Stable 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, as well as protease resistant dual agonist peptides directed against GLP-1R and GIPR, methods of making these peptides, as well as compositions comprising the protease resistant peptides and methods of treatment utilizing these peptides.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 529,271, filed July 27, 2023, and U.S. Provisional Patent Application No. 63 / 540,427, filed September 26, 2023.

[0002] Merging of sequence lists A sequence list in ST.26 XML format entitled 2950-17_PCT_ST26, comprising 575,627 bytes, was created on July 26, 2024, prepared in accordance with 37 CFR 1.822 to 1.824, and filed with this application on July 26, 2024, the full text of which is incorporated herein by reference. Technical Field

[0003] This invention relates to variants and stable forms of glucagon-like peptide-1 (GLP-1), methods of application and production thereof, and also to dual agonists targeting GLP-1R and GIPR and their delivery via in vivo delivery platforms (such as genetically modified bacteria) to deliver therapeutic activity directly to the mucosa of animals in need. Background Technology

[0004] Type 2 diabetes is a disease caused by hyperglycemia due to insulin resistance and relative insulin deficiency. Type 2 diabetes (T2DM) is the most common type of diabetes and is characterized by a variety of interrelated metabolic disorders. Although various treatments such as tirzepatide, semaglutide, liraglutide, GLP-1, GIP (7DTY_P), Medi7219, and exenatide (to name just a few) are available for T2DM, treatment options for domestic cats (…) Felis catus Treatment for other species still needs further improvement. 。

[0005] The use of peptide therapeutics in the treatment of type 2 diabetes mellitus (T2DM) is becoming increasingly widespread. Oral administration represents a significant medical advancement, but it faces challenges such as gastrointestinal instability and low absorption efficiency into the circulation. Exenatide, telposide, smegglutide, and liraglutide are administered subcutaneously, while Medi7219 and J229 are administered orally. Drugs designed for T2DM can function as single or dual / triple agonists. See Pechenov et al., “Development of anorally delivered GLP-1 receptor agonist through peptide engineering and drug delivery to treat chronic disease,” Scientific Reports 11(1):22521 (2021).

[0006] GLP-1 (a proglucagon-derived peptide produced by intestinal L cells) and GIP (produced by K cells) are both used to treat type 2 diabetes. Both GLP-1 and GIP stimulate insulin secretion; GLP-1 delays gastric emptying, while GIP does not. Exenatide is a GLP-1 analog, originally discovered in the saliva of the Gila monster, and shares 53% amino acid identity with GLP-1 (Bond, “Exenatide (Byetta) as a novel treatment option for type 2 diabetes mellitus,”). Proceedings (Baylor University. Medical Center) 19(3):281–284 (2006)). Liraglutide (4APD_A) shares 97% sequence identity with GLP-1, and the addition of its C16 fatty acid side chain promotes the binding of the drug to circulating serum albumin (Garber, “Long-acting glucagon-likepeptide 1 receptor agonists: a review of their efficacy and tolerability,” Diabetes Care 34(Supplement 2): S279-84 (2011)). Furthermore, as used in exenatide, replacing the 8th alanine with glycine (GLP-1-Gly8) significantly enhances insulin secretion (Lin et al., “Oral Delivery of Pentameric Glucagon-Like Peptide-1 by Recombinant Lactobacillus in DiabeticRats,”). PloS One11(9):e0162733 (2016)).

[0007] The GIPR and GLP-1R receptors each contain an N-terminal extracellular domain (ECD), a central domain consisting of seven transmembrane α-helices, and a C-terminal cytoplasmic domain that mediates intracellular signal transduction through physical binding to G proteins. Thilborpeptide agonists targeting GIPR and GLP-1 exhibit similar peptide-receptor binding interfaces, but structural changes are observed in the extracellular loops (ECL1, ECL3) and transmembrane (TM1, TM3) regions. The N-terminus of thiolborpeptide shows good overlap in both receptors and interacts with the central transmembrane domain, leading to conformational rearrangement and receptor activation. The Tyr-1 to Met-14 residues of thiolborpeptide agonists do not contact the extracellular domain of the receptor. Hydrophobic interactions are believed to be involved in the binding process.

[0008] The GIP binding interface spans the C-terminal region of the peptide's α-helix, which contains Asp-15 to Lys-30 residues that interact with the receptor's extracellular domain, such as... Figure 2 As shown. Figure 2 As shown, the C-terminus of the GLP-1 agonist interacts with its extracellular domain, while the N-terminus of GLP-1 interacts with the transmembrane domain of the GLP-1R receptor. (Image source: Cong et al., "Molecular insights into ago-allosteric modulation of the human glucagon-like peptide-1 receptor") Nature Communications , 12 (1):3763 (2021).

[0009] Helical peptides expose hydrophobic residues to the ECD, indicating that their binding is primarily dominated by hydrophobic interactions. Structural studies of the GLP-1 peptide bound to the ECD confirmed that the C-terminal α-helix region of GLP-1 is located within a binding cleft at the N-terminus of the ECD. The hydrophobic surface of GLP-1 constitutes the main body of the interaction with the ECD and may be a key factor in ECD / peptide affinity. The receptor's ECD folds down toward the TMD to stabilize the complex. The formation of polar network rearrangement indicates that GLP-1 triggers the activation of GLP-1R. After receptor activation, the three-layered structure of the polar network rearranges as follows: (i) central polar network, (ii) HETX motif polar network, (iii) TM2-6-7-helix 8 polar network (see [link to relevant documentation]). Figure 2(Cong et al. (2021)). The receptor ECD recognizes and “captures” the C-terminal portion (residues 15-30) of the agonist, after which the N-terminal portion of the peptide moves and docks into the TMD. During this process, the highly conserved PLLG motif of the receptor located in the middle of helix VI destabilizes and undergoes a conformational change. The base of helix VI moves outward 10-20 Å, forming an angle of 40-60° with helix VII, thereby creating space in the intracellular portion of the receptor for binding of the cytoplasmic protein complex.

[0010] GLP-1 (7-37), GIP (1-42), exenatide (1-39), telzopatide (1-39), smegglutide, liraglutide, J211, Medi7219 (to name only) (and a few others) have all been studied in detail. The N-terminus of telzopatide shows higher homology with GIP peptide, while the C-terminus is more similar to exenatide (30-39 aa). See Nauck and D'Alessio, “Tirzepatide, adual 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 , 21 (1): 169 (2022), and Wang, “Designing aDual GLP-1R / GIPR Agonist from Tirzepatide: Comparing Residues Between Tirzepatide, GLP-1, and GIP,” Drug Design, Development and Therapy16 :1547–1559 (2022). The major circulating GIP peptide is the GIP(1–42) variant, which is truncated at the N-terminus by DPP-4 to generate GIP(3–42). GIP(1–42) is derived from the precursor protein proGIP via prohormone convertase (PC), but also contains the PC2 cleavage motif, which can be cleaved at Gly31, Lys32, and Lys33 sites. After cleavage, two peptides are generated: GIP(1–30)-NH2 and GIP(34–42). The C-terminated GIP(1–30)-NH2 peptide appears to have similar agonist properties to the full-length GIP(1–42) (Alaña 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 al., “Molecular interactions of full-length and truncated GIPpeptides with the GIP receptor - A comprehensive review,” Peptides125 :170224 (2020)). The last 12 residues of GIP (1-42) were observed to be in a disordered state, neither binding to the ECD nor to residues near the TMD, indicating that these residues are of little importance to GIPR binding and receptor activation. Furthermore, the presence of the C-terminus was observed to affect the overall stability of the GIP peptide, and the presence of the C-terminus enhanced the agonistic activity of the N-terminated truncated GIP peptide compared to the same N-terminated truncated GIP peptide lacking the C-terminus.

[0011] GLP-1 is a 30-amino acid peptide secreted by intestinal L cells after food intake. It exists in two equivalent forms: GLP-1(7-36)-NH2 and GLP-1(7-37). GLP-1(7-36) is more abundant. GLP-1(7-36) binds to and activates the GLP-1 receptor (GLP-1R), thereby exerting its regulatory function.

[0012] Several modification methods have been proposed in this field to engineer telpoide by replacing non-natural AIBs with natural amino acids and reducing its proteolytic sensitivity. Telpoide is a GIP analog, and Wang et al. (2022) proposed that Gly could be a possible option for AIB modification (Wang (2022)). However, while this modification enhances the peptide's binding affinity to the GIPR receptor, it reduces its binding affinity to GLP-1R. Importantly, glycine is readily cleaved by the proteolytic enzyme elastase (Wang (2022)).

[0013] GLP-1 (7-37) has a histidine (His) at position 7, which is crucial for activating the GLP-1R receptor and maintaining its insulin-secreting activity. 7 Replace with Trp 7 It reduces the binding affinity of GLP-1 and significantly decreases its activity; while the N-terminus deletion of Tyr in GIP reduces the binding affinity of GLP-1 and significantly decreases its activity. 1 It will also significantly reduce its activity. Tyr 1 and Ile 7 GIP peptides are key to activating the GLPR receptor. They deliver Thr from GLP-1. 7 The introduction of tyr peptide may reduce its GIP activity. The MAR709 peptide uses Tyr derived from GIP.10 and Ile 12 Ile 12 Tyr plays an important role in GLPR receptor activation. Tyr is used in telpolide. 10 and Ile 12 Tyr in GLP-1 19 Replacing with Ala reduces the binding affinity and activity of GLP-1 agonists. Aib13 of telpolide appears to reduce its GLP-1 activity without affecting its GIP activity (Wang (2022)).

[0014] GLP-1 is an incretin hormone secreted by L cells in the intestine that stimulates the pancreas to secrete insulin in a glucose-dependent manner. See Chia and Egan, “Incretin-Based Therapies in Type 2 Diabetes Mellitus,” J Clin Endocrinol Metab. 93(10):3703–3716 (2008), and Muller et al., “Glucagon-like peptide 1 (GLP-1),” Mol Metab 30:72-130 (2019). Exogenously provided GLP-1 analogues have been used to treat type 2 diabetes. They have been shown to play an important role in increasing β-cell mass and have potent antidiabetic effects associated with weight loss. See Baggio and Drucker, “Biology of incretins: GLP-1 and GIP,” Gastroenterology 132(6):2131-57(2007).

[0015] The amino acid sequence of GLP-1 (SEQ ID NO:18) is highly conserved in mammals, exhibiting 100% sequence identity in human, mouse, rat, sheep, goat, cattle, hamster, cat, and dog (to name only) species (data not shown). GLP-1 is an endogenous ligand for the GLP-1 receptor and shares 53% sequence identity with exenatide (a long-acting GLP-1 receptor agonist). 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 its two N-terminal amino acids. GLP-1 also has many sites that are easily hydrolyzed by digestive proteases such as 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 also) Figure 1 ).

[0016] Inactive full-length GLP-1 (1-37) is processed into two active cycling forms: GLP-1 (7-37) and GLP-1 (7-36) amide, the latter being the most abundant form in the blood. Both forms of GLP-1 have very short half-lives due to their susceptibility to digestion by dipeptidyl peptidase-IV (DPP-IV) in serum. GLP-1 also contains aromatic residues (Phe12, Tyr19, Phe28, and Trp31), which are sensitive to chymotrypsin, pepsin, and / or neprilysin. Figure 1 A summary of known cleavage sites is provided. 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.

[0017] GLP-1 has been reported to have a half-life of less than 2 minutes, and its concentration returns to baseline levels within 90 minutes after subcutaneous injection, making systemic administration difficult. See Kieffer. et al. , “Degradation of glucose-dependentinsulinotropic polypeptide and truncated glucagon-like peptide 1 in vitro and in vivo by dipeptidyl peptidase IV,” Endocrinology 136(8):3585-96(1995). Therefore, the treatment of type 2 diabetes requires DPP-IV resistant GLP-1 analogs.

[0018] Peptides possess low toxicity and high specificity, making them suitable therapeutic agents for 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 susceptible to proteolytic degradation during storage or oral administration, and often 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 required, leading to decreased patient compliance (Hamman and Steenekamp, ​​2011). Therefore, there is an urgent need to develop improved GLP-1 peptides and more economical and patient-friendly drug delivery methods that are low in toxicity and highly specific, resistant to degradation, and orally administered.

[0019] Several patent publications have addressed modified GLP-1 peptides. US Patent Publication 2018 / 0162920 (published June 14, 2018, Revell and Bednarek) (Publication No. 920) provides protease-resistant lipotropic GLP-1 analogs through selective, strategically targeted lipolation and α-functionalized amino acid substitution in the peptide. More specifically, Publication No. 920 discloses the lipolation of certain lysine or cysteine ​​residues. The engineered GLP-1 peptide in this application does not contain lysine or cysteine. US Patent 7,847,063 (published December 7, 2010, Sugita et al.) provides recombinant GLP-1 derivatives. International Patent Publication 2020 / 023388 (published January 30, 2020, Alsina-Fernandez et al.) describes a dosing regimen for a GIP / GLP1 co-agonist. U.S. Patent 6,620,910 (granted September 16, 2003, by Calas et al.) provides a modified GLP-1 peptide containing lysine and susceptible to trypsin cleavage.

[0020] This application provides and includes GLP-1 derivatives that strongly bind to GLP-1R receptors in humans, cats, and other mammals, and are modified to provide substantial resistance to the proteolytic activity of digestive enzymes such as DPP-IV, neprilysin, α-chymotrypsin, trypsin, elastase, or pepsin. This application also reports modifications to the telpoide sequence to retain its dual agonist properties: replacing non-natural amino acids (e.g., α-aminoisobutyric acid) with naturally occurring encoding amino acids, and making the dual agonist peptide resistant to intestinal peptidases. This application provides and includes peptides combining glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) activity for the treatment of diabetes. GLP-1 and GIP co-stimulate insulin secretion and produce an incretin effect. GLP-1R and GIPR are expressed on pancreatic β-cells, and activation of these receptors potently stimulates insulin secretion even in cases of mild hyperglycemia. Summary of the Invention

[0021] This application generally relates to novel engineered glucagon-like peptide-1 (eGLP-1) peptides or pharmaceutically acceptable salts, solvates, and / or other forms thereof, corresponding pharmaceutical compositions, and methods and / or uses of said eGLP-1 peptides for treating metabolic diseases and / or related disorders. Furthermore, this application also generally relates to novel engineered dual-agonist peptides or pharmaceutically acceptable salts, solvates, and / or other forms thereof, corresponding pharmaceutical compositions, and methods and / or uses of said dual-agonist peptides for treating metabolic diseases and / or related disorders.

[0022] This application provides and includes engineered glucagon-like peptide-1 (eGLP-1) in one form, comprising the amino acid sequence of formula I: HGEGTSESDVSX 12 X 13 X 14 EGQAAQEX 22 X 23 AX 25 X 26 VDGX(I) (SEQ ID NO:1), where X 12 = S or Q; X 13 = S, Q, or Y; X 14 = I or L; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 = I or V; and X 30 = R or S.

[0023] More specifically, this application includes an engineered glucagon-like peptide-1 (eGLP-1) comprising the amino acid sequence of any of SEQ ID NO:2 to SEQ ID NO:382. More specifically, this application includes an engineered glucagon-like peptide-1 (eGLP-1) comprising the amino acid sequence of formula III (SEQ ID NO:382) or any of SEQ ID NO:372 to SEQ ID NO:380.

[0024] In some aspects, this application includes an engineered nucleic acid vector encoding engineered glucagon-like peptide-1 (eGLP-1), said engineered glucagon-like peptide-1 (eGLP-1) comprising the amino acid sequence of formula I. This application also includes one or more vectors encoding one or more eGLP-1 peptides of formula I. This application also includes concatenators of engineered glucagon-like peptide-1 (eGLP-1) comprising the amino acid sequence of formula I (SEQ ID NO:1). In some aspects, said engineered nucleic acid vector encodes engineered glucagon-like peptide-1 (eGLP-1), said engineered glucagon-like peptide-1 (eGLP-1) comprising the amino acid sequences of any of SEQ ID NO:14 and combinations thereof. In some respects, the engineered nucleic acid vector encodes engineered glucagon-like peptide-1 (eGLP-1), which comprises an amino acid sequence of formula III (SEQ ID NO:382) or any of SEQ ID NO:372 to SEQ ID NO:380.

[0025] In some aspects, this application includes a nucleic acid expression cassette comprising a promoter for transcriptional expression, a nucleic acid sequence encoding a peptide of formula I (SEQ ID NO:1) eGLP-1, and translation and transcription termination sequences. In some aspects, the translation termination sequence comprises one or more stop codons. In some aspects, the translation termination sequence comprises a terminal stop codon and one or more in-frame or out-of-frame stop codons located at the 3' end of the terminal stop codon. In some aspects, the nucleic acid expression cassette encodes engineered glucagon-like peptide-1 (eGLP-1), which comprises amino acid sequences and combinations thereof from any of SEQ ID NO:14. In some aspects, the nucleic acid expression cassette encodes engineered glucagon-like peptide-1 (eGLP-1), which comprises amino acid sequences and combinations thereof from SEQ ID NO:14 to SEQ ID NO:14. In some aspects, the nucleic acid expression cassette encodes engineered glucagon-like peptide-1 (eGLP-1), which comprises amino acid sequences and combinations thereof from Formula III (SEQ ID NO:382) to SEQ ID NO:372 to SEQ ID NO:380.

[0026] This application further includes and provides a method for preparing an engineered glucagon-like peptide-1 (eGLP-1) polypeptide having the amino acid sequence of Formula I, the method comprising culturing host cells transformed with an expression vector encoding the eGLP-1 polypeptide under conditions allowing eGLP-1 expression, and recovering eGLP-1. This application also includes a method for preparing an engineered glucagon-like peptide-1 (eGLP-1) polypeptide having the amino acid sequences of any one of the following or combinations thereof, the method comprising culturing host cells transformed with an expression vector encoding the eGLP-1 polypeptide under conditions allowing eGLP-1 expression, and recovering eGLP-1. It also includes a method for preparing an engineered glucagon-like peptide-1 (eGLP-1) polypeptide having an amino acid sequence of formula III (SEQ ID NO:382) or any of SEQ ID NO:372 to SEQ ID NO:380 or a combination thereof, the method comprising culturing host cells transformed with an expression vector encoding an eGLP-1 polypeptide under conditions that allow eGLP-1 expression, and recovering eGLP-1.

[0027] In some aspects, this application provides a host cell comprising a polynucleotide sequence encoding an engineered glucagon-like peptide-1 (eGLP-1) polypeptide having the amino acid sequence of Formula I (SEQ ID NO:1). In some aspects, the host cell comprises a polynucleotide sequence encoding an engineered glucagon-like peptide-1 (eGLP-1) polypeptide having the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO:14 or a combination thereof. In some aspects, the host cell comprises a polynucleotide sequence encoding an engineered glucagon-like peptide-1 (eGLP-1) polypeptide having the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO:14 or a combination thereof. In other respects, the eGLP-1 polypeptide is encoded having an amino acid sequence of formula III (SEQ ID NO:382) or any of SEQ ID NO:372 to SEQ ID NO:380, or a combination thereof.

[0028] This application further includes a method for treating or preventing a disease, comprising administering to a subject requiring treatment an effective amount of an engineered glucagon-like peptide-1 (eGLP-1) polypeptide having the amino acid sequence of Formula I (SEQ ID NO:1), wherein the disease or disorder is selected from the group consisting of lipodystrophy, dyslipidemia, hyperlipidemia, overweight, obesity, hypothalamic amenorrhea, Alzheimer's disease, leptin deficiency, fatty liver disease, diabetes, type 1 diabetes, type 2 diabetes, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), metabolic syndrome X, metabolic dysfunction-associated steatohepatitis (MASH), and Huntington's disease.

[0029] This application includes a method for treating or preventing a disease or condition caused by or characterized by hypoglycemia or impaired insulin release, the method comprising administering to a subject requiring treatment an effective amount of an engineered glucagon-like peptide-1 (eGLP-1) polypeptide having the amino acid sequence of Formula I. In some aspects, the method for treating or preventing a disease or condition caused by or characterized by hypoglycemia or impaired insulin release includes administering to a subject requiring treatment an effective amount of an engineered glucagon-like peptide-1 (eGLP-1) polypeptide having the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO:14 or a combination thereof. In some aspects, the eGLP-1 polypeptide comprises the amino acid sequence of SEQ ID NO:3 to SEQ ID NO:14. In some respects, the eGLP-1 polypeptide comprises or has a sequence of formula III (SEQ ID NO:382) or any of SEQ ID NO:372 to SEQ ID NO:380, or a combination thereof.

[0030] This application includes a method for treating or preventing a disease or condition caused by or characterized by hypoglycemia or impaired insulin release, the method comprising administering to a subject requiring treatment an effective amount of host cells converted to express an engineered glucagon-like peptide-1 (eGLP-1) polypeptide having the amino acid sequence of Formula I (SEQ ID NO:1). In some aspects, this application also includes a method for treating or preventing a disease or condition caused by or characterized by hypoglycemia or impaired insulin release, the method comprising administering to a subject requiring treatment an effective amount of host cells converted to express an engineered glucagon-like peptide-1 (eGLP-1) polypeptide having the amino acid sequences of SEQ ID NO:2 to SEQ ID NO:14 and combinations thereof. In some aspects, the eGLP-1 polypeptide comprises the amino acid sequences of SEQ ID NO:3 to SEQ ID NO:14. In some respects, the eGLP-1 polypeptide comprises the amino acid sequence of formula III (SEQ ID NO:382) or any of SEQ ID NO:372 to SEQ ID NO:380.

[0031] This application further includes and provides: administering a composition comprising an engineered glucagon-like peptide-1 (eGLP-1) polypeptide having the amino acid sequence of Formula I (SEQ ID NO:1) for the treatment or prevention of diseases or conditions caused by or characterized by hypoglycemia or impaired insulin release. In some aspects, this application includes and provides administering a composition comprising an engineered glucagon-like peptide-1 (eGLP-1) polypeptide having the amino acid sequence of Formula I (SEQ ID NO:1) for glycemic control, promoting insulin production, reducing A1c, increasing β-cell mass, promoting weight loss, or reducing overweight. Furthermore, the eGLP-1 polypeptide provided herein can also be used to treat related disorders. In some aspects, administering an eGLP-1 polypeptide having the amino acid sequence cited from SEQ ID NO:14 or a combination thereof for glycemic control, promoting insulin production, reducing A1c, increasing β-cell mass, promoting weight loss, or reducing overweight, or a combination thereof. In some aspects, the application of an eGLP-1 polypeptide having the amino acid sequence of Formula III (SEQ ID NO:382) or any of SEQ ID NO:372 to SEQ ID NO:380, or a combination thereof, is for glycemic control, promoting insulin production, reducing A1c, promoting β-cell mass, promoting weight loss or reducing overweight, or a combination thereof. In one aspect, the composition is a cell expressing an engineered glucagon-like peptide-1 (eGLP-1) polypeptide having the amino acid sequence of Formula I, a polypeptide having the amino acid sequences of SEQ ID NO:2 to SEQ ID NO:14, or a polypeptide having the amino acid sequence of Formula III (SEQ ID NO:382) or any of SEQ ID NO:372 to SEQ ID NO:380, or a combination thereof.

[0032] This application further includes and provides: an administration composition as a direct feeding microorganism (DFM) for the treatment or prevention of conditions caused by or characterized by overweight in patients, and for the treatment of obesity, morbid obesity, obesity-related inflammation, obesity-related gallbladder disease, obesity-induced sleep apnea, metabolic syndrome, prediabetes, 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, said composition comprising an engineered glucagon-like peptide-1 (eGLP-1) polypeptide having the amino acid sequence of Formula I (SEQ ID NO:1), a polypeptide having the amino acid sequences of SEQ ID NO:2 to SEQ ID NO:14, or a polypeptide having the amino acid sequence of Formula III (SEQ ID NO:382) or any of SEQ ID NO:372 to SEQ ID NO:380, or a combination thereof. In some aspects, a composition comprising an engineered glucagon-like peptide-1 (eGLP-1) polypeptide is a direct-feed microbial (DFM) composition comprising bacteria transformed with nucleic acids encoding engineered glucagon-like peptide-1 (eGLP-1) comprising the amino acid sequence of Formula I (SEQ ID NO: 1), having an amino acid sequence from [reference source not found] to [reference source not found], or having an amino acid sequence of Formula III (SEQ ID NO: 382) or any of SEQ ID NO: 372 to SEQ ID NO: 380, or a combination thereof, of a polypeptide. In some aspects, the eGLP-1 polypeptide comprises an amino acid sequence from [reference source not found] to [reference source not found]. In one or more aspects, the composition comprising an engineered glucagon-like peptide-1 (eGLP-1) polypeptide is an engineered composition comprising bacteria that are nucleic acid engineered to secrete eGLP-1 comprising the amino acid sequence of formula I (SEQ ID NO:1), having the amino acid sequences of SEQ ID NO:2 to SEQ ID NO:14, or having the amino acid sequence of formula III (SEQ ID NO:382) or any of SEQ ID NO:372 to SEQ ID NO:380, or a combination thereof. In some aspects, host cells are engineered to secrete eGLP-1 by plasmid-based expression or chromosome integration.

[0033] This application provides and includes, in one form, an engineered polypeptide, particularly an engineered dual agonist polypeptide, comprising the amino acid sequence of formula II (SEQ ID NO: 111): Y X2E GT X6X7S DX 10 SIX13 X 14 DX 16 IAQX 20 AX 22 VQX 25 X 26 IAGGPSS GA PP(I) Where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 = L, K, H, or I; X 16 = K, R, H, or V; X 20 = K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V.

[0034] This application further includes and provides an engineered polypeptide comprising the amino acid sequence of formula II (SEQ ID NO: 111), wherein X2 = V; X6 = P or S; X7 = C or E; X 10 = C or E; X 13 = S;X 14 = L, H, or I; X 16 = H or V; X 20 = H or N; X 22 = A or P; X 25 = P or I; and X 26 = L or V.

[0035] More specifically, this application includes dual agonist polypeptides comprising the amino acid sequence of any of SEQ ID NO:147. In some aspects, this application includes dual agonist polypeptides comprising the amino acid sequence of any of SEQ ID NO:113 to SEQ ID NO:116. In some aspects, this application includes dual agonist polypeptides comprising the amino acid sequence of any of SEQ ID NO:147. In some aspects, this application includes dual agonist polypeptides comprising the amino acid sequence of formula IV (SEQ ID NO:383) or SEQ ID NO:381.

[0036] This application further includes and provides a separated polynucleotide encoding an engineered polypeptide, said engineered polypeptide comprising the amino acid sequence of formula II (SEQ ID NO: 111): Where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 = L, K, H, or I; X 16 = K, R, H, or V; X 20 = K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V.

[0037] This application includes isolated polynucleotides encoding dual agonist polypeptides comprising the amino acid sequence of any of SEQ ID NO:147, any of SEQ ID NO:113 to SEQ ID NO:116, any of SEQ ID NO:147, or formula IV (SEQ ID NO:383) or SEQ ID NO:381.

[0038] This application also includes and provides a nucleic acid expression cassette comprising one or more of the following: a nucleic acid sequence containing a transcription promoter; a nucleic acid sequence encoding a polypeptide, said polypeptide comprising the amino acid sequence of formula II (SEQ ID NO: 111), wherein X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 =L, K, H, or I; X 16 = K, R, H, or V; X 20 = K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V; a nucleic acid sequence containing a translation terminator; and a nucleic acid sequence containing a transcription terminator. In some aspects, the nucleic acid sequence encodes a dual agonist polypeptide containing an amino acid sequence of formula IV (SEQ ID NO:383) or SEQ ID NO:381.

[0039] This application further includes and provides a method for preparing engineered peptides, said engineered peptides comprising the amino acid sequence of formula II (SEQ ID NO: 111): Y X2E GT X6X7S DX 10 SIX 13 X 14 DX 16 IAQX 20 AX 22 V QX 25 X 26 IAGGPSSGAPP(I), where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 = L, K, H, or I; X 16 = K, R, H, or V; X 20 = K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V; the method includes culturing host cells transformed with an expression vector encoding an engineered peptide under conditions that allow expression of the engineered peptide, and recovering the engineered peptide.

[0040] This application further includes and provides a pharmaceutical composition comprising an engineered polypeptide and a carrier, said engineered polypeptide comprising the amino acid sequence of formula II (SEQ ID NO:111).

[0041] This application further includes and provides a pharmaceutical composition comprising a recombinant host cell containing a polynucleotide encoding an engineered polypeptide of formula II (SEQ ID NO: 111).

[0042] This application further includes and provides a host cell containing a polynucleotide encoding an engineered polypeptide, said engineered polypeptide comprising the amino acid sequence of formula II (SEQ ID NO: 111): Y X2E GT X6X7S DX 10 S IX 13 X 14 DX 16 IAQX 20 AX 22 VQX 25 X 26IAGGPSSGAPP(I), where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 = L, K, H, or I; X 16 = K, R, H, or V; X 20 =K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V.

[0043] This application further includes and provides a host cell comprising a vector containing a polynucleotide encoding an engineered polypeptide comprising the amino acid sequence of formula II (SEQ ID NO: 111): Y X2E GT X6X7S DX 10 SIX 13 X 14 DX 16 IAQX 20 AX 22 VQX 25 X 26 IAGGPSSGAPP(I), where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 =L, K, H, or I; X 16 = K, R, H, or V; X 20 = K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V.

[0044] This application further includes and provides a transformed cell comprising a nucleic acid expression cassette, the nucleic acid expression cassette comprising one or more of the following: 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): Y X2E GT X6X7S DX 10 SIX 13 X 14 DX 16 IAQX 20 AX22 VQX 25 X 26 IAGGPSSGAPP(I), where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 = L, K, H, or I; X 16 = K, R, H, or V; X 20 = K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V; nucleic acid sequences containing translation terminators; and nucleic acid sequences containing transcription terminators.

[0045] This application further includes and provides a genetically engineered plant or a portion thereof comprising a recombinant nucleic acid, wherein the recombinant nucleic acid encodes an engineered polypeptide of formula II (SEQ ID NO: 111): Y X2E GT X6X7S DX 10 SIX 13 X 14 DX 16 IA QX 20 AX 22 VQX 25 X 26 IAGGPSSGAPP(I), where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 = L, K, H, or I; X 16 = K, R, H, or V; X 20 = K, R, H, N; X 22 =F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V.

[0046] This application further includes and provides methods for treating or preventing conditions caused by or characterized by overweight in subjects, as well as for treating obesity, morbid obesity, obesity-related inflammation, obesity-related gallbladder disease, obesity-induced sleep apnea, metabolic syndrome, prediabetes, insulin resistance, glucose intolerance, type 2 diabetes, type 1 diabetes, hypertension, atherosclerotic dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke, or microvascular disease, the method comprising administering an effective amount of the engineered peptide, pharmaceutical composition, or cells provided herein to a subject in need of treatment.

[0047] This application further includes and provides methods for treating or preventing diseases or conditions caused by or characterized by hypoglycemia or impaired insulin release, including administering an effective amount of the engineered peptide, pharmaceutical composition, or cells described herein to a subject in need of treatment.

[0048] This application further includes and provides a pharmaceutical composition comprising an engineered polypeptide comprising an amino acid sequence of formula II (SEQ ID NO: 111).

[0049] This application further includes and provides a pharmaceutical composition comprising an engineered polypeptide containing an amino acid sequence selected from any one of SEQ ID NO:112 to SEQ ID NO:148. In some aspects, the pharmaceutical composition comprises a dual agonist polypeptide containing an amino acid sequence selected from any one of SEQ ID NO:113 to SEQ ID NO:116.

[0050] This application further includes and provides direct-feed microorganisms (DFM), which comprise bacteria transformed with nucleic acids encoding engineered polypeptides comprising the amino acid sequence of formula II (SEQ ID NO: 111): Y X2E GT X6X7S DX 10 SIX 13 X 14 DX 16 IAQX 20 AX 22 VQX 25 X 26 IAGGPS SG APP(I), where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 = L, K, H, or I; X 16= K, R, H, or V; X 20 = K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 =L or V. Attached Figure Description

[0051] This invention is disclosed with reference to the accompanying drawings, wherein: Figure 1 This is a diagram showing the proteolytic cleavage sites and enzymes in wild-type GLP-1.

[0052] Figure 2 This diagram illustrates a comparison of the ECD conformations of GLP-1 and GLP-1R upon binding. As shown, the C-terminal region of the GLP-1 agonist interacts with the extracellular domain of the GLP-1R receptor, while the N-terminal region of GLP-1 interacts with the transmembrane domain of the GLP-1R receptor. (Adapted from Cong et al., 2021) Figure 3 a.

[0053] Figure 3 This is a diagram illustrating the rearrangement of the GLP-1R polar network induced by GLP-1 binding. In the activated GLP-1R receptor, the three-layered structure of the polar network is rearranged as follows: (i) central polar network, (ii) HETX motif polar network, and (iii) TM2-6-7-helix 8 polar network. (Adapted from Cong et al., 2021, Figure 4.)

[0054] Figure 4 is a diagram illustrating the superimposed structures of reported peptides binding to GLP-1R (A) and GIPR (B) receptors. (A) shows the superimposed GLP-1R receptors, with different colors representing different complexes formed with the following reported peptides: telpoglutide (7FIM-red), GLP-1 (6X18-pink, 6VCB-blue), Exendin-P5 (6B3J-green), truncated peptide agonist (5NX2-yellow), Exendin-4 (7LLL-cyan), proglucagon (7LLY-white), peptide-20 (7VBH-grey), non-acylated telpoglutide (7VBI-brown), taspoglutide (7KI1-purple), and smegglutide (7KI0-dark blue-green). (B) GIPR complexes and their structural superpositions that combine reported peptides, including non-acylated telpoide (7VAB-red), telpoide (7RBT-blue, 7FIY-grey), GIP (7RA3-white, 7DTY-cyan) and peptide-20 (7FIN-yellow).

[0055] Figure 5 illustrates the cryo-electron microscopy (cryo-EM) structures of hGLP1 and HGIP combined with hGLP-1R. A and C show the cryo-EM structures of the complexes formed by hGLP1 and hGIP combined with hGLP-1R and hGIPR, respectively. B and D show the modeled complexes formed by hGLP1 and hGIP combined with hGLP-1R and hGIPR, respectively.

[0056] Figure 6 Sequence alignment of the cat and human GLP-1R receptors. Boxes represent binding pocket residues and differences in binding pocket residues between cats and humans. Boxes represent conserved binding pocket residues. The binding pocket residues in cGIPR are P91, W92, L145, Y146, and K198 (numbered based on SEQ ID NO:221).

