection, or transdermally.

25. The method of claim 19, 20, 21 or 22, wherein the subject is human, cat, dog, or non-human primate.

26. A pharmaceutical composition comprising a dual agonist polypeptide comprising an amino acid sequence selected from any of SEQ ID NO:391 to SEQ ID NO:394.

27. 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:387 to SEQ ID NO:389.

28. A direct fed microbial (DFM) comprising bacteria transformed with a nucleic acid encoding one or more engineered dual agonist polypeptide of Formula VIII (SEQ ID NO:401) or Formula IX (SEQ ID NO:402) and / or encoding one or more engineered Glucagon-Like Peptide 1 (eGLP-1) comprising the amino acid sequence of Formula V (SEQ ID NO:398), Formula VI (SEQ ID NO:399) or Formula VII (SEQ ID NO:400).

29. The direct feed microbial (DFM) of claim 28, comprising bacteria transformed with a nucleic acid encoding one or more engineered dual agonist polypeptide selected from SEQ ID NO:391 to SEQ ID NO:394 and / or encoding one or more engineered Glucagon-Like Peptide I (eGLP-1) polypeptide selected from any of SEQ ID NO:387 to SEQ ID NO:389.

30. The DFM of claim 28 or 29, wherein said bacteria comprise a strain selected from the group consisting of Bacillus, Lactobacillus, Lactococcus, Enterococcus, Salmonella, and combinations thereof.

31. The engineered polypeptide of claim 5, 6, 7, 8, 9 or 10, wherein the engineered polypeptide is covalently linked to an albumin binding domain (ABD) sequence.

32. The engineered polypeptide covalently linked to an albumin binding domain (ABD) sequence of claim 31, wherein the albumin binding domain (ABD) sequence comprises SEQ ID NO:384, SEQ IDAtty. Docket No. 2950-42 PCT47.NO:385 or SEQ ID NO:386 or comprises the variant albumin binding domain (ABD) sequence of claim 27 or 28.

33. The engineered polypeptide covalently linked to an albumin binding domain (ABD) sequence of claim 31, wherein the engineered dual agonist polypeptide sequence comprises the amino acid sequence of any of SEQ ID NO:391 to SEQ ID NO:394 or wherein the eGLP-1 polypeptide sequence comprises the amino acid sequence of any of SEQ ID NO:387 to SEQ ID NO:389.

34. An engineered dual agonist or GLP-1 (eGLP-1) polypeptide covalently linked to a variant albumin binding domain (ABD) sequence, wherein the engineered polypeptide sequence is selected from Formula I, Formula II, Formula III, Formula IV and the variant albumin binding domain (ABD) sequence is selected from SEQ ID NO:395, SEQ ID NO:396 and SEQ ID NO:397.

35. The engineered dual agonist or GLP-1 (eGLP-1) polypeptide covalently linked to a variant albumin binding domain (ABD) sequence of claim 32, wherein the engineered polypeptide sequence is selected from SEQ ID NOs:1-14, SEQ ID NOs:372-380, SEQ ID NOs:112-147 and SEQ ID NO:

381.

36. A pharmaceutical composition comprising the engineered polypeptide of claim 31, 32, 33, 34 or 35 and a carrier.

37. An isolated polynucleotide encoding variant albumin binding domain of claim 1 or 2.

38. An isolated polynucleotide encoding the engineered polypeptide of claim 1, 32, 33, 34 or 35.

39. A host cell comprising the polynucleotide of claim 37 or 38.

40. The host cell of claim 39, wherein said cell is a bacterial cell, plant cell, yeast cell, or algae cell.

41. A kit comprising the engineered polypeptide of claim 31, the pharmaceutical composition of claim 36, or the host cell of claim 39, and a carrier.

42. 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 polypeptide of claim 31, 32, 33, 34 or 35, a pharmaceutical composition of claim 36, or a host cell of claim 39.

43. The method of claim 42, wherein the dual agonist polypeptide sequence comprises the amino acid sequence of any one of SEQ ID NO:391 to SEQ ID NO:394, or wherein the engineered Glucagon-LikeAtty. Docket No. 2950-42 PCT57.Peptide 1 (eGLP-1) sequence comprises the amino acid sequence of any one of SEQ ID NO:387 to SEQ ID NO:389.

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 1 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 claim 31, 32, 33, 34 or 35, a pharmaceutical composition of claim 36, or a host cell of claim 39.

45. The method of claim 44, wherein the dual agonist polypeptide sequence comprises the amino acid sequence of any one of SEQ ID NO:391 to SEQ ID NO:394, or wherein the engineered Glucagon-Like Peptide 1 (eGLP-1) sequence comprises the amino acid sequence of any one of SEQ ID NO:387 to SEQ ID NO:389.

46. The method of claim 42, 43, 44 or 45, wherein the disease or condition is diabetes or obesity.

47. The method of claim 42, 43, 44 or 45, wherein the dual agonist polypeptide, the eGLP-1 polypeptide or the pharmaceutical composition is administered orally, by injection, or transdermally.

48. The method of claim 42, 43, 44 or 45, wherein the subject is human, cat, dog, or non-human primate.

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