Synthetic GLP-1r binding proteins, manufacture, and uses thereof

Abstract

The present disclosure provides synthetic glucagon-like peptide-1 receptor (GLP-1R) binding proteins, compositions containing such binding proteins, and methods of making and using such binding proteins, including their uses in treating GLP-1R dysfunction and / or a GLP-1R-mediated disease or disorder.

Description

SYNTHETIC GLP-1R BINDING PROTEINS, MANUFACTURE, AND USES THEREOFFIELD

[0001] The disclosure relates generally to synthetic GLP-1 receptor (GLP-1R) binding proteins, their manufacture and use in the treatment of various GLP-1 R-mediated disorders, including endocrine, metabolic, cardiovascular, and / or neurodegenerative disorders.BACKGROUND

[0002] The modulation of GLP-1 receptor activity is believed to be associated with a number disorders, including endocrine and / or metabolic disorders (e.g. type 2 diabetes mellitus, obesity, pancreatic dysfunction), gastrointestinal disorders (e.g.. motility disorders, e.g., irritable bowel syndrome, e.g., irritable bowel disease), organ dysfunction (e.g., renal dysfunction, reduced kidney function, e.g., hepatic dysfunction, e.g., nonalcoholic steatohepatitis, nonalcoholic fatty liver disease), cardiovascular disorders (e.g, heart attack, stroke), and neurodegenerative disorders (e.g.. Parkinson’s. Alzheimer’s). See. e.g., Zhao et al. (2021 ) FRONT. ENDOCRINOL. 12:721 135; see also Kokkoraskis et al. (2023) METABOLISM 148: 155689; Ussher et a / . (2023) NAT. REV. CARDIO. 20:463 and Peng W et al. (2022) AGING DIS. 13:468.

[0003] It has been discovered that GLP-1 receptor activity' is mediated by an incretin hormone called glucagon-like peptide (GLP-1), which is a processing fragment of proglucagon, a glucagon precursor. See. e.g., Holst, JJ (2019) FRONT. ENDOCRINOL. 10:260. Incretin hormones, including GLP-1, are peptide hormones secreted from the small intestine in response to the presence of glucose following food ingestion. Id. GLP-1 has been found to stimulate insulin secretion, inhibit glucagon secretion, reduce appetite and food intake, and maintain its activity in patients with type 2 diabetes mellitus. Id.

[0004] The utility of GLP-1, however, has been limited by its short half-life in humans. This issue has been addressed by the development of inhibitors of the enzyme dipeptidyl peptidase 4 (DPP-4), which is associated with the rapid degradation of GLP-1, and by long- acting synthetic analogs of GLP-1 and / or agonists of GLP-1 R that can resist DPP-4 degradation. Id.

[0005] Currently seven GLP-1R agonists, namely, semaglutide (including high dose semaglutide), dulaglutide, liraglutide (including high-dose liraglutide), lixisenatide, andexenatide (twice daily and extended release) are currently available in the United States. These GLP-1R agonists are administered for improving glycemic control as an adjunctive therapy to diet and exercise in patients with type 2 diabetes mellitus and in patients with refractory diabetes or those intolerant to oral medications. See Latif W. et al. (2023), STATPEARLS: TREASURE ISLAND (FL): STATPEARLS PUBLISHING; updated 2023 Mar 27. Certain of the approved GLP-1R agonists are also approved: (i) to reduce the risk of adverse cardiovascular events in patients with type II diabetes and cardiovascular disease (e.g. semaglutide); (ii) to improve HbAlc in patients with increased risk of cardiovascular events (e.g., dulaglutide); (iii) to manage weight in patients (12 and older) with a body mass index (BMI) of 30 or greater (e.g., high dose liraglutide); (iv) for combination with long acting insulin (e.g, lixisenatide) and (v) for weight loss / weight management (e.g.. high-dose semaglutide).

[0006] Despite the advances made to date, GLP-1R agonists face several challenges and limitations, including adverse side effect profiles, scalability, manufacturability, and stability'. See, e.g., Wang L etal., (2022) SIGNAL TRANSDUCT. TARGET THER. 7:48; Zhao etal., (2021), supra. For example, adverse side effects of GLP-1R agonists include gastrointestinal distress and discomfort including nausea, vomiting, diarrhea, and stomach pain. Other side effects can include hypoglycemia (especially in combination with one or more other diabetes medications), reduced appetite, headaches, pancreatitis, and, though rare, instances of thyroid tumor have occurred. See Zhao et al. (2021) supra,' see also Romera et al. (2018) DIABETES THER. 10:5. Another side effect is weight loss that can occur with higher dosages of the GLP-1R agonists. Although beneficial in weight loss reduction, e.g.. in treating obesity, the other side effects can limit the tolerability of high doses of the GLP-1R agonists. See, e.g., Romera (2018) supra.