[0057] Figure 7 This is a diagram illustrating the activity of GLP-1 according to one aspect of this specification.

[0058] Figure 8 This is a sequence alignment of reported and engineered GLP-1 analogs.

[0059] Figure 9 shows the sequences of GLP-1 and GLP-1-Gly8 modified to remove the DPP-4 protease cleavage site.

[0060] Figure 10 Cross-species sequence alignment of the GIPR receptor for humans (SEQ ID NO:220), cats (SEQ ID NO:221), rats (SEQ ID NO:222), and mice (SEQ ID NO:223). Asterisks indicate conserved binding pocket residues.

[0061] Figure 11 This is a diagram illustrating GLP-1 activity according to one aspect of this specification. The hGLP-1 peptide (green) docks with the hGLP-1R receptor (yellow) and superimposes onto the selected template 6x18 hGLP-1 (blue) / hGLP-1R (red).

[0062] Figure 12 The diagram shows the docking of telposide (green) with the hGLP-1R receptor (white) and superposition on the selected template 7FIM telposide (blue) / hGLP-1R (red).

[0063] Figure 13 This demonstrates the superposition of a homologous cGIP structure onto an hGIP cryo-electron microscopy structure. Figure 13A shows the hGIP and cGIP sequences (rows 1 and 2), related peptides in the prior art, and the peptides in this application. Figure 13 B showed the 18th position ( Figure 13 A diagram illustrating the differences in single residues (selected in box A). cGIP is shown in red, and hGIP in blue. hGIP residue 18His is shown in yellow, and cGIP 18Arg is shown in green.

[0064] Figure 14: (A) The cGLP-1 (yellow) peptide docks with the cGLP-1R receptor (white) and superimposes onto a template (6x18) selected for the hGLP-1 (green) / hGLP-1R (blue) complex. (B) Different binding pose residues between humans and cats are shown in cGLP-1R (magenta) and hGLP-1R (cyan).

[0065] Figure 15 (A) Residues around AIB2 of telpoeptide (Leu393, Leu397, Glu396 and Thr400) interact with hGLP-1R; (B) Residues around AIB2 (Leu375, Lys374, Ala359 and Glu355) interact with cGLP-1R.

[0066] Figure 16: Conformational analysis of telpoide analogs after docking with cGLP-1R reveals differences in binding conformation. (A, B) Mutations in peptide-4 and 8 (cyan) in telpoide (blue) indicate that the N-terminus of the peptide has lost its helical structure, while in (C) peptide-6 (cyan) is far from the binding region.

[0067] Figure 17 This is a diagram illustrating the GIP activity according to one aspect of this specification.

[0068] Figure 18 This is a graph showing the effect of the test material (Lactobacillus reuteri with various peptides or expressed peptides) on weekly body weight on day 40.

[0069] Figure 19 This study demonstrates the effect of BEP009 on fat content in a mouse DIO study on day 39, as measured by EchoMRI. The solvent was evaluated and compared with liraglutide, BEP009, the LR3632 chassis, and engineered strains BE105ENLR139 (1X GLP1 C1.1 BEP-009 sequence) and BE105ENLR181 (5x GLP1 C1.1 BEP-009 sequence), with liraglutide as a control.

[0070] Figure 20The effects of GLP-1 peptides and constructs (including BEP009-ABD) on weekly body weight were demonstrated on day 82. The solvent was compared with liraglutide, BEP009, LR3632 chassis, and engineered strain BE105ENLR181 (5x GLP1 C1.1 BEP-009 sequence), with liraglutide as a control.

[0071] Figure 21 The effects of the test materials and peptides on weekly blood glucose levels in the DIO model up to day 78 were demonstrated. The solvent was compared with liraglutide, BEP009, BEP-009-ABD fusion, LR3632 chassis, and engineered strain BE105ENLR181 (5x GLP1C1.1 BEP-009 sequence), with liraglutide as a control.

[0072] Figure 22 The effect on fasting blood glucose / AUC was assessed in an OGTT test on day 75. The solvent was compared with liraglutide, BEP009, BEP-009-ABD fusion, LR3632 chassis, and engineered strain BE105ENLR181 (5x GLP1 C1.1BEP-009 sequence), with liraglutide as a control.

[0073] Figure 23 The effect on fat content was assessed using EchoMRI on day 81. The solvent was compared with liraglutide, BEP009, BEP-009-ABD fusion, LR3632 chassis, and engineered strain BE105ENLR181 (5x GLP1 C1.1BEP-009 sequence), with liraglutide as a control.

[0074] Figure 24 The effect on lean body mass was shown on day 81 using EchoMRI. The solvent was compared with liraglutide, BEP009, BEP-009-ABD fusion, LR3632 chassis, and engineered strain BE105ENLR181 (5x GLP1 C1.1 BEP-009 sequence), with the liraglutide group serving as a control. The change in lean body mass (%) is plotted in the figure.

[0075] Figure 25 The effects of the test material and peptides on bi-weekly food intake (standardized to solvent control) are shown on day 84. The percentage change in food intake (days 43 to 48) is plotted in the figure.

[0076] Figure 26 This study showcases GLP-1R cell assays for various eGLP-1 peptides and dual agonist peptides, as well as the eGLP-1-ABD fusion peptide. GLP-1, telposide, and liraglutide were used as positive controls for GLP-1R activation.

[0077] Throughout the views, corresponding reference numerals indicate the relevant parts. The examples listed herein are intended to illustrate several embodiments of the invention, but should not be construed in any way as limiting the scope of the invention. Detailed Implementation

[0078] definition: The term “about” as used in this article refers to ±10%.

[0079] The terms “comprising,” “including,” “having,” and variations thereof mean “including, but not limited to,” “including.” It should be understood that wherever an aspect is described herein with the expression “comprising,” similar aspects expressed as “consisting of” and / or “substantially consisting of” are also provided.

[0080] The term "composed of" means "including and limited to".

[0081] The term "basically composed of" means that a composition, method, or structure may include additional ingredients, steps, and / or portions, provided that such additional ingredients, steps, and / or portions do not substantially alter the basic and novel features of the claimed composition, method, or structure.

[0082] Unless the context clearly specifies otherwise, the singular form used herein includes the plural reference. For example, "a compound" or "at least one compound" can include multiple compounds, including mixtures thereof.

[0083] In this application, various embodiments of this disclosure may be presented in the form of a range. It should be understood that the description in the form of a range is for convenience and brevity only and should not be construed as a rigid limitation on the scope of this disclosure. Therefore, the description of a range should be regarded as having specifically disclosed all possible subranges within that range as well as individual numerical values. For example, when describing a range such as "1 to 6", it should be regarded as having specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, such as: 1, 2, 3, 4, 5, 6. This rule applies regardless of the width of the range.

[0084] In this document, all references to numerical ranges are intended to include any referenced numbers (fractions or integers) within the indicated range. The expressions “between” the first and second indicators and “to” the first indicator are used interchangeably in this document and are intended to include the first and second indicators and all fractions and integers between them.

[0085] As used in this article, the term "method" refers to the manner, means, techniques, and procedures for accomplishing a particular task, including but not limited to those known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry, and medicine, or those that can be easily developed from known manner, means, techniques, and procedures.

[0086] Units, prefixes, and symbols are all in the form accepted by the International System of Units (SI). Numerical ranges include the endpoints that define the range. Unless otherwise stated, amino acid sequences are written from left to right, from the amino group to the carboxyl group. The headings provided herein are not intended to limit any aspect of this disclosure and should be understood in conjunction with the entire specification. Therefore, the complete definitions of the following terms should be found in the full text of the specification.

[0087] As used herein, “treatment” or “management” refers to a method of achieving a beneficial or anticipated outcome. For the purposes of this application, a beneficial or anticipated outcome includes, but is not limited to, relieving symptoms and / or reducing the severity of symptoms and / or preventing the worsening of symptoms associated with the disease or condition. In one aspect, “treatment” or “management” includes one or more of the following: (a) suppressing the disease or condition (e.g., reducing one or more symptoms caused by the disease or condition, and / or reducing the severity of the disease or condition); (b) slowing or halting the progression of one or more symptoms associated with the disease or condition (e.g., stabilizing the disease or condition, delaying the worsening or progression of the disease or condition); and (c) alleviating the disease or condition, for example, by causing the clinical symptoms to subside, improving the disease state, delaying disease progression, improving quality of life, and / or prolonging survival.

[0088] As used herein, “therapeutic effective amount” or “effective amount” means an amount that is capable of effectively evoking the desired biological or medical response, including an amount of a compound sufficient to produce a therapeutic effect on a disease when administered to a subject for the treatment of that disease. Effective amounts can vary depending on the compound, the disease and its severity, and factors such as the age and weight of the subject being treated. Effective amounts can include a range of amounts. As understood in the art, an effective amount can be one or more doses, meaning that a single or multiple administrations may be required to achieve the desired therapeutic endpoint. An effective amount can be considered in the context of administration of one or more therapeutic agents, and a single agent may be considered to be administered at an effective amount if its combination with one or more other agents can or does achieve the desired or beneficial effect. The appropriate dose of any compound administered in combination may optionally be reduced due to the combined effects of the compounds (e.g., additive or synergistic effects).

[0089] As used herein, "fatty acid" refers to a non-branched alkyl acid with a carbon chain length of at least six carbon atoms, such as 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more carbon atoms. Fatty acids may contain one, two, three or more carboxylic acid groups. Fatty acids may also contain other functional groups, such as, but not limited to, amide groups and benzene rings. Exemplary fatty acids include hexanoic acid, octanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, 1,6-adipic acid, 1,8-octanoic acid, 1,10-sebacic acid, 1,12-dodecanoic acid, 1,14-tetradecanoic acid, 1,16-hexadecanoic acid, and 1,18-octadecanoic acid.

[0090] The term "subject" means any subject who requires treatment with the peptides or polypeptides provided herein, particularly mammalian subjects. Mammal subjects include, but are not limited to, humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, dairy cows, apes, monkeys, orangutans, and chimpanzees. In one aspect, the subject is a human subject. In another aspect, the subject is a domesticated animal. In another aspect, the subject is a cat or dog.

[0091] As described herein, “α-methyl-functionalized amino acids” refer to amino acids in which a methyl (CH3) substituent is attached to the first (α) carbon atom. α-methyl-functionalized amino acids encompass any one of the 20 naturally occurring amino acids. As stated throughout, α-methyl-functionalized amino acids can substitute for any naturally occurring amino acid in a peptide. The term “natural” amino acid refers to one of the 20 standard amino acids present in biologically generated proteins.

[0092] peptides This application generally relates to novel engineered glucagon-like peptide-1 (eGLP-1) polypeptides or pharmaceutically acceptable salts, solvates and / or other forms thereof, corresponding pharmaceutical compositions, and methods and / or uses of the eGLP-1 polypeptides for the treatment of diabetes, obesity and other metabolic disorders.

[0093] Specifically, this application relates to compounds of formula (I) or pharmaceutically acceptable salts, solvates, and / or other forms thereof, corresponding pharmaceutical compositions, and methods and / or uses for treating diabetes, obesity, and other metabolic disorders. More specifically, this application relates to polypeptides having the amino acid sequences of SEQ ID NO:1 to SEQ ID NO:14. In some aspects, the eGLP-1 polypeptide comprises the amino acid sequences of SEQ ID NO:3 to SEQ ID NO:14. In some aspects, the eGLP-1 polypeptide comprises the amino acid sequences of formula III (SEQ ID NO:382) or SEQ ID NO:372 to SEQ ID NO:380.

[0094] Table 1: Engineered GLP-1 Sequences

[0095] This application also includes and provides a nucleic acid molecule encoding a polypeptide having an amino acid sequence having an error! No reference source found to SEQ ID NO:14, and a transformed cell having a nucleic acid molecule encoding a polypeptide having an amino acid sequence having SEQ ID NO:2 to SEQ ID NO:14.

[0096] This disclosure provides and includes an engineered glucagon-like peptide-1 (eGLP-1) polypeptide comprising the amino acid sequence of formula I (SEQ ID NO:1): HGEGTSESDVSX 12 X 13 X 14 EGQAAQEX 22 X 23 AX 25 X 26 VDGX 30 (I) Among them, X 12 = S or Q, X 13 = S, Q, or Y, X 14 = I or L, X 22 = V, I, or F, X 23 = V or I, X 25 = V, I, or W, X 26 = I or V, and X 30 = R or S. As used throughout this application, the eGLP-1 polypeptide comprises a sequence of thirty (30) amino acids, wherein subscripts indicate the amino acid sequence from the amino terminus to the carboxyl terminus. As provided herein and will be discussed in detail below, the polypeptide may be concatenated to comprise two, three, four, five or more repeating units of the core dGLP-1 sequence of Formula I. This application also includes and provides for the addition of amino acids at the amino or carboxyl terminus. In some respects, the carboxyl terminus comprises an amide.

[0097] In one respect, the eGLP-1 polypeptide comprises the amino acid sequence of formula I, wherein X 12 = Q, X 13 = S, X 14 = I or L, X 22 = V, I, or F, X 23 = V or I, X 25 = V, I, or W, X 26 = I or V, and X30 = R (SEQ ID NO:1). In one aspect, the eGLP-1 polypeptide comprises the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO:14. In some aspects, the eGLP-1 polypeptide comprises the amino acid sequence of SEQ ID NO:3 to SEQ ID NO:14. In some aspects, the eGLP-1 polypeptide comprises the amino acid sequence of formula III (SEQ ID NO:382) or any one of SEQ ID NO:372 to SEQ ID NO:380.

[0098] As provided in this article, the eGLP-1 peptide is modified to eliminate the protease recognition sites of common proteolytic enzymes such as dipeptidyl peptidase 4 (DPP-IV or DPP-4, Gene ID: 1803), enkephalinase (membrane metalloendopeptidase (MME), Gene ID: 4311, see also Gene ID: 79258), serine protease α-chymotrypsin, trypsin, elastase or pepsin family acidic proteases, thereby exhibiting substantial resistance to proteolytic degradation.

[0099] This application generally relates to novel engineered dual agonist peptides or pharmaceutically acceptable salts, solvates, and / or other forms thereof, corresponding pharmaceutical compositions, and methods and / or uses of said dual agonist peptides for treating diabetes, obesity, and other metabolic disorders. In this document, the terms "dual agonist peptide" and "engineered peptide" are used interchangeably, referring to a peptide of formula II (SEQ ID NO: 111). More specifically, "dual agonist peptide" and "engineered peptide" are peptides of SEQ ID NO: 112 to SEQ ID NO: 147, or polymerized peptides thereof, including peptides of SEQ ID NO: 164 to SEQ ID NO: 207. In some aspects, this application includes dual agonist peptides comprising the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116, or polymerized peptides thereof, including peptides of SEQ ID NO: 164 to SEQ ID NO: 207. Furthermore, it also includes dual agonist peptides comprising the amino acid sequence of formula IV (SEQ ID NO: 383) or SEQ ID NO: 381. Typically, the dual agonist polypeptide is associated with telposide (LY3298176, SEQ ID NO:208), a GIP analog and a single-molecule bifunctional (GIP and GLP-1 receptor) long-acting agonist. The maximum sequence identity between telposide and the dual agonist polypeptide of this application is 95%.

[0100] Specifically, this application relates to compounds of formula (I) or pharmaceutically acceptable salts, solvates and / or other forms thereof, corresponding pharmaceutical compositions, and methods and / or uses for treating diabetes, obesity and other metabolic disorders.

[0101] Y X2E GT X6X7S DX 10 SIX 13 X 14 DX 16 IAQX 20 AX 22 VQWLIAGGPSS GA PP Formula (II) (SEQ ID NO: 111) Where X2 = V or K, X6 = F, P or S, X7 = T, C or E, X 10 = Y, C, or E, X 13 = A, S, Y, N, I, L, R, V or K, X 14 = L, K, H or I, X 16 = K, R, H or V, X 20 = K, R, H, N, X 22 = F, A, P, X 25 = W, P, K, H or I, and X 26 = L or V.

[0102] As used throughout this application, the dual agonist polypeptide comprises a sequence of thirty (37) amino acids, wherein subscripts indicate the amino acid sequence from the amino terminus to the carboxyl terminus. As provided herein and discussed in detail below, the polypeptide may be two, three, four, five or more repeating units tandemly comprising the core dual agonist polypeptide sequence of Formula II. This specification also includes and provides for amino acids added at the amino or carboxyl terminus. In some respects, the carboxyl terminus comprises an amide.

[0103] In some aspects, the dual agonist polypeptide comprises a polypeptide having the amino acid sequence of any one of SEQ ID NO:111 to SEQ ID NO:147. In some aspects, the dual agonist polypeptide comprises a polypeptide having the amino acid sequence of any one of SEQ ID NO:113 to SEQ ID NO:116. The dual agonist polypeptides of this application and their sequences are listed in Table 6. In other aspects, the dual agonist polypeptide comprises a polypeptide having the amino acid sequence of Formula IV (SEQ ID NO:383) or SEQ ID NO:381.

[0104] Table 6: Dual Agonist Peptides

[0105] In some respects, the dual agonist peptide comprises a peptide of formula (II), wherein X2 = V, X6 = S, X7 = C, X 10 = C or E, X 13 = S, X 14 = I, X 16 = V, X 20 = H or N, X 22 = A, X 25 = I, and X 26 = V.

[0106] In addition, the invention includes dual agonist polypeptides that further contain proline at the carboxyl terminus (position 38). On the other hand, the dual agonist polypeptide further contains two additional amino acids at the carboxyl terminus: proline and serine (PS). The presence of these terminal proline and serine residues at the carboxyl terminus is consistent with other therapeutically active dual agonist polypeptides known in the art (e.g., telopeptide (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 some aspects, the dual agonist polypeptide contains the amino acid sequence of any one of SEQ ID NO:123 to SEQ ID NO:147. In other aspects, the dual agonist polypeptide contains a carboxyl-terminal amide.

[0107] In some aspects, the dual agonist polypeptide, pharmaceutically acceptable salt, solvate, or other form, and the corresponding pharmaceutical composition comprise the amino acid sequence of any one of SEQ ID NO:111 to SEQ ID NO:147. In some aspects, the dual agonist polypeptide, pharmaceutically acceptable salt, solvate, or other form, and the corresponding pharmaceutical composition comprise the amino acid sequence of any one of SEQ ID NO:113 to SEQ ID NO:116. In some aspects, the polypeptide, pharmaceutically acceptable salt, solvate, or other form, and the corresponding pharmaceutical composition comprise the amino acid sequence of any one of SEQ ID NO:145 to SEQ ID NO:147. In other aspects, the polypeptide, pharmaceutically acceptable salt, solvate, or other form, and the corresponding pharmaceutical composition comprise the amino acid sequence of any one of SEQ ID NO:142 to SEQ ID NO:144. In still other aspects, the polypeptide, pharmaceutically acceptable salt, solvate, or other form, and the corresponding pharmaceutical composition comprise the amino acid sequence of any one of SEQ ID NO:137 to SEQ ID NO:141. In a further aspect, the polypeptide, pharmaceutically acceptable salt, solvate, or other form, and the corresponding pharmaceutical composition comprise the amino acid sequence of any one of SEQ ID NO:127 to SEQ ID NO:136. In some aspects, the polypeptide, pharmaceutically acceptable salt, solvate, or other form, and the corresponding pharmaceutical composition comprise the amino acid sequence of any one of SEQ ID NO:123 to 127.

[0108] As provided herein, in some respects, dual-agonist peptides are modified to resist proteolytic degradation by means of modifications that eliminate one or more protease recognition sites against common proteolytic enzymes such as dipeptidyl peptidase 4 (DPP-IV or DPP-4, Gene ID: 1803), enkephalinase (membrane metalloendopeptidase (MME), Gene ID: 4311, see also Gene ID: 79258), serine proteases α-chymotrypsin, trypsin, elastase, or the pepsin family of acidic proteases. In some respects, protease-resistant dual-agonist peptides comprise the peptides listed in Tables 10, 11, and 12. In other respects, dual-agonist peptides comprise protease-resistant peptides further modified to improve stability, including the dual-agonist peptides listed in Table 13.

[0109] In this document, the term "substantial resistance" refers to a peptide exhibiting a lower level of degradation compared to wild-type unmodified GLP-1 peptides (wt-GLP-1), or a lower level of degradation compared to telpoeptide. In some respects, eGLP-1 peptides maintain substantially the same receptor selectivity as their corresponding wt-GLP-1 peptides. In some respects, the eGLP-1 peptides provided herein exhibit enhanced receptor potency compared to wt-GLP-1 peptides. eGLP-1 peptides exhibiting "substantial resistance" to proteolytic degradation, for example, retain at least about 50% of their integrity after exposure to the enzyme, for a specific period of time, under conditions where the enzyme is normally active (e.g., suitable pH, temperature, and other environmental conditions). The eGLP-1 peptides provided herein exhibit substantial resistance to proteolytic degradation for time periods of at least 4 hours, at least 8 hours, at least 12 hours, or at least 24 hours. In some respects, these dual-agonist peptides maintain substantially the same receptor selectivity as their corresponding telpoeptide peptides. In some respects, the dual agonist peptides provided herein exhibit enhanced receptor potency compared to telposide peptides. The dual agonist peptides possessing “substantial resistance” to proteolytic degradation, for example, can maintain at least about 50% integrity for a specific period of time after exposure to the enzyme, under conditions where the enzyme is normally active (e.g., suitable pH, temperature, and other environmental conditions). The dual agonist peptides provided herein possess substantial resistance to proteolytic degradation for time periods of at least 4 hours, at least 8 hours, at least 12 hours, or at least 24 hours.

[0110] In some respects, under conditions where the enzyme is normally active, after exposure to the enzyme, at least 60% of the eGLP-1 peptide remains intact for a specific period of time. In other respects, under conditions where the enzyme is normally active, after exposure to the enzyme, the eGLP-1 peptide retains 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% integrity over a period of at least 4 hours, at least 8 hours, at least 12 hours, or at least 24 hours.

[0111] In some respects, under conditions where the enzyme is normally active, after exposure to the enzyme, at least 60% of the dual agonist peptide remains intact for a specific period of time. In other respects, under conditions where the enzyme is normally active, after exposure to the enzyme, the dual agonist peptide retains 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% integrity over a period of at least 4 hours, at least 8 hours, at least 12 hours, or at least 24 hours.

[0112] polymer Peptide multimerization has been shown to provide enhanced activity and better in vivo stability. See Sheard et al., "Peptide Multimerization as Leads for Therapeutic Development," Biologics 2(1):15-33 (2022) 。 As reported by Lin et al., polymerized GLP-1 analogs can be expressed as either bacterial secretory or bacterial surface-type. See Lin et al., “Oral Delivery of Pentameric Glucagon-Like Peptide-1 by Recombinant Lactobacillus in Diabetic Rats,” PLoS ONE 11(9):e0162733 (2016). Polymers containing terminal trypsin-sensitive amino acids can be digested by intestinal trypsin to produce active monomeric GLP-1 analogs. Ibid.

[0113] This application also provides and includes multimers of the eGLP-1 peptides provided herein. The multimers comprise multiple copies of the eGLP-1 peptide, up to a maximum of five copies (e.g., 5-fold copies). Multimers of the eGLP-1 peptides in Table 2 are listed in SEQ ID NO:32 to SEQ ID NO:67. Also included are the linker-separated multimers of the eGLP-1 peptides provided herein, as discussed below. The multimers of the eGLP-1 peptides can be expressed in host cells using appropriate nucleic acid vectors employing methods known in the art. In some aspects, the multimerized eGLP-1 peptides can be purified from host cells for therapeutic purposes. In other aspects, the multimerized eGLP-1 peptides can be provided as oral compositions expressing transgenic host cells of the multimers, as described by Lin et al. Notably, the eGLP-1 peptides of SEQ ID NO:32 to SEQ ID NO:67 contain a trypsin-sensitive site at the carboxyl terminus of each inner peptide of their multimers. Therefore, it is believed that upon exposure to trypsin, the eGLP-1 polypeptide monomers of SEQ ID NO:3 to SEQ ID NO:382 are released. In some aspects thereof, multimers of the eGLP-1 polypeptide of Formula III (SEQ ID NO:382) or any of SEQ ID NO:372 to SEQ ID NO:380 are also conceived and provided.

[0114] This application also provides and includes multimers of the dual agonist peptides provided herein. The multimers comprise multiple copies of the dual agonist peptide, up to a maximum of five copies (e.g., 5-fold copies). Multimers of the dual agonist peptides in Table 6 are shown in SEQ ID NO:148 to SEQ ID NO:207. In some aspects, multimers of the dual agonist peptides in Table 6 are shown in SEQ ID NO:164 to SEQ ID NO:167. Also included are multimers of dual agonist peptides separated by linkers, such as those discussed below. The multimers of the dual agonist peptides can be expressed in host cells using methods known in the art, utilizing appropriate nucleic acid vectors. In some aspects, the multimerized dual agonist peptides can be purified from host cells for therapeutic purposes. In other aspects, the multimerized dual agonist peptides can be provided as oral compositions of transgenic host cells expressing the multimers, as described by Lin et al. Notably, the dual agonist peptides of SEQ ID NO:148 to SEQ ID NO:207 contain a trypsin-sensitive site at the carboxyl terminus of each internal peptide. Therefore, it is believed that upon exposure to trypsin, a dual agonist polypeptide monomer containing the amino acid sequences of SEQ ID NO:111 to SEQ ID NO:147 is released.

[0115] Connectors and Fusion Some of the eGLP-1 peptides described herein are fusion proteins comprising the amino acid sequence of the eGLP-1 peptide according to Formula I or any of SEQ ID NO:3 to SEQ ID NO:14, and the multimeric eGLP-1 peptide of SEQ ID NO:32 to SEQ ID NO:67, and one or more additional domains or amino acid sequences. In some respects, the additional domains or amino acid sequences include, but are not limited to, one or more of a linker, an expression peptide tag for purification, a hinge, or an Fc domain. Linkers, peptide tags, hinges, and Fc domains are well known to those skilled in the art and can be incorporated into the eGLP-1 peptides described herein, and their potency, activity, and efficacy in treating hypoglycemic conditions (e.g., type 2 diabetes) can be tested according to the methods provided herein without extensive experimental procedures. As described herein, linkers, peptide tags, hinges, and Fc linkers can be added to the N-terminus or C-terminus of the eGLP-1 peptide of this application, or both.

[0116] In some respects, the eGLP-1 peptide can be directly or via a linker sequence (including one or more linkers or linker sequences described herein) linked to an additional domain or peptide sequence. In some respects, a dual agonist peptide can be directly or via a linker sequence (including one or more linkers or linker sequences described herein) linked to an additional domain or peptide sequence. In some respects, the additional domain or peptide sequence can function or is capable of: stabilizing, prolonging half-life, providing protection (e.g., preventing degradation, processing, or clearance), or directing the engineered peptide to a specific location, cell type, tissue, organ, or site in the body, for example, by binding to a receptor or ligand. In some respects, the additional domain or peptide sequence may be an albumin-binding domain. Examples of peptide-ABD fusion peptides are provided, described, and evaluated in the embodiments herein. Furthermore, or in another respect, transferrin receptor antibodies or peptides can be linked to the fusion peptide. This addition is intended to improve brain bioavailability and can be achieved via flexible linkers, etc., as described herein. Transferrin receptors are also present in the gut and mediate or promote the systemic transport of intestinal secretory peptides, as described by Rue et al. (Rue, L et al. (2023) Pharmaceutics 15, 1748) and Meister et al. (Meister SW (2020) Int J Mol Sci 21, 2999).

[0117] Some of the dual agonist peptides described herein are fusion proteins comprising the amino acid sequence of a dual agonist peptide according to Formula II (SEQ ID NO: 111) or any of SEQ ID NO: 112 to SEQ ID NO: 147, and one or more additional domains or amino acid sequences. In some aspects, the fusion protein comprises a multimer of a dual agonist peptide according to SEQ ID NO: 113 to SEQ ID NO: 116 or SEQ ID NO: 164 to SEQ ID NO: 167. In some aspects, the additional domains or amino acid sequences include, but are not limited to, a linker, an expression peptide tag for purification, a hinge, or one or more of an Fc domain. Linkers, peptide tags, hinges, and Fc domains are well known to those skilled in the art and can be incorporated into the dual agonist peptides described herein, and their potency, activity, and efficacy in treating hypoglycemic conditions (e.g., type 2 diabetes) can be tested according to the methods provided herein without extensive experimental procedures. As provided herein, linkers, peptide tags, hinges, and Fc linkers can be added to the N-terminus or C-terminus of the dual agonist peptides of this application, or both.

[0118] Linkers used in the various eGLP-1 peptides or dual agonist peptides described herein can facilitate the formation of desired structures. In some respects, peptide linkers may contain 1-50 amino acids, 1-25 amino acids, 25-50 amino acids, or 30-50 amino acids. Generally, longer linkers are associated with higher activity (greater flexibility) but may also reduce stability due to greater peptide exposure. Linkers may contain, for example, (Gly-Ser)n residues, where n is at least 1 and at most, for example, an integer such as 4, 5, 6, 10, or higher, and optionally, some Glu or Lys residues may be dispersed within the linker to improve solubility. Alternatively, some linkers may not contain any serine residues, for example, when the linker requires O-linked glycosylation.

[0119] Suitable linker examples include the GS linker. Because the GS linker does not share significant homology with known proteins, it is considered biologically inert and unlikely to produce novel morphological effects or significant antigenicity. The length and amino acid sequence of the linker can be easily selected and optimized.

[0120] In some respects, the connector contains (GGGGS)n, where n is 1, 2, 3, or 4. For example, certain specific linkers have the following amino acid sequences: 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), GGGGGS GGGGSA (SEQ ID NO:74), GGGGGS GGGGS 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.

[0121] In some respects, the connector contains (GGSS)n (SEQ ID NO:224), where n is 1, 2, 3, 4 to 10. For example, some specific linkers have the following amino acid sequences: 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 GGSSGGSS 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 GGSSGGSS GGSS GGSS (SEQ ID NO:231), GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS (SEQ ID NO:231) NO:232), GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS (SEQ ID NO:233). As described herein, this linker can be linked via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:111 to SEQ ID NO:147. In some aspects, this linker can be linked via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:113 to SEQ ID NO:116. In some aspects, this linker can be linked via a peptide bond to the amino or carboxyl terminus of the amino acid sequences in SEQ ID NO:127 to SEQ ID NO:147.

[0122] In some respects, the connector contains GSGGS (SEQ ID NO:234), where n is 1, 2, 3, 4 to 10. For example, some specific linkers have the following amino acid sequences: GSGGS GSGGS (SEQ ID NO:235), GSGGS GSGGS GSGGS (SEQ ID NO:236), GSGGS GSGGS GSGGS GSGGS (SEQ ID NO:237), GSGGS GSGGS GSGGS GSGGSGSGGS (SEQ ID NO:238), GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS (SEQ ID NO: 239), GSGGSGSGGS GSGGS GSGGS GSGGS GSGGS GSGGS (SEQ ID NO: 240), GSGGS GSGGS GSGGS GSGGSGSGGS GSGGS GSGGS GSGGS (SEQ ID NO: 241), GSGGS GSGGS GSGGS GSGGS GSGGS GSGGSGSGGS GSGGS GSGGS (SEQ ID NO: 240) NO:242), or GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGSGSGGS GSGGS GSGGS (SEQ ID NO:243). As described herein, this linker can be linked via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:111 to SEQ ID NO:147. In some aspects, this linker can be linked via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:113 to SEQ ID NO:116. In some aspects, this linker can be linked via a peptide bond to the amino or carboxyl terminus of the amino acid sequences in SEQ ID NO:127 to SEQ ID NO:147.

[0123] In some respects, the connector contains GGGS (SEQ ID NO:244), where n is 1, 2, 3, 4 to 10. For example, some specific linkers have the following amino acid sequences: 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:248) NO:249), GGGS GGGS GGGS GGGS GGGSGGGS 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 GGGSGGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS (SEQ ID NO:253). As described herein, this linker can be linked via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:111 to SEQ ID NO:147. In some aspects, this linker can be linked via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:113 to SEQ ID NO:116. In some aspects, this linker can be linked via a peptide bond to the amino or carboxyl terminus of the amino acid sequences in SEQ ID NO:127 to SEQ ID NO:147.

[0124] In some respects, the connector contains GGSG (SEQ ID NO:254), where n is 1, 2, 3, 4 to 10. For example, some specific linkers have the following amino acid sequences: 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:258) NO:259), GGSG GGSG GGSG GGSG GGSGGGSG 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 GGSGGGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG (SEQ ID NO:263). As described herein, this linker can be linked via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:111 to SEQ ID NO:147. In some aspects, this linker can be linked via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:113 to SEQ ID NO:116. In some aspects, this linker can be linked via a peptide bond to the amino or carboxyl terminus of the amino acid sequences in SEQ ID NO:127 to SEQ ID NO:147.

[0125] In some respects, the connector contains GGSGG (SEQ ID NO:264), where n is 1, 2, 3, 4 to 10. For example, some specific linkers have the following amino acid sequences: GGSGG GGSGG (SEQ ID NO:265), GGSGG GGSGG GGSGG (SEQ ID NO:266), GGSGG GGSGG GGSGG GGSGG (SEQ ID NO:267), GGSGG GGSGG GGSGG GGSGGGGSGG (SEQ ID NO:268), GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG (SEQ ID NO:269), GGSGGGGSGG GGSGG GGSGG GGSGG GGSGG GGSGG (SEQ ID NO:270), GGSGG GGSGG GGSGG GGSGGGGSGG GGSGG GGSGG GGSGG (SEQ ID NO:271), GGSGG GGSGG GGSGG GGSGG GGSGG GGSGGGGSGG GGSGG GGSGG (SEQ ID NO:272), or GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG (SEQ ID NO:273). As described herein, the linker can be attached via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:111 to SEQ ID NO:147. In some aspects, the linker can be attached via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:113 to SEQ ID NO:116. In some aspects, the linker can be attached via a peptide bond to the amino or carboxyl terminus of the amino acid sequences in SEQ ID NO:127 to SEQ ID NO:147.

[0126] In some respects, the connector contains GGSSG (SEQ ID NO:274), where n is 1, 2, 3, 4 to 10. For example, some specific linkers have the following amino acid sequences: GGSSG GSGSG (SEQ ID NO: 275), GGSSG GSGSG GSGSG (SEQ ID NO: 276), GGSSG GSGSG GSGSG GSGSG (SEQ ID NO: 277), GGSSG GSGSG GSGSG GSGSGGSGSG (SEQ ID NO: 278), GGSSG GSGSG GSGSG GSGSG GSGSG GSGSG (SEQ ID NO: 279), GSGSGGSGSG GSGSG GSGSG GSGSG GSGSG GSGSG (SEQ ID NO: 280), GGSSG GSGSG GSGSG GSGSGGSGSG GSGSG GSGSG GSGSG (SEQ ID NO: 281), GGSSG GSGSG GSGSG GSGSG GSGSG GSGSGGSGSG GSGSG GSGSG (SEQ ID NO: 280) NO:282), or GGSSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSGGSGSG GSGSG GSGSG (SEQ ID NO:283). As described herein, this linker can be attached via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:111 to SEQ ID NO:147. In some aspects, this linker can be attached via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:113 to SEQ ID NO:116. In some aspects, this linker can be attached via a peptide bond to the amino or carboxyl terminus of the amino acid sequences in SEQ ID NO:127 to SEQ ID NO:147.

[0127] In some respects, the connector contains GSGGG (SEQ ID NO:284), where n is 1, 2, 3, 4 to 10. For example, some specific linkers have the following amino acid sequences: GSGGG GSGGG (SEQ ID NO:285), GSGGG GSGGG GSGGG (SEQ ID NO:286), GSGGG GSGGG GSGGG GSGGG (SEQ ID NO:287), GSGGG GSGGG GSGGG GSGGGGSGGG (SEQ ID NO:288), GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG (SEQ ID NO: 289), GSGGGGSGGG GSGGG GSGGG GSGGG GSGGG GSGGG (SEQ ID NO: 290), GSGGG GSGGG GSGGG GSGGGGSGGG GSGGG GSGGG GSGGG (SEQ ID NO: 291), GSGGG GSGGG GSGGG GSGGG GSGGG GSGGGGSGGG GSGGG GSGGG (SEQ ID NO:292), or GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGGGG (SEQ ID NO:293). As described herein, the linker can be attached via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:111 to SEQ ID NO:147. In some aspects, the linker can be attached via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:113 to SEQ ID NO:116.

[0128] In some respects, the connector contains GGGSG (SEQ ID NO:294), where n is 1, 2, 3, 4 to 10. For example, some specific linkers have the following amino acid sequences: GGGSG GGGSG (SEQ ID NO:295), GGGSG GGGSG GGGSG (SEQ ID NO:296), GGGSG GGGSG GGGSG GGGSG (SEQ ID NO:297), GGGSG GGGSG GGGSG GGGSGGGGSG (SEQ ID NO:298), GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG (SEQ ID NO:299), GGGSGGGGSG GGGSG GGGSG GGGSG GGGSG GGGSG (SEQ ID NO:300), GGGSG GGGSG GGGSG GGGSGGGGSG GGGSG GGGSG GGGSG (SEQ ID NO:301), GGGSG GGGSG GGGSG GGGSG GGGSG GGGSGGGGSG GGGSG GGGSG (SEQ ID NO:302), or GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG (SEQ ID NO:303). As described herein, the linker can be attached via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:111 to SEQ ID NO:147. In some aspects, the linker can be attached via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:113 to SEQ ID NO:116. In some aspects, the linker can be attached via a peptide bond to the amino or carboxyl terminus of the amino acid sequences in SEQ ID NO:127 to SEQ ID NO:147.

[0129] In some respects, the connector contains GSSSG (SEQ ID NO:304), where n is 1, 2, 3, 4 to 10. For example, some specific linkers have the following amino acid sequences: GSSSG GSSSG (SEQ ID NO:305), GSSSG GSSSG GSSSG (SEQ ID NO:306), GSSSG GSSSG GSSSG GSSSG (SEQ ID NO:307), GSSSG GSSSG GSSSGGSSSG 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 GSSSGGSSSG GSSSG GSSSG GSSSG GSSSG GSSSG (SEQ ID NO:311), GSSSG GSSSG GSSSG GSSSGGSSSG GSSSG GSSSG GSSSG GSSSG (SEQ ID NO:311) NO:312), or GSSSG GSSSG GSSSG GSSSG GSSSGGSSSG GSSSG GSSSG GSSSG GSSSG (SEQ ID NO:313). As described herein, this linker can be attached via a peptide bond to the amino or carboxyl terminus of any amino acid sequence of any of SEQ ID NO:111 to SEQ ID NO:147. In some aspects, the linker can be attached via a peptide bond to the amino or carboxyl terminus of any amino acid sequence of any of SEQ ID NO:113 to SEQ ID NO:116. In some aspects, the linker can be attached via a peptide bond to the amino or carboxyl terminus of the amino acid sequences of SEQ ID NO:127 to SEQ ID NO:147.

[0130] In some respects, the connector contains GSSSS (SEQ ID NO:314), where n is 1, 2, 3, 4 to 10. For example, some specific linkers have the following amino acid sequences: GSSSS GSSSS (SEQ ID NO:315), GSSSS GSSSS GSSSS (SEQ ID NO:316), GSSSS GSSSS GSSSS GSSSS (SEQ ID NO:317), GSSSS GSSSS GSSSS GSSSSGSSSS (SEQ ID NO:318), GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS (SEQ ID NO: 319), GSSSSGSSSS GSSSS GSSSS GSSSS GSSSS GSSSS (SEQ ID NO: 320), GSSSS GSSSS GSSSS GSSSSGSSSS GSSSS GSSSS GSSSS (SEQ ID NO: 321), GSSSS GSSSS GSSSS GSSSS GSSSS GSSSSGSSSS GSSSS GSSSS (SEQ ID NO: 321) NO:322), or GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSSGSSSS GSSSS GSSSS (SEQ (SEQ ID NO:323). As described herein, the linker can be linked via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:111 to SEQ ID NO:147. In some aspects, the linker can be linked via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:113 to SEQ ID NO:116. In some aspects, the linker can be linked via a peptide bond to the amino or carboxyl terminus of the amino acid sequences in SEQ ID NO:127 to SEQ ID NO:147.

[0131] In some respects, the connector contains GGGGS (SEQ ID NO:324), where n is 1, 2, 3, 4 to 10. For example, some specific linker amino acid sequences: 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:328) ID NO:329), GGGGSGGGGS GGGGS GGGGS GGGGS GGGGS GGGGS (SEQ ID NO:330), GGGGS GGGGS GGGGS GGGGSGGGGS GGGGS GGGGS GGGGS (SEQ ID NO:331), or GGGGS GGGGS GGGGS GGGGS GGGGS GGGGSGGGGS GGGGS GGGGS (SEQ ID NO:332). As described herein, this linker can be attached via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:111 to SEQ ID NO:147. In some aspects, this linker can be attached via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:113 to SEQ ID NO:116. In some aspects, this linker can be attached via a peptide bond to the amino or carboxyl terminus of the amino acid sequences in SEQ ID NO:127 to SEQ ID NO:147.

[0132] In some respects, the connector contains AAAGG (SEQ ID NO:333), where n is 1, 2, 3, 4 to 10. For example, some specific linkers have the following amino acid sequences: AAAGG AAAGG (SEQ ID NO:334), AAAGG AAAGG AAAGG (SEQ ID NO:335), AAAGG AAAGG AAAGG AAAGG (SEQ ID NO:336), AAAGG AAAGG AAAGG AAAGGAAAGG (SEQ ID NO:337), AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG (SEQ ID NO:337) NO:338), AAAGGAAAGG AAAGG AAAGG AAAGG AAAGG AAAGG (SEQ ID NO:339), AAAGG AAAGG AAAGG AAAGGAAAGG AAAGG AAAGG AAAGG (SEQ ID NO:340), AAAGG AAAGG AAAGG AAAGG AAAGG AAAGGAAAGG AAAGG AAAGG (SEQ ID NO:339) (SEQ ID NO:341), or ARGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG (SEQ ID NO:342). As described herein, the linker can be attached via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:111 to SEQ ID NO:147. In some aspects, the linker can be attached via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:113 to SEQ ID NO:116.

[0133] In some respects, the connector contains GGSAAAGG (SEQ ID NO:343), where n is 1, 2, 3, 4 to 10. For example, some specific linkers have the following amino acid sequences: GGSAAAGG GGSAAAGG (SEQ ID NO: 344), GGSAAAGGGGSAAAGG GGSAAAGG (SEQ ID NO: 345), GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG (SEQ ID NO: 346), GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG (SEQ ID NO: 346) NO:347), GGSAAAGGGGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG (SEQ ID NO:348), GGSAAAGG GGSAAAGGGGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG (SEQ ID NO:349), GGSAAAGG GGSAAAGGGGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG (SEQ ID NO:347) (SEQ ID NO:350), GGSAAAGGGGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG (SEQ ID NO:351), or GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG (SEQ ID NO:352). As described herein, the linker can be attached via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:111 to SEQ ID NO:147. In some aspects, the linker can be attached via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:113 to SEQ ID NO:116. In some aspects, the linker can be attached via a peptide bond to the amino or carboxyl terminus of the amino acid sequences in SEQ ID NO:127 to SEQ ID NO:147.

[0134] In some respects, eGLP-1 peptides or dual agonist peptides may 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), and isopeptag (TDKDMTITFTNKKDAE, SEQ ID NO:89). NO:89), Myc (EQKLISEEDL, SEQ ID NO:90), NE-Tag (TKENPRSNQEESYDDNES, SEQ ID NO:91), PolyGlutamate-tag (EEEEEEE, SEQ ID NO:92), Poly Arginine-tag (RRRRRRRR, SEQ ID NO:93), Rho1D4-tag (TETSQVAPA, SEQ ID NO:94), SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP, SEQ ID NO:95), Sdytag (DPIVMIDNDKPIT, SEQ ID NO:96), SH3 (STVPVAPPRRRRRG, SEQ ID NO:97), Snooptag (KLGDIEFIKVNK, SEQ ID NO:98), Softag 1 (SLAELLNAGLGGS, SEQ IDNO:99), Softag 3 (TQDPSRVG, SEQ ID NO:100), Spot-tag (PDRVRAVSHWSS, SEQ ID NO:101), Spytag (AHIVMVDAYKPTK, SEQ ID NO:102), S-tag (KETAAAKFERQHMDS,The following tags were used: SEQ ID NO:103, Strep-tag (WSHPQFEK, SEQ ID NO:104), T7tag (MASMTGGQQMG, SEQ ID NO:105), TC-tag (EVHTNQDPLD, SEQ ID NO:106), Ty-tag (CCPGCC, SEQ ID NO:107), VSV-tag (YTDIEMNRLGK, SEQ ID NO:108), Xpress-tag (DLYDDDDK, SEQ ID NO:109), and HiBit (VSGWRLFKKIS, SEQ ID NO:110). The nucleic acid sequence encoding the eGLP-1 peptide or dual agonist peptide was fused within the frame to the peptide tag, thereby incorporating the peptide tag into the amino or carboxyl terminus of the eGLP-1 peptide or dual agonist peptide.

[0135] As provided herein, the tag can be attached via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:111 to SEQ ID NO:147. In some aspects, the tag can be attached via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:114 to SEQ ID NO:116. In some aspects, the tag can be attached via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:127 to SEQ ID NO:147. The tag can be attached via a peptide bond to the amino or carboxyl terminus of any of the amino acid sequences in SEQ ID NO:2 to SEQ ID NO:14, or any of SEQ ID NO:32 to SEQ ID NO:67, or of Formula I (SEQ ID NO:1), or of Formula III (SEQ ID NO:382), or of any of SEQ ID NO:372 to SEQ ID NO:380. In other aspects, the tag can be attached via a peptide bond to the linker provided above, and then to the amino or carboxyl terminus of the dual agonist polypeptide provided herein.

[0136] Importantly, the effects of adding linkers, peptide tags, hinges, and Fc linkers can be evaluated using a three-dimensional model of the GLP-1R receptor structural molecular docking method described below.

[0137] Modification This application also includes and provides esterified eGLP-1 peptides or esterified dual agonist peptides. Compared with corresponding non-esterified eGLP-1 peptides or esterified dual agonist peptides, the esterified eGLP-1 peptides or esterified dual agonist peptides may exhibit enhanced properties, such as a longer in vivo half-life.

[0138] "Lipidification" refers to the process of directly or indirectly covalently linking one or more fatty acids or polyethylene glycols to the eGLP-1 peptide or dual agonist peptide described herein. In some respects, the lipid moiety is covalently linked via an amino- or carboxyl-terminus. In other respects, the lipid moiety is covalently linked via internal amino acids such as arginine, glutamine, aspartic acid, glutamic acid, tyrosine, histidine, threonine, and serine.

[0139] Esterified eGLP-1 peptides or dual agonist peptides are referred to as esterified peptides. The covalent linkage process can convert a carboxylic acid to another functional group, such as a secondary amide, or it can occur on another functional group present on the fatty acid, while retaining the carboxylic acid present in the original fatty acid. The covalent linkage of one or more fatty acids can be directly linked to the eGLP-1 peptide or dual agonist peptide provided herein, or indirectly linked via a divalent linker motif located between the one or more fatty acids and the eGLP-1 peptide or dual agonist peptide. The divalent linker motif can contain one or more amino acids, polyethylene glycol (PEG), or combinations thereof. The PEG-containing linker motif may further contain other functional groups, such as amides, depending on the needs of covalent linkage. The linker motif containing one or more amino acids can be linked via a C-terminus, N-terminus, side chain, or any combination thereof.

[0140] "Polyethylene glycol" or "PEG" is a polyether monovalent group with the general formula -(O-CH2-CH2). n -OH; or a divalent polyether group, with the general formula -(O-CH2-CH2). n -O-, where n is an integer greater than 1. When PEG is followed by a number, it indicates the number of repeating units in that part. For example, PEG3 can correspond to the divalent group of the formula -(O-CH2-CH2)3-O-, while PEG8 can correspond to the monovalent group of the formula -(O-CH2-CH2)8-OH.

[0141] PEG is prepared by polymerization of ethylene oxide, and commercially available products have molecular weights ranging from 300 Daltons (Da) to 10,000,000 Da. Low molecular weight PEG is typically provided as pure oligomers, referred to as monodisperse, homogeneous, or discrete oligomers. These oligomers are used in certain aspects of this application. In some aspects, the PEG is PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12, PEG18, or PEG24. In other aspects, the PEG is PEG2, PEG6, or PEG24.

[0142] Polynucleotides This specification provides and includes nucleic acids encoding an eGLP-1 polypeptide of Formula I. In some aspects, the eGLP-1 polypeptide encoded by the nucleic acid comprises the amino acid sequence of any one of SEQ ID NO:1 to SEQ ID NO:14. In some aspects, the eGLP-1 polypeptide encoded by the nucleic acid comprises the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO:14. In some aspects, the eGLP-1 polypeptide encoded by the nucleic acid comprises the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO:14, or Formula III, or SEQ ID NO:382, or any one of SEQ ID NO:372 to SEQ ID NO:380.

[0143] This specification provides and includes nucleic acids encoding a dual agonist polypeptide of formula II (SEQ ID NO: 111). In some aspects, the dual agonist polypeptide encoded by the nucleic acid comprises the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147. In some aspects, the dual agonist polypeptide encoded by the nucleic acid comprises the amino acid sequence of any one of SEQ ID NO: 113 to SEQ ID NO: 116. In some aspects, the dual agonist polypeptide encoded by the nucleic acid comprises the amino acid sequence of any one of formula IV (SEQ ID NO: 383) or SEQ ID NO: 381.

[0144] The term “polynucleotide” or “nucleotide” as used in this article is intended to cover both single nucleic acids and multiple nucleic acids, referring to isolated nucleic acid molecules or constructs such as messenger RNA (mRNA) or plasmid DNA (pDNA).

[0145] The term "nucleic acid" refers to any one or more nucleic acid fragments, such as DNA or RNA fragments, present in a polynucleotide. When used to describe nucleic acids or polynucleotides, the term "isolated" means a nucleic acid molecule, DNA, or RNA that has been isolated from its natural environment. For example, for the purposes of this disclosure, a recombinant polynucleotide encoding an eGLP-1 polypeptide or a dual agonist polypeptide contained in a vector is considered isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in a heterologous host cell or recombinant polynucleotides purified (partially or substantially purified) from other polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of this disclosure. According to this disclosure, isolated polynucleotides or nucleic acids also include synthetically produced such molecules. Furthermore, polynucleotides or nucleic acids may contain regulatory elements, such as promoters, enhancers, ribosome binding sites, or transcription termination signals.

[0146] The term "vector" refers to a construct capable of delivering one or more target genes or sequences into a host cell and expressing one or more genes or sequences in certain respects within the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors bound to cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells. This application also includes and provides eukaryotic viral vectors. Bacterial and eukaryotic viral vectors provide simplified delivery of eGLP-1 peptides or dual-agonist peptide sequences into host cells.

[0147] In this article, the term "host cell" refers to a cell or population of cells that carries or is capable of carrying recombinant nucleic acids encoding an eGLP-1 peptide or a dual agonist peptide. Host cells can be prokaryotic cells (e.g., *Escherichia coli*) or eukaryotic cells, such as fungal cells (e.g., yeast cells, such as *Saccharomyces cerevisiae*). Saccharomyces cerivisiae Pichia pastoris () Pichia pastoris ) or millet wine schizophytes ( Schizosaccharomyces pombe ( ), and various animal cells, such as insect cells (e.g., Sf-9) or mammalian cells (e.g., HEK293F, CHO, COS 7, NIH-3T3), and plant cells. See Chunfeng et al., “Expression of cholera toxin B-lumbrokinase fusion protein in Pichia pastoris--the use of transmucosal carriers in the delivery of therapeutic proteins to protect rats against thrombosis,” Appl. Biochem. Biotechnol 169(2):636-50(2013).

[0148] The term “composition” or “pharmaceutical composition” refers to a composition containing the eGLP-1 peptide or dual agonist peptide provided herein, and, for example, a pharmaceutically acceptable carrier, excipient, or diluent, for administration to a subject in need of treatment (e.g., a mammalian subject receiving treatment for a hypoglycemic condition such as type 2 diabetes).

[0149] The term "pharmaceutically acceptable" means a composition that, within reasonable medical judgment, is suitable for contact with tissues of humans and other mammals without causing excessive toxicity or other complications, and has a reasonable benefit / risk ratio.

[0150] "Effective dose" refers to the amount of a peptide containing an eGLP-1 peptide or a dual agonist peptide, as described herein, administered to a subject as a single dose or as part of a series of doses, which is effective in treating, for example, type 2 diabetes. For example, an effective dose is defined as one or more of the following outcomes following administration: prevention or regulation of hyperglycemia, promotion of insulin synthesis, increase in β-cell mass, reduction or maintenance of weight (e.g., prevention of weight gain), reduction of food intake, regulation of gastric acid secretion, or regulation of gastric emptying. This dose may be a fixed dose for all subjects or may vary depending on the subject's weight, health status and physical condition, required level of glycemic control, peptide formulation, professional assessment of the medical condition, and other relevant factors.

[0151] The term "subject" refers to any subject who requires treatment with the eGLP-1 peptide or dual agonist peptide provided herein, particularly mammalian subjects. Mammal subjects include, but are not limited to, humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, dairy cows, apes, monkeys, orangutans, chimpanzees, and other non-human primates. In one respect, a subject is a human subject. In another respect, a subject is a cat.

[0152] As used herein, the term "subject in need" refers to an individual who requires treatment, such as a subject diagnosed with a metabolic disease or disorder. In some respects, this subject may have a hypoglycemic condition or be susceptible to hypoglycemia, such as type 2 diabetes. Subjects in need of treatment include those with metabolic diseases or disorders that can be alleviated through measures such as: controlling food intake, weight loss, energy metabolism, blood glucose levels, insulin levels and / or insulin secretion, positive inotropic effects, reduction of catabolism, delayed gastric emptying, obesity, diabetes and diabetes-related conditions, and hepatic steatosis-related inflammation and damage. Such conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, insulin resistance and related disorders such as polycystic ovary syndrome, obesity, and any type of diabetes (including type 1 diabetes, type 2 diabetes, and gestational diabetes).

[0153] Preparation method This specification provides a method for preparing an eGLP-1 peptide or dual agonist peptide by any suitable method. For example, the eGLP-1 peptide or dual agonist peptide provided herein can be recombinantly produced using convenient vector / host cell combinations well known to those skilled in the art. Several methods are currently available for the recombinant production of eGLP-1 peptides or dual agonist peptides. Typically, a polynucleotide sequence encoding the eGLP-1 peptide or dual agonist peptide is inserted into a suitable expression vector, for example, a vector containing the elements required for transcription and translation insertion. The nucleic acid encoding the eGLP-1 peptide or dual agonist peptide is inserted into the vector at the correct reading frame. The expression vector is then transfected into a suitable host cell that will express the eGLP-1 peptide or dual agonist peptide. Suitable host cells include, but are not limited to, bacterial, yeast, or mammalian cells. Various commercially available host expression vector systems can be used to express the eGLP-1 peptide or dual agonist peptide described herein.

[0154] Recombinant expression of the eGLP-1 peptide or dual agonist peptide described herein can be achieved by constructing an expression vector containing a polynucleotide encoding the peptide disclosed herein. Once the polynucleotide encoding the eGLP-1 peptide or dual agonist peptide is obtained, a vector for producing the peptide can be prepared using recombinant DNA techniques well known in the art.

[0155] As will be recognized, due to codon degeneracy, the nucleic acid sequences encoding eGLP-1 peptides or dual agonist peptides may differ. Since amino acids can be encoded by different codons, the same amino acid can be transported to the ribosome by several different tRNAs. However, significant biases exist in the use of synonymous codons in both prokaryotes and eukaryotes, including biases between codons recognized by the same transfer RNA and biases between codon sets recognized by different synonymous tRNAs. See Bulmer, “Coevolution of codon usage and transfer RNA abundance,” Nature 325:728-730 (1987). Depending on the organism, this application conceives and includes different nucleic acids encoding the eGLP-1 polypeptide provided herein.

[0156] Codon and expression optimization methods known in the art can optimize multiple parameters and factors, including codon usage (e.g., codon fitness index [CAI], effective codon count [ENc], relative synonym codon usage rate [RSCU], and synonym codon usage order [SCUO]), codon pairs, tRNA usage (e.g., tRNA fitness index [tAI]), GC content, ribosome binding site (RBS), hidden stop codons, motif avoidance, restriction site removal, mRNA secondary structure of the gene (e.g., mRNA free energy), and hydrophobicity index optimization. For example, see Sharp et al., “The Codon Adaptation Index—a measure of directional synonymous codon usage bias, and its potential applications,” Nucleic Acids Research, 15(3), 1281-1295 (1987); U.S. Patent No. 8,326,547 (issued December 4, 2012, Liu et al.); and U.S. Patent Publication No. 2021 / 0366574 (published November 25, 2021, Fan). Many online tools for codon optimization have been developed, such as DNAWorks (helixweb.nih.gov / dnaworks / ), Jcat (www.jcat.de / ), Syntheticgenedesigner (userpages.umbc.edu / ~wug1 / codon / sgd / ), GeneDesign (genedesign.org / ), Gene Designer2.0 (www.dna20.com / resources / genedesigner), OPTIMIZER (genomes.urv.es / OPTIMIZER), Visualgenedeveloper (www.visualgenedeveloper.net / ), Eugene (bioinformatics.ua.pt / eugene), and mRNA. Optimizer (website: bioinformatics.ua.pt / software / mRNA-optimiser), COOL (website: bioinfo.bti.a-star.edu.sg / COOL / ), and D-Tailor (website: sourceforge.net / projects / dtailor / ).

[0157] Once the eGLP-1 peptide or dual agonist peptide is produced via recombinant expression, it can be purified using any protein purification method known in the art, such as chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly affinity chromatography for specific antigens after protein A treatment, and sizing column chromatography), centrifugation, differential solubility assays, or any other standard protein purification technique. In some aspects, the eGLP-1 peptide or dual agonist peptide may have a peptide tag at the amino or carboxyl terminus for purification, detection, or both. In some aspects, the peptide tag contains a protease cleavage site to allow removal of the peptide tag by proteolysis, thereby generating the eGLP-1 peptide or dual agonist peptide.

[0158] Alternatively, the eGLP-1 peptides or dual agonist peptides provided herein can be chemically synthesized using methods well known to those skilled in the art, such as solid-phase synthesis as described by Merrifield (1963, J. Am. Chem. Soc. 85: 2149-2154). Solid-phase peptide synthesis can be achieved, for example, using automated synthesizers and standard reagents. The preparation of eGLP-1 peptides or dual agonist peptides using chemical synthesis methods allows for the incorporation of non-standard amino acids, including but not limited to L-amino acids and α-methyl amino acids.

[0159] Formulations and Compositions In some respects, the eGLP-1 peptides or dual agonist peptides provided herein possess one or more of the following criteria: acceptable solubility, ease of formulation, plasma stability (e.g., resistance to proteolysis), and improved pharmacokinetic properties. In some respects, the disclosed eGLP-1 peptides or dual agonist peptides are soluble in standard buffers over a wide pH range.

[0160] In some respects, the disclosed eGLP-1 peptides or dual agonist peptides exhibit acceptable stability against proteases in serum or plasma. Common degradation products of natural GLP-1 (wild-type GLP-1 or wtGLP-1) include DPP IV, pepsin, trypsin, chymotrypsin, enkephalinase, and elastase cleavage products. These cleavage products are produced by the action of proteases present in plasma or the digestive tract (for orally administered compositions). In contrast, the eGLP-1 peptides or dual agonist peptides provided herein are modified to eliminate or reduce proteolysis, thereby improving stability and ultimately achieving higher sustained plasma concentrations.

[0161] This application further provides compositions, such as pharmaceutical compositions, comprising an effective amount of the eGLP-1 polypeptide or dual agonist polypeptide provided herein, formulated for the treatment of metabolic diseases, such as obesity and diabetes. This application also provides and includes pharmaceutical compositions comprising host cells converted with a polynucleotide encoding the eGLP-1 polypeptide or dual agonist polypeptide provided herein. In some 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 SEQ ID NO:382, or SEQ ID NO:372 to SEQ ID NO:380. In some 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 some aspects, the host cells express an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO:14. In some aspects, the host cell expresses a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO:111 to SEQ ID NO:147, SEQ ID NO:148, or SEQ ID NO:164 to SEQ ID NO:207. In some aspects, the host cell expresses a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO:113 to SEQ ID NO:116. In some aspects, the host cell expresses a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO:383 or SEQ ID NO:381.

[0162] The pharmaceutical compositions provided herein can be formulated for specific routes of administration, such as oral, parenteral, and rectal administration. Furthermore, the pharmaceutical compositions of this application can be formulated into solid forms (including capsules, tablets, pills, granules, powders, or suppositories) or liquid forms (including solutions, suspensions, or emulsions). The pharmaceutical compositions can undergo routine pharmaceutical processing, such as sterilization, and / or may contain conventional inert diluents, lubricants, or buffers, as well as adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, and buffers.

[0163] The compositions provided herein can be formulated according to known methods. Suitable preparation methods are described, for example, in the following literature: Remington's Pharmaceutical Sciences, 23rd edition, A Dejare editor, Elsevier-Saunders, Mosby, Churchill, Easton, Pa. (2021), the entire text of which is incorporated herein by reference. The compositions can be in various forms, including but not limited to aqueous solutions, emulsions, gels, suspensions, lyophilized forms, or any other forms known in the art. Furthermore, the compositions may contain pharmaceutically acceptable additives, such as carriers, diluents, binders, stabilizers, and preservatives. Once formulated, the compositions of this application can be administered directly to a subject. In some aspects, the formulated compositions are provided as dry compositions that will be suspended or dissolved in a liquid carrier (typically an aqueous carrier) prior to administration.

[0164] The carriers that can be used in the compositions of this application are well known in the art, including but not limited to thyroglobulin, albumin (such as human serum albumin), tetanus toxoid, and polyamino acids (such as polylysine, polyglutamic acid, etc.). Various aqueous carriers can be used, such as water, buffered water, 0.8% physiological saline, 0.3% glycine, hyaluronic acid, etc. The compositions can be sterilized using conventional, well-known sterilization techniques, or can be sterile filtered. The resulting compositions can be packaged directly for use, or lyophilized; the lyophilized formulation must be mixed with a sterile solution before administration. The compositions may, as needed, contain pharmaceutically acceptable excipients to approximate physiological conditions, such as pH adjusters and buffers, osmotic pressure regulators, wetting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitol monolaurate, triethanolamine oleate, etc.

[0165] In some respects, the pharmaceutical compositions provided herein containing eGLP-1 peptides or dual agonist peptides or host cells engineered to express eGLP-1 peptides or dual agonist peptides (APIs) can be formulated into tablets and gelatin capsules, comprising the API active ingredient and the following components: a) diluents, such as lactose, glucose, sucrose, mannitol, sorbitol, cellulose, and / or glycine; b) lubricants, such as silica, talc, stearic acid, and their magnesium or calcium salts, and / or polyethylene glycol; for tablets, may also contain c) binders, such as magnesium aluminum silicate, starch paste, gelatin, tragacanth gum, methylcellulose, sodium carboxymethyl cellulose, and / or polyvinylpyrrolidone; and, if desired, may also contain: d) disintegrants, such as starch, agar, alginic acid, or their sodium salts or effervescent mixtures; and / or e) absorbents, colorants, flavoring agents, and sweeteners. Tablets can be prepared using film coating or enteric coating methods known in the art.

[0166] Compositions suitable for oral administration include an effective amount of the compound of this application, which may be in the form of tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard capsules or soft capsules, chewable tablets, syrups or elixirs. Compositions intended for oral administration may be prepared according to any pharmaceutical composition preparation method known in the art, and such compositions may contain one or more agents selected from sweeteners, flavoring agents, coloring agents, and preservatives to provide a pharmaceutically sound and palatable formulation. Tablets comprise the active ingredient mixed with non-toxic, pharmaceutically acceptable excipients suitable for tablet production. These excipients may, for example, be inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrants such as corn starch or alginic acid; binders such as starch, gelatin, or gum arabic; and lubricants such as magnesium stearate, stearic acid, or talc. Tablets may be uncoated or coated using known techniques to delay their disintegration and absorption in the gastrointestinal tract, thereby providing a longer duration of action. For example, sustained-release materials such as glyceryl monostearate or glyceryl distearate can be used. Oral formulations can be in the form of hard gelatin capsules, in which the active ingredient is mixed with an inert solid diluent (such as calcium carbonate, calcium phosphate, or kaolin); or in the form of soft gelatin capsules, in which the active ingredient is mixed with water or an oily medium (such as peanut oil, liquid paraffin, or olive oil).

[0167] host cells This application further provides and includes host cells transformed with a polynucleotide encoding a peptide having the amino acid sequence of Formula I. In one aspect, the polynucleotide is an engineered vector comprising a polynucleotide encoding a peptide having the amino acid sequence of Formula I. In some 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 the amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In some 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 some 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.

[0168] This application further provides and includes host cells transformed with a polynucleotide encoding a peptide having the amino acid sequence of Formula II (SEQ ID NO: 111). In one aspect, the polynucleotide is an engineered vector comprising a polynucleotide encoding a peptide having the amino acid sequence of Formula II (SEQ ID NO: 111). In some aspects, the engineered vector encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 111 to SEQ ID NO: 147. In some 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 a polymerized polypeptide comprising 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.

[0169] The expression vector is prepared using standard methods. Typically, the expression vector includes a promoter for transcriptional expression, a nucleic acid sequence encoding a peptide comprising formula I, and a transcription terminator. In some aspects, the nucleic acid sequence encoding the eGLP-1 polypeptide ends with a stop codon to terminate translation. 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 vector encodes a polypeptide comprising the amino acid sequences of any one of SEQ ID NO:32 to SEQ ID NO:67. In some aspects, the expression vector encodes an eGLP-1 polypeptide comprising the amino acid sequences of any one of SEQ ID NO:2 to SEQ ID NO:14. In some aspects, the expression vector encodes an eGLP-1 polypeptide comprising the amino acid sequences of any one of SEQ ID NO:3 to SEQ ID NO:14. In some aspects, the expression vector encodes an eGLP-1 polypeptide comprising the amino acid sequences of any one of SEQ ID NO:3 to SEQ ID NO:14. In some aspects, the polypeptide may further include one or more sequences for secretion or membrane localization. In some aspects, the eGLP-1 polypeptide may further include an expression peptide tag for detection or purification.

[0170] 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 some 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 a polymerized polypeptide comprising 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 some 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 some 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 a further aspect, the engineered vector encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NO:123 to SEQ ID NO:127.

[0171] The expression vector is prepared using standard methods. Typically, the expression vector contains a transcriptional expression promoter, a nucleic acid sequence encoding a peptide containing a dual agonist polypeptide of formula II (SEQ ID NO:111), and a transcription terminator. In some aspects, the nucleic acid sequence encoding the dual agonist polypeptide ends with a stop codon to terminate translation. Suitable formula II amino acid sequences include the amino acid sequences of SEQ ID NO:112 to SEQ ID NO:147. In some aspects, suitable formula II amino acid sequences include the amino acid sequences of any one of SEQ ID NO:113 to SEQ ID NO:116. Other suitable formula II amino acid sequences include the amino acid sequences of any one of SEQ ID NO:164 to SEQ ID NO:167. Other suitable formula II amino acid sequences include the amino acid sequences of SEQ ID NO:148 to SEQ ID NO:207. In some aspects, these polypeptides may further include one or more sequences for secretion or membrane localization. In some aspects, the dual agonist polypeptide may further include an expression peptide tag for detection or purification.

[0172] The expression vectors described herein are introduced into host cells using standard methods. Applicable cells include bacterial cells, plant cells, yeast cells, or algal cells. At least one copy of the recombinant nucleic acid is stably introduced into the host cell. In some cases, two or more copies of the recombinant nucleic acid are introduced into the host cell.

[0173] In some respects, recombinant nucleic acids are integrated into bacterial cells or other host cells to achieve stable expression of the desired eGLP-1 peptide or dual agonist peptide described herein. In one respect, this integration occurs within the host cell chromosome. In some respects, the recombinant nucleic acid integrates randomly into the host cell chromosome. In other respects, the integration of the recombinant nucleic acid is targeted, for example, by using transposases and appropriate targeting sequences.