[0007] In addition to side effects that can be experienced in patients, the currently approved GLP-1R agonists contain non-natural amino acids and / or lipid modifications, which can impact the cost of scale-up, manufacturing, and formulation. See. e.g., Ding Y. et al. (2020) AMINO ACIDS 52: 1207). Furthermore, the GLP-1R agonists can be unstable and can be challenging to engineer to facilitate tissue-targeted agonism.

[0008] Accordingly, despite the advances made to date, there remains a need for GLP-1R agonists with improved safety, efficacy, potency, manufacturability', scalability, and stability.SUMMARY

[0009] The disclosure is based, in part, upon the discovery' of synthetic GLP-1R binding proteins that specifically bind GLP-1R and modulate the GLP-1R axis, for example, by causing or enhancing downstream signaling to, among other things, activate cyclic AMP. The GLP-1R binding proteins described and provided herein have specific binding activity7for GLP-1R, have the ability7to agonize downstream GLP-lR-mediated signaling activity7, and, among other things, are thermally and chemically stable, resistant to oxidation, resistant to protease degradation, deamination, and glycosylation, and have millimolar (mM)-level solubility. The binding proteins can also be conjugated (e.g., via chemical conjugation or as a fusion protein) to an effector.

[0010] Accordingly, the disclosure provides, among other things, sy thetic GLP-1R binding proteins, methods of making such binding proteins, and methods of using such proteins to treat a disease or disorder mediated by the GLP-1R axis.

[0011] In one aspect, the disclosure provides a synthetic GLP-1R binding protein, comprising: (a) an amino acid sequence from 30 amino acids to 95 amino acids in length; (b) a net negative charge when present in phosphate buffered saline (PBS); (c) a binding affinity7for GLP-1R stronger than 1 pM: and (d) a stability profile such that the protein (i) retains at least 90% binding affinity to GLP-1R upon cooling to room temperature after thermal denaturation at 95°C in PBS for at least about five minutes relative to the protein prior to thermal denaturation; (ii) retains at least 90% binding affinity to GLP-1R after incubation for 16 hours at 37°C of incubation in PBS relative to the protein under the same conditions prior to incubating; and / or (iii) retains at least 90% binding affinity7to GLP-1R in PBS following chemical denaturation in 4 M urea for 1 hour at room temperature relative to the protein prior to chemical denaturation.

[0012] In another aspect, the disclosure provides a synthetic GLP-1R binding protein, comprising (a) an amino acid sequence from 30 amino acids to 95 amino acids in length;(b) a net negative charge in phosphate buffered saline (PBS); (c) a binding affinity for GLP-1R stronger than 1 pM; (d) at least one alpha helix; (e) at least three beta sheets; (f) at least three amino acid loops, where a first loop having a first amino acid sequence connects a terminal amino acid (e.g., a C-terminal amino acid) of a first alpha helix to a terminal amino acid (e.g., an N-terminal amino acid) of a first beta sheet, and a second loop having a second amino acid sequence connects a second, terminal amino acid (e.g., a C-terminalamino acid) of the first beta sheet to a terminal amino acid (e.g., an N-terminal amino acid) of a second beta sheet, and a third loop having a third amino acid sequence connects a third, terminal amino acid (e.g., a C-terminal amino acid) of the second beta sheet to a terminal amino acid (e g., an N-terminal amino acid) of a third beta sheet; and (g) a hydrophobic core defined by at least two hydrophobic amino acids present in at least one of the alpha helices and / or at least one of the beta sheets.

[0013] In certain embodiments, the synthetic GLP-1R binding protein comprises an optional fourth amino acid loop and / or an optional fourth beta sheet. In certain embodiments, the synthetic GLP-1R binding protein comprises an optional fourth amino acid loop and / or an optional fourth beta sheet, wherein the fourth loop has a fourth amino acid sequence and connects a fourth, terminal amino acid (e.g., a C-terminal amino acid) of the fourth loop to a terminal amino acid (e.g., an N-terminal amino acid) of the fourth beta sheet.

[0014] In certain embodiments, the synthetic GLP-1R binding protein comprises one or more of the following features: (a) free of tryptophan amino acids; (b) free of methionine amino acids; (c) free of lysine amino acids; (d) does not comprise an unpaired cysteine amino acid when cysteine amino acids are present in the protein; (e) free of glycosylation sites; (!) free of protease cleavage sites; and (g) soluble up to at least 1 mM in PBS at 4°C for one month.