[0174] In some respects, the host cell for transformation is a yeast cell. In other respects, the yeast cell is Pichia pastoris (…). Pichia pastoris Yeast cells of strain ( ). See Chunfeng et al., 2013. In other respects, the host cells for transformation are selected from the genus *Bacillus* ( ). Bacillus Lactobacillus ( ) Lactobacillus Lactococcus spp. Lactococcus Salmonella ( Salmonella ) and Enterococcus spp. Enterococcus Lactobacilli are bacterial cells composed of the genus *Lactobacillus*. In one respect, *Lactobacillus gasseri* (…) Lactobacillus gasseri See Lin et al., “Oral Delivery of Pentameric Glucagon-Like Peptide-1 by Recombinant Lactobacillus in Diabetic Rats,” PloS One , 11 (9); e0162733 (2011) and (2016 and Duan et al., “Engineered commensalbacteria reprogram intestinal cells into glucose-responsive insulin-secreting cells for the treatment of diabetes,” Diabetes 64(5):1794-803(2015). On the other hand, the lactobacillus is *Lactobacillus plantarum* ( Lactobacillus plantarum ). See Luo et al., “Antidiabetic effect of an engineered bacterium Lactobacillus plantarum-pMG36e -GLP-1 in monkeymodel,” Synth. Syst. Biotechnol 6(4):272-282(2021).

[0175] This application also includes and provides transformed plant cells. In some respects, the recombinant nucleic acids are integrated into the genome of the plant cells. In other respects, the recombinant nucleic acids are integrated into the chloroplasts of the plant cells. As provided herein, the transformed plant cells can regenerate into plants or plant parts, including seeds.

[0176] Direct feeding microorganisms (DFM), also commonly known as probiotics, refer to microorganisms that can colonize the gastrointestinal tract of an animal and produce beneficial effects on that animal. In some respects, the microorganisms can be bacterial species, such as those belonging to the genera *Bacillus*, *Lactobacillus*, *Lactococcus*, *Salmonella*, and *Enterococcus*. In other respects, the microorganisms can also be yeasts or molds. These microorganisms can be administered to animals orally or via mucosal routes, or, in the case of poultry, to fertilized eggs, i.e., intraovarian administration.

[0177] In some aspects of this application, the 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 sequence encodes a polypeptide comprising the amino acid sequences of any one of SEQ ID NO:32 to SEQ ID NO:67. In some aspects, the polynucleotide sequence encodes an eGLP-1 polypeptide comprising the amino acid sequences of any one of SEQ ID NO:2 to SEQ ID NO:14. In some aspects, the polynucleotide sequence encodes an eGLP-1 polypeptide comprising the amino acid sequences of any one of SEQ ID NO:3 to SEQ ID NO:14.

[0178] In certain aspects of this application, DFM can be a host cell transformed with a multinucleotide sequence encoding a dual agonist polypeptide of formula II (SEQ ID NO: 111). DFM is characterized by its general safety (even being labeled Generally Regarded as Safe, GRAS), and most DFMs do not exhibit natural antibiotic resistance. Probiotics and DFM provide an attractive delivery method that can deliver therapeutic amounts of the dual agonist polypeptide described herein.

[0179] Suitable amino acid sequences of formula II include the amino acid sequences of SEQ ID NO:112 to SEQ ID NO:147. In some aspects, the polynucleotide sequence encodes a dual agonist polypeptide comprising the amino acid sequences of any one of SEQ ID NO:113 to SEQ ID NO:116. In other aspects, the polynucleotide sequence encodes a dual agonist polypeptide comprising the amino acid sequences of any one of SEQ ID NO:164 to SEQ ID NO:167. In other aspects, the polynucleotide sequence encodes a dual agonist polypeptide comprising the amino acid sequences of any one of SEQ ID NO:148 to SEQ ID NO:207. In some aspects, the polynucleotide sequence encodes a dual agonist polypeptide comprising the amino acid sequences of any one of SEQ ID NO:123 to SEQ ID NO:147.

[0180] As described herein, DFM host cells are engineered to express the eGLP-1 peptide on the bacterial surface or in a secretory form. DFM is generally considered safe (even recognized as “GRAS”), and most DFMs do not exhibit natural antibiotic resistance. Probiotics and DFM provide an attractive delivery method for delivering therapeutic amounts of the eGLP-1 peptide described herein.

[0181] DFM as a delivery system Suitable strains for use as DFM provide an attractive and useful starting point for the production of the eGLP-1 peptide or dual agonist peptide described in this application. In addition to being suitable for the production of purified eGLP-1 peptides or dual agonist peptides, the transformed DFM can also be used as an in vivo delivery system for the synthesis and delivery of the eGLP-1 peptide or dual agonist peptide described herein for the treatment of metabolic disorders.

[0182] Direct-feed strains (engineered DFMs or eDFMs) engineered (e.g., genetically modified) to express eGLP-1 peptides or dual agonist peptides can serve as a delivery system to continuously deliver therapeutically effective amounts of eGLP-1 peptides or dual agonist peptides directly to the host. In some aspects, eDFMs deliver therapeutic amounts of eGLP-1 peptides or dual agonist peptides directly to the gastrointestinal tract. In other aspects, the delivery system is a live recombinant engineered DFM, such as bacteria, that can multiply within the host, and even, in some cases, colonize, and deliver eGLP-1 peptides or dual agonist peptides directly to the subject requiring treatment. Therefore, eDFMs, utilizing suitable vectors and nucleic acid systems known in the art, provide an improved delivery platform and system for the rapid and efficient expression of heterologous eGLP-1 peptides or dual agonist peptides as described in this application. The eGLP-1 peptide suitable for eDFMs is a peptide having the amino acid sequence of Formula I. In some aspects, eDFMs express peptides comprising the amino acid sequences of SEQ ID NO:1 to SEQ ID NO:14. In other respects, eDFM expresses a polypeptide comprising the amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In some respects, 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 some respects, eDFM expresses an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO:14.

[0183] The dual agonist polypeptide suitable for eDFM is a polypeptide having the amino acid sequence of Formula II (SEQ ID NO: 111). In some aspects, eDFM expresses a polypeptide comprising the amino acid sequences of SEQ ID NO: 112 to SEQ ID NO: 147. In other aspects, eDFM expresses a dual agonist polypeptide comprising the amino acid sequences of any one of SEQ ID NO: 113 to SEQ ID NO: 116. In other aspects, eDFM expresses a dual agonist polypeptide comprising the amino acid sequences of any one of SEQ ID NO: 164 to SEQ ID NO: 167. In other aspects, eDFM expresses a dual agonist polypeptide comprising the amino acid sequences of any one of SEQ ID NO: 148 to SEQ ID NO: 207. In some aspects, eDFM expresses a dual agonist polypeptide comprising the amino acid sequences of any one of SEQ ID NO: 123 to SEQ ID NO: 147.

[0184] As provided in this article, eDFM can express the desired peptide on the eDFM surface, or it can be engineered to secrete the eGLP-1 peptide or dual agonist peptide into the environment (e.g., the gastrointestinal tract).

[0185] In some respects, DFM can be freeze-dried. See Kwon. et al. , “Oral delivery of humanbiopharmaceuticals, autoantigens and vaccine antigens bioencapsulated implant cells,” Adv Drug. Deliv. Rev. 65(6):782-99(2013).

[0186] As the host cell of the production system The production of recombinant proteins in microbial cells is an important aspect of the modern biotechnology industry. Intracellular expression of heterologous proteins in host cells is widely used; these proteins are typically isolated from cultures that produce host cells. The eGLP-1 peptide or dual agonist peptide described in this application can be expressed by a plasmid transfected into bacterial cells, or by a coding sequence integrated into the host bacterial genome.

[0187] Furthermore, recent advances in the secretory expression of recombinant proteins have prompted the scientific and industrial communities to apply secretory-capable bacteria to protein production. Using secretory host cells, synthesized eGLP-1 peptides or dual agonist peptides can be directly secreted and accumulated in extracellular culture media. Secreted eGLP-1 peptides or dual agonist peptides enable cost-effective downstream purification processes. Moreover, secretion does not require, or does not require, lysis of host cells, thus simplifying the production and purification process (if necessary). Simultaneously, the secretory expression of eGLP-1 peptides or dual agonist peptides prevents the accumulation of target eGLP-1 peptides or dual agonist peptides within host cells, which can limit cell growth and yield, lead to cytotoxicity, and cause protein misfolding. See Mergulhao et al., “Recombinant protein secretion in Escherichia coli “ Biotechnol Adv 23(3):177 202 (2005); Song et al., “Improving Protein Production on the Level of Regulation of both Expression and Secretion Pathways in Bacillus subtilis “ J Microbiol Biotechnol 25(7):963 77 (2015).

[0188] Reagent test kit In some aspects, this application provides and includes a pharmaceutical kit comprising engineered glucagon-like peptide-1 (eGLP-1) having the amino acid sequence of Formula I (e.g., an active pharmaceutical ingredient (API)). In other aspects, the kit comprises a recombinant host cell containing a polynucleotide encoding engineered glucagon-like peptide-1 (eGLP-1) (as an API). In some aspects, in addition to the API, the pharmaceutical composition further comprises an excipient selected from binders, coating agents, colorants, disintegrants, fragrances, flow aids, lubricants, preservatives, adsorbents, and carriers.

[0189] In some aspects, this application provides and includes a pharmaceutical kit comprising an engineered dual agonist polypeptide (e.g., an active pharmaceutical ingredient (API)) having an amino acid sequence of Formula II (SEQ ID NO: 111). In other aspects, the kit comprises a recombinant host cell containing a polynucleotide encoding the dual agonist polypeptide (as the API). In some aspects, in addition to the API, the pharmaceutical composition further comprises an excipient selected from binders, coating agents, colorants, disintegrants, fragrances, flow aids, lubricants, preservatives, adsorbents, and carriers.

[0190] Treatment The eGLP-1 peptide or dual agonist peptide described herein can achieve one or more of the following effects: prevention or regulation of hyperglycemia, promotion of insulin synthesis, inhibition of glucagon synthesis, increase of β-cell mass, weight loss or maintenance of weight (e.g., prevention of weight gain), reduction of food intake, regulation of gastric acid secretion, or regulation of gastric emptying.

[0191] This specification provides and includes methods for treating hypoglycemic conditions (e.g., type 2 diabetes), including administering the eGLP-1 peptide disclosed herein to a subject requiring treatment. Additionally, the eGLP-1 peptide for treating hypoglycemic conditions (e.g., type 2 diabetes) is also provided. Furthermore, the use of the eGLP-1 peptide provided herein in the manufacture of a medicament for treating hypoglycemic conditions (e.g., type 2 diabetes) is also provided. In some aspects, the method of treating hypoglycemic conditions (e.g., type 2 diabetes) includes administering the eGLP-1 peptide or a dual agonist peptide disclosed herein to a subject requiring treatment, which reduces the level of hemoglobin A1c (HbA1c).

[0192] The term "HbA1c" refers to the product of non-enzymatic glycosylation of the B chain of hemoglobin. Those skilled in the art are familiar with methods for measuring HbA1c levels. HbA1c values ​​are of paramount importance in monitoring diabetes treatment. Because its formation depends primarily on blood glucose levels and red blood cell lifespan, HbA1c serves as a "glucose memory," reflecting average blood glucose levels over the past 4-6 weeks. Diabetic patients with consistently well-controlled HbA1c levels (i.e., HbA1c < 6.5% of total hemoglobin in the sample) through intensive diabetes treatment show significantly better prevention of diabetic microvascular complications.

[0193] This application provides and includes a treatment method comprising: providing an eGLP-1 polypeptide comprising an amino acid sequence comprising any one of SEQ ID NO:1 to SEQ ID NO:14 or a combination thereof, for preventing, slowing the progression of, delaying or treating metabolic disorders. In other aspects, the treatment method comprises providing a therapeutic amount of a polypeptide comprising an amino acid sequence comprising any one of SEQ ID NO:32 to SEQ ID NO:67. In other aspects, the treatment method comprises providing a therapeutic amount of an eGLP-1 polypeptide comprising an amino acid sequence comprising any one of SEQ ID NO:2 to SEQ ID NO:14. In some aspects, the treatment method comprises providing a therapeutic amount of an eGLP-1 polypeptide comprising an amino acid sequence comprising any one of SEQ ID NO:3 to SEQ ID NO:14.

[0194] This application provides and includes a treatment method 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 fasting blood glucose, postprandial blood glucose, and / or glycated hemoglobin HbA1c. In other aspects, the treatment method comprises providing a therapeutic amount of a polypeptide comprising the amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In other aspects, the treatment method 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 some aspects, the treatment method 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.

[0195] This application provides and includes a treatment method 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 the progression, delaying or reversing the progression of impaired glucose tolerance, impaired fasting glucose, insulin resistance and / or metabolic syndrome to type 2 diabetes. In other aspects, the treatment method comprises providing a therapeutic amount of a polypeptide comprising the amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In other aspects, the treatment method 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 some aspects, the treatment method 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.

[0196] This application provides and includes a treatment method 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 the prevention, slowing of progression, delay, or treatment of diabetic complications selected from the group consisting of: insulin resistance, glucose intolerance, elevated fasting blood glucose, prediabetes, type 1 diabetes, type 2 diabetes, gestational diabetes, hypertension, dyslipidemia, or a combination thereof. In other aspects, the treatment method comprises providing a therapeutic amount of a polypeptide comprising the amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In other aspects, the treatment method 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 other aspects, the treatment method 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.

[0197] This application provides and includes a treatment method comprising: providing an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:1 to SEQ ID NO:14 or a combination thereof, for reducing weight, body fat or a combination thereof, or preventing an increase in weight, body fat or a combination thereof, or promoting a decrease in weight, body fat or a combination thereof. In other aspects, the treatment method comprises providing a therapeutic amount of a polypeptide comprising the amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In other aspects, the treatment method 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 some aspects, the treatment method 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.

[0198] This application provides and includes a treatment method comprising: providing an eGLP-1 polypeptide comprising the amino acid sequence of any one of SEQ ID NO:1 to SEQ ID NO:14 or a combination thereof, for preventing or treating the degeneration of pancreatic β cells and / or for improving and / or restoring or protecting the function of pancreatic β cells and / or restoring pancreatic insulin secretion function.

[0199] This application provides and includes a treatment method 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 the prevention, mitigation, delay, or treatment of a disease or condition caused by abnormal accumulation of liver or ectopic fat. In other aspects, the treatment method comprises providing a therapeutic amount of a polypeptide comprising the amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In other aspects, the treatment method 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 some aspects, the treatment method 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.

[0200] This application provides and includes a treatment method 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 treatment method comprises providing a therapeutic amount of a polypeptide comprising the amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In other aspects, the treatment method 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 some aspects, the treatment method 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.

[0201] This application provides and includes a treatment method 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 the prevention, slowing progression, delay, or treatment of new-onset diabetes mellitus (NODAT) and / or post-transplant metabolic syndrome (PTMS). In other aspects, the treatment method comprises providing a therapeutic amount of a polypeptide comprising the amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In other aspects, the treatment method 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 some aspects, the treatment method 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.

[0202] This application provides and includes a treatment method 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 the prevention, delay, or reduction of new-onset diabetes mellitus (NODAT), PTMS-related complications, or both, including microvascular and macrovascular disease and events, graft rejection, infection, and death. In other aspects, the treatment method comprises providing a therapeutic amount of a polypeptide comprising the amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In other aspects, the treatment method 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 some aspects, the treatment method 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.

[0203] This application provides and includes a treatment method 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-related conditions. In other aspects, the treatment method comprises providing a therapeutic amount of a polypeptide comprising the amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In other aspects, the treatment method 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 some aspects, the treatment method 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.

[0204] The eGLP-1 peptide described in this article can be used for glycemic control, promoting insulin production, increasing β-cell mass, and promoting weight loss or reducing overweight. Furthermore, the eGLP-1 peptide described in this article can also be used to treat related disorders. Examples of related disorders include, but are not limited to: insulin resistance, glucose intolerance, prediabetes, elevated fasting blood glucose, hypertension, dyslipidemia (or a combination of these metabolic risk factors), glucagonoma, cardiovascular diseases such as congestive heart failure, atherosclerosis, arteriosclerosis, coronary artery disease or peripheral artery disease, stroke, respiratory dysfunction, or kidney disease.

[0205] This application provides and includes a treatment method comprising: providing a dual agonist polypeptide comprising an amino acid sequence of any one of SEQ ID NO:111 to SEQ ID NO:147 or a combination thereof, for preventing, slowing the progression of, delaying or treating metabolic disorders. In some aspects, the method provides a dual agonist polypeptide comprising an amino acid sequence of any one of SEQ ID NO:113 to SEQ ID NO:116 or a multimer of any one of SEQ ID NO:164 to SEQ ID NO:167. In some aspects, the method provides a dual agonist polypeptide comprising an amino acid sequence of any one of SEQ ID NO:148 to SEQ ID NO:207.

[0206] This application provides and includes a treatment method comprising: providing a dual agonist polypeptide comprising an amino acid sequence comprising any one of SEQ ID NO:111 to SEQ ID NO:147 or a combination thereof, for improving glycemic control and / or for reducing fasting blood glucose, postprandial blood glucose, and / or glycated hemoglobin HbA1c. In some aspects, the method of improving glycemic control comprises providing a dual agonist polypeptide comprising an amino acid sequence comprising any one of SEQ ID NO:113 to SEQ ID NO:116. In some aspects, the method of improving glycemic control comprises providing a dual agonist polypeptide comprising an amino acid sequence comprising any one of SEQ ID NO:127 to SEQ ID NO:147 or a combination thereof. In some aspects, the method of improving glycemic control comprises providing a dual agonist polypeptide comprising an amino acid sequence comprising any one of SEQ ID NO:148 to SEQ ID NO:207 or a combination thereof.

[0207] This application provides and includes a treatment method comprising: providing a dual agonist polypeptide comprising an 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 the progression of impaired glucose tolerance, impaired fasting glucose, insulin resistance and / or metabolic syndrome to type 2 diabetes. In some aspects, a method for preventing, slowing, delaying or reversing the progression of impaired glucose tolerance, impaired fasting glucose, insulin resistance and / or metabolic syndrome to type 2 diabetes is provided, the method being implemented by providing a peptide comprising an amino acid sequence of any one of SEQ ID NO:113 to SEQ ID NO:116. In one aspect, a method for preventing, slowing, delaying or reversing the progression of impaired glucose tolerance, impaired fasting glucose, insulin resistance and / or metabolic syndrome to type 2 diabetes is provided, the method being implemented by providing a peptide comprising an amino acid sequence of any one of SEQ ID NO:127 to SEQ ID NO:147. Furthermore, a method for providing a dual agonist polypeptide comprising an amino acid sequence of any one of SEQ ID NO:148 to SEQ ID NO:207 is also included.

[0208] This application provides and includes a treatment method comprising: providing a dual agonist polypeptide comprising an amino acid sequence comprising any one of SEQ ID NO:111 to SEQ ID NO:147 or a combination thereof, for the prevention, slowing of progression, delay, or treatment of a condition or disorder selected from the group consisting of diabetic complications. In some aspects, the method comprises providing a dual agonist polypeptide comprising an amino acid sequence comprising any one of SEQ ID NO:113 to SEQ ID NO:116. In some aspects, the method comprises providing a dual agonist polypeptide comprising an amino acid sequence comprising any one of SEQ ID NO:127 to SEQ ID NO:147. In a further aspect, the method comprises providing a dual agonist polypeptide comprising an amino acid sequence comprising any one of SEQ ID NO:148 to SEQ ID NO:207.

[0209] This application provides and includes a treatment method comprising: providing a dual agonist polypeptide comprising the amino acid sequences of SEQ ID NO:111 to SEQ ID NO:147 or a combination thereof, for reducing weight and / or body fat, or preventing weight and / or body fat gain, or promoting weight and / or body fat reduction. In some aspects, this application includes a treatment method comprising: providing a dual agonist polypeptide comprising the amino acid sequences of SEQ ID NO:113 to SEQ ID NO:116 or a combination thereof, for reducing weight and / or body fat, or preventing weight and / or body fat gain, or promoting weight and / or body fat reduction.

[0210] This application further provides a treatment method 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 weight and / or body fat, or preventing weight and / or body fat gain, or promoting weight and / or body fat reduction. In some aspects, a method for reducing weight and / or body fat, or preventing weight and / or body fat gain, or promoting weight and / or body fat reduction, 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 weight and / or body fat, or preventing weight and / or body fat gain, or promoting weight and / or body fat reduction, comprising providing a polypeptide having the amino acid sequence of any one of SEQ ID NO:127 to SEQ ID NO:147.

[0211] This application provides and includes a treatment method 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 pancreatic β-cell degeneration and / or for improving and / or restoring or protecting pancreatic β-cell function and / or restoring insulin secretion function. In some aspects, the dual agonist polypeptide comprises the amino acid sequence of any one of SEQ ID NO:113 to SEQ ID NO:116, for preventing or treating pancreatic β-cell degeneration and / or for improving and / or restoring or protecting pancreatic β-cell function and / or restoring insulin secretion function. In some aspects, the dual agonist polypeptide comprises the amino acid sequence of any one of SEQ ID NO:127 to SEQ ID NO:147. In some aspects, this application includes and provides a dual agonist polypeptide having the amino acid sequence of any one of SEQ ID NO:148 to SEQ ID NO:207, for preventing or treating pancreatic β-cell degeneration and / or for improving and / or restoring or protecting pancreatic β-cell function and / or restoring pancreatic insulin secretion function.

[0212] This application provides and includes a treatment method 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 the prevention, mitigation, delay, or treatment of a disease or condition caused by abnormal accumulation of liver or ectopic fat. In some aspects, this application includes 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 the prevention, mitigation, delay, or treatment of a disease or condition caused by abnormal accumulation of liver or ectopic fat. In some aspects, this application includes a dual agonist polypeptide comprising the amino acid sequence of any one of SEQ ID NO:164 to SEQ ID NO:167 or a combination thereof, for the prevention, mitigation, delay, or treatment of a disease or condition caused by abnormal accumulation of liver or ectopic fat. This application provides and includes a treatment method 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 the prevention, mitigation, delay, or treatment of a disease or condition caused by abnormal accumulation of liver or ectopic fat. This application provides and includes a treatment method comprising: providing a dual agonist polypeptide comprising an amino acid sequence of any one of SEQ ID NO:148 to SEQ ID NO:207 or a combination thereof, for the prevention, mitigation, delay or treatment of a disease or condition caused by abnormal accumulation of liver or ectopic fat.

[0213] This application provides and includes a treatment method comprising: providing a dual agonist polypeptide comprising the amino acid sequences of any one of SEQ ID NO:111 to SEQ ID NO:147 or combinations thereof, for maintaining and / or improving insulin sensitivity, and / or for treating or preventing hyperinsulinemia and / or insulin resistance. In some aspects, this application includes a dual agonist polypeptide comprising the amino acid sequences of any one of SEQ ID NO:113 to SEQ ID NO:116 or combinations thereof, for maintaining and / or improving insulin sensitivity, and / or for treating or preventing hyperinsulinemia and / or insulin resistance. In some aspects, this application includes a dual agonist polypeptide comprising the amino acid sequences of any one of SEQ ID NO:164 to SEQ ID NO:167 or combinations thereof, for maintaining and / or improving insulin sensitivity, and / or for treating or preventing hyperinsulinemia and / or insulin resistance. In some aspects, the polypeptide comprises the amino acid sequences of SEQ ID NO:127 to SEQ ID NO:147. This application provides and includes a treatment method comprising providing a dual agonist polypeptide comprising an 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 insulin sensitivity, and / or for treating or preventing hyperinsulinemia and / or insulin resistance.

[0214] This application provides and includes a treatment method comprising: providing a dual agonist polypeptide comprising an amino acid sequence or combination thereof of any one of SEQ ID NO:111 to SEQ ID NO:147 for the prevention, slowing of progression, delay, or treatment of new-onset diabetes mellitus (NODAT) and / or post-transplant metabolic syndrome (PTMS). In some aspects, this application includes a dual agonist polypeptide comprising an amino acid sequence or combination thereof of any one of SEQ ID NO:113 to SEQ ID NO:116 for the prevention, slowing of progression, delay, or treatment of new-onset diabetes mellitus (NODAT) and / or post-transplant metabolic syndrome (PTMS). In some aspects, this application includes a dual agonist polypeptide comprising an amino acid sequence or combination thereof of any one of SEQ ID NO:164 to SEQ ID NO:167 for the prevention, slowing of progression, delay, or treatment of new-onset diabetes mellitus (NODAT) and / or post-transplant metabolic syndrome (PTMS). In some aspects, the polypeptide comprises an amino acid sequence of SEQ ID NO:127 to SEQ ID NO:147. This application provides and includes a treatment method comprising providing a dual agonist polypeptide comprising an amino acid sequence of any one of SEQ ID NO:148 to SEQ ID NO:207 or a combination thereof for the prevention, slowing of progression, delay or treatment of new-onset diabetes mellitus (NODAT) and / or post-transplant metabolic syndrome (PTMS).

[0215] This application provides and includes a treatment method comprising: providing a dual agonist polypeptide comprising an amino acid sequence of any one of SEQ ID NO:111 to SEQ ID NO:147 or a combination thereof, for the prevention, delay, or reduction of NODAT and / or PTMS-related complications, including microvascular and macrovascular disease and events, transplant rejection, infection, and death. In some aspects, this application includes a dual agonist polypeptide comprising an amino acid sequence of any one of SEQ ID NO:113 to SEQ ID NO:116 or a combination thereof, for the prevention, delay, or reduction of NODAT and / or PTMS-related complications, including microvascular and macrovascular disease and events, transplant rejection, infection, and death. In some aspects, the polypeptide comprises an amino acid sequence of any one of SEQ ID NO:164 to SEQ ID NO:167. In some aspects, the polypeptide comprises an amino acid sequence of any one of SEQ ID NO:127 to SEQ ID NO:147. This application provides and includes a treatment method comprising providing a dual agonist polypeptide comprising an amino acid sequence of any one of SEQ ID NO:148 to SEQ ID NO:207 or a combination thereof, for the prevention, delay or reduction of NODAT and / or PTMS-related complications, including microvascular and macrovascular diseases and events, transplant rejection, infection and death.

[0216] This application provides and includes a treatment method comprising providing a dual agonist polypeptide comprising an amino acid sequence or combination thereof of any one of SEQ ID NO:111 to SEQ ID NO:147 for treating hyperuricemia and hyperuricemia-related conditions. In some aspects, this application includes a dual agonist polypeptide comprising an amino acid sequence or combination thereof of any one of SEQ ID NO:113 to SEQ ID NO:116 for treating hyperuricemia and hyperuricemia-related conditions. In some aspects, this application includes a dual agonist polypeptide comprising an amino acid sequence or combination thereof of any one of SEQ ID NO:164 to SEQ ID NO:167 for treating hyperuricemia and hyperuricemia-related conditions. In some aspects, the polypeptide comprises the amino acid sequences of SEQ ID NO:127 to SEQ ID NO:147. This application provides and includes a treatment method comprising providing a dual agonist polypeptide comprising an amino acid sequence or combination thereof of any one of SEQ ID NO:148 to SEQ ID NO:207 for treating hyperuricemia and hyperuricemia-related conditions.

[0217] This application provides and includes a treatment method comprising providing a dual agonist polypeptide comprising an amino acid sequence or combination thereof of any one of SEQ ID NO:111 to SEQ ID NO:147 for the prevention, delay, or reduction of neurodegenerative diseases. In some aspects, this application includes a dual agonist polypeptide comprising an amino acid sequence or combination thereof of any one of SEQ ID NO:113 to SEQ ID NO:116 for the prevention, delay, or reduction of neurodegenerative diseases. In some aspects, this application includes a dual agonist polypeptide comprising an amino acid sequence or combination thereof of any one of SEQ ID NO:164 to SEQ ID NO:167 for the prevention, delay, or reduction of neurodegenerative diseases. In some aspects, the polypeptide comprises an amino acid sequence of SEQ ID NO:127 to SEQ ID NO:147. This application provides and includes a treatment method comprising providing a dual agonist polypeptide comprising an amino acid sequence or combination thereof of any one of SEQ ID NO:148 to SEQ ID NO:207 for the prevention, delay, or reduction of neurodegenerative diseases (NDD). In some aspects, methods of treating NDD include providing a polypeptide comprising the amino acid sequences of SEQ ID NO:127 to SEQ ID NO:147. In some aspects, providing a dual agonist polypeptide for treating NDD comprises providing a host cell expressing a polypeptide comprising SEQ ID NO:111 to SEQ ID NO:147. In some aspects, providing a dual agonist polypeptide for treating NDD comprises providing a host cell expressing a polypeptide comprising any one of SEQ ID NO:113 to SEQ ID NO:116 or SEQ ID NO:164 to SEQ ID NO:167. In some aspects, the host cell expresses a polypeptide comprising SEQ ID NO:127 to SEQ ID NO:147. In some aspects of NDD treatment, the host cell is directly fed microorganisms expressing the polypeptides described herein. In some aspects, NDD treatment is a combination therapy for treating hypoglycemia.

[0218] In some aspects, this application provides methods for treating, preventing, delaying, or alleviating Alzheimer's disease (AD) or its symptoms, 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 some aspects, the method for treating, preventing, delaying, or alleviating Alzheimer's disease (AD) or its symptoms comprises 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 some aspects, the method for treating, preventing, delaying, or alleviating Alzheimer's disease (AD) or its symptoms comprises 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 some aspects, the AD is late-onset AD (also known as sporadic AD). In some aspects, this application provides a method for reducing amyloid plaque burden, reducing tau protein phosphorylation, or both, in a subject requiring treatment, prevention, delay, or alleviation of Alzheimer's disease (AD) or its symptoms. In some aspects, treatment methods for Alzheimer's disease (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 preventing the loss of brain insulin receptors and synapses. In some aspects, treatment methods for AD also 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 preventing or mitigating cognitive impairment. In some aspects, treatment methods for 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 some aspects, treatment methods for 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. In some aspects of this application, treatment methods for AD include providing a polypeptide comprising the amino acid sequence of SEQ ID NO:127 to SEQ ID NO:147. In some aspects, providing a dual agonist polypeptide comprises providing a host cell expressing a polypeptide comprising the amino acid sequence of any one of SEQ ID NO:111 to SEQ ID NO:147. In some aspects, providing a dual agonist polypeptide includes providing a host cell expressing a polypeptide comprising the amino acid sequence of any one of SEQ ID NO:113 to SEQ ID NO:116. In some aspects, providing a dual agonist polypeptide includes providing a host cell expressing a polypeptide comprising any one of SEQ ID NO:164 to SEQ ID NO:167.In some respects, the host cell expresses a polypeptide comprising SEQ ID NO:127 to SEQ ID NO:147. In some respects, the host cell is directly fed with microorganisms expressing the polypeptides described herein. In some respects, the treatment of AD is a combination therapy for the treatment of hypoglycemia.

[0219] In some aspects, this application provides methods for treating, preventing, delaying, or alleviating the symptoms of amyotrophic lateral sclerosis (ALS), comprising providing a therapeutic amount of a dual agonist polypeptide comprising an amino acid sequence comprising any one of SEQ ID NO:111 to SEQ ID NO:147 or a combination thereof. In some aspects, the method for treating, preventing, delaying, or alleviating the symptoms of ALS comprises providing a therapeutic amount of a dual agonist polypeptide comprising an amino acid sequence comprising any one of SEQ ID NO:113 to SEQ ID NO:116. In some aspects, the method for treating, preventing, delaying, or alleviating the symptoms of ALS comprises providing a therapeutic amount of a dual agonist polypeptide comprising an amino acid sequence comprising any one of SEQ ID NO:164 to SEQ ID NO:167. In some aspects, a treatment for ALS comprises providing a polypeptide comprising an amino acid sequence comprising SEQ ID NO:127 to SEQ ID NO:147. In some aspects, providing a dual agonist polypeptide for treating ALS comprises providing a host cell expressing a polypeptide comprising SEQ ID NO:111 to SEQ ID NO:147. In some respects, the host cells express peptides comprising SEQ ID NO:127 to SEQ ID NO:147. In some respects of ALS treatment, the host cells are directly fed microorganisms expressing the peptides described herein. In some respects, ALS treatment is a combination therapy for treating hypoglycemia.

[0220] In some aspects, this application provides methods for treating, preventing, delaying, or alleviating Parkinson's disease (PD) or its symptoms, including 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 some aspects, this application includes 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 treating, preventing, delaying, or alleviating Parkinson's disease (PD) or its symptoms, including providing a therapeutic amount of the dual agonist polypeptide. In some aspects, a method of treating PD includes providing a polypeptide comprising the amino acid sequence of SEQ ID NO:164 to SEQ ID NO:167. In some aspects, a method of treating PD includes providing a polypeptide comprising the amino acid sequence of SEQ ID NO:127 to SEQ ID NO:147. In some aspects, providing a dual agonist polypeptide for treating PD includes providing a host cell expressing a polypeptide comprising any one of SEQ ID NO:111 to SEQ ID NO:147. In some aspects, providing a dual agonist polypeptide for the treatment of PD includes providing a host cell expressing a polypeptide comprising any one of SEQ ID NO:113 to SEQ ID NO:116. In some aspects, the host cell expresses a polypeptide comprising SEQ ID NO:127 to SEQ ID NO:147. In some aspects of PD treatment, the host cell is a microorganism directly fed to express the polypeptide as described herein. In some aspects, PD treatment is a combination therapy for the treatment of hypoglycemia.

[0221] The dual-agonist peptides described herein can be administered for glycemic control, promoting insulin production, increasing β-cell mass, and promoting weight loss or reducing overweight. Furthermore, the dual-agonist peptides described herein can be used to treat related disorders. Examples of related disorders include, but are not limited to: insulin resistance, glucose intolerance, prediabetes, elevated fasting blood glucose, hypertension, dyslipidemia (or a combination of these metabolic risk factors), glucagonoma, cardiovascular diseases such as congestive heart failure, atherosclerosis, arteriosclerosis, coronary artery disease or peripheral artery disease, stroke, respiratory dysfunction, or kidney disease.

[0222] As used herein, “treatment” refers to a method for achieving beneficial or anticipated clinical outcomes. As provided herein, the beneficial or anticipated clinical outcomes of the disclosed eGLP-1 peptide or dual agonist peptide include, but are not limited to: stabilizing serum glucose and insulin levels, increasing β-cell mass, or improving, mitigating, stabilizing, or inhibiting weight gain. “Treatment” includes, in some respects, both therapeutic interventions and preventative measures. Those requiring treatment include individuals who already have the disease and those who need to prevent it. “Treatment” refers to improving glycemic control in type 2 diabetes and does not necessarily mean a complete cure of the condition.

[0223] The eGLP-1 peptide or dual agonist peptide provided herein can be administered via, for example, oral, parenteral, inhalation, or topical application. The term "parenteral" as used herein includes, for example, intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. Another example of a form of administration is an injection, particularly an injection for intravenous or intra-arterial injection or infusion. The eGLP-1 peptide or dual agonist peptide provided herein can be administered as a single dose or multiple doses. In some aspects, the eGLP-1 peptide or dual agonist peptide is configured for transdermal administration. In some aspects, the eGLP-1 peptide or dual agonist peptide is administered orally or subcutaneously.

[0224] Parenteral preparations can be a single bolus dose, an infusion, or a loading bolus followed by a maintenance dose. These compositions can be administered at specific fixed or variable intervals, such as once daily, or as needed, such as based on the patient's self-monitored blood glucose levels. Dosing regimens can also be adjusted to achieve the best desired effect (e.g., therapeutic or prophylactic effect).

[0225] Those skilled in the art can readily determine the dosage of the eGLP-1 peptide or dual agonist peptide. Factors influencing the route of administration and corresponding dosage of the eGLP-1 peptide or dual agonist peptide include, but are not limited to, disease severity (e.g., degree of obesity), the subject's medical history, and the age, height, weight, health status, and physical condition of the subject receiving treatment. Similarly, the dosage of the eGLP-1 peptide or dual agonist peptide will depend on the route of administration and whether the subject receives a single or multiple doses of the agent. In some respects, the eGLP-1 peptide or dual agonist peptide provided herein can be administered once daily by injection.

[0226] In the treatment methods described in this application, oral treatment with DFM expressing the eGLP-1 peptide or dual agonist peptide provided herein is also provided and included.

[0227] The eGLP-1 peptide or dual agonist peptide of this application provides and covers a method for treating conditions caused by or characterized by overweight and for treating obesity, morbid obesity, obesity-related inflammation, obesity-related gallbladder disease, obesity-induced sleep apnea, metabolic syndrome, prediabetes, 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, by administering the compound of claim 1 to the subject in an amount sufficient to treat conditions caused by or characterized by overweight and for treating obesity, morbid obesity, obesity-related inflammation, obesity-related gallbladder disease, obesity-induced sleep apnea, metabolic syndrome, prediabetes, 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 treatment method includes providing a therapeutic amount of a polypeptide comprising the amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In other aspects, the treatment method includes 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 some aspects, the treatment method includes 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. This application provides a treatment method comprising providing a dual agonist polypeptide having the amino acid sequences of SEQ ID NO:111 to SEQ ID NO:147. In some aspects, this application includes administering 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. In some aspects, the polypeptide comprises the amino acid sequences of SEQ ID NO:127 to SEQ ID NO:147. In other aspects, the dual agonist polypeptide comprises the amino acid sequence of any one of SEQ ID NO:148 to SEQ ID NO:207.

[0228] The eGLP-1 peptide or dual agonist peptide compound described herein may be used to prevent weight gain or promote weight loss. As used herein, “prevention” means curbing or reducing weight gain compared to an untreated condition, and does not necessarily mean a complete cessation of weight gain. The peptide may lead to reduced food intake and / or increased energy expenditure, resulting in observed weight changes. In some respects, independent of its effects on weight, the eGLP-1 peptide or dual agonist peptide provided herein has beneficial effects on circulating glucose levels, glucose tolerance, and / or circulating cholesterol levels (capable of reducing circulating LDL levels and increasing the HDL / LDL ratio). Therefore, the eGLP-1 peptide or dual agonist peptide provided herein may be used directly or indirectly to treat any disease caused by or characterized by overweight, such as treating and / or preventing obesity, morbid obesity, obesity-related inflammation, obesity-related gallbladder disease, and obesity-induced sleep apnea.

[0229] This application provides and includes the use of eGLP-1 peptides or dual agonist peptides for the treatment of prediabetes, insulin resistance, glucose intolerance, type 2 diabetes, type 1 diabetes, hypertension or atherogenic dyslipidemia (or a combination of two or more of these metabolic risk factors), atherosclerosis, arteriosclerosis, coronary artery disease, peripheral artery disease, stroke, and microvascular disease. In some respects, the role in these conditions may stem from or be related to their effects on body weight, or it may be unrelated to their effects on body weight. In some respects, eGLP-1 peptides or dual agonist peptides are used to treat obesity.

[0230] The eGLP-1 peptide or dual agonist peptide provided herein is a compound suitable for medical treatment methods, particularly for treating the aforementioned conditions. The eGLP-1 peptide or dual agonist peptide provided herein can be used to prepare a medicament for treating the aforementioned conditions. In other aspects, the medicament comprises a therapeutic amount of a peptide containing the amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67. In other aspects, the medicament comprises a therapeutic amount of an eGLP-1 peptide containing the amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO:14. In some aspects, the medicament comprises a therapeutic amount of an eGLP-1 peptide containing the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO:14.

[0231] This application provides a treatment method comprising providing a dual agonist polypeptide having the amino acid sequences of SEQ ID NO:111 to SEQ ID NO:147. In some aspects, the treatment method comprises providing a dual agonist polypeptide having the amino acid sequences of any one of SEQ ID NO:113 to SEQ ID NO:147. In some aspects, the polypeptide comprises the amino acid sequences of SEQ ID NO:127 to SEQ ID NO:147. In other aspects, the dual agonist polypeptide comprises the amino acid sequences of any one of SEQ ID NO:148 to SEQ ID NO:207.

[0232] Combination therapy In some respects, the eGLP-1 peptide described herein can be administered in combination with one or more other therapies. In some respects, the eGLP-1 peptide can be used as part of a combination therapy with agents used to treat diabetes, obesity, dyslipidemia, or hypertension.

[0233] Add-on therapies may include one or more existing standard therapies for type 2 diabetes or other hypoglycemic conditions, or new therapies. In some respects, one or more add-on therapies may include, but are not limited to, blood glucose monitoring, dietary modifications, exercise, insulin, thiazolidinediones, sulfonylureas, incretins, metformin, glibenclamides, dipeptidyl peptidase-4 inhibitors, bile acid sequestrants, or any combination thereof.

[0234] sequence SEQ ID NO:1 Formula I HGEGTSESDVSXXXEGQAAQEXXAXXVDGX SEQ ID NO:2 C1.1 HGEGTFTSDVSSYLEGQAAQEFIAWLVDGR SEQ ID NO:3 C1.2 HGEGTSESDVSSYLEGQAAQEFIAWLVDGR SEQ ID NO:4 C1.3 HGEGTSESDVSSQLEGQAAQEFIAWLVDGR SEQ ID NO:5 C1.4 HGEGTSESDVSSQIEGQAAQEFIAWLVDGR SEQ ID NO:6 C1.5 HGEGTSESDVSQSIEGQAAQEFIAWLVDGR SEQ ID NO:7 C1.6 HGEGTSESDVSQSIEGQAAQEVIAWLVDGR SEQ ID NO:8 C1.7 HGEGTSESDVSQSIEGQAAQEIVAWLVDGR SEQ ID NO:9 C1.8 HGEGTSESDVSQSIEGQAAQEIVAIVVDGR SEQ ID NO:10C1.9 HGEGTSESDVSQSIEGQAAQEIVAVIVDGR SEQ ID NO:11C1.10HGEGTSESDVSQSIEGQAAQEVIAVIVDGR SEQ ID NO:12C1.8.1HGEGTSESDVSQSIEGQAAQEIVAIVVDGS SEQ ID NO:13C1.9.1HGEGTSESDVSQSIEGQAAQEIVAVIVDGS SEQ ID NO:14C1.10.1HGEGTSESDVSQSIEGQAAQEVIAVIVDGS SEQ ID NO:15hGLP1RMAGAPGPLRLAVLLLGMVGRAGPRPQGATVSLWETVQKWREYRRQCQRSLTEDPPPATDLFCNRTFDEYACWPDGEPGSFVNVSCPWYLPWASSVPQGHVYRFCTAEGLWLQKDNSSLPWRDLSECEESKRGERSWGEEQLLFLYIIYTVAYALSFSALVIASAILLGFRHLHCTRNYIHLNLFASFILRALSVFIKDAALKWMYSTAAQQHQWDGLLSYQDSLSCRLVFLLMQYCVAANYYWLLVEGVYLYTLLAFSVFSEQWIFRLYVSIGWGVPLLFVVPWGIVKILYEDEGCWTRNSNMNYWLIIRLPILFAIGVNFLIFVRVICIVVSKLKANLMCKTDIKCRLAKSTLTLIPLLGTHEVIFAFVMDEHARGTLRFIKLFTELSFTSFQGLMVAILYCFVNNEVQLEFRKSWERWRLEHLHIQRDSSMKPLKCPTSSLSSGATAGSSMYTATCQASCS SEQ ID NO:16cGLP1RMAGAPCLALLLLGAVGRAGPRPQGATVSLSETVQKWREYRHQCQRFLTEAPPPATGLFCNRTFDEYACWPDGLPGSFVNVSCPWYLPWASSVLQGHVYRFCTAE GLWLRQDNSSLPWRNLSECEESKRGERSSPEEQLLSFSIIYTVGYTLSFSALVIASAILLSFRHLHCTRNYIHLNLFASFILRALSVFIRDAVLKWMYSTAPQQHQWDGLLSYQDSLGC RLVFLLMQYCVAANYYWLLVEGVYLYTLLAFSVFSEQRIFRLYLSIGWGVPLLFVIPWGIVKYLYEDEGCWTRNSNMNYWLIIRLPILFAIGVNFLIFVRVICIVVSKLKANLMCKTD IKCRLAKSTLTLIPLLGTHEVVFAFVMDEHARGTLRFIKLFTELSFTSFQGLMVAILYCFVNNEVQMEFRRSWERWRLKHLHIQRDSSMKPLKCPTSSLTSGGTVGSSVYAASCQASCS SEQ ID NO:17Exendin-4HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS SEQ ID NO:18GLP-1HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR SEQ ID NO:19GLP-1-Gly8HGEGTFTSDVSSYLEGQAAKEFIAWLVKGR SEQ ID NO:20Trp 5xGLP-1HGEGTFTSDVSSYLEGQAAQEFIAWLVDGR SEQ ID NO:21 Liraglutide HAEFTFTSDVSSYLEGQAAKEFIAWLVRGRG (Hexadecanoyl group linked to lysine via glutamate spacer) SEQ ID NO:22 Smegglutinin HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG (K20 conjugated with smegglutinin, PubChem id 90016781) SEQ ID NO:23J211 H A EGS F TSDV SSFLEGEAA K E F IA F VV K GG (K26 is α-K) SEQ ID NO:24J229 H A EGS F TSDV S S F LEGEAAKE F IA F VV K GG (K26 is lipidated) SEQ ID NO:25Medi7219HaEGSfTSDVsSKLEGEAAkEfIAKVVEGG (Lowercase letters are α-methyl, US2018 / 0162920 SEQ ID 263) SEQ ID NO:26GLP1_A8GHGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG SEQ ID NO:27GLP1_K20QHGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG SEQ ID NO:28GLP1_K28DHGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG SEQ ID NO:29Hld_SEMAADIISTIGDLVKWIIDTVNKFKK SEQ ID NO:305xGLP1trp-ABDconMHGEGTFTSDVSSYLEGQAAQEFIAWLVDGRHGEGTFTSDVSSYLEGQAAQEFIAWLVDGRHGEGTFTSDVSSYLEGQAAQEFIAWLVDGRHGEGTFTSDVSSYLEGQAAQEFIAWLVDGRHGEGTFTSDVSSYLEGQAAQEFIAWLVDGRGAPVPYPDPLEPRGGGGSGGGGSGGGGSLKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA

[0235] SEQ ID NO:315xGLP1trpHGEGTFTSDVSSYLEGQAAQEFIAWLVDGRHGEGTFTSDVSSYLEGQAAQEFIAWLVDGRHGEGTFTSDVSSYLEGQAAQEFIAWLVDGRHGEGTFTSDVSSYLEGQAAQEFIAWLVDGRHGEGTFTSDVSSYLEGQAAQEFIAWLVDGRHGEGTFTSDVSSYLEGQAAQEFIAWLVDGRGAPVPYPDPLEPR SEQ ID NO:32>C1.2-2xHGEGTSESDVSSYLEGQAAQEFIAWLVDGXHGEGTSESDVSSYLEGQAAQEFIAWLVDG (X = R or K) SEQ ID NO:33>C1.2-3xHGEGTSESDVSSYLEGQAAQEFIAWLVDGXHGEGTSESDVSSYLEGQAAQEFIAWLVDGXHGEGTSESDVSSYLEGQAAQEFIAWLVDG (X = R or K) SEQ ID NO:34>C1.2-4xHGEGTSESDVSSYLEGQAAQEFIAWLVDGXHGEGTSESDVSSYLEGQAAQEFIAWLVDGXHGEGTSESDVSSYLEGQAAQEFIAWLVDGXHGEGTSESDVSSYLEGQAAQEFIAWLVDG (X =R or K) SEQ ID NO:35>C1.2-5xHGEGTSESDVSSYLEGQAAQEFIAWLVDGXHGEGTSESDVSSYLEGQAAQEFIAWLVDGXHGEGTSESDVSSYLEGQAAQEFIAWLVDGXHGEGTSESDVSSYLEGQAAQEFIAWLVDGXHGEGTSESDVSSYLEGQAAQEFIAWLVDG (X = R or K) SEQ ID NO:36>C1.3-2xHGEGTSESDVSSQLEGQAAQEFIAWLVDGXHGEGTSESDVSSQLEGQAAQEFIAWLVDG (X = R or K) SEQ ID NO:37>C1.3-3xHGEGTSESDVSSQLEGQAAQEFIAWLVDGXHGEGTSESDVSSQLEGQAAQEFIAWLVDGXHGEGTSESDVSSQLEGQAAQEFIAWLVDG (X = R or K) SEQ ID NO:38>C1.3-4xHGEGTSESDVSSQLEGQAAQEFIAWLVDGXHGEGTSESDVSSQLEGQAAQEFIAWLVDGXHGEGTSESDVSSQLEGQAAQEFIAWLVDGXHGEGTSESDVSSQLEGQAAQEFIAWLVDG (X =R or K) SEQ ID NO:39>C1.3-5xHGEGTSESDVSSQLEGQAAQEFIAWLVDGXHGEGTSESDVSSQLEGQAAQEFIAWLVDGXHGEGTSESDVSSQLEGQAAQEFIAWLVDGXHGEGTSESDVSSQLEGQAAQEFIAWLVDGXHGEGTSESDVSSQLEGQAAQEFIAWLVDG (X = R or K) SEQ ID NO:40>C1.4-2xHGEGTSESDVSSQIEGQAAQEFIAWLVDGXHGEGTSESDVSSQIEGQAAQEFIAWLVDG (X = R or K) SEQ ID NO:41>C1.4-3xHGEGTSESDVSSQIEGQAAQEFIAWLVDGXHGEGTSESDVSSQIEGQAAQEFIAWLVDGXHGEGTSESDVSSQIEGQAAQEFIAWLVDG (X = R or K) SEQ ID NO:42>C1.4-4xHGEGTSESDVSSQIEGQAAQEFIAWLVDGXHGEGTSESDVSSQIEGQAAQEFIAWLVDGXHGEGTSESDVSSQIEGQAAQEFIAWLVDGXHGEGTSESDVSSQIEGQAAQEFIAWLVDG (X =R or K) SEQ ID NO:43>C1.4-5xHGEGTSESDVSSQIEGQAAQEFIAWLVDGXHGEGTSESDVSSQIEGQAAQEFIAWLVDGXHGEGTSESDVSSQIEGQAAQEFIAWLVDGXHGEGTSESDVSSQIEGQAAQEFIAWLVDGXHGEGTSESDVSSQIEGQAAQEFIAWLVDG (X = R or K) SEQ ID NO:44>C1.5-2xHGEGTSESDVSQSIEGQAAQEFIAWLVDGXHGEGTSESDVSQSIEGQAAQEFIAWLVDG (X = R or K) SEQ ID NO:45>C1.5-3xHGEGTSESDVSQSIEGQAAQEFIAWLVDGXHGEGTSESDVSQSIEGQAAQEFIAWLVDGXHGEGTSESDVSQSIEGQAAQEFIAWLVDG (X = R or K) SEQ ID NO:46>C1.5-4xHGEGTSESDVSQSIEGQAAQEFIAWLVDGXHGEGTSESDVSQSIEGQAAQEFIAWLVDGXHGEGTSESDVSQSIEGQAAQEFIAWLVDGXHGEGTSESDVSQSIEGQAAQEFIAWLVDG (X = R or K) SEQ ID NO:47>C1.5-5xHGEGTSESDVSQSIEGQAAQEFIAWLVDGXHGEGTSESDVSQSIEGQAAQEFIAWLVDGXHGEGTSESDVSQSIEGQAAQEFIAWLVDGXHGEGTSESDVSQSIEGQAAQEFIAWLVDGXHGEGTSESDVSQSIEGQAAQEFIAWLVDG (X = R or K) SEQ ID NO:48>C1.6-2xHGEGTSESDVSQSIEGQAAQEVIAWLVDGXHGEGTSESDVSQSIEGQAAQEVIAWLVDG (X = R or K) SEQ ID NO:49>C1.6-3xHGEGTSESDVSQSIEGQAAQEVIAWLVDGXHGEGTSESDVSQSIEGQAAQEVIAWLVDGXHGEGTSESDVSQSIEGQAAQEVIAWLVDG (X = R or K) SEQ ID NO:50>C1.6-4xHGEGTSESDVSQSIEGQAAQEVIAWLVDGXHGEGTSESDVSQSIEGQAAQEVIAWLVDGXHGEGTSESDVSQSIEGQAAQEVIAWLVDGXHGEGTSESDVSQSIEGQAAQEVIAWLVDG (X = R or K) SEQ ID NO:51>C1.6-5xHGEGTSESDVSQSIEGQAAQEVIAWLVDGXHGEGTSESDVSQSIEGQAAQEVIAWLVDGXHGEGTSESDVSQSIEGQAAQEVIAWLVDGXHGEGTSESDVSQSIEGQAAQEVIAWLVDGXHGEGTSESDVSQSIEGQAAQEVIAWLVDG (X = R or K) SEQ ID NO:52>C1.7-2xHGEGTSESDVSQSIEGQAAQEIVAWLVDGXHGEGTSESDVSQSIEGQAAQEIVAWLVDG (X = R or K) SEQ ID NO:53>C1.7-3xHGEGTSESDVSQSIEGQAAQEIVAWLVDGXHGEGTSESDVSQSIEGQAAQEIVAWLVDGXHGEGTSESDVSQSIEGQAAQEIVAWLVDG (X = R or K) SEQ ID NO:54>C1.7-4xHGEGTSESDVSQSIEGQAAQEIVAWLVDGXHGEGTSESDVSQSIEGQAAQEIVAWLVDGXHGEGTSESDVSQSIEGQAAQEIVAWLVDGXHGEGTSESDVSQSIEGQAAQEIVAWLVDG (X = R or K) SEQ ID NO:55>C1.7-5xHGEGTSESDVSQSIEGQAAQEIVAWLVDGXHGEGTSESDVSQSIEGQAAQEIVAWLVDGXHGEGTSESDVSQSIEGQAAQEIVAWLVDGXHGEGTSESDVSQSIEGQAAQEIVAWLVDGXHGEGTSESDVSQSIEGQAAQEIVAWLVDG (X = R or K) SEQ ID NO:56>C1.8-2xHGEGTSESDVSQSIEGQAAQEIVAIVVDGXHGEGTSESDVSQSIEGQAAQEIVAIVVDG (X = R or K) SEQ ID NO:57>C1.8-3xHGEGTSESDVSQSIEGQAAQEIVAIVVDGXHGEGTSESDVSQSIEGQAAQEIVAIVVDGXHGEGTSESDVSQSIEGQAAQEIVAIVVDG (X = R or K) SEQ ID NO:58>C1.8-4xHGEGTSESDVSQSIEGQAAQEIVAIVVDGXHGEGTSESDVSQSIEGQAAQEIVAIVVDGXHGEGTSESDVSQSIEGQAAQEIVAIVVDGXHGEGTSESDVSQSIEGQAAQEIVAIVVDG (X =R or K) SEQ ID NO:59>C1.8-5xHGEGTSESDVSQSIEGQAAQEIVAIVVDGXHGEGTSESDVSQSIEGQAAQEIVAIVVDGXHGEGTSESDVSQSIEGQAAQEIVAIVVDGXHGEGTSESDVSQSIEGQAAQEIVAIVVDGXHGEGTSESDVSQSIEGQAAQEIVAIVVDG (X = R or K) SEQ ID NO:60>C1.9-2xHGEGTSESDVSQSIEGQAAQEIVAVIVDGXHGEGTSESDVSQSIEGQAAQEIVAVIVDG (X = R or K) SEQ ID NO:61>C1.9-3xHGEGTSESDVSQSIEGQAAQEIVAVIVDGXHGEGTSESDVSQSIEGQAAQEIVAVIVDGXHGEGTSESDVSQSIEGQAAQEIVAVIVDG (X = R or K) SEQ ID NO:62>C1.9-4xHGEGTSESDVSQSIEGQAAQEIVAVIVDGXHGEGTSESDVSQSIEGQAAQEIVAVIVDGXHGEGTSESDVSQSIEGQAAQEIVAVIVDGXHGEGTSESDVSQSIEGQAAQEIVAVIVDG (X =R or K) SEQ ID NO:63>C1.9-5xHGEGTSESDVSQSIEGQAAQEIVAVIVDGXHGEGTSESDVSQSIEGQAAQEIVAVIVDGXHGEGTSESDVSQSIEGQAAQEIVAVIVDGXHGEGTSESDVSQSIEGQAAQEIVAVIVDGXHGEGTSESDVSQSIEGQAAQEIVAVIVDG (X = R or K) SEQ ID NO:64>C1.10-2xHGEGTSESDVSQSIEGQAAQEVIAVIVDGXHGEGTSESDVSQSIEGQAAQEVIAVIVDG (X = R or K) SEQ ID NO:65>C1.10-3xHGEGTSESDVSQSIEGQAAQEVIAVIVDGXHGEGTSESDVSQSIEGQAAQEVIAVIVDGXHGEGTSESDVSQSIEGQAAQEVIAVIVDG (X = R or K) SEQ ID NO:66>C1.10-4xHGEGTSESDVSQSIEGQAAQEVIAVIVDGXHGEGTSESDVSQSIEGQAAQEVIAVIVDGXHGEGTSESDVSQSIEGQAAQEVIAVIVDGXHGEGTSESDVSQSIEGQAAQEVIAVIVDG (X =R or K) SEQ ID NO:67>C1.10-5xHGEGTSESDVSQSIEGQAAQEVIAVIVDGXHGEGTSESDVSQSIEGQAAQEVIAVIVDGXHGEGTSESDVSQSIEGQAAQEVIAVIVDGXHGEGTSESDVSQSIEGQAAQEVIAVIVDGXHGEGTSESDVSQSIEGQAAQEVIAVIVDG (X = R or K) SEQ ID NO:68E-tagGAPVPYPDPLEPR SEQ ID NO:69G GGGGS GGGGS GGGGS GGGGSA SEQ ID NO:70A PPGGS GGGGS GGGGS GGGGSA SEQ ID NO:71GTGGGGS GGGGS GGGGS GGGGSA SEQ ID NO:72GGGGGS GGGGS GGGGS GGGGSA SEQ ID NO:73GGGGGSA SEQ ID NO:74GGGGGSGGGGSA SEQ ID NO:75GGGGGSGGGGS GGGGSA SEQ ID NO:76G KGGGS GGGGS GGGGS GGGGSA SEQ ID NO:77GGGGGS GGGGS GGGGS GGGGSA SEQ ID NO:78G GGGG GGGG GGGG GGGG A SEQ ID NO:79Alfa-tagSRLEEELRRRLTE SEQ ID NO:80Avi-tagGLNDIFEAQKIEWHE SEQ ID NO:81C-tagEPEA SEQ ID NO:82Calmodulin-tagKRRWKKNFIAVSAANRFKKISSSGAL SEQ ID NO:83DogtagDIPATYEFTDGKHYITNEPIPPK SEQ ID NO:84E-tagGAPVPYPDPLEPR SEQ ID NO:85FLAGDYKDDDDK SEQ ID NO:86G4TEELLSKNYHLENEVARLKK SEQ ID NO:87HAYPYDVPDYA SEQ ID NO:88HisHHHHHH SEQ ID NO:89IsopeptagTDKDMTITFTNKKDAE SEQ ID NO:90MycEQKLISEEDL SEQ ID NO:91NE-TagTKENPRSNQEESYDDNES SEQ ID NO:92Poly Glutamate-tagEEEEEEE SEQ ID NO:93Poly Arginine-tagRRRRRRR SEQ ID NO:94Rho1D4-tagTETSQVAPA SEQ ID NO:95SBP-tagMDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP SEQ ID NO:96SdytagDPIVMIDNDKPIT SEQ ID NO:97SH3STVPVAPPRRRRG SEQ ID NO:98SnooptagKLGDIEFIKVNK SEQ ID NO:99Softag 1SLAELLNAGLGGS SEQ ID NO:100Softag 3TQDPSRVG SEQ ID NO:101Spot-tagPDRVRAVSHWSS SEQ ID NO:102SpytagAHIVMVDAYKPTK SEQ ID NO:103S-tagKETAAAKFERQHMDS SEQ ID NO:104Strep-tagWSHPQFEK SEQ ID NO:105T7tagMASMTGGQQMG SEQ ID NO:106TC-tagEVHTNQDPLD SEQ ID NO:107Ty-tagCCPGCC SEQ ID NO:108VSV-tagYTDIEMNRLGK SEQ ID NO:109Xpress-tagDLYDDDDK SEQ ID NO:110HiBitVSGWRLFKKIS SEQ ID NO:111 Type 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 I; X16 = K, R, H or V; X20 = K, R, H, N; X22 = F, A, P; X25 = W, P, K, H or I; X26 = L or V.