[0015] In certain embodiments, the synthetic GLP-1R binding protein has a binding affinity from between about 10 pM to about 0.001 nM; about 10 pM to about 0.01 nM, about 7.5 pM to about 0.75 nM; about 5 pM to about 0.5 nM; about 2.5 pM to about 0.25 nM; about 1 pM to about 1 nM; about 0.75 pM to about 1 nM, about 0.5 pM to about 1 nM; about 0.25 pM to about 1 nM; about 0. 10 pM to about 1 nM; about 75 nM to about 1 nM; about 50 nM to about 1 nM; about 25 nM to about 1 nM; about 10 nM to about 1 nM; or about 5 nM to about 1 nM. The synthetic GLP-1R binding protein can have a binding affinity7stronger than about 10 pM, about 7.5 pM, about 5 pM, about 2.5 pM, about 1 pM, about 0.75 pM, about 0.5 pM, about 0.25 pM, about 0. 1 pM, about 75 nM. about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, about 0.1 nM, about 0.01 nM, or about 0.001 nM.

[0016] In certain embodiments, the N-terminus of the first alpha helix in the synthetic GLP-1R binding protein is preceded by one or more N-terminal amino acids, and / or the C- terminus of the third beta sheet or, when present, the C-terminus of the fourth beta sheet in the synthetic GLP-1R binding protein is followed by one or more C-terminal amino acids.

[0017] In certain embodiments, the synthetic GLP-1 R binding protein comprises from about 30 to about 90 amino acids in length, from about 35 amino acids to about 85 amino acids in length, from about 35 amino acids to about 75 amino acids in length, from about 35 amino acids to about 65 amino acids in length, from about 35 amino acids to about 55 amino acids in length, from about 35 amino acids to about 50 amino acids in length, from about 40 amino acids to about 85 amino acids in length, from about 40 amino acids to about 75 amino acids in length, from about 40 amino acids to 65 amino acids in length, from about 45 amino acids to about 70 amino acids in length, from about 45 amino acids to about 65 amino acids in length, from about 45 amino acids to about 60 amino acids in length, from about 50 amino acids to about 75 amino acids, from about 50 amino acids to about 70 amino acids in length, from about 50 amino acids to about 65 amino acids in length, from about 60 amino acids to about 70 amino acids in length, or from about 60 amino acids to about 65 amino acids in length. In certain embodiments, the synthetic GLP- 1R binding protein comprises 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 amino acids.

[0018] In certain embodiments, the synthetic GLP-1R binding protein comprises one or more of the following: (a) the first alpha helix contains at least one hydrophobic amino acid, wherein, optionally, one or more of the at least one hydrophobic amino acids is not solvent accessible; (b) the first, second, third, and / or fourth beta sheet each contains at least two or three hydrophobic amino acids, wherein, optionally, one or more of the at least two or three hydrophobic amino acids is not solvent accessible; (c) the first alpha helix contains at least one or two solvent accessible amino acids; (d) the first, second, third, and / or fourth beta sheet contains at least one or two solvent accessible amino acids; (e) the first alpha helix contains at least one or two solvent accessible amino acids; and (f) the first and / or second and / or third and / or fourth loop contains at least one hydrophobic amino acid. In some embodiments, a GLP-1R binding protein comprises (a) the first, second, third, and / or fourth beta sheets, each contains at least two hydrophobic amino acids; (b) the first, second, third, and, when present, fourth beta sheet each contains at least one solvent accessibleamino acid; (c) the first, second, third and, when present, fourth beta sheet each contains at least two hydrophobic and one solvent accessible amino acids; (d) the first alpha helix contains at least four solvent accessible amino acids; and / or (e) the first, second, third, and / or fourth loop contains at least one hydrophobic amino acid.

[0019] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI. L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 71, wherein DI, D2, D3, and D4 independently comprise any of the following combinations: (a) DI comprises an amino acid sequence of SEQ ID NO: 84, wherein XI is H, L, Y, I, a peptide bond, or is absent; X2 is A, G, S, T, D, E, P, H, V, N, a peptide bond, or is absent; X3 is E, a peptide bond, or is absent; X4 is G, A, a peptide bond, or is absent; X5 is T. P, V. H, G. Y, a peptide bond, or is absent; X6 is F. A, a peptide bond, or is absent; XI 1 is S or R; X13 is Y or I; XI 6 is R or V; XI 8 is A, D, or L; X19 is S, I, E, V, or L; X20 is R or I; X21 is S or F; X23 is L, I, or V; X25 is I, L, Y, or A; X27 is Q or L; X28 is L, G, N, or Q; and X29 is R or G; (b) D2 comprises an amino acid sequence of X36X37X38X39LD, wherein X36 is F or T; X37 is S, V, or L; X38 is L, F, Y, or R; and X39 is D or V; (c) D3 comprises an amino acid sequence of X46X47TX49X50D, wherein X46 is E, V, or D; X47 is L or P; X49 is V or F; and X50 is T or I; and (d) D4 comprises an amino acid sequence of X57X58X59VX61, wherein X57 is T, K, or R; X58 is T, F, K, L, or R; X59 is T. V, F, R, or H; and X61 is Q, S, T, or H. In certain embodiments, the synthetic GLP-1R binding protein comprises LI, which comprises an amino acid sequence of DGX33TX35, wherein X33 is I, G, V, K, or F; and X35 is D or G; and / or L2, which comprises an amino acid sequence of X42X43X44X45, wherein X42 is L, D, or E; X43 is L, Y, or E; X44 is T or F; and X45 is G or F; and / or L3. which comprises an amino acid sequence of NX53X54X55E, wherein X53 is N or T; X54 is T or F; and X55 is G or L. .