[0236] SEQ ID NO:112 7FIM_V2 / A13YVEGTFTSDYSIALDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO:113 7FIM_V2 / S13YVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO:114 7FIM_V2 / Y13YVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO:115 7FIM_V2 / I13YVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO:116 7FIM_K_R YVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPS SEQ ID NO:117 7FIM_V2 / Q13YVEGTFTSDYSIQLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO:118 7FIM_V2 / L13YVEGTFTSDYSILLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO:119 7FIM_V2 / R13YVEGTFTSDYSIRLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO:120 7FIM_K2 / V13YKEGTFTSDYSIVLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO:1217FIM_K2 / K13YKEGTFTSDYSIKLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO:1227FIM_R_HYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPS SEQ ID NO:123C1YVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPS SEQ ID NO:124C1.1YVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPS SEQ ID NO:125C1.2YVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPS SEQ ID NO:126C1.3YVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:127C1.4YVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:128C1.4.1.1YVEGTPCSDCSISLDHIAQKAPVQPVIAGGPSSGAPPPS SEQ ID NO:129C1.4.1.2YVEGTPCSDCSISLDKIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:130C1.4.1.3YVEGTPCSDCSISLDKIAQKAPVQPVIAGGPSSGAPPPS SEQ ID NO:131C1.4.2.1YVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:132C1.4.2.2YVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPS SEQ ID NO:133C1.4.2.3YVEGTSCSDCSISLDHIAQHAAVQKVIAGGPSSGAPPPS SEQ ID NO:134C1.4.2.4YVEGTSCSDCSISKDHIAQHAAVQKVIAGGPSSGAPPPS SEQ ID NO:135C1.4.2.5YVEGTSCSDCSISKDKIAQHAAVQKVIAGGPSSGAPPPS SEQ ID NO:136C1.4.2.6YVEGTSCSDCSISKDKIAQKAAVQKVIAGGPSSGAPPPS SEQ ID NO:137C1.4.1YVEGTSCSDCSISHDKIAQKAAVQKVIAGGPSSGAPPPS SEQ ID NO:138C1.4.2YVEGTSCSDCSISHDKIAQKAAVQHVIAGGPSSGAPPPS SEQ ID NO:139C1.4.3YVEGTSCSDCSISHDKIAQKAAVQIVIAGGPSSGAPPPS SEQ ID NO:140C1.4.4YVEGTSCSDCSISHDVIAQKAAVQIVIAGGPSSGAPPPS SEQ ID NO:141C1.4.5YVEGTSCSDCSISIDVIAQKAAVQIVIAGGPSSGAPPPS SEQ ID NO:142C1.4.5.1YVEGTSESDCSISIDVIAQKAAVQIVIAGGPSSGAPPPS SEQ ID NO:143C1.4.5.2YVEGTSCSDESISIDVIAQKAAVQIVIAGGPSSGAPPPS SEQ ID NO:144C1.4.5.3YVEGTSESDESISIDVIAQKAAVQIVIAGGPSSGAPPPS SEQ ID NO:145C1.4.5.1.1YVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO:146C1.4.5.2.1YVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO:147C1.4.5.3.1YVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO:1487FIM_V2 / S13_2xYVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO:1497FIM_V2 / S13_3xYVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO:1507FIM_V2 / S13_4xYVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO:1517FIM_V2 / S13_5xYVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSISLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO:1527FIM_V2 / Y13_2xYVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO:1537FIM_V2 / Y13_3xYVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO:1547FIM_V2 / Y13_4xYVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO:1557FIM_V2 / Y13_5xYVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO:1567FIM_V2 / I13_2xYVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO:1577FIM_V2 / I13_3xYVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO:1587FIM_V2 / I13_4xYVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO:1597FIM_V2 / I13_5xYVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPPXYVEGTFTSDYSIILDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO:1607FIM_K_R_2xYVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPS SEQ ID NO:1617FIM_K_R_3xYVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPS SEQ ID NO:1627FIM_K_R_4xYVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPS SEQ ID NO:1637FIM_K_R_5xYVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDRIAQRAFVQWLIAGGPSSGAPPPS SEQ ID NO:1647FIM_V2 / S13 / H16 / H20_2xYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPS SEQ ID NO:1657FIM_V2 / S13 / H16 / H20_3xYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPS SEQ ID NO:1667FIM_V2 / S13 / H16 / H20_4xYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPS SEQ ID NO:1677FIM_V2 / S13 / H16 / H20_5xYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPSXYVEGTFTSDYSISLDHIAQHAFVQWLIAGGPSSGAPPPS SEQ ID NO:168C1_2xYVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPS SEQ ID NO:169C1_3xYVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPS SEQ ID NO:170C1_4xYVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPS SEQ ID NO:171C1_5xYVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDYSISLDHIAQHAPVQWLIAGGPSSGAPPPS SEQ ID NO:172C1.1_2xYVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPS SEQ ID NO:173C1.1_3xYVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPS SEQ ID NO:174C1.1_4xYVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPS SEQ ID NO:175C1.1_5xYVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQWLIAGGPSSGAPPPS SEQ ID NO:176C1.2_2xYVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPS SEQ ID NO:177C1.2_3xYVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPS SEQ ID NO:178C1.2_4xYVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPS SEQ ID NO:179C1.2_5xYVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPLIAGGPSSGAPPPS SEQ ID NO:180C1.3_2xYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:181C1.3_3xYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:182C1.3_4xYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:183C1.3_5xYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPTSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:184C1.4_2xYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:185C1.4_3xYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:186C1.4_4xYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:187C1.4_5xYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTPCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:188C1.4.2.1_2xYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:189C1.4.2.1_3xYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:190C1.4.2.1_4xYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:191C1.4.2.1_5xYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAPVQPVIAGGPSSGAPPPS SEQ ID NO:192C1.4.2.2_2xYVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPS SEQ ID NO:193C1.4.2.2_3xYVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPS SEQ ID NO:194C1.4.2.2_4xYVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPS SEQ ID NO:195C1.4.2.2_5xYVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPSXYVEGTSCSDCSISLDHIAQHAAVQPVIAGGPSSGAPPPS SEQ ID NO:196C1.4.5.1.1_2xYVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO:197C1.4.5.1.1_3xYVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO:198C1.4.5.1.1_4xYVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO:199C1.4.5.1.1_5xYVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDCSISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO:200C1.4.5.2.1_2xYVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO:201C1.4.5.2.1_3xYVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO:202C1.4.5.2.1_4xYVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO:203C1.4.5.2.1_5xYVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSCSDESISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO:204C1.4.5.3.1_2xYVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO:205C1.4.5.3.1_3xYVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO:206C1.4.5.3.1_4xYVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO:207 C1.4.5.3.1_5xYVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPSXYVEGTSESDESISIDVIAQNAAVQIVIAGGPSSGAPPPS SEQ ID NO:208 Tirzepatide (7FIM) YAEGTFTSDYSIALDKIAQKAFVQWLIAGGPSSGAPP SEQ ID NO:209 Peptide_20 HXQGTFTSDKSKYLDERAAQDFVQWLLDGG PSSGAPPPS (X = AIB) SEQ ID NO:210 Peptide_19 YXEGTFTSDYSIYLDKQAAXEFVNWLLAGGPSAPPPSK (X = AIB) SEQ ID NO:211 Semaglutide HXEGTFTSDVSSYLEGQAAKEFIAWLVRGRG SEQ ID NO:212 Exendin-4 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS SEQ ID NO:213 7DTY_P (GIP) YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ SEQ ID NO:214 MEDI7219 HaEGSfTSDVsSKLEGEAAkEfIAKVVEGG (lowercase letters are α-methyl, US_2018_0162920_A1_263) SEQ ID NO:215 J211 HaEGSfTSDVsSfLEGEAAkEfIAfVVkGG (US_2018_0162920_A1_2) SEQ ID NO:216 J229 HaEGSfTSDVsSFLEGEAAkEfIAFVVKGG (US_2018_0162920_A1_2) SEQ ID NO:217 wtGLP-1 haegtftsdvssylegqaakefiawlvkgrg SEQ ID NO:218hGLP1RMAGAPGPLRLAVLLLGMVGRAGPRPQGATVSLWETVQKWREYRRQCQRSLTEDPPPATDLFCNRTFDEYACWPDGEPGSFVNVSCPWYLPWASSVPQGHVYRFCTAEGLWLQKDNSSLPWRDLSECEESKRGERSWGEEQLLFLYIIYTVAYALSFSALVIASAILLGFRHLHCTRNYIHLNLFASFILRALSVFIKDAALKWMYSTAAQQHQWDGLLSYQDSLSCRLVFLLMQYCVAANYYWLLVEGVYLYTLLAFSVFSEQWIFRLYVSIGWGVPLLFVVPWGIVKILYEDEGCWTRNSNMNYWLIIRLPILFAIGVNFLIFVRVICIVVSKLKANLMCKTDIKCRLAKSTLTLIPLLGTHEVIFAFVMDEHARGTLRFIKLFTELSFTSFQGLMVAILYCFVNNEVQLEFRKSWERWRLEHLHIQRDSSMKPLKCPTSSLSSGATAGSSMYTATCQASCS SEQ ID NO:219cGLP1Rmagapsplclallllgavgragprpqgatvslsetvqkwreyrhqcqrflteapppatglfcnrtfdeyacwpdglpgsfvnvscpwylpwassvlqghvyrfctaeglwlrqdnsslpwrnlseceeskrgersspeeqllsfsiiytvgytlsfsalviasaillsfrhlhctrnyihlnlfasfilralsvfirdavlkwmystapqqhqwdgllsyqdslgcrlvfllmqycvaanyywllvegvylytllafsvfseqrifrlylsigwgvpllfvipwgivkylyedegcwtrnsnmnywliirlpilfaigvnflifvrvicivvsklkanlmcktdikcrlakstltlipllgthevvfafvmdehargtlrfiklftelsftsfqglmvailycfvnnevqmefrrswerwrlkhlhiqrdssmkplkcptssltsggtvgssvyaascqascs SEQ ID NO:220hGIPRMTTSPILQLLLRLSLCGLLLQRAETGSKGQTAGELYQRWERYRRECQETLAAAEPPSGLACNGSFDMYVCWDYAAPNATARASCPWYLPWHHHVAAGFVLRQCGSDGQWGLWRDHTQCENPEKNEAFLDQRLILERLQVMYTVGYSLSLATLLLALLILSLFRRLHCTRNYIHINLFTSFMLRAAAILSRDRLLPRPGPYLGDQALALWNQALAACRTAQIVTQYCVGANYTWLLVEGVYLHSLLVLVGGSEEGHFRYYLLLGWGAPALFVIPWVIVRYLYENTQCWERNEVKAIWWIIRTPILMTILINFLIFIRILGILLSKLRTRQMRCRDYRLRLARSTLTLVPLLGVHEVVFAPVTEEQARGALRFAKLGFEIFLSSFQGFLVSVLYCFINKEVQSEIRRGWHHCRLRRSLGEEQRQLPERAFRALPSGSGPGEVPTSRGLSSGTLPGPGNEASRELESYC SEQ ID NO:221cGIPRMPNGPPWQLFLPLLWSWGPLLRRAETGSVGQTAGELYQRWERYRRECRETLEAVDPPAGLACNGSFDMYVCWDYAAPNATARASCPWYLPWHSHVATGFVLRHCGSDGQWGPWRDHSQCEDPEKNGAFQDQRLILERLQVMYTVGYSVSLATLLLALLILSFFRRLRCTRNYIHINLFTSFMLRAAAILTRDRLLPPPGPYPGDQAPVLWKPALAACRTAQIVTQYCVGANYTWLLVEGVYLHSLLVLVGGSEGGHFRCYVFLGWGAPALFVIPWVIVRYLYENTQCWERNEVKAIWWIIRTPILVTISINFLIFIRILGILVSKLRTRQMRCPDYRLRLARSTLTLVPLLGVHEVVFAPVTEEQARGALRFAKLGFEIFLSSFQGFLVGVVYCFVNKEVQAEIRRCWHRCRLRHSLGEERRQPPERASRTPPTGSGPRPVATDRTPSLGALPGPGNEASRGLESHC SEQ ID NO:222rGIPRMPLRLLLLLLWLWGLSLQRAETDSEGQTTGELYQRWERYGWECQNTLEATEPPSGLACNGSFDMYACWNYTAANTTARVSCPWYLPWYRQVAAGFVFRQCGSDGQWGSWRDHTQCENPEKNGAFQDQKLILERLQVVYTVGYSLSLATLLLALLILSLFRRLHCTRNYIHMNLFTSFMLRAGAILTRDQLLPPLGPYTGNQTPTLWNQALAACRTAQILTQYCVGANYTWLLVEGVYLHHLLVVVRRSEKGHFRCYLLLGWGAPALFVIPWVIVRYLYENTQCWERNEVKAIWWIIRTPILITILINFLIFIRILGILVSKLRTRQMRCPDYRLRLARSTLTLMPLLGVHEVVFAPVTEEQAEGSLRFAKLAFEIFLSSFQGFLVSVLYCFINKEVQSEIRRLRLSLQEQCPRPHLGQAPRAVPLSSAPQEAAIRNALPSGMLHVPGDEVLESYC SEQ ID NO:223mGIPRMPLRLLLLLLWLWGLQWAETDSEGQTTTGELYQRWEHYGQECQKMLETTEPPSGLACNGSFDMYACWNYTAANTTARVSCPWYLPWFRQVSAGFVFRQCGSDGQWGSWRDHTQCENPEKNGAFQDQTLILERLQIMYTVGYSLSLTTLLLALLILSLFRRLHCTRNYIHMNLFTSFMLRAAAILTRDQLLPPLGPYTGDQAPTPWNQALAACRTAQIMTQYCVGANYTWLLVEGVYLHHLLVIVGRSEKGHFRCYLLLGWGAPALFVIPWVIVRYLRENTQCWERNEVKAIWWIIRTPILITILINFLIFIRILGILVSKLRTRQMRCPDYRLRLARSTLTLVPLLGVHEVVFAPVTEEQVEGSLRFAKLAFEIFLSSFQGFLVSVLYCFINKEVQSEIRQGWRHRRLRLSLQEQRPRPHQELAPRAVPLSSACREAAVGNALPSGMLHVPGDEVLESYC SEQ ID NO:224GGSS SEQ ID NO:225GGSS GGSS SEQ ID NO:226GGSS GGSS GGSS SEQ ID NO:227GGSS GGSS GGSS GGSS SEQ ID NO:228GGSS GGSS GGSS GGSS GGSS SEQ ID NO:229GGSS GGSS GGSS GGSS GGSS GGSS SEQ ID NO:230GGSS GGSS GGSS GGSS GGSS GGSS GGSS SEQ ID NO:231GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS SEQ ID NO:232GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS SEQ ID NO:233GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS GGSS SEQ ID NO:234GSGGS SEQ ID NO:235GSGGS GSGGS SEQ ID NO:236GSGGS GSGGS GSGGS SEQ ID NO:237GSGGS GSGGS GSGGS GSGGS SEQ ID NO:238GSGGS GSGGS GSGGS GSGGS GSGGS SEQ ID NO:239GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS SEQ ID NO:240GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS SEQ ID NO:241GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS SEQ ID NO:242GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS SEQ ID NO:243GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGS GSGGSGSGGS SEQ ID NO:244GGGS SEQ ID NO:245GGGS GGGS SEQ ID NO:246GGGS GGGS GGGS SEQ ID NO:247GGGS GGGS GGGS GGGS SEQ ID NO:248GGGS GGGS GGGS GGGS GGGS SEQ ID NO:249GGGS GGGS GGGS GGGS GGGS GGGS SEQ ID NO:250GGGS GGGS GGGS GGGS GGGS GGGS GGGS SEQ ID NO:251GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS SEQ ID NO:252GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS SEQ ID NO:253GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS GGGS SEQ ID NO:254GGSG SEQ ID NO:255GGSG GGSG SEQ ID NO:256GGSG GGSG GGSG SEQ ID NO:257GGSG GGSG GGSG GGSG SEQ ID NO:258GGSG GGSG GGSG GGSG GGSG SEQ ID NO:259GGSG GGSG GGSG GGSG GGSG GGSG SEQ ID NO:260GGSG GGSG GGSG GGSG GGSG GGSG GGSG SEQ ID NO:261GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG SEQ ID NO:262GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG SEQ ID NO:263GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG GGSG SEQ ID NO:264GGSGG SEQ ID NO:265GGSGG GGSGG SEQ ID NO:266GGSGG GGSGG GGSGG SEQ ID NO:267GGSGG GGSGG GGSGG GGSGG SEQ ID NO:268GGSGG GGSGG GGSGG GGSGG GGSGG SEQ ID NO:269GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG SEQ ID NO:270GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG SEQ ID NO:271GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG SEQ ID NO:272GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG SEQ ID NO:273GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGG GGSGGGGSGG SEQ ID NO:274GSGSG SEQ ID NO:275GSGSG GSGSG SEQ ID NO:276GSGSG GSGSG GSGSG SEQ ID NO:277GSGSG GSGSG GSGSG GSGSG SEQ ID NO:278GSGSG GSGSG GSGSG GSGSG GSGSG SEQ ID NO:279GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG SEQ ID NO:280GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG SEQ ID NO:281GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG SEQ ID NO:282GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG SEQ ID NO:283GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSG GSGSGGSGSG SEQ ID NO:284GSGGG SEQ ID NO:285GSGGG GSGGG SEQ ID NO:286GSGGG GSGGG GSGGG SEQ ID NO:287GSGGG GSGGG GSGGG GSGGG SEQ ID NO:288GSGGG GSGGG GSGGG GSGGG GSGGG SEQ ID NO:289GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG SEQ ID NO:290GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG SEQ ID NO:291GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG SEQ ID NO:292GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG SEQ ID NO:293GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGG GSGGGGSGGG SEQ ID NO:294GGGSG SEQ ID NO:295GGGSG GGGSG SEQ ID NO:296GGGSG GGGSG GGGSG SEQ ID NO:297GGGSG GGGSG GGGSG GGGSG SEQ ID NO:298GGGSG GGGSG GGGSG GGGSG GGGSG SEQ ID NO:299GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG SEQ ID NO:300GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG SEQ ID NO:301GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG SEQ ID NO:302GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG SEQ ID NO:303GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSG GGGSGGGGSG SEQ ID NO:304GSSSG SEQ ID NO:305GSSSG GSSSG SEQ ID NO:306GSSSG GSSSG GSSSG SEQ ID NO:307GSSSG GSSSG GSSSG GSSSG SEQ ID NO:308GSSSG GSSSG GSSSG GSSSG GSSSG SEQ ID NO:309GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG SEQ ID NO:310GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG SEQ ID NO:311GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG SEQ ID NO:312GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG SEQ ID NO:313GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSG GSSSGGSSSG SEQ ID NO:314GSSSS SEQ ID NO:315GSSSS GSSSS SEQ ID NO:316GSSSS GSSSS GSSSS SEQ ID NO:317GSSSS GSSSS GSSSS GSSSS SEQ ID NO:318GSSSS GSSSS GSSSS GSSSS GSSSS SEQ ID NO:319GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS SEQ ID NO:320GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS SEQ ID NO:321GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS SEQ ID NO:322GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS SEQ ID NO:323GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSS GSSSSGSSSS SEQ ID NO:324GGGGS SEQ ID NO:325GGGGS GGGGS SEQ ID NO:326GGGGS GGGGS GGGGS SEQ ID NO:327GGGGS GGGGS GGGGS GGGGS SEQ ID NO:328GGGGS GGGGS GGGGS GGGGS GGGGS SEQ ID NO:329GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS SEQ ID NO:330GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS SEQ ID NO:331GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS SEQ ID NO:332GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS SEQ ID NO:333AAAGG SEQ ID NO:334AAAGG AAAGG SEQ ID NO:335AAAGG AAAGG AAAGG SEQ ID NO:336AAAGG AAAGG AAAGG AAAGG SEQ ID NO:337AAAGG AAAGG AAAGG AAAGG AAAGG SEQ ID NO:338AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG SEQ ID NO:339AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG SEQ ID NO:340AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG SEQ ID NO:341AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG SEQ ID NO:342AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGG AAAGGAAAGG SEQ ID NO:343GGSAAAGG SEQ ID NO:344GGSAAAGG GGSAAAGG SEQ ID NO:345GGSAAAGG GGSAAAGG GGSAAAGG SEQ ID NO:346GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG SEQ ID NO:347GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG SEQ ID NO:348GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG SEQ ID NO:349GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGGGGSAAAGG SEQ ID NO:350GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGGGGSAAAGG GGSAAAGG SEQ ID NO:351GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGGGGSAAAGG GGSAAAGG GGSAAAGG SEQ ID NO:352GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGGGGSAAAGG GGSAAAGG GGSAAAGG GGSAAAGG SEQ ID NO:353DPIVMIDNDKPITSdytag SEQ ID NO:354STVPVAPPRRRRGSH3 SEQ ID NO:355KLGDIEFIKVNKSnooptag SEQ ID NO:356SLAELLNAGLGGSSoftag 1 SEQ ID NO:357TQDPSRVGSoftag 3 SEQ ID NO:358PDRVRAVSHWSSSpot-tag SEQ ID NO:359AHIVMVDAYKPTKSpytag SEQ ID NO:360KETAAAKFERQHMDSS-tag SEQ ID NO:361WSHPQFEKStrep-tag SEQ ID NO:362MASMTGGQQMGT7tag SEQ ID NO:363EVHTNQDPLDTC-tag SEQ ID NO:364CCPGCCTy-tag SEQ ID NO:365YTDIEMNRLGKVSV-tag SEQ ID NO:366DLYDDDDKXpress-tag SEQ ID NO:367VSGWRLFKKISHiBit SEQ ID NO:368YAEGTFISDYSIAMDKIRQQDFVNWLLAQKGKKNDWK Implementation plan for engineered glucagon-like peptide-1: 1. An engineered glucagon-like peptide-1 (eGLP-1) comprising the following amino acid sequence: Formula I (SEQ ID NO:1): HGEGTSESDVSX 12 X 13 X 14 EGQAAQEX 22 X 23 AX 25 X 26 VDGX 30 (I) Where X 12 = S or Q; X 12 = S, Q, or Y; X 14 = I or L; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 =I or V; and X 30 = R or S, Or Formula III (SEQ ID NO:382): X1G EGTSESDVSX 12 X 13 X 14 EX 16 QAX 19 X 20 EX 22 X 23 AX 25 X 26 VDGX 30 (III) Where X1 = H or Y; X 12 = S, Q, A, or M; X 13 = S, Q, Y, or M; X 14 = I or L; X16 = G or M; X 19 = A or T; X 20 = Q, V, or D; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 = I or V; and X 30 = R or S.

[0237] 2. Implementation of eGLP-1 according to scheme 1, wherein X 12 = Q;X 13 = S;X 14 = I or L; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 = I or V; and X 30 = R.

[0238] 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 SEQ ID NO:372 to SEQ ID NO:380.

[0239] 4. The eGLP-1 polypeptide of embodiment 1, wherein the polypeptide comprises two, three, four, or five copies of the eGLP-1 polypeptide of formula I or formula III.

[0240] 5. The eGLP-1 polypeptide of embodiment 4, wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NO:32 to SEQ ID NO:67.

[0241] 6. The eGLP-1 of any one of embodiments 1 to 5, wherein the eGLP-1 comprises a C-terminal amide.

[0242] 7. The eGLP-1 of any one of embodiments 1 to 6, wherein the eGLP-1 comprises one or more α-methyl amino acids.

[0243] 8. The eGLP-1 of any one of embodiments 1 to 7, wherein the eGLP-1 further comprises a lipid moiety covalently bonded to an amino terminus, a carboxyl terminus, or an amino acid of formula I.

[0244] 9. The eGLP-1 of any one of embodiments 1 to 8, wherein the eGLP-1 has substantial resistance to protein hydrolytic degradation.

[0245] 10. The eGLP-1 of embodiment 9, wherein the eGLP-1 has substantial resistance to degradation by DPP-IV, enkephalinase, α-chymotrypsin, trypsin, elastase or pepsin.

[0246] 11. The eGLP-1 of embodiment 9, wherein the eGLP-1 has substantial resistance to degradation by DPP-IV and enkephalinase.

[0247] 12. The eGLP-1 of embodiment 9, wherein the eGLP-1 has substantial resistance to degradation by α-chymotrypsin, trypsin, elastase or pepsin.

[0248] 13. The eGLP-1 of any one of embodiments 1 to 13, wherein the eGLP-1 retains at least the same receptor potency as the corresponding wild-type unmodified GLP-1.

[0249] 14. The eGLP-1 of any one of embodiments 1 to 14, wherein the eGLP-1 retains at least the same receptor selectivity as the corresponding wild-type unmodified GLP-1.

[0250] 15. The eGLP-1 of any one of embodiments 1 to 14, wherein the eGLP-1 exhibits enhanced receptor potency compared to the corresponding wild-type unmodified GLP-1.

[0251] Polynucleotides 16. A polynucleotide encoding engineered glucagon-like peptide-1 (eGLP-1), said engineered glucagon-like peptide-1 (eGLP-1) comprising the following amino acid sequence: Formula I (SEQ ID NO:1): HGEGTSESDVSX 12 X 13 X 14 EGQAAQEX 22 X 23 AX 25 X 26 VDGX 30 (I) Where X 12 = S or Q; X 12 = S, Q, or Y; X 14 = I or L; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 =I or V; and X 30= R or S, Or Formula III (SEQ ID NO:382): X1G EGTSESDVSX 12 X 13 X 14 EX 16 QAX 19 X 20 EX 22 X 23 AX 25 X 26 VDGX 30 (III) Where X1 = H or Y; X 12 = S, Q, A, or M; X 13 = S, Q, Y, or M; X 14 = I or L; X 16 = G or M; X 19 = A or T; X 20 = Q, V, or D; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 = I or V; and X 30 = R or S.

[0252] 17. The polynucleotide of embodiment 16, wherein the polynucleotide is isolated.

[0253] 18. An engineered vector comprising a polynucleotide encoding engineered glucagon-like peptide-1 (eGLP-1), said engineered glucagon-like peptide-1 (eGLP-1) comprising the following amino acid sequence: Formula I: HGEGTSESDVSX 12 X 13 X 14 EGQAAQEX 22 X 23 AX 25 X 26 VDGX 30 (I) Where X 12 = S or Q; X 12 = S, Q, or Y; X 14 = I or L; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X26 =I or V; and X 30 = R or S, Or Formula III (SEQ ID NO:382): X1G EGTSESDVSX 12 X 13 X 14 EX 16 QAX 19 X 20 EX 22 X 23 AX 25 X 26 VDGX 30 (III) Where X1 = H or Y; X 12 = S, Q, A, or M; X 13 = S, Q, Y, or M; X 14 = I or L; X 16 = G or M; X 19 = A or T; X 20 = Q, V, or D; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 = I or V; and X 30 = R or S.

[0254] 19. A nucleic acid expression cassette comprising one or more of the following: Nucleic acid sequences containing transcription promoters; Nucleic acid sequences encoding polypeptides containing the following amino acid sequence: Formula I HGEGTSESDVSX 12 X 13 X 14 EGQAAQEX 22 X 23 AX 25 X 26 VDGX 30 (I) Where X 12 = S or Q; X 12 = S, Q, or Y; X 14 = I or L; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 =I or V; and X30 = R or S, Or Formula III (SEQ ID NO:382): X1G EGTSESDVSX 12 X 13 X 14 EX 16 QAX 19 X 20 EX 22 X 23 AX 25 X 26 VDGX 30 (III) Where X1 = H or Y; X 12 = S, Q, A, or M; X 13 = S, Q, Y, or M; X 14 = I or L; X 16 = G or M; X 19 = A or T; X 20 = Q, V, or D; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 = I or V; and X 30 = R or S; Nucleic acid sequences containing translation terminators; and Nucleic acid sequences containing transcription terminators.

[0255] 20. The nucleic acid expression cassette of embodiment 19, wherein the polypeptide further comprises one or more in-frame amino acid sequences, the in-frame amino acid sequences comprising a secretion signal sequence, a peptide tag, or a combination thereof.

[0256] 21. A method for preparing engineered glucagon-like peptide-1 (eGLP-1) polypeptide, wherein the engineered glucagon-like peptide-1 (eGLP-1) polypeptide comprises the following amino acid sequence: Formula I HGEGTSESDVSX 12 X 13 X 14 EGQAAQEX 22 X 23 AX 25 X 26 VDGX 30 (I) Where X 12 = S or Q; X 12= S, Q, or Y; X 14 = I or L; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 =I or V; and X 30 = R or S, Or Formula III (SEQ ID NO:382): X1G EGTSESDVSX 12 X 13 X 14 EX 16 QAX 19 X 20 EX 22 X 23 AX 25 X 26 VDGX 30 (III) Where X1 = H or Y; X 12 = S, Q, A, or M; X 13 = S, Q, Y, or M; X 14 = I or L; X 16 = G or M; X 19 = A or T; X 20 = Q, V, or D; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 = I or V; and X 30 = R or S; The method includes culturing host cells transformed with an expression vector encoding the eGLP-1 peptide under conditions that allow expression of the eGLP-1 peptide, and recovering the eGLP-1 peptide.

[0257] 22. A pharmaceutical composition comprising: an engineered glucagon-like peptide-1 (eGLP-1) polypeptide, said engineered glucagon-like peptide-1 (eGLP-1) polypeptide comprising the following amino acid sequence: Formula I: HGEGTSESDVSX 12 X 13 X 14 EGQAAQEX 22 X 23 AX 25 X 26 VDGX 30 (I) Where X 12 = S or Q; X 12 = S, Q, or Y; X 14 = I or L; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 =I or V; and X 30 = R or S; Or Formula III (SEQ ID NO:382): X1G EGTSESDVSX 12 X 13 X 14 EX 16 QAX 19 X 20 EX 22 X 23 AX 25 X 26 VDGX 30 (III) Where X1 = H or Y; X 12 = S, Q, A, or M; X 13 = S, Q, Y, or M; X 14 = I or L; X 16 = G or M; X 19 = A or T; X 20 = Q, V, or D; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 = I or V; and X 30 = R or S; And the carrier.

[0258] 23. A pharmaceutical composition comprising a recombinant host cell containing a polynucleotide encoding engineered glucagon-like peptide-1 (eGLP-1).

[0259] 24. The pharmaceutical composition of embodiment 23, further comprising a carrier.

[0260] 25. A host cell comprising a polynucleotide encoding engineered glucagon-like peptide-1 (eGLP-1), said engineered glucagon-like peptide-1 (eGLP-1) comprising the following amino acid sequence: Formula I: HGEGTSESDVSX12 X 13 X 14 EGQAAQEX 22 X 23 AX 25 X 26 VDGX 30 (I) Where X 12 = S or Q; X 12 = S, Q, or Y; X 14 = I or L; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 =I or V; and X 30 = R or S, Or Formula III (SEQ ID NO:382): X1G EGTSESDVSX 12 X 13 X 14 EX 16 QAX 19 X 20 EX 22 X 23 AX 25 X 26 VDGX 30 (III) Where X1 = H or Y; X 12 = S, Q, A, or M; X 13 = S, Q, Y, or M; X 14 = I or L; X 16 = G or M; X 19 = A or T; X 20 = Q, V, or D; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 = I or V; and X 30 = R or S.

[0261] 26. A host cell comprising a vector containing a polynucleotide encoding engineered glucagon-like peptide-1 (eGLP-1), said engineered glucagon-like peptide-1 (eGLP-1) comprising an amino acid sequence of the following formula: Formula I: HGEGTSESDVSX 12 X13 X 14 EGQAAQEX 22 X 23 AX 25 X 26 VDGX 30 (I) Where X 12 = S or Q; X 12 = S, Q, or Y; X 14 = I or L; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 =I or V; and X 30 = R or S, Or Formula III (SEQ ID NO:382): X1G EGTSESDVSX 12 X 13 X 14 EX 16 QAX 19 X 20 EX 22 X 23 AX 25 X 26 VDGX 30 (III) Where X1 = H or Y; X 12 = S, Q, A, or M; X 13 = S, Q, Y, or M; X 14 = I or L; X 16 = G or M; X 19 = A or T; X 20 = Q, V, or D; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 = I or V; and X 30 = R or S.

[0262] 27. A transformed cell comprising a nucleic acid expression cassette, said nucleic acid expression cassette comprising one or more of the following: Nucleic acid sequences containing transcription promoters; Nucleic acid sequences encoding polypeptides containing the following amino acid sequence: Formula I: HGEGTSESDVSX 12 X13 X 14 EGQAAQEX 22 X 23 AX 25 X 26 VDGX 30 (I) Where X 12 = S or Q; X 12 = S, Q, or Y; X 14 = I or L; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 =I or V; and X 30 = R or S, Or Formula III (SEQ ID NO:382): X1G EGTSESDVSX 12 X 13 X 14 EX 16 QAX 19 X 20 EX 22 X 23 AX 25 X 26 VDGX 30 (III) Where X1 = H or Y; X 12 = S, Q, A, or M; X 13 = S, Q, Y, or M; X 14 = I or L; X 16 = G or M; X 19 = A or T; X 20 = Q, V, or D; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 = I or V; and X 30 = R or S; Nucleic acid sequences containing translation terminators; and Nucleic acid sequences containing transcription terminators.

[0263] 28. The transformed cell of embodiment 27, wherein the cell is a bacterial cell, plant cell, yeast cell, or algal cell.

[0264] 29. The transformed cells of embodiment 26 or 28, wherein the cells contain two or more copies of the recombinant nucleic acid.

[0265] 30. A transformed cell according to embodiment 28 or any one of 28, wherein the bacterial cell comprises integrating the nucleic acid expression cassette into the bacterial chromosome.

[0266] 31. The transformed cell of embodiment 30, wherein the integration into the bacterial chromosome is integration into the transposase locus.

[0267] 32. The transformed cells of embodiment 28, wherein the cells are yeast cells.

[0268] 33. The transformed cells of embodiment 32, wherein the yeast cells are Pichia pastoris (… Pichia pastoris ) cells of the strain.

[0269] 34. The transformed cells of embodiment 28, wherein the cells are selected from Bacillus spp. ( Bacillus Lactobacillus ( ) Lactobacillus Salmonella ( Salmonella Lactococcus spp. Lactococcus ), Enterococcus spp. Enterococcus Bacterial cells of the genus Bacteria, which are grouped into groups of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 ...

[0270] 35. The transformed cell of embodiment 28, wherein the transformed cell is a plant cell.

[0271] 36. The transformed cell of embodiment 36, wherein the recombinant nucleic acid is integrated into the chloroplasts of the plant cell.

[0272] 37. The transformed cell of embodiment 36, wherein the recombinant nucleic acid is integrated into the genome of the plant cell.

[0273] 38. A genetically engineered plant or a portion thereof, comprising a recombinant nucleic acid encoding engineered glucagon-like peptide-1 (eGLP-1) according to embodiment 1.

[0274] 39. The genetically engineered plant or a portion thereof of embodiment 38, wherein the portion thereof is a plant seed.

[0275] 40. The genetically engineered plant or a portion thereof according to embodiment 38, wherein the recombinant nucleic acid is integrated into the chloroplasts of the plant or the portion thereof.

[0276] 41. The genetically engineered plant or a portion thereof according to embodiment 38, wherein the recombinant nucleic acid is integrated into the genome of the plant cell.

[0277] 42. A kit comprising a pharmaceutical composition according to any one of embodiments 22 to 41.

[0278] 43. The kit according to 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 a portion thereof of any one of embodiments 38 to 41, and a vector.

[0279] 44. A method for treating or preventing conditions caused by or characterized by overweight in a subject, and for treating obesity, morbid obesity, obesity-related inflammation, obesity-related gallbladder disease, obesity-induced sleep apnea, metabolic syndrome, prediabetes, insulin resistance, glucose intolerance, type 2 diabetes, type 1 diabetes, hypertension, atherosclerotic dyslipidemia, atherosclerosis, arteriosclerosis, coronary artery disease, peripheral artery disease, stroke, or microvascular disease, said method comprising administering to the subject requiring treatment an effective amount of eGLP-1 of any one of embodiments 1 to 15, the pharmaceutical composition of embodiment 22, or the host cell of embodiment 25.

[0280] 45. A method of treating or preventing a disease or condition caused by or characterized by hypoglycemia or impaired insulin release, comprising administering to a subject in need of treatment an effective amount of any one of embodiments 1 to 15 of eGLP-1, the pharmaceutical composition of embodiment 22, or the host cell of embodiment 25.

[0281] 46. ​​The method of implementation scheme 44 or 45, wherein the disease or condition is diabetes or obesity.

[0282] 47. The method of implementation scheme 46, wherein the disease or condition is type 2 diabetes.

[0283] 48. The method of any one of embodiments 44 to 47, wherein the administration further improves glycemic control, provides weight control, improves β-cell function and mass, reduces gastric acid secretion and gastric emptying rate, or any combination thereof.

[0284] 49. The method of any one of embodiments 44 to 48, wherein the eGLP-1 or the pharmaceutical composition is administered orally, by injection or transdermally.

[0285] 50. The method of any one of embodiments 44 to 48, wherein the host cell is administered orally.

[0286] 51. The method of any one of embodiments 44 to 47, wherein the eGLP-1 or a pharmaceutical composition thereof is administered orally.

[0287] 52. The method of embodiment 49, wherein the injection is administered subcutaneously or intravenously.

[0288] 53. The method of any one of embodiments 44 to 52, wherein the eGLP-1 or the pharmaceutical composition is administered once daily.

[0289] 54. The method of any one of implementation schemes 44 to 53, further comprising the application of one or more additional therapies.

[0290] 55. The method of implementation scheme 54, wherein the additional therapy includes blood glucose monitoring, dietary modification, exercise, insulin, thiazolidinediones, sulfonylureas, incretins, metformin, glibenclamides, dipeptidyl peptidase-4 inhibitors, bile acid sequestrants, or any combination thereof.

[0291] 56. The method of any one of implementation schemes 44 to 55, wherein the subject is a human, a non-human primate, a cat, or a dog.

[0292] 57. The method of any one of implementation schemes 44 to 56, wherein the subject is a cat.

[0293] 58. A pharmaceutical composition comprising engineered glucagon-like peptide-1 of the following formula: Formula I: HGEGTSESDVSX 12 X 13 X 14 EGQAAQEX 22 X 23 AX 25 X 26 VDGX 30 (I) Where X 12 = S or Q; X 12 = S, Q, or Y; X 14 = I or L; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 =I or V; and X 30 = R or S, Or Formula III (SEQ ID NO:382): X1G EGTSESDVSX 12 X 13 X 14 EX 16 QAX 19 X 20 EX 22 X 23 AX 25 X26 VDGX 30 (III) Where X1 = H or Y; X 12 = S, Q, A, or M; X 13 = S, Q, Y, or M; X 14 = I or L; X 16 = G or M; X 19 = A or T; X 20 = Q, V, or D; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 = I or V; and X 30 = R or S.

[0294] 59. A pharmaceutical composition comprising engineered glucagon-like peptide-1 (eGLP-1), said engineered glucagon-like peptide-1 (eGLP-1) comprising an amino acid sequence selected from any one of SEQ ID NO:1 to SEQ ID NO:14.

[0295] 60. The pharmaceutical composition of embodiment 58 or 59, wherein the composition is a lyophilized composition of transformed plant cells or transformed bacterial cells.

[0296] 61. A direct-feed microorganism (DFM) comprising bacteria transformed with nucleic acid encoding engineered glucagon-like peptide-1 (eGLP-1), said engineered glucagon-like peptide-1 (eGLP-1) comprising the following amino acid sequence: Formula I: HGEGTSESDVSX 12 X 13 X 14 EGQAAQEX 22 X 23 AX 25 X 26 VDGX 30 (I) Where X 12 = S or Q; X 12 = S, Q, or Y; X 14 = I or L; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 =I or V; and X 30 = R or S, Or Formula III (SEQ ID NO:382): X1G EGTSESDVSX 12 X 13 X 14 EX 16 QAX 19 X 20 EX 22 X 23 AX 25 X 26 VDGX 30 (III) Where X1 = H or Y; X 12 = S, Q, A, or M; X 13 = S, Q, Y, or M; X 14 = I or L; X 16 = G or M; X 19 = A or T; X 20 = Q, V, or D; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 = I or V; and X 30 = R or S.

[0297] 62. The direct feeding microorganism (DFM) of embodiment 61, wherein the eGLP-1 polypeptide comprises an amino acid sequence selected from any one of SEQ ID NO:1 to SEQ ID NO:14, SEQ ID NO:32 to SEQ ID NO:67, or SEQ ID NO:372 to SEQ ID NO:380.

[0298] 63. The direct feeding microorganism (DFM) of embodiment 61, wherein the eGLP-1 polypeptide comprises an amino acid sequence selected from any one of SEQ ID NO: 2 to 14.

[0299] 64. The direct feeding microorganism (DFM) of embodiment 61, wherein the eGLP-1 polypeptide comprises an amino acid sequence selected from any one of SEQ ID NO: 3 to 14.

[0300] 65. The DFM of any one of embodiments 61 to 64, wherein the bacteria comprises strains selected from the group consisting of Bacillus, Lactobacillus, Lactococcus, Salmonella, Enterococcus, and combinations thereof.

[0301] 66. The DFM of embodiment 65, wherein the transforming bacteria is Bacillus subtilis ( Bacillus subtilis ) strain.

[0302] 67. The DFM of embodiment 66, wherein the transformed Bacillus subtilis strain and the DFM further comprise one or more Bacillus amyloliquefaciens strains (…). B. amyloliquefaciens ) strain.

[0303] 68. The DFM of embodiment 66, wherein the transformed Bacillus subtilis bacterial strain and the DFM further comprise one or more anaerobic cellulose-decomposing bacteria.

[0304] 69. The DFM of embodiment 68, wherein one or more anaerobic cellulose-decomposing bacteria are members of the phylum Bacillota.

[0305] 70. The DFM of embodiment 69, wherein the Bacillus phylum member is *Ruminococcus flavinatus* (… Ruminococcus flavefaciens ) bacterial strains.

[0306] 71. The DFM of embodiment 68, wherein one or more anaerobic cellulose-decomposing bacteria are Bacteroides ( Bacteroides )member.

[0307] 72. The DFM of embodiment 71, wherein the Bacteroides member is Bacteroides fragilis ( Bacteroides fragilis ).

[0308] 73. The DFM of embodiment 63, wherein the bacterial strain is Lactobacillus ( ). Lactobacillus ) strain.

[0309] 74. The DFM of embodiment 73, wherein the lactobacillus strain is Lactobacillus gasseri ( Lactobacillus gasseri ) strain.

[0310] 75. The DFM of embodiment 73, wherein the Lactobacillus strain is Lactobacillus plantarum ( Lactobacillus plantarum ) strain.

[0311] 76. The DFM of any one of embodiments 61 to 75, wherein the DFM is freeze-dried.

[0312] Implementation plan for engineered dual-agonist peptides: Implementation Scheme 1. An engineered polypeptide comprising the following amino acid sequence: Formula II (SEQ ID NO:111): Y X2E GT X6X7S DX 10 SIX13 X 14 DX 16 IAQX 20 AX 22 VQX 25 X 26 IAGGPSS GA PP(I) in X2 = V or K; [Telbopeptide = A] X6 = F, P, or S; [Telborpeptide = F] X7 = T, C, or E; [Telborpeptide = T] X 10 = Y, C, or E; [Telbopeptide = Y] X 13 = A, S, Y, N, I, L, R, V, or K; [Telbopeptide = A] X 14 = L, K, H, or I; [Telbopeptide = L] X 16 = K, R, H, or V; [Telbopeptide = K] X 20 = K, R, H, N; [Telbopeptide = K] X 22 = F, A, P; [Telbopeptide = F] X 25 = W, P, K, H, or I; and [Telbopeptide = W] X 26 = L or V, Or formula IV (SEQ ID NO:383): Y X2E GT X6X7S DX 10 SIX 13 X 14 DX 16 IAX 19 X 20 AX 22 VQX 25 X 26 IAGGPSS GA PP (IV). Where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 = L, K, H, or I; X 16 = K, R, H, or V; X19 = Q or V; X 20 = K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V.

[0313] Implementation Scheme 2. The engineered peptide of Implementation Scheme 1, wherein X2 = V; X6 = P or S; X7 = C or E; X 10 = C or E; X 13 = S;X 14 = L, H, or I; X 16 = H or V; X 20 = H or N; X 22 = A or P; X 25 = P or I; and X 26 = L or V.

[0314] Implementation Scheme 3. The engineered peptide of Implementation Scheme 1, wherein X2 = V; X6 = S; X7 = C; X 10 = C or E; X 13 = S;X 14 = I;X 16 = V;X 20 = H or N; X 22 = A;X 25 = I; and X 26 = V.

[0315] Implementation Scheme 4. The engineered peptide of Implementation Scheme 1, which further contains proline at position 38.

[0316] Implementation Scheme 5. The engineered peptide of Implementation Scheme 4, which further contains serine at position 39.

[0317] Implementation Scheme 6. The engineered polypeptide of Implementation Scheme 1, comprising the amino acid sequence of any one of SEQ ID NO:111 to SEQ ID NO:147 or SEQ ID NO:381.

[0318] Implementation Scheme 7. The engineered polypeptide of Implementation Scheme 6, wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NO:113 to SEQ ID NO:116.

[0319] Implementation Scheme 8. The engineered polypeptide of Implementation Scheme 6, wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NO:142 to SEQ ID NO:144.

[0320] Implementation Scheme 9. The engineered polypeptide of Implementation Scheme 6, wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NO:137 to SEQ ID NO:141.

[0321] Implementation Scheme 10. The engineered polypeptide of Implementation Scheme 6, wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NO:127 to SEQ ID NO:136.

[0322] Implementation Scheme 11. The engineered polypeptide of Implementation Scheme 6, wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NO:123 to SEQ ID NO:127.

[0323] Implementation Scheme 12. An engineered polypeptide of any one of Implementation Schemes 1 to 11, wherein the engineered polypeptide comprises a C-terminal amide.

[0324] Implementation Scheme 13. An engineered polypeptide of any one of Implementation Schemes 1 to 11, wherein the polypeptide comprises two, three, four, or five copies of a polymer 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.

[0325] Implementation Scheme 14. The engineered polypeptide of Implementation Scheme 13, wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NO:148 to SEQ ID NO:207.

[0326] Implementation Scheme 15. An engineered polypeptide of any one of Implementation Schemes 1 to 11, wherein the engineered polypeptide comprises one or more α-methyl amino acids.

[0327] Implementation Scheme 16. An engineered polypeptide of any one of Implementation Schemes 1 to 11, wherein the engineered polypeptide further comprises a lipid moiety covalently bonded to an amino-terminus, a carboxyl-terminus, or an amino acid of Formula II (SEQ ID NO: 111) or Formula IV (SEQ ID NO: 383).

[0328] Implementation Scheme 17. An engineered polypeptide of any one of Implementation Schemes 1 to 11, wherein the engineered polypeptide has substantial resistance to protein hydrolysis degradation.

[0329] Implementation Scheme 18. The engineered polypeptide of Implementation Scheme 17, wherein the engineered polypeptide has substantial resistance to degradation by DPP-IV, enkephalinase, α-chymotrypsin, trypsin, elastase or pepsin.

[0330] Implementation Scheme 19. An engineered polypeptide of any one of Implementation Schemes 1 to 16, wherein the engineered polypeptide retains at least the same receptor potency as the corresponding wild-type unmodified polypeptide.

[0331] Implementation Scheme 20. An engineered polypeptide of any one of Implementation Schemes 1 to 16, wherein the engineered polypeptide retains at least the same receptor potency as telpolide.

[0332] Implementation Scheme 21. An engineered polypeptide of any one of Implementation Schemes 1 to 16, wherein the engineered polypeptide retains at least the same receptor selectivity as the corresponding wild-type unmodified polypeptide.

[0333] Implementation Scheme 22. An engineered peptide of any one of Implementation Schemes 1 to 16, wherein the engineered peptide exhibits enhanced receptor efficacy compared to the corresponding wild-type unmodified peptide.

[0334] Implementation Scheme 23. An isolated polynucleotide encoding an engineered polypeptide, said engineered polypeptide comprising the following amino acid sequence: Formula II (SEQ ID NO:111): Where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 = L, K, H, or I; X 16 = K, R, H, or V; X 20 = K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V, Or formula IV (SEQ ID NO:383): Y X2E GT X6X7S DX 10 SIX 13 X 14 DX 16 IAX 19 X 20 AX 22 VQX 25 X 26 IAGGPSS GA PP (IV). Where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14= L, K, H, or I; X 16 = K, R, H, or V; X 19 = Q or V; X 20 = K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V.

[0335] Implementation Scheme 24. An engineered carrier comprising the polynucleotide of Implementation Scheme 23.

[0336] Implementation Scheme 25. A nucleic acid expression kit comprising one or more of the following: Nucleic acid sequences containing transcription promoters; Nucleic acid sequences encoding polypeptides containing the following amino acid sequence: Formula II (SEQ ID NO:111): Where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 = L, K, H, or I; X 16 = K, R, H, or V; X 20 = K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V, Or formula IV (SEQ ID NO:383): Y X2E GT X6X7S DX 10 SIX 13 X 14 DX 16 IAX 19 X 20 AX 22 VQX 25 X 26 IAGGPSS GA PP (IV). Where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 = L, K, H, or I; X 16 = K, R, H, or V; X 19 = Q or V; X 20= K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V; Nucleic acid sequences containing translation terminators; and Nucleic acid sequences containing transcription terminators.

[0337] Implementation Scheme 26. The nucleic acid expression cassette of Implementation Scheme 25, wherein the polypeptide further comprises one or more in-frame amino acid sequences, the in-frame amino acid sequences comprising a secretion signal sequence, a peptide tag, or a combination thereof.

[0338] Implementation Scheme 27. A method for preparing engineered peptides, said engineered peptides comprising the following amino acid sequence: Formula II (SEQ ID NO:111) Y X2E GT X6X7S DX 10 SIX 13 X 14 DX 16 IAQX 20 AX 22 VQX 25 X 26 IAGGPSS GA PP(I). Where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 = L, K, H, or I; X 16 = K, R, H, or V; X 20 = K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V, Or formula IV (SEQ ID NO:383): Y X2E GT X6X7S DX 10 SIX 13 X 14 DX 16 IAX 19 X 20 AX 22 VQX 25 X 26 IAGGPSS GA PP (IV). Where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 = L, K, H, or I; X 16 = K, R, H, or V; X 19 = Q or V; X 20 = K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V; The method includes culturing host cells transformed with an expression vector encoding the engineered peptide under conditions that allow expression of the engineered peptide, and recovering the engineered peptide.

[0339] Implementation Scheme 28. A pharmaceutical composition comprising an engineered polypeptide and a carrier, said engineered polypeptide comprising an amino acid sequence of formula II (SEQ ID NO: 111) or formula IV (SEQ ID NO: 383).

[0340] Implementation Scheme 29. A pharmaceutical composition comprising a recombinant host cell containing a polynucleotide encoding an engineered polypeptide of formula II (SEQ ID NO:111) or formula IV (SEQ ID NO:383).

[0341] Implementation scheme 30. The pharmaceutical composition of implementation scheme 29 further comprises a carrier.

[0342] Implementation Scheme 31. A host cell comprising a polynucleotide encoding an engineered polypeptide, said engineered polypeptide comprising the following amino acid sequence: Formula II (SEQ ID NO:111): Y X2E GT X6X7S DX 10 SIX 13 X 14 DX 16 IAQX 20 AX 22 VQX 25 X 26 IAGGPSS GA PP(I). Where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 = L, K, H, or I; X16 = K, R, H, or V; X 20 = K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V, Or formula IV (SEQ ID NO:383): Y X2E GT X6X7S DX 10 SIX 13 X 14 DX 16 IAX 19 X 20 AX 22 VQX 25 X 26 IAGGPSS GA PP (IV). Where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 = L, K, H, or I; X 16 = K, R, H, or V; X 19 = Q or V; X 20 = K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V.

[0343] Implementation Scheme 32. A host cell comprising a vector, the vector comprising a polynucleotide encoding an engineered polypeptide, said engineered polypeptide comprising an amino acid sequence of the following formula: Formula II (SEQ ID NO:111): Y X2E GT X6X7S DX 10 SIX 13 X 14 DX 16 IAQX 20 AX 22 VQX 25 X 26 IAGGPSS GA PP(I). Where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13= A, S, Y, N, I, L, R, V or K; X 14 = L, K, H, or I; X 16 = K, R, H, or V; X 20 = K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V, Or formula IV (SEQ ID NO:383): Y X2E GT X6X7S DX 10 SIX 13 X 14 DX 16 IAX 19 X 20 AX 22 VQX 25 X 26 IAGGPSS GA PP (IV). Where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 = L, K, H, or I; X 16 = K, R, H, or V; X 19 = Q or V; X 20 = K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V.

[0344] Implementation Scheme 33. A transformed cell comprising a nucleic acid expression cassette, said nucleic acid expression cassette comprising one or more of the following: Nucleic acid sequences containing transcription promoters; Nucleic acid sequences encoding polypeptides containing the following amino acid sequence: Formula II (SEQ ID NO:111): Y X2E GT X6X7S DX 10 SIX 13 X 14 DX 16 IAQX 20 AX 22 VQX 25 X 26 IAGGPSS GA PP(I). Where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 = L, K, H, or I; X 16 = K, R, H, or V; X 20 = K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V, Or formula IV (SEQ ID NO:383): Y X2E GT X6X7S DX 10 SIX 13 X 14 DX 16 IAX 19 X 20 AX 22 VQX 25 X 26 IAGGPSS GA PP (IV). Where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 = L, K, H, or I; X 16 = K, R, H, or V; X 19 = Q or V; X 20 = K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V; Nucleic acid sequences containing translation terminators; and Nucleic acid sequences containing transcription terminators.

[0345] Implementation Scheme 34. The transformed cells of Implementation Scheme 33, wherein the cells are bacterial cells, plant cells, yeast cells or algal cells.

[0346] Implementation Scheme 35. Transformed cells of Implementation Scheme 32 or 33, wherein the cells contain two or more copies of the recombinant nucleic acid.

[0347] Implementation Scheme 36. The transformed cell of any one of Implementation Schemes 32 to 35, wherein the cell comprises integration into the bacterial chromosome.

[0348] Implementation Scheme 37. The transformed cells of Implementation Scheme 36, wherein the integration in the bacterial chromosome is integration into the transposase locus.

[0349] Implementation scheme 38. The transformed cells of any one of implementation schemes 32 to 35, wherein the cells are yeast cells.

[0350] Implementation Scheme 39. The transformed cells of Implementation Scheme 38, wherein the yeast cells are Pichia pastoris ( Pichia pastoris ) cells of the strain.

[0351] Implementation Scheme 40. The transformed cells of any one of Implementation Schemes 32 to 35, wherein the cells are selected from the genus Bacillus ( Bacillus Lactobacillus ( ) Lactobacillus Lactococcus spp. Lactococcus ), Enterococcus spp. Enterococcus Bacterial cells of the genus Bacteria, which are grouped into groups of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 ...

[0352] Implementation Scheme 41. The transformed cells of any one of Implementation Schemes 32 to 35, wherein the transformed cells are plant cells.

[0353] Implementation Scheme 42. The transformed cells of Implementation Scheme 41, wherein the recombinant nucleic acid is integrated into the chloroplasts of the plant cells.

[0354] Implementation Scheme 43. The transformed cells of Implementation Scheme 41, wherein the recombinant nucleic acid is integrated into the genome of the plant cells.

[0355] Implementation Scheme 44. A genetically engineered plant or a portion thereof, comprising a recombinant nucleic acid encoding an engineered polypeptide of the following formula: Formula II (SEQ ID NO:111): Y X2E GT X6X7S DX 10 SIX 13 X 14 DX 16 IAQX 20 AX 22 VQX 25 X 26 IAGGPSS GA PP(I). Where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 = L, K, H, or I; X 16= K, R, H, or V; X 20 = K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V, Or formula IV (SEQ ID NO:383): Y X2E GT X6X7S DX 10 SIX 13 X 14 DX 16 IAX 19 X 20 AX 22 VQX 25 X 26 IAGGPSS GA PP (IV). Where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 = L, K, H, or I; X 16 = K, R, H, or V; X 19 = Q or V; X 20 = K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V.

[0356] Implementation Scheme 45. The genetically engineered plant or a portion thereof of Implementation Scheme 44, wherein the portion thereof is a plant seed.

[0357] Implementation Scheme 46. The genetically engineered plant or part thereof of Implementation Scheme 44 or 45, wherein the recombinant nucleic acid is integrated into the chloroplasts of the plant or part thereof.

[0358] Implementation Scheme 47. The genetically engineered plant or a portion thereof of Implementation Scheme 46, wherein the recombinant nucleic acid is integrated into the genome of the plant cell.

[0359] Implementation Scheme 48. A kit comprising the pharmaceutical composition of Implementation Scheme 28 or Implementation Scheme 29.

[0360] Implementation scheme 49. The kit of implementation scheme 48, which contains the host cell and vector of implementation scheme 31.

[0361] method Implementation Scheme 50. A method for treating or preventing conditions caused by or characterized by overweight in a subject, and for treating obesity, morbid obesity, obesity-related inflammation, obesity-related gallbladder disease, obesity-induced sleep apnea, metabolic syndrome, prediabetes, insulin resistance, glucose intolerance, type 2 diabetes, type 1 diabetes, hypertension, atherosclerotic dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke, or microvascular disease, comprising administering to the subject in need of treatment an effective amount of an engineered polypeptide of any one of Implementation Schemes 1 to 22, a pharmaceutical composition of Implementation Schemes 28 to 30, or a cell of Implementation Schemes 31 to 47.

[0362] Implementation Scheme 51. A method for treating or preventing a disease or condition caused by or characterized by hypoglycemia or impaired insulin release, comprising administering to a subject in need of treatment an effective amount of an engineered polypeptide of any one of Implementation Schemes 1 to 22, a pharmaceutical composition of Implementation Schemes 28 to 30, or a cell of Implementation Schemes 31 to 47.

[0363] Implementation scheme 52. The method of implementation scheme 50, wherein the disease or condition is diabetes or obesity.

[0364] Implementation scheme 53. The method of implementation scheme 52, wherein the disease or condition is type 2 diabetes.

[0365] Implementation Scheme 54. The method of any one of Implementation Schemes 50 to 53, wherein the administration further improves glycemic control, provides weight control, improves β-cell function and quantity, reduces gastric acid secretion and gastric emptying rate, or any combination thereof.

[0366] Implementation Scheme 55. The method of any one of Implementation Schemes 50 to 54, wherein the engineered polypeptide or the pharmaceutical composition is administered orally, by injection or transdermally.

[0367] Implementation Scheme 56. The method of any one of Implementation Schemes 50 to 54, wherein the host cell is administered orally.

[0368] Implementation Scheme 57. The method of Implementation Scheme 50, wherein the engineered polypeptide or a pharmaceutical composition thereof is administered orally.

[0369] Implementation Scheme 58. The method of Implementation Scheme 55, wherein the injection is administered subcutaneously or intravenously.

[0370] Implementation Scheme 59. The method of any one of Implementation Schemes 50 to 58, wherein the engineered polypeptide or the pharmaceutical composition is administered once daily.

[0371] Implementation Scheme 60. The method of any one of Implementation Schemes 50 to 59, further comprising the application of one or more additional therapies.

[0372] Implementation Scheme 61. The method of Implementation Scheme 60, wherein the additional therapy includes blood glucose monitoring, dietary modification, exercise, insulin, thiazolidinediones, sulfonylureas, incretins, metformin, glibenclamides, dipeptidyl peptidase-4 inhibitors, bile acid sequestrants, or any combination thereof.

[0373] Implementation scheme 62. The method of any one of implementation schemes 50 to 61, wherein the subject is a human, a non-human primate, a cat, or a dog.

[0374] Implementation Scheme 63. A pharmaceutical composition comprising an engineered polypeptide comprising the amino acid sequence of Formula II (SEQ ID NO: 111).

[0375] Implementation Scheme 64. A pharmaceutical composition comprising an engineered polypeptide comprising an amino acid sequence selected from any one of SEQ ID NO:112 to SEQ ID NO:147 or SEQ ID NO:381.

[0376] Implementation Scheme 65. The pharmaceutical composition of Implementation Scheme 64, wherein the composition is a lyophilized composition of transformed plant cells or transformed bacterial cells.

[0377] Implementation Scheme 66. A direct-feed microorganism (DFM) comprising bacteria transformed with nucleic acids encoding an engineered polypeptide, said engineered polypeptide comprising the following amino acid sequence: Formula II (SEQ ID NO:111): Y X2E GT X6X7S DX 10 SIX 13 X 14 DX 16 IAQX 20 AX 22 VQX 25 X 26 IAGGPSS GA PP(I). Where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 = L, K, H, or I; X 16 = K, R, H, or V; X 20 = K, R, H, N; X 22= F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V, Or formula IV (SEQ ID NO:383): Y X2E GT X6X7S DX 10 SIX 13 X 14 DX 16 IAX 19 X 20 AX 22 VQX 25 X 26 IAGGPSS GA PP (IV). Where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 = L, K, H, or I; X 16 = K, R, H, or V; X 19 = Q or V; X 20 = K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 = L or V.

[0378] Implementation Scheme 67. Direct-feed microorganisms (DFM) of Implementation Scheme 66, wherein the engineered polypeptide comprises an amino acid sequence selected from any one of SEQ ID NO:112 to SEQ ID NO:17 or SEQ ID NO:148 to SEQ ID NO:207, or SEQ ID NO:381.

[0379] Implementation Scheme 68. The DFM of Implementation Scheme 66, wherein the bacteria comprises strains selected from the group consisting of Bacillus, Lactobacillus, Lactococcus, Enterococcus, and combinations thereof.

[0380] Implementation Scheme 69. The DFM of Implementation Scheme 68, wherein the transforming bacteria is Bacillus subtilis ( Bacillus subtilis ) strain.

[0381] Implementation Scheme 70. The DFM of Implementation Scheme 68, wherein the transformed Bacillus subtilis bacteria and the DFM further comprise one or more Bacillus amyloliquefaciens species ( B. amyloliquefaciens ) strain.

[0382] Implementation Scheme 71. The DFM of Implementation Scheme 68, wherein the transformed Bacillus subtilis bacteria and the DFM further comprise one or more anaerobic cellulose-decomposing bacteria.

[0383] Implementation Scheme 72. The DFM of Implementation Scheme 68, wherein one or more anaerobic cellulose-decomposing bacteria are members of the Bacillota phylum.

[0384] Implementation Scheme 73. The DFM of Implementation Scheme 72, wherein the Bacillus phylum member is *Ruminococcus flavinatus* (… Ruminococcus flavefaciens ) bacterial strains.

[0385] Implementation Scheme 74. The DFM of Implementation Scheme 68, wherein one or more anaerobic cellulose-decomposing bacteria are Bacteroides ( Bacteroides )member.

[0386] Implementation Scheme 75. The DFM of Implementation Scheme 74, wherein the Bacteroides member is Bacteroides fragilis ( Bacteroides fragilis ).

[0387] Implementation scheme 76. The DFM of implementation scheme 68, wherein the bacterial strain is a Lactobacillus strain.

[0388] Implementation Scheme 77. DFM of Implementation Scheme 76, wherein the lactobacillus strain is Lactobacillus gasseri ( Lactobacillus gasseri ) strain.

[0389] Implementation Scheme 78. The DFM of Implementation Scheme 76, wherein the lactobacillus strain is Lactobacillus plantarum ( Lactobacillus plantarum ) strain.

[0390] Implementation Scheme 79. The DFM of any one of Implementation Schemes 67 to 78, wherein the DFM is freeze-dried.

[0391] While this disclosure has been described with reference to preferred embodiments, those skilled in the art will understand that various modifications may be made to this disclosure without departing from its scope, and equivalents may be substituted for elements therein to suit particular circumstances. Therefore, this disclosure is not intended to be limited to the specific embodiments disclosed as the best mode for carrying out this disclosure, but rather to cover all embodiments falling within the scope and spirit of the appended claims.

[0392] Example Example 1 Homology modeling The three-dimensional crystal structure of the human GLP-1R (hGLP-1R) complex was obtained from the Protein Data Bank (PDB) database (Berman et al., "The Protein Data Bank,"). Nucleic Acids Research , 28 (1), 235–242 (2000) 。 The PDB database contains 48 peptide complexes that bind to the hGLP-1R receptor. Eleven of these 48 complex structures were selected for superimposition of the receptor-binding complexes, as shown in Table 2.

[0393] Table 2: Selected PDB structures for overlay and template selection

[0394] The overlay results show that all 11 complex models are well aligned with an rmsd of 1.659 Å between them, as shown in Figure 4.

[0395] Based on factors such as full-length structure, peptide complexation, resolution, and publication year, the hGLP-1R / hGLP1 binding complex structure (RCSB PDB - 6X18) was selected. The hGLP-1R / hGLP1 binding complex model was designed to normalize and benchmark the modeling parameters of the cat receptor. The final superposition model is shown in Figure 4.

[0396] Based on the high similarity between human and feline GLP-1R receptors, the cGLP-1R structure was modeled using PDBid: 6X18. A composite feline receptor structure (cGLP-1R / cGLP) model was designed using baseline parameters of the human complex (e.g., hGLP-1R / hGLP).

[0397] The model was validated by examining stereochemical properties using a Ramachandran plot, and sequence alignment was performed to check the similarity between human and cat receptors and their respective peptides.

[0398] Both cat and human GLP-1R receptors showed 92% sequence identity. Further analysis after sequence alignment indicated that the binding pocket residues were highly conserved. Figure 6 The differences between humans and cats are reflected in three residues (Leu144-Phe, Tyr145-Ser, and Lys197-Arg). Two residues (Pro90 and Trp91 of the ECD) are absent in cats.

[0399] Standardized docking parameters Molecular docking of hGLP-1 and hGIP peptides with the hGLP-1R receptor was performed. Binding posture analysis of the hGLP-1R / hGLP-1 complexes showed that the peptides had the same binding posture, with overall rmsd of 0.030 Å and 0.795 Å, respectively. Most side chain orientations, spatial distances, and interactions were preserved in both complexes.

[0400] The N-terminus of GLP-1 peptides interacts with transmembrane residues, such as His7-Q234 (TM3), Ala8-L388 / L384 (TM7), Glu9-L388 / R190 (TM2) / Y152 (TM1), Gly10-N300, Thr11-R380 (TM7), Phe12-L388 (TM7) / L141 / L144 (TM1), Thr1 3-K197 (TM2), Ser14-N300 (ECL2), Asp15-R380, Val16-L201, and Ser17-T298 / R299 (ECL2) / Y205 (TM2) [for clarity, peptide residues are represented by three-letter codes] (Zhao et al., 2022), while the C-terminus forms a peptide-protein interaction network with the extracellular domain (ECD) of the hGLP-1R receptor, such as Figure 2 As shown.

[0401] Sequence comparison of GLP-1 endogenous ligands in vertebrates A co-model of the GLP-1R receptor was constructed, and endogenous ligand docking was performed. Model construction showed that the cGLP-1R / cGLP1 co-model and the hGLP-1R / hGLP1 template had identifiable similarity. Binding poses of the cat receptor superimposed on its respective template were analyzed. The cGLP-1R and hGLP-1R co-models complexed with GLP-1 showed good alignment, and the side chain orientation was preserved (data not shown).

[0402] Design of GLP-1 analogs using ligand-based methods Exenatide has lower sensitivity to DDP-4, resulting in a longer half-life. Liraglutide shares 97% sequence identity with GLP-1. It has an additional C16 fatty acid side chain. This extends its duration of action to 24 hours, allowing the peptide to be injected once daily. See Garber, “Long-acting glucagon-like peptide 1 receptoragonists: a review of their efficacy and tolerability,” Diabetes Care , 34 (Supplement 2): S279-84 (2011).

[0403] In GLP1[Gly 8 In the analogues, the change from Ala to Gly significantly improved resistance to DPP-4 protein hydrolysis inactivation. (Lin et al., "Oral Delivery of Pentameric Glucagon-Like Peptide-1 by Recombinant Lactobacillus in Diabetic Rats") PloS One , 11 (9); e0162733 (2011). This modification has also been observed in exenatide. GLP-1-Glycine was considered in this paper. 8 The sequence was used to prepare GLP-1 analogs and attempt to make them resistant to proteases. GLP-1 contains Lys residues at positions 26 and 34, which are important proteolytic cleavage sites for trypsin. Literature review showed that replacing Lys at position 26 with Gln and Lys at position 34 with Asp significantly improved resistance to trypsin (Figure 9).

[0404] Furthermore, we introduced a multiple modification method by analyzing the proteolytic cleavage sites of different peptides. See [link to related documentation] Figure 1 To design the peptides, both subcutaneously and orally administered peptides were analyzed, with the aim of modifying peptides resistant to cleavage by a variety of proteolytic enzymes.

[0405] Peptides were designed by mutating specific residues. Further, each peptide underwent energy minimization, molecular docking, and subsequent conformational sampling. Table 3 lists the variations of the engineered peptides, their docking energies, and cleavage site residues in sequence. In-depth analysis of each peptide revealed that they bind to the same interaction pocket, maintain their helical structure, and exhibit higher affinity for both receptors.

[0406] Table 3: GLP-1 engineered analogues, their modifications, cleavage sites, and binding energies with hGLP-1R and cGLP-1R

[0407] Peptides C1.8, C1.9, and C1.10 were selected for further engineering. Arg was introduced to impart trypsin resistance at position 36. 36 To Ser 36 The changes were as follows. The newly designed peptides C.1.8.1, C.1.9.1 and C.1.10.1 all exhibited trypsin resistance, and their energy scores were also comparable (Table 4).

[0408] Table 4: Designed GLP-1 analogs, their modifications, cleavage sites, and binding energies with hGLP-1R and cGLP-1R

[0409] GLP1 activity assay Clonal ChemiBrite cells expressing the human GLP-1 receptor were prepared by stably transfecting HEK293 cells with ChemiBrite clytin, the GLP-1 receptor, and a promiscuous G protein that couples the receptor to the calcium signaling pathway. These cells were stability-tested and can be directly used for luminescent analysis of GLP-1 receptor agonists, antagonists, and modulators.

[0410] Figure 7 Representative data are presented for the activation of the GLP-1 receptor stably expressed in HEK293 cells induced by GLP-1 (7-36) using a luminescent calcium flux assay. HEK293 cells expressing GLP-1R were incubated at room temperature with 10 µM coelenterate-h for 3 h. Calcium flux in response to the indicated ligands was then measured in 96-well plates using a FLIPR Tetra equipped with an ICCD camera at a final concentration of 0.5% DMSO. Following the manufacturer's protocol, the luminescent signal obtained in the experiments was 260,000 RLU (relative light units), measured by area under the curve (AUC) within 80 seconds of agonist addition. Parental cells (Cat. No. HTSHEK-2L) were also tested to determine the specificity of the obtained signal.

[0411] The peptides described in this specification have undergone GLP-1 activity testing both in vitro and in vivo.

[0412] Evaluation of the efficacy of engineered GLP-1 peptide in type 2 and type 1 diabetic mouse and rat models The efficacy of GLP-1 peptides was evaluated in type 2 and type 1 diabetic mouse and rat models by administration in the form of peptides or in the form of direct-feed microorganisms (DFM) incorporating an expression vector of the GLP-1 peptide described in this specification (“therapeutic peptide and host cells”). In vivo activity was assessed by injection (subcutaneous or intravenous) or oral administration. Alternatively, host cells expressing GLP-1 from Bacillus, Lactobacillus, Lactococcus, Salmonella, and Enterococcus can be transformed and screened using conventional methods for oral administration in DFM form.

[0413] Using different models of type 2 and type 1 diabetes (including diet-induced obesity (DIO) in type 2 diabetes), ob - / ob - and db - / db -The efficacy of therapeutic peptides and host cell candidates for GLP-1 delivery was evaluated in mice and rats. See, for example, the internet URL www.jax.org / news-and-insights / jax-blog / 2015 / july / choosing-among-type-ii-diabetes-mouse-models. Obesity is one of the leading risk factors for diabetes in humans, cats, and other species. Similar to humans, certain mouse strains become obese when fed a high-fat or so-called “Western” diet. Among the many mouse strains, C57BL / 6 is one of the most sensitive to this diet-induced obesity (DIO), developing severe obesity, exacerbated glucose intolerance, moderate insulin resistance, and elevated glucose levels. Therefore, DIO is a commonly used model for studying prediabetes and diabetes-related metabolic syndrome in humans. Similarly, obesity ( Lep ob ) and diabetes ( Lepr db The homozygous mutant mouse was the earliest characterized diabetic mouse model and remains a popular choice for diabetes researchers. Leptin is a hormone that regulates appetite; the leptin gene (… Lep ) or its receptor ( Lepr Mutations in this gene can induce uncontrolled eating (overeating), which can lead to obesity and frank diabetes.

[0414] Table 5 below shows an example study design for evaluating the efficacy of engineered candidates in a DIO type 2 diabetes model. Test samples were delivered via direct injection or oral gavage to the engineered candidate or host cells expressing the engineered candidate (e.g., DFM). In short, the study included 3 or 4 groups, each consisting of 12 DIO mice (Jackson Labs) fed a high-fat diet (HFD) for 16 weeks. Group 1 served as a negative control, receiving a sham dose. Group 2 received liraglutide daily intraperitoneally for 12 weeks. Group 3 received 1 x 10⁻⁶ liters of liraglutide via oral gavage in response to DFM treatment. 9 Unmodified host cells, CFU / mouse / day, volume 0.2–0.4 ml, for 12 weeks, served as a negative control. Group 4 was the experimental group, where the engineered peptide was delivered via: injection of peptide composition (subcutaneous or intravenous), oral formulation, or oral gavage containing DFM from transformed host cells expressing the engineered peptide. Orally delivered DFM was transformed and thus delivered at a rate of 1 × 10⁻⁶. 9 GLP-1 was delivered via CFU / mouse / day, in a volume of 0.2–0.4 ml, for 12 weeks. The following parameters were measured in all mice: 1. Weight, food intake, weekly 2. Body composition, weeks 0, 4, and 8 3. OGTT, Week 6 4. Non-fasting blood glucose, weekly 5. Blood (serum) samples (5 equal aliquots each) for testing insulin, adipokines, clinical chemistry, liver function, and blood lipids, at weeks 2, 4, 6, and 8. 6. Fecal samples, weekly, for microbiome analysis. 7. Final autopsy—Intestines, pancreas, liver, adipose tissue, and muscle were collected and placed in RNA latex for qPCR; additional samples were taken for histopathological / immunohistochemical analysis. Table 5: Study design for evaluating engineered candidates in the DIO type 2 diabetes model

[0415] Example 2: Homologous Modeling The three-dimensional crystal structure of the human GLP-1R (hGLP-1R) and GIPR (hGIPR) complex was obtained from the Protein Data Bank (PDB) database (Berman et al., "The Protein Data Bank,"). Nucleic Acids Research 28(1):235–242 (2000)) 。 The PDB database contains 48 peptide complexes that bind to the hGLP-1R receptor. Eleven of these complex structures were selected for receptor-binding complex superposition, as shown in Table 7.

[0416] Table 7: Selected PDB structures for overlay and template selection

[0417] The superposition results showed that all 11 complex models overlapped well, with an RMSD of 1.659 Å between them. Figure 4A As shown. Similarly, PDB contains nine hGIPR complex structures that bind to peptides (Table 7), and the rmsd of all nine binding complex structures is 0.995 Å. Figure 4B As shown.

[0418] Based on factors such as full-length structure, peptide complexation, resolution, and publication year, the hGLP-1R-hGLP1 binding complex structure (RCSB PDB - 6X18) and the hGIPR-hGIP binding complex structure (RCSB PDB - 7DTY) were selected. These structures were used as templates for designing the cGIPR complex. The hGLP-1R / hGLP1 and hGIPR / hGIP binding complex models were designed to normalize and benchmark the cat receptor modeling parameters. The final model is shown in Figure 5.

[0419] Based on the high similarity between human and feline GLP-1R and GIPR receptors, the cGLP-1R structure was modeled using PDBid: 6X18, and the cGIPR structure was modeled using PDB Ids: 7DTY and 7RA3. Using the baseline parameters of human complexes (e.g., hGLP-1R / hGLP, hGIPR / hGIP), composite feline receptor structures (cGLP-1R / cGLP, cGIPR / GIP) were designed.

[0420] The model was validated by examining the stereochemical properties using a Laplace plot. Sequence alignment was performed to check the similarity between the human and cat receptors and their respective peptides.

[0421] Both cat and human GLP-1R receptors showed 92% sequence identity. Further analysis after sequence alignment indicated that the binding pocket residues were highly conserved. Figure 6 The differences between humans and cats are reflected in three residues (Leu144-Phe, Tyr145-Ser, and Lys197-Arg). Two residues (Pro90 and Trp91 of the ECD) are absent in cats. Figure 6 ).

[0422] Similarly, cGIPR and hGIPR share 84.65% sequence identity, and cross-species analysis shows that binding pocket residues are conserved in humans, rats, mice, and cats. Figure 10 Because the cat sequence is short, it lacks six binding pocket residues (67Met, Tyr68, Tyr87, Leu88, Pro89, and Trp90). Figure 10 ).