[0020] In certain embodiments, the synthetic GLP-1R binding protein comprises an amino acid, X62, wherein X62 is L or S, and wherein X62 may or may not be a C-terminal amino acid. In some embodiments, the synthetic GLP-1R binding protein comprises a C-terminal amino acid of X62, wherein X62 is L or S. That is, in some embodiments, X62 is not a C- terminal amino acid. In some such embodiments, one or more additional amino acids can be present following X62 (e.g., bound, e.g, via a peptide bond) to amino acid X62.

[0021] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 71. wherein XI is H, L, Y, I, a peptide bond, or is absent; X2 is A, G, S, T, D, E, P, H, V, N, a peptide bond, or is absent; X3 is E, a peptide bond, or is absent; X4 is G, A, a peptide bond, or is absent; X5 is T, P, V, H, G, Y, a peptide bond, or is absent; X6 is F, A, a peptide bond, or is absent; Xll is S or R; X13 is Y or I; X16 is R or V; X18 is A. D, or L; X19 is S, I, E, V, or L; X20 is R or I; X21 is S or F; X23 is L. I. or V; X25 is I, L, Y, or A; X27 is Q or L; X28 is L, G, N. or Q; X29 is R or G; X33 is I, G, V, K, or F; X35 is D or G; X36 is F or T; X37 is S, V, or L; X38 is L, F, Y, or R; X39 is D or V; X42 is L, D, or E; X43 is L, Y, or E; X44 is T or F; X45 is G or F; X46 is E. V, or D; X47 is L or P; X49 is V or F; X50 is T or I; X53 is N or T; X54 is T or F; X55 is G or L; X57 is T, K. or R; X58 is T. F, K. L, or R; X59 is T, V. F, R. or H; X61 is Q, S, T, or H; and X62 is L or S.

[0022] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 2-29 and 31- 70.

[0023] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI. L2. and L3 are loops 1, 2. and 3, respectively; and (ii) an amino acid of SEQ ID NO: 72, wherein DI, D2, D3, and D4 independently comprise any of the following combinations: (a) DI comprises an amino acid sequence of SEQ ID NO: 85, wherein XI is H. L, Y. I, a peptide bond, or is absent; X2 is A, G, S, T, D, E, P, H, V, N. a peptide bond, or is absent; X3 is E, a peptide bond, or is absent; X4 is G, a peptide bond, or is absent; X5 is T, P, V, H, G, Y, a peptide bond, or is absent; X6 is F, A, a peptide bone, or is absent; XI 3 is Y or I; XI 8 is A, D, or L; XI 9 is I, S, or E; X20 is R or I; X23 is I, L, or V; X28 is L or G; and X29 is R or G; (b) D2 comprises an amino acid sequence of SEQ ID NO: 97, wherein X38 is L or Y; (c) D3 comprises an amino acid sequence of SEQ ID NO: 99, wherein X47 is P or L; and (d) D4 comprises an amino acid sequence of X57X58X59VX61, wherein X57 is T, K, or R; X58 is F, T, or L; X59 is T, H, V, or R; and X61 is Q, S, or T. In certain embodiments, the synthetic GLP-1R binding protein comprises LI, which comprises an amino acid sequence of DGX33TX35, wherein X33 is I, G, V. K, or F; and X35 is D or G; and / or L2, which comprises an amino acid sequence ofX42X43TG, wherein X42 is D, L, or E; and X43 is L or Y; and / or L3, which comprises an amino acid sequence of SEQ ID NO: 105.