[0423] Example 3: Standardized docking parameters The hGLP-1 and hGIP peptides were molecularly docked with the hGLP-1R and hGIPR receptors, respectively. Binding posture analysis of the hGLP-1R / hGLP-1 and hGIPR / hGIP complexes showed that the peptides had the same binding posture, with overall rmsd of 0.030 Å and 0.795 Å, respectively. Most of the side chain orientations, spatial distances, and interactions in both complexes were preserved.

[0424] The N-terminus of GLP-1 peptides interacts with transmembrane residues, such as His7-Q234 (TM3), Ala8-L388 / L384 (TM7), Glu9-L388 / R190 (TM2) / Y152 (TM1), Gly10-N300, Thr11-R380 (TM7), Phe12-L388 (TM7) / L141 / L144 (TM1), Thr1 3-K197 (TM2), Ser14-N300 (ECL2), Asp15-R380, Val16-L201, and Ser17-T298 / R299 (ECL2) / Y205 (TM2) [for clarity, peptide residues are represented by three-letter codes] (Zhao et al., 2022), while the C-terminus forms a peptide-protein interaction network with the extracellular domain (ECD) of the hGLP-1R receptor, such as Figure 11 As shown.

[0425] Docking of telpoeptide with hGLP1R showed that the peptide binds in the same region as the previously reported hGLP-1R receptor, with an RMSD of 1.205 Å. Peptide-protein interaction analysis revealed that the N-terminus of telpoeptide interacts with the following GLP-1R residues: Tyr1-Q234, AIB2-L388, Glu3-Y152 / R190, Phe6-Y148 / L388 / L141 / L144, Thr7-T298, Ser8-N300, Asp9-R380, Tyr10-Y145 / L141 / L201, Ser11-Y205 / T298, and AIB13-L141 [for clarity, peptide residues are represented by three-letter codes] (e.g., ...). Figure 12 As shown in the figure, the C-terminus interacts with the receptor's ECD.

[0426] Similar studies were conducted on the hGIPR receptor via molecular docking of hGIP and telpokines. Telpokines is a dual agonist of GLP-1R and GIPR. Docking of telpokines revealed the following interactions: Tyr1-Q234 / W296, AIB2-L374 / I378, Glu3-R183 / Y145, Thr5-R300, Phe6-Y141 / L134 / L137 / L374 / L378, Thr7-R190, Ser8-N290, Asp9-R370, Tyr10-Q138 / R196, Ser11-E288, and AIB13-R131 (Zhao et al., 2022).

[0427] Example 4: Sequence comparison of endogenous ligands (GIP and GLP-1) in cats and humans cGIP and hGIP share 97% sequence identity. The only residue difference occurs at position 18 (His-Arg). Figure 13 Furthermore, the 2QKH peptide structure selected as the modeling template for the cGIP peptide deviated from hGIP by 0.982 Å. However, GLP-1 showed no difference between cat and human sequences. Compared to telpoeptide, GIP showed 8 positional differences between the two species, while GLP-1 showed 13 positional differences between the two species.

[0428] A complex model of the cGLP-1R and cGIPR receptors was constructed and docked using their respective endogenous ligands and telpoeptide. Model construction showed that the cGLP-1R / cGLP-1 co-model and the hGLP-1R / hGLP-1 template had identifiable similarity. The binding posture of the cat receptor after superposition with its respective template was analyzed. The cGLP-1R and hGLP-1R co-models complexed with GLP-1 showed good alignment and preserved side chain orientation. As shown in Figure 14, three residues differed in the binding pockets of the two species.

[0429] Example 5: Doping of telpoeptide into cat receptors Doping telpoeptide with cGLP-1R and cGIPR receptors confirmed that it binds to the same interaction pocket as the human receptors. Spatial distance and interaction were preserved. The rmsd of the telpoeptide-bound hGLP-1R and hGIPR complexes were observed to be 0.846 Å and 1.588 Å, respectively.

[0430] Example 6: Modification of telpoeptide Replacement of non-natural AIB amino acids Based on the alignment of reported peptide sequences, residues 2 and 13 of telpoeptide were modified from non-natural amino acids (AIBs) to natural amino acids. Telpoeptide, peptide-20, peptide-19, smegglutinin, and exendin-4 were docked with hGLP-1R and cGLP-1R receptors, and their binding energies were analyzed (see Table 8).

[0431] Table 8: Reported docking binding energies of peptides with hGLP-1R and cGLP-1R

[0432] The peptide with the highest calculated binding affinity for hGLP-1R is peptide-20, while the peptide with the highest calculated binding affinity for cGLP-1R is Exendin-4.

[0433] Residues surrounding AIB in human and cat GLP-1R receptors were analyzed. In hGLP-1R, Leu was observed. 393 Leu 397Glu 396 and Thr 400 The residues are located in AIB 2 (Around peptide residues, see Figure 15(A)); in the cGLP-1R receptor, AIB 2 Leu was observed around 375 Lys 374 Ala 359 and Glu 355 Residues (Fig. 15(B)).

[0434] Thilborpeptide-related peptides were prepared by replacing non-natural amino acids with natural amino acids to protect them from proteolytic enzyme attack. Thilborpeptide is a GIP analog, and the literature indicates that Gly could be a possible option for AIB modification (Wang, 2022). However, this modification reduces the peptide's binding affinity to GLP-1R while increasing its binding to the GIPR receptor. Replacing AIB with Gly also introduces a cleavage site for the proteolytic enzyme elastase (Wang, 2022).

[0435] The 7th amino acid residue of GLP-1 (7–37) is His, which is considered essential for the activation of the GLP-1 receptor and the maintenance of insulinotropic activity. 7 Replace with Trp 7 It reduces the binding affinity of GLP-1 and significantly decreases its activity; while the N-terminal Tyr of GIP... 1 The absence of Tyr significantly reduces its activity. 1 and Ile 7 These are key residues in the GIP peptide that activate the GIPR receptor. Therefore, it is predicted that Thr in GLP-1 will... 7 The introduction of tyrpeptide may reduce its GIP activity. Tyr in GIP 10 and Ile 12 Used for MAR709 peptide, Ile 12 It plays an important role in the activation of the GIP receptor. Thilbortide utilizes Tyr... 10 and Ile 12 Tyr in GLP-1 19 Replacing with Ala reduces the binding affinity and activity of GLP-1 agonists. It has been reported that the Ala in telpolide... 13 It reduces its GLP-1 activity without affecting its GIP activity (Wang, 2022).

[0436] AIB was modified into native amino acids in telpoide using a ligand-based approach. Different peptide sequences were analyzed using the proposed peptide-receptor model, and then amino acid combinations in the telpoide peptide sequence were mutated (see Table 9). For each modified peptide, conformational sampling and molecular docking were performed on both hGLP-1R and cGLP-1R receptors.

[0437] As shown in Table 9, the docking scores and top-ranked postures of the modified peptides were visually examined. Peptides exhibiting variations as in peptides 2 and 3 bound in the same posture as telpoeptide, and both peptides showed better binding affinity for both hGLP-1R and cGLP-1R. Conversely, analogs exhibiting variations as in peptides 4 and 8 lost their helicity at the N-terminus of the peptide when targeting cGLP-1R (Fig. 16(A, B)), but showed better interaction with hGLP-1R. Peptide 6 failed to bind in the binding pocket and showed a tendency to migrate outward from the receptor. Figure 16C Similarly, peptides 5 and 7 lose their helicality and cannot bind in the same manner as telpoide. Modifications to peptides 9 and 10 are susceptible to the effects of the proteolytic enzyme trypsin.

[0438] Table 9: Modifications at positions 2 and 13 of telpopeptide

[0439] Modification of DPP-4 and trypsin-sensitive sites To protect the modified telpoeptide from degradation by serum and gastrointestinal proteases, the telpoeptide sequence was further modified by substituting native amino acids at predicted vulnerable sites at multiple stages, after considering each proteolytic enzyme individually. The cleavage sites of the proteolytic enzymes are as follows: Figure 1 As shown.

[0440] Peptide 3 shown in Table 9 was selected for further modification, with cleavage site analysis performed after each modification. Subsequent changes led to the evolution of the peptides in Table 9, which showed resistance to DPP-4 and trypsin, but remained sensitive to chymotrypsin cleavage at positions 1, 14, 16, and 20 (see Table 10).

[0441] Table 10: Modification of telpoeptide in stage 1

[0442] Modification of chymotrypsin cleavage sites in DPP-4 and trypsin-resistant peptides In stage 2, after the modifications shown in Table 10, the peptides exhibited resistance to DPP-4, but the cleavage sites of trypsin (sites 16 and 20) and chymotrypsin (sites 1 and 14) remained intact. Molecular docking studies of hGLP-1R and cGLP-1R showed that the modified peptides interacted within the same binding pocket, but their helicity was disrupted (see Table 11).

[0443] Table 11: Modification of telpoeptide in stage 2 and its docking scores with hGLP-1R and cGLP-1R

[0444] To prevent the designed peptides from unwinding, additional substitutions were introduced into the peptides listed in Table 11. These peptides retained their helical nature.

[0445] Table 12: Peptide modifications that remove protease-sensitive sites

[0446] Further modifications were introduced to peptide C1.4.2.6 in Table 12 to improve peptide stability (see Table 13).

[0447] Table 13: Modified peptides with improved stability

[0448] The binding affinity of the peptide was assessed by calculating its binding energies to human and cat receptors, and the results are listed in Table 14. As described herein, the peptide exhibits resistance to different proteolytic enzyme cleavage sites, and its binding energies are closer to those of telpoide.

[0449] Table 14: Docking scores with hGLP-1R and cGLP-1R

[0450] The peptides of telpolide are coupled to the C20 fatty acid moiety via a linker to Lys20 (avoiding trypsin cleavage and promoting binding to serum albumin), making them difficult to express and deliver via bacterial systems (e.g., Lactobacillus). To enable their delivery via Lactobacillus systems, Lys20 is... 20 Mutate into Asn 20 This leads to the sixth stage series (see Table 15).

[0451] Table 15: Thipoeptide modification in stage 6 and its docking scores with hGLP-1R and cGLP-1R.

[0452]

[0453] Peptides C1.4.5.1.1, C1.4.5.2.1, and C1.4.5.3.1 were engineered as part of Phase 6 (see Table 15). These engineered peptides are resistant to DPP-4, chymotrypsin, and trypsin, and their binding energies are comparable to those of telpolide.

[0454] Example 7: Assay of Dual Agonist Activity GIP-1 Clonal ChemiBrite cells expressing the human GLP-1 receptor were prepared by stably transfecting HEK293 cells with ChemiBrite clytin, the GLP-1 receptor, and a hybrid G ​​protein that couples the receptor to the calcium signaling pathway. These cells were stability-tested and can be directly used for luminescent assays of GLP-1 receptor agonists, antagonists, and modulators. Similar in vitro methods for assessing GLP-1 activity are known in the art.

[0455] Figure 24 Representative data are presented for the activation of the GLP-1 receptor stably expressed in HEK293 cells induced by GLP-1 (7-36) using a luminescent calcium flux assay. HEK293 cells expressing GLP-1R were incubated at room temperature with 10 µM coelenterate-h for 3 h, and then calcium flux in response to the indicated ligands was measured in 96-well plates using a FLIPR Tetra equipped with an ICCD camera at a final concentration of 0.5% DMSO. The luminescent signal obtained in this experiment was 260,000 RLU (relative light units), measured by area under the curve (AUC) within 80 seconds of agonist addition, according to the manufacturer's protocol. Parental cells (Cat. No. HTSHEK-2L) were also tested to determine the specificity of the obtained signal.

[0456] The peptides described in this specification have undergone GLP-1 activity testing both in vitro and in vivo.

[0457] GIPR activity Clonal CHO-K1 / GIP / Gα15 cells expressing the human GIP receptor were prepared by stably transfecting CHO cells with the human GIPR receptor and a hybrid G ​​protein that couples the receptor to the calcium signaling pathway. These cells were stability-tested and can be directly used for luminescent analysis of GIPR agonists. Similar in vitro methods for determining GLP-1 activity are known in the art.

[0458] Figure 17Representative data on the activation of the stably expressed GIPR receptor in CHO cells induced by GIP using a luminescent calcium flux assay are presented. Cells were loaded with calcium-4 before stimulation with the agonist GIP. Intracellular calcium changes were normalized and measured using FLIPR. Relative fluorescence units (RFUs) were normalized and logarithmically plotted against the cumulative GIP dose (mean ± standard deviation, n = 2). In this representative example, the effect of GIP on the EC50 of cells is shown. 50 The value is 0.12 µM.

[0459] The GIPR activity of the dual agonist peptides was tested using a luminescence assay, and the results are listed in Table 16. Each concentration ranges from 1 x 10⁻⁶. -9 Six replicate tests were performed for M (nM) to 1000 nM, and the average RLU and activity were evaluated and presented at a concentration of 1 µM.

[0460] Table 16: GIPR activity measured by luminescence detection method

[0461] Evaluation of the efficacy of engineered dual agonist peptides in mouse and rat models of type 2 and type 1 diabetes. The efficacy of dual-agonist peptides was evaluated in type 2 and type 1 diabetic mouse and rat models by administration in the form of peptides or in the form of direct-feed microorganisms (DFM) incorporating expression vectors for dual-agonist peptides as described in this specification (“therapeutic peptides and host cells”). In vivo activity was assessed by injection (subcutaneous or intravenous) or oral administration. Alternatively, host cells from Bacillus, Lactobacillus, Lactococcus, Salmonella, and Enterococcus species expressing dual-agonist peptides could be transformed and screened using conventional methods for oral administration in DFM form.

[0462] Using different models of type 2 and type 1 diabetes (including diet-induced obesity (DIO) in type 2 diabetes), ob - / ob - and db - / db -The efficacy of therapeutic peptides and host cell candidates delivering dual agonist peptides was evaluated in mice and rats. See, for example, the internet URL www.jax.org / news-and-insights / jax-blog / 2015 / july / choosing-among-type-ii-diabetes-mouse-models. Obesity is one of the leading risk factors for diabetes in humans, cats, and other species. Similar to humans, certain mouse strains become obese when fed a high-fat or so-called “Western” diet. Among the many mouse strains, C57BL / 6 is one of the most sensitive to this diet-induced obesity (DIO), developing severe obesity, exacerbated glucose intolerance, moderate insulin resistance, and elevated glucose levels. Therefore, DIO is a commonly used model for studying prediabetes and diabetes-related metabolic syndrome in humans. Similarly, obesity ( Lep ob ) and diabetes ( Lepr db The homozygous mutant mouse was the earliest characterized diabetic mouse model and remains a popular choice for diabetes researchers. Leptin is a hormone that regulates appetite; the leptin gene (… Lep ) or its receptor ( Lepr Mutations in this substance can induce uncontrolled eating (overeating), which can lead to obesity and overt diabetes.

[0463] Table 17 below shows an example study design for evaluating the efficacy of engineered candidates in a DIO type 2 diabetes model. Test samples were delivered via direct injection or oral gavage to the engineered candidate or host cells expressing the engineered candidate (e.g., DFM). In short, the study included 3 or 4 groups, each consisting of 12 DIO mice (Jackson Labs) fed a high-fat diet (HFD) for 16 weeks. Group 1 served as a negative control, receiving a sham dose. Group 2 received daily intraperitoneal administration of telpolide for 12 weeks. Group 3 received 1 x 10⁻⁶ DFM treatment via oral gavage. 9 Unmodified host cells, CFU / mouse / day, volume 0.2–0.4 ml, for 12 weeks, served as a negative control. Group 4 was the experimental group, where the engineered peptide was delivered via injection of peptide compositions (subcutaneous or intravenous), oral formulation, or oral gavage containing DFM from transformed host cells expressing the engineered peptide. Orally delivered DFM was transformed and thus delivered at a rate of 1 x 10⁻⁶. 9 GLP1 was delivered via CFU / mouse / day, in a volume of 0.2–0.4 ml, for 12 weeks. The following parameters were measured in all mice: 8. Weight, food intake, weekly 9. Body composition, weeks 0, 4, and 8 10. OGTT, Week 6 11. Non-fasting blood glucose, weekly 12. Blood (serum) samples (5 equal aliquots each) for testing insulin, adipokines, clinical chemistry, liver function, and blood lipids, at weeks 2, 4, 6, and 8. 13. Fecal samples, weekly, for microbiome analysis. 14. Final autopsy—Intestines, pancreas, liver, adipose tissue, and muscle were collected and placed in RNA latex for qPCR; additional samples were taken for histopathological / immunohistochemical analysis. Table 17: Study Design for Evaluating Engineered Candidates in a DIO Type 2 Diabetes Model

[0464] Example 8 Design and generation of fusion peptides—albumin-binding domain Glucagon-like peptide-1 (GLP-1) is considered a potentially effective peptide for treating type 2 diabetes mellitus (T2DM); however, its extremely short half-life limits its clinical application. The albumin-binding domain (ABD), with high affinity for human serum albumin (HSA), is a suitable scaffold for prolonging the half-life of therapeutic peptides and proteins. Previous studies have shown that the albumin-binding domain can prolong the half-life of native glucagon-like peptide-1 (Tan H et al. (2021) Eur J Pharmacol 890:173650; doi.org / 10.1016 / j.ejphar.2020.173650). The engineered GLP-1 peptides and dual-agonist peptides presented and described in this article are fused with ABD sequences.

[0465] Naturally occurring ABD is a small triple-helix protein domain (Nilvebrant and Hober (2013) CompStruct Biotechnol J 6, 1-8). G148-ABD3 (GA3), expressed by Streptococcus G148 strain, is one of the best-characterized domains and has been extensively developed for prolonging the half-life of therapeutic peptides or proteins (Nilvebrant and Hober (2013) Comp Struct Biotechnol J 6, 1-8; Stork et al. (2007) Prot Eng Des Sel 20, 569-576; Gapizov et al. (2019) Biotechnol Appl Biochem 66, 617-625). Inspired by the promising applications of GA3 in prolonging half-life, numerous variants have been designed to further improve the pharmacodynamic and pharmacokinetic properties of fusion chaperones. ABD035, screened using phage display technology, showed an apparent affinity of 50-50 fM for HSA, which is several orders of magnitude higher than that of wild-type GA3 (1.2 nM) (Jonsson et al. (2008) Prot Eng Des Sel 21, 515-527). ABDCon, a variant designed using a shared sequence design method, showed an affinity of 75 pM for HSA (Jacobs et al. (2015) Prot Eng Des Sel 28, 385-393).

[0466] The sequences of GA3, ABD035, and ABDCon albumin-binding domain (ABD) were aligned, and the results are shown below (gap positions in the aligned sequences are indicated by "-"; amino acid differences are shown in bold): GA3LAEAKVLANRELDKYGV-SDYYKNLINNAKTVEGVKALIDEILAALP (SEQ ID NO:384) ABD035LAEAKVLANRELDKYGV-SDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO:385) ABDConLKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA--(SEQ ID NO:386) By generating ABD fusions of GLP-1 peptides and dual agonist peptides, fusion constructs comprising Glp-1-peptide- / -linker- / -ABD and Glp-1-peptide- / -linker- / -ABD constructs were obtained. These peptides were linked to the exemplary ABD sequence ABD035 (SEQ ID NO:385). The resulting ABD fusion constructs and basic peptide sequences are shown below (ABD sequences are shown in bold).

[0467] Various

[0468] Example 9 Efficacy evaluation of engineered Lactobacillus reuteri strains and Glp-1 peptides (BEP009 and BEP009-ABD) in DIO mice The efficacy of the GLP-1 peptide and the GLP-1 peptide-ABD fusion was evaluated in type 2 and type 1 diabetic mouse models by administration either in the form of a peptide or in the form of a direct-feed microbial (DFM) formulation incorporating an expression vector of the GLP-1 peptide described herein (“therapeutic peptide and host cells”). Specifically, a diet-induced obesity (DIO) model was used. In vivo activity was assessed by intraperitoneal injection.

[0469] Objective: To evaluate the efficacy of engineered Lactobacillus reuteri 3632 delivering one copy of GLP-1 C1.1 peptide (SEQ ID NO:2) (named 1X BEP009) and five copies of GLP-1 C1.1 peptide (SEQ ID NO:2) (named 5XBEP009) in a diet-induced obesity (DIO) mouse model. Liraglutide was used as a positive control to validate the DIO model. GLP-1 C1.1 peptide (SEQ ID NO:2) (named BEP009) administered via intraperitoneal injection served as the injection peptide control, while Lactobacillus reuteri 3632 served as the chassis control.

[0470] The research design is shown in Table 18 below: Table 18

[0471] Liraglutide: A short-acting GLP-1 agonist used to treat type 2 diabetes and obesity.

[0472] BEP009: A GLP-1 version with trypsin and DPPIV resistance.

[0473] BE105ENLR139: Lactobacillus reuteri strain 3632 that integrates a single copy of BEP009 (1X BEP009).

[0474] BE105ENLR181: Lactobacillus reuteri strain 3632 that integrates five copies of BEP009 (5XBEP009) linked through a trypsin-sensitive site.

[0475] Among numerous mouse strains, C57BL / 6 is one of the most sensitive to diet-induced obesity (DIO), developing severe obesity, exacerbated glucose intolerance, moderate insulin resistance, and elevated glucose levels. Therefore, DIO is a commonly used model for studying prediabetes and diabetes-related metabolic syndrome in humans.

[0476] This study used 6-week-old C57BL / 6J mice that were obese at 0 weeks. Mice were fed a high-fat diet (HFD). Body weight was measured weekly. Resting glucose was measured twice weekly. Food intake was assessed every two weeks. The assessments and aspects of the clinical mouse study included the following: Part 1 of the study involved an oral glucose tolerance test (OGTT) on day 33 and an EchoMRI on day 39. Subsequent study adjustments included administration of BEP009-ABD to group 5 mice starting on day 48. Furthermore, adjustments to the study starting on day 48 included dose escalation of both BEP009 and BEP009-ABD. From day 48, fecal pellet colonization was assessed (to evaluate Lactobacillus reuteri colonization). In subsequent parts of the study, an OGTT was performed on day 75 and an EchoMRI on day 81. Administration of the test substance was discontinued on day 85. The study ended on day 99, with assessments performed on both the OGTT and EchoMRI on day 99.

[0477] Figure 18 The effects of the experimental materials on weekly body weight on day 40 were shown. Liraglutide showed a sustained reduction in body weight compared to the solvent control group. I / P injection of BEP009 had a slight effect on body weight from day 19 compared to the solvent control group. In this study, *Lactobacillus reuteri* 3632 and engineered strains BE105ENLR139 (1X GLP1) and BE105ENLR181 (5X GLP1) had minimal or no effect on body weight.

[0478] The effect of BEP009 on fat content was assessed in an EchoMRI test on day 39. Results are as follows: Figure 19 As expected, liraglutide reduced body fat percentage in the EchoMRI test on day 39 compared to the solvent control group. BEP009 had minimal effect on body fat percentage in the EchoMRI test on day 39 compared to the solvent control group. Chassis LR3632 and engineered strains BE105ENLR139 and BE105ENLR181 also showed no effect on body fat percentage in the EchoMRI test on day 39 compared to the solvent control group.

[0479] Initial phases of this study indicate that the DIO model performed as expected. The control peptide, liraglutide, significantly reduced weekly body weight and also significantly reduced resting and fasting blood glucose levels, as well as the AUC in the OGTT. BEP-009 slightly reduced weekly body weight and had no significant effect on blood glucose. BEP-009 may be rapidly cleared by the kidneys, preventing it from reaching therapeutic concentrations. This observation supports the hypothesis that BEP-009 requires sustained high circulating concentrations to reach the hypothalamus and induce weight loss.

[0480] Based on preliminary results, the study protocol was adjusted and implemented on day 48. Group 3: Starting on day 48, the dose of BEP009 was increased to 2X for the first 3 weeks and to 4X for the following 2 weeks. Group 5: Given the minimal effect of BEP009 on weekly body weight and its lack of effect on AUC or fasting blood glucose levels in the OGTT on day 33, suggesting that BEP009 may be rapidly cleared by the kidneys, we hypothesized that adding ABD could prolong its half-life. This prolongation might allow BEP009 to reach therapeutic concentrations, thereby affecting body weight and blood glucose levels. To verify this hypothesis, we discontinued BE105ENLR139 (1X GLP1) on day 47 and began daily administration of BEP009-ABD (eGLP-1-ABD fusion construct) from day 48, at a dose of 2X for the first 3 weeks and 4X for the following 2 weeks. We also began collecting fecal samples weekly to understand the colonization dynamics of the engineered strain. Groups 1, 2, 4, and 6: No significant changes were observed.

[0481] The results of the assessment of the effects of GLP-1 peptides and constructs, including BEP009-ABD, on weekly body weight on day 82 were determined, such as... Figure 20 As shown. As expected, daily administration of liraglutide effectively reduced HFD-induced weight gain compared to the solvent control group. Daily administration of BEP009 slightly reduced HFD-induced weight gain compared to the solvent control group. Daily administration of BEP009-ABD effectively reduced HFD-induced weight gain compared to the solvent control group, and this effect was comparable to that of liraglutide. Chassis administration resulted in slightly higher weight gain than the solvent control group. The engineered strain had no significant effect on HFD-induced weight gain compared to the solvent control group.

[0482] The effect on weekly blood glucose levels was measured until day 78. Results are as follows: Figure 21 As shown. As expected, liraglutide showed a trend toward lowering blood glucose levels throughout the study compared to the solvent control group, although this effect was more pronounced after day 50. Compared to the solvent control group, BEP009-ABD showed a significant reduction in blood glucose immediately after administration on day 48, and this effect persisted until the end of the study.

[0483] On day 75, changes in fasting blood glucose / AUC were assessed in the OGTT trial. Results are as follows: Figure 22 As expected, liraglutide significantly reduced fasting blood glucose and AUC in the OGTT on day 75 compared to the solvent control group. The effect of i / p injection of BEP009 on AUC and fasting blood glucose was slight in the OGTT on day 75 compared to the solvent control group. BEP009-ABD reduced fasting blood glucose and AUC in the OGTT on day 75 compared to the solvent control group, with a reduction effect slightly better than or comparable to liraglutide. Compared to the solvent control group, *Lactobacillus reuteri* 3632 and engineered strain BE105ENLR181 (5x GLP1) showed slightly increased fasting blood glucose and AUC in the OGTT on day 75.

[0484] The effect on fat content was assessed on day 81 using EchoMRI. Results are as follows: Figure 23 As expected, liraglutide reduced fat content in EchoMRI on day 81 compared to the solvent control group. i / p injection of BEP009 showed a slight reduction in fat content in EchoMRI on day 81 compared to the solvent control group. BEP009-ABD reduced fat content in EchoMRI on day 81 compared to the solvent control group, with a slightly better reduction than BEP009. Neither *Lactobacillus reuteri* 3632 nor the engineered strain BE105ENLR181 (5x GLP1) showed a reduction in fat content compared to the solvent control group.

[0485] On day 81, the effect of EchoMRI on lean body mass was also assessed, and the results were as follows: Figure 24 As expected, liraglutide reduced lean body mass in the EchoMRI test on day 81 compared to the solvent control group. I / P injection of BEP009 slightly increased lean body mass in the EchoMRI test on day 81 compared to the solvent control group. BEP009-ABD reduced lean body mass in the EchoMRI test on day 81 compared to the solvent control group, and this reduction was comparable to that of liraglutide. *Lactobacillus reuteri* 3632 and engineered strain BE105ENLR181 (5x GLP1) showed a slight increase in lean body mass compared to the solvent control group.

[0486] The effects of the test materials and peptides on bi-weekly food intake (normalized with solvent control) on day 84 were determined, and the results are as follows: Figure 25As expected, daily administration of liraglutide effectively reduced cumulative food intake (total grams / animal / day) compared to the solvent control group. Daily administration of BEP009 showed a reduction in cumulative food intake (total grams / animal / day) compared to the solvent control group. Daily administration of BEP009-ABD also effectively reduced cumulative food intake (total grams / animal) compared to the solvent control group, with effects comparable to liraglutide but weaker than BEP009. Administration of the chassis strain had no effect on cumulative food intake compared to the solvent control group. Administration of the engineered strain resulted in a slight increase in cumulative food intake compared to the solvent control group.

[0487] The results of this study indicate that liraglutide showed significant reductions in all assessed parameters. BEP-009 peptide itself slightly reduced weekly body weight, fat and lean body mass, and fasting blood glucose. The peptide-ABD fusion BEP-009-ABD significantly reduced weekly body weight, bi-weekly food intake, fat and lean body mass, and resting and fasting blood glucose. Adding ABD achieved optimal systemic concentrations for best therapeutic effect. BEP009-ABD had a more significant effect on glucose than on body weight, suggesting that higher doses may be needed to achieve weight loss. While its effect on glucose was comparable to or better than liraglutide, its effect on body weight was weaker. This may be due to inefficient crossing of the blood-brain barrier or retrograde diffusion from the blood-brain barrier into the systemic circulation, thus hindering concentration in the brain. However, compared to liraglutide, BEP009-ABD had similar or better glucose activity, suggesting that its moderate ...

Claims

1. An engineered dual agonist polypeptide comprising the following amino acid sequence: Formula II (SEQ ID NO:111): Y X2E G T X6X7S D X 10 S I X 13 X 14 D X 16 I A Q X 20 A X 22 V Q X 25 X 26 I A G G P S S G A PP(I) in X2 = V or K; [Telbopeptide = A] X6 = F, P, or S; [Telborpeptide = F] X7 = T, C, or E; [Telborpeptide = T] X 10 = Y, C, or E; [Telbopeptide = Y] X 13 = A, S, Y, N, I, L, R, V, or K; [Telbopeptide = A] X 14 = L, K, H, or I; [Telbopeptide = L] X 16 = K, R, H, or V; [Telbopeptide = K] X 20 = K, R, H, N; [Telbopeptide = K] X 22 = F, A, P; [Telbopeptide = F] X 25 = W, P, K, H, or I; and [Telbopeptide = W] X 26 = L or V, Or formula IV (SEQ ID NO:383): Y X2E G T X6X7S D X 10 S I X 13 X 14 D X 16 I A X 19 X 20 A X 22 V Q X 25 X 26 I A G G P S S G A PP(IV), Where X2 = V or K; X6 = F, P or S; X7 = T, C or E; X 10 = Y, C, or E; X 13 = A, S, Y, N, I, L, R, V or K; X 14 =L, K, H, or I; X 16 = K, R, H, or V; X 19 = Q or V; X 20 = K, R, H, N; X 22 = F, A, P; X 25 = W, P, K, H, or I; and X 26 =L or V.

2. The engineered dual agonist polypeptide of claim 1, wherein... X2 = V; X6 = P or S; X7 = C or E; X 10 = C or E; X 13 = S;X 14 = L, H, or I; X 16 = H or V; X 20 = H or N; X 22 =A or P; X 25 = P or I; and X 26 = L or V.

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

381.

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

116.

5. An engineered glucagon-like peptide-1 (eGLP-1) polypeptide comprising the following amino acid sequence: Formula I: H G E G T S E S D V S X 12 X 13 X 14 E G Q A A Q E X 22 X 23 A X 25 X 26 V D G X 30 (I) in X 12 = S or Q; X 12 = S, Q, or Y; X 14 = I or L; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 = I or V; and X 30 = R or S, Or Formula III (SEQ ID NO:382): X1G E G T S E S D V S X 12 X 13 X 14 E X 16 Q A X 19 X 20 E X 22 X 23 A X 25 X 26 V D G X 30 (III), Where X1 = H or Y; X 12 = S, Q, A, or M; X 13 = S, Q, Y, or M; X 14 = I or L; X 16 = G or M; X 19 = A or T; X 20 = Q, V, or D; X 22 = V, I, or F; X 23 = V or I; X 25 = V, I, or W; X 26 = I or V; and X 30 = R or S.

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

380.

7. The engineered polypeptide of claim 1 or claim 5, wherein the engineered polypeptide has substantial resistance to protein hydrolytic degradation.

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

9. The engineered polypeptide of claim 1 or claim 5, wherein the engineered polypeptide has substantial resistance to degradation by DPP-IV, enkephalinase, α-chymotrypsin, trypsin, elastase or pepsin.

10. An isolated polynucleotide encoding: Engineered dual agonist peptides comprising the amino acid sequence of formula II (SEQ ID NO:111) or formula IV (SEQ ID NO:383); and / or Engineered glucagon-like peptide-1 (eGLP-1) polypeptide containing the amino acid sequence of formula I (SEQ ID NO:1) or formula III (SEQ ID NO:382).

11. A pharmaceutical composition comprising: Engineered dual agonist peptides comprising the amino acid sequence of formula II (SEQ ID NO:111) or formula IV (SEQ ID NO:383); and / or Engineered glucagon-like peptide-1 (eGLP-1) polypeptides comprising the amino acid sequence of formula I (SEQ ID NO:1) or formula III (SEQ ID NO:382); And the carrier.

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

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

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

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

16. A method for treating or preventing conditions caused by or characterized by overweight in a subject, and for treating obesity, morbid obesity, obesity-related inflammation, obesity-related gallbladder disease, obesity-induced sleep apnea, metabolic syndrome, prediabetes, insulin resistance, glucose intolerance, type 2 diabetes, type 1 diabetes, hypertension, atherosclerotic dyslipidemia, atherosclerosis, arteriosclerosis, coronary artery disease, peripheral artery disease, stroke, or microvascular disease, comprising administering to the subject in need of treatment an effective amount of the engineered dual agonist polypeptide of claim 1 and / or the engineered glucagon-like peptide-1 (eGLP-1) of claim 5, the pharmaceutical composition of claim 11, or the host cell of claim 12.

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

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

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

20. A pharmaceutical composition comprising an engineered glucagon-like peptide-1 (eGLP-1) polypeptide, said engineered glucagon-like peptide-1 (eGLP-1) polypeptide comprising an amino acid sequence selected from any one of SEQ ID NO:112 to SEQ ID NO:147 or SEQ ID NO:

381.

21. A pharmaceutical composition comprising an engineered glucagon-like peptide-1 (eGLP-1) polypeptide, said engineered glucagon-like peptide-1 (eGLP-1) polypeptide comprising an amino acid sequence selected from any one of SEQ ID NO:1 to SEQ ID NO:14 or SEQ ID NO:32 to SEQ ID NO:

67.

22. A direct-feed microorganism (DFM) comprising bacteria transformed with nucleic acids, said nucleic acids encoding an engineered dual agonist polypeptide of formula II or formula IV, and / or an engineered glucagon-like peptide-1 (eGLP-1) encoding an amino acid sequence comprising formula I or formula III.

23. The DFM of claim 22, wherein the bacteria comprises strains selected from the group consisting of Bacillus, Lactobacillus, Lactococcus, Enterococcus, Salmonella, and combinations thereof.

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