[0024] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 72. wherein XI is H, L, Y, I, a peptide bond, or is absent; X2 is A, G, S, T, D, E, P, H, V, N, a peptide bond, or is absent; X3 is E, a peptide bond, or is absent; X4 is G, a peptide bond, or is absent; X5 is T, P, V, H, G, Y, a peptide bond, or is absent; X6 is F, A, a peptide bond, or is absent; XI 3 is Y or I; XI 8 is A, D, or L; XI 9 is I, S, or E; X20 is R or I; X23 is I, L. or V; X28 is L or G; X29 is R or G; X33 is I, G, V, K, or F; X35 is D or G; X38 is L or Y; X42 is D, L, or E; X43 is L or Y; X47 is P or L; X57 is T, K, or R; X58 is F, T, or L; X59 is T, H, V, or R; and X61 is Q, S, or T.

[0025] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 2, 6-9, 11-17, 20, 23, 28-32, 37-40. 42. 44. and 46-70.

[0026] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 73, wherein DI, D2, D3, and D4 independently comprise any of the following combinations: (a) DI comprises an amino acid sequence of SEQ ID NO: 86. wherein XI is H, L, or Y; X2 is A, G, S, T, D, E, P, H, V, or N; X5 is T, P, V, H, G, or Y; X6 is F or A; X18 is A or D; X19 is I, S, or E; X20 is R or I; X23 is I or L; X28 is L or G; X29 is R or G; (b) D2 comprises an amino acid sequence of SEQ ID NO: 97, wherein X38 is L or Y; (c) D3 comprises an amino acid sequence of SEQ ID NO: 100; and (d) D4 comprises an amino acid sequence of X57X58X59VX61. wherein X57 is T, K, or R; X58 is F or T; X59 is T or H; and X61 is Q, S, or T. In certain embodiments, the synthetic GLP-1R binding protein comprises LI, which comprises an amino acid sequence of DGX33TX35, wherein X33 is I or V; X35 is D or G; and / or L2, which comprises an amino acid sequence of X42X43TG, wherein X42 is D, L. or E; and X43 is L or Y; and / or L3, which comprises an amino acid sequence of SEQ ID NO: 105.

[0027] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 73, wherein XI is H, L, or Y; X2 is A,G, S, T, D, E, P, H, V, or N; X5 is T, P, V, H, G, or Y; X6 is F or A; XI 8 is A or D; X19 is I. S, or E; X20 is R or I; X23 is I or L; X28 is L or G; X29 is R or G; X33 is I or V; X35 is D or G; X38 is L or Y; X42 is D, L, or E; X43 is L or Y; X57 is T, K, or R; X58 is F or T; X59 is T or H; and X61 is Q, S, or T.

[0028] In another aspect, the disclosure provides a synthetic GLP- 1 R binding protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 2, 6, 8, 9, 11- 14, 20, 28, 37, and 46-59.

[0029] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary- structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI. D2, D3, and D4 are domains 1, 2, 3. and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 74, wherein DI, D2, D3, and D4 independently comprise any of the following combinations: (a) DI comprises an amino acid sequence of SEQ ID NO: 87, wherein XI is H. I. a peptide bond, or is absent; X13 is Y or I; X18 is D, A, or L; X19 is S or I; X23 is V, I, or L; X28 is L or G; (b) D2 comprises an amino acid sequence of SEQ ID NO: 98; (c) D3 comprises an amino acid sequence of SEQ ID NO: 100; and (d) D4 comprises an amino acid sequence of X57X58X59VQ, wherein X57 is T or K; X58 is T or F; and X59 is T, V, or R. In certain embodiments, the synthetic GLP-1R binding protein comprises LI, which comprises an amino acid sequence of SEQ ID NO: 103, wherein X33 is K, I, F, or G; and / or L2, which comprises an amino acid sequence of SEQ ID NO: 104; and / or L3, which comprises an amino acid sequence of SEQ ID NO: 105.

[0030] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 74, wherein XI is H, I, a peptide bond, or is absent; X13 is Y or I; X18 is D, A, or L; X19 is S or I; X23 is V, I, or L; X28 is L or G; X33 is K. I. F, or G; X57 is T or K; X58 is T or F; and X59 is T. V, or R.

[0031] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 15-17, 23, 29, 31, 38, 40, 42, and 60.

[0032] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LL L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acidof SEQ ID NO: 75, wherein DI, D2, D3, and D4 independently comprise any of the following combinations: (a) DI comprises an amino acid sequence of SEQ ID NO: 88. wherein XI is H, a peptide bond, or is absent; X2 is A, a peptide bond, or is absent; X3 isE, a peptide bond, or is absent; X4 is G, a peptide bond, or is absent; X5 is T, a peptide bond, or is absent; X6 is F, a peptide bond, or is absent; XI 3 is Y or I; XI 8 is A or L; XI 9 is S or I; X23 is L or I; and X28 is L or G; (b) D2 comprises an amino acid sequence of SEQ ID NO: 98; (c) D3 comprises an amino acid sequence of SEQ ID NO: 100; and (d) D4 comprises an amino acid sequence of X57X58TVQ, wherein X57 is T or K; and X58 is T,F, or L. In certain embodiments, the synthetic GLP-1R binding protein comprises LI, which comprises an amino acid sequence of SEQ ID NO: 166, wherein X33 is I or G; and / or L2, which comprises an amino acid sequence of X42LTG. wherein X42 is L or D; and / or L3, which comprises an amino acid sequence of SEQ ID NO: 105.

[0033] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 75, wherein XI is H, a peptide bond, or is absent; X2 is A, a peptide bond, or is absent; X3 is E, a peptide bond, or is absent; X4 is G, a peptide bond, or is absent; X5 is T, a peptide bond, or is absent; X6 is F, a peptide bond, or is absent; XI 3 is Y or I; XI 8 is A or L; XI 9 is S or I; X23 is L or I; X28 is L or G; X33 is I or G; X42 is L or D; X57 is T or K; and X58 is T, F, or L.

[0034] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 30, 39, 44, and 61-70.

[0035] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4 respectively, and LI. L2. and L3 are loops 1, 2. and 3 respectively; and (ii) an amino acid of SEQ ID NO: 76, wherein DI, D2, D3, and D4 independently comprise any of the following combinations: (a) DI comprises an amino acid sequence of SEQ ID NO: 89, wherein XI is H. L, Y. a peptide bond, or is absent; X2 is A, G. S, T, D, E, P, H, V, or N; and X13 is Y or I; (b) D2 comprises an amino acid sequence of SEQ ID NO: 98; (c) D3 comprises an amino acid sequence of SEQ ID NO: 99, wherein X47 is P or L; and (d) D4 comprises an amino acid sequence of SEQ ID NO: 102, wherein X57 is T or K. In certain embodiments, the synthetic GLP-1R binding protein comprises LI, which comprises anamino acid sequence of SEQ ID NO: 166, wherein X33 is I or G; and / or L2, which comprises an amino acid sequence of SEQ ID NO: 104; and / or L3, which comprises an amino acid sequence of SEQ ID NO: 105.

[0036] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 76, wherein XI is H, L, Y, a peptide bond, or absent; X2 is A, G, S, T, D, E, P, H, V, or N; X13 is Y or I; X33 is I or G; X47 is P or L; and X57 is T or K.

[0037] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 2, 7-9, 29, 32, 40. 46-54, and 60.

[0038] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 77, wherein DI, D2, D3, and D4 independently comprise any of the following combinations: (a) DI comprises an amino acid sequence of SEQ ID NO: 90. wherein XI is H, L, Y, a peptide bond, or is absent; X2 is A, G, S, T, D, E, P, H, V, or N; and XI 3 is Y or I; (b) D2 comprises an amino acid sequence of SEQ ID NO: 98; (c) D3 comprises an amino acid sequence of SEQ ID NO: 99, wherein X47 is L or P; and (d) D4 comprises an amino acid sequence of SEQ ID NO: 102. wherein X57 is T or K. In certain embodiments, the synthetic GLP-1R binding protein comprises LI, which comprises an amino acid sequence of SEQ ID NO: 166, wherein X33 is I or G; and / or L2, which comprises an amino acid sequence of SEQ ID NO: 104; and / or L3, which comprises an amino acid sequence of SEQ ID NO: 105.

[0039] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 77. wherein XI is H. L, Y, a peptide bond, or is absent; X2 is A, G, S, T, D, E, P, H, V, or N; XI 3 is Y or I; X33 is I or G; X47 is L or P; and X57 is T or K.

[0040] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 2, 7-9, 29, 32, 40, 46-54, and 60.

[0041] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary’ structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 78, wherein DI, D2, D3, and D4 independently comprise any of the following combinations: (a) DI comprises an amino acid sequence of SEQ ID NO: 91. wherein XI is H or Y; X2 is A, G. S, P, or V; X4 is G or A; XI 1 is S or R; X13 is Y or I; XI 6 is R or V; XI 8 is A or L; XI 9 is S, I, E, V, or L; X20 is R or I; X21 is S or F; X23 is L or I; X25 is I, L, Y, or A; X27 is Q or L; X28 is L, G, N, or Q; and X29 is R or G; (b) D2 comprises an amino acid sequence of X36X37X38X39LD, wherein X36 is F or T; X37 is S, V, or L; X38 is L. F, Y, or R; and X39 is D or V; (c) D3 comprises an amino acid sequence of X46X47TX49X50D, wherein X46 is E, D, or V; X47 is L or P; X49 is V or F; and X50 is T or I; and (d) D4 comprises an amino acid sequence of X57X58X59VX61, wherein X57 is T, K. or R; X58 is T, F, L, K, or R; X59 is T, H, or F; and X61 is Q, T, or H. In certain embodiments, the synthetic GLP-1R binding protein comprises LI. which comprises an amino acid sequence of SEQ ID NO: 166. wherein X33 is I or G; and / or L2. which comprises an amino acid sequence of X42X43X44X45, wherein X42 is L or D; X43 is L or E; X44 is T or F; and X45 is G or F; and / or L3, which comprises an amino acid sequence of NX53X54X55E, wherein X53 is N or T; X54 is T or F; and X55 is G or L.

[0042] In certain embodiments, the synthetic GLP-1R binding protein comprises an amino acid, X62, wherein X62 is L or S, and wherein X62 may or may not be a C-terminal amino acid. In some embodiments, the synthetic GLP-1R binding protein comprises a C-terminal amino acid of X62, wherein X62 is L or S. That is, in some embodiments, X62 is not a C- terminal amino acid. In some such embodiments, one or more additional amino acids can be present following X62 (e.g., bound, e.g., via a peptide bond) to amino acid X62.

[0043] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 78. wherein XI is H or Y; X2 is A, G. S, P, or V; X4 is G or A; XI 1 is S or R; X13 is Y or I; X16 is R or V; X18 is A or L; X19 is S, I, E, V, or L; X20 is R or I; X21 is S or F; X23 is L or I; X25 is I, L, Y, or A; X27 is Q or L; X28 is L, G, N, or Q; X29 is R or G; X33 is I or G; X36 is F or T; X37 is S, V, or L; X38 is L. F, Y, or R; X39 is D or V; X42 is L or D; X43 is L or E; X44 is T or F; X45 is G or F; X46 is E. D, or V; X47 is L or P; X49 is V or F; X50 is T or I; X53 is N or T; X54 isT or F; X55 is G or L; X57 is T, K, or R; X58 is T, F, L, K, or R; X59 is T, H, or F; X61 is Q, T, or H; and X62 is L or S.

[0044] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 2-7, 9-11, 18- 22, 24-27, 30-47, 51 , and 53.

[0045] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI. L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 79, wherein DI. D2, D3, and D4 independently comprise any of the following combinations: (a) DI comprises an amino acid sequence of SEQ ID NO: 92, wherein XI is H or Y; X2 is A, G, S, P, or V; X23 is I or L; X28 is L or G; and X29 is R or G; (b) D2 comprises an amino acid sequence of SEQ ID NO: 98; (c) D3 comprises an amino acid sequence of SEQ ID NO: 101. wherein X46 is E or D; and X47 is L or P; and (d) D4 comprises an amino acid sequence of X57X58X59VQ, wherein X57 is T, K, or R; X58 is F or T; and X59 is T or H. In certain embodiments, the synthetic GLP-1R binding protein comprises LI, which comprises an amino acid sequence of SEQ ID NO: 166. wherein X33 is I or G; and / or L2, which comprises an amino acid sequence of SEQ ID NO: 104; and / or L3, which comprises an amino acid sequence of SEQ ID NO: 105.

[0046] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 79, wherein XI is H or Y; X2 is A, G, S, P, or V; X23 is I or L; X28 is L or G; X29 is R or G; X33 is I or G; X46 is E or D; X47 is L or P; X57 is T, K, or R; X58 is F or T; and X59 is T or H.

[0047] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 2, 5-7, 9, 11. 32, 37, 40, 42, 46, 47, 51, and 53.

[0048] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 80, wherein DI, D2, D3, and D4 independently comprise any of the following combinations: (a) DI comprises an amino acid sequence of SEQ ID NO: 93.wherein XI is H or Y; X2 is A, G, S, P, or V; (b) D2 comprises an amino acid sequence of SEQ ID NO: 98; (c) D3 comprises an amino acid sequence of SEQ ID NO: 99. wherein X47 is L or P; and (d) D4 comprises an amino acid sequence of SEQ ID NO: 102, wherein X57 is T or K. In certain embodiments, the synthetic GLP-1R binding protein comprises LI, which comprises an amino acid sequence of SEQ ID NO: 166, wherein X33 is I or G; and / or L2, which comprises an amino acid sequence of SEQ ID NO: 104; and / or L3, which comprises an amino acid sequence of SEQ ID NO: 105.

[0049] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 80, wherein XI is H or Y; X2 is A, G,S, P, or V; X33 is I or G; X47 is L or P; and X57 is T or K.

[0050] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 2, 7, 9, 32, 40, 46, 47, 51, and 53.

[0051] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI. D2, D3, and D4 are domains 1, 2, 3. and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 81, wherein DI, D2, D3, and D4 independently comprise any of the following combinations: (a) DI comprises an amino acid sequence of SEQ ID NO: 94, wherein X13 is Y or I; X16 is R or V; X18 is A or L; X19 is S, I, E, V, or L; X20 is R or I; X23 is L or I; X25 is I, L, Y, or A; X27 is Q or L; X28 is L, G, or Q; and X29 is R or G; (b) D2 comprises an amino acid sequence of X36X37X38X39LD, wherein X36 is F or T; X37 is S, V, or L; X38 is L, F, Y, or R; X39 is D or V; (c) D3 comprises an amino acid sequence of X46X47TVX50D, wherein X46 is E. D, or V; X47 is L or P; X50 is T or I; and (d) D4 comprises an amino acid sequence of X57X58X59VX61. wherein X57 isT, K, or R; X58 is T, F, L, K, or R; X59 is T, H, or F; and X61 is Q or T. In certain embodiments, the synthetic GLP-1R binding protein comprises LI, which comprises an amino acid sequence of SEQ ID NO: 166, wherein X33 is I or G; and / or L2, which comprises an amino acid sequence of X42X43X44X45. wherein X42 is L or D; X43 is L or E; X44 is T or F; and X45 is G or F; and / or L3, which comprises an amino acid sequence of NX53X54X55E, wherein X53 is N or T; X54 is T or F; and X55 is G or L.

[0052] In certain embodiments, the synthetic GLP-1R binding protein comprises an amino acid, X62, wherein X62 is L or S, and wherein X62 may or may not be a C-terminal amino acid. In some embodiments, the synthetic GLP-1R binding protein comprises a C-terminal amino acid of X62, wherein X62 is L or S. That is, in some embodiments, X62 is not a C- terminal amino acid. In some such embodiments, one or more additional amino acids can be present following X62 (e.g., bound, e.g., via a peptide bond) to amino acid X62.

[0053] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 81. wherein X13 is Y or I: X16 is R or V: X18 is A or L; X19 is S, I, E, V, or L; X20 is R or I; X23 is L or I; X25 is I, L, Y, or A; X27 is Q or L; X28 is L, G, or Q; X29 is R or G; X33 is I or G; X36 is F or T; X37 is S, V, or L; X38 is L ,F, Y, or R; X39 is D or V; X42 is L or D; X43 is L or E; X44 is T or F; X45 is G or F; X46 is E, D, or V; X47 is L or P; X50 is T or I; X53 is N or T; X54 is T or F; X55 is G or L; X57 is T. K, or R; X58 is T, F. L, K. or R; X59 is T. H, or F; X61 is Q or T; and X62 is L or S.

[0054] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 2-7, 10, 11, 18- 22, 24, 27, and 30-45.

[0055] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI. D2, D3, and D4 are domains 1, 2, 3. and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 82, wherein DI, D2, D3, and D4 independently comprise any of the following combinations: (a) DI comprises an amino acid sequence of SEQ ID NO: 95, wherein X23 is I or L; X28 is L or G; and X29 is R or G; (b) D2 comprises an amino acid sequence of SEQ ID NO: 98; (c) D3 comprises an amino acid sequence of SEQ ID NO: 101, wherein X46 is E or D; and X47 is L or P; and (d) D4 comprises an amino acid sequence of X57X58X59VQ, wherein X57 is T, K, or R; X58 is F or T; and X59 is T or H. In certain embodiments, the synthetic GLP-1R binding protein comprises LI, which comprises an amino acid sequence of SEQ ID NO: 166. wherein X33 is I or G; and / or L2, which comprises an amino acid sequence of SEQ ID NO: 104; and / or L3, which comprises an amino acid sequence of SEQ ID NO: 105.

[0056] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 82. X23 is I or L; X28 is L or G; X29 is R or G; X33 is I or G; X46 is E or D; X47 is L or P; X57 is T, K, or R; X58 is F or T; and X59 is T or H.

[0057] In another aspect, the disclosure provides a synthetic GLP- 1 R binding protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 2, 5-7, 11, 32, 37, 40, and 42.

[0058] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary- structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI. D2, D3, and D4 are domains 1, 2, 3. and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 83, wherein DI, D2, D3, and D4 independently comprise any of the following combinations: (a) DI comprises an amino acid sequence of SEQ ID NO: 96; (b) D2 comprises an amino acid sequence of SEQ ID NO: 98; (c) D3 comprises an amino acid sequence of SEQ ID NO: 99, wherein X47 is L or P; and (d) D4 comprises an amino acid sequence of SEQ ID NO: 102, wherein X57 is T or K. In certain embodiments, the synthetic GLP-1R binding protein comprises LI, which comprises an amino acid sequence of SEQ ID NO: 166, wherein X33 is I or G; and / or L2, which comprises an amino acid sequence of SEQ ID NO: 104; and / or L3, which comprises an amino acid sequence of SEQ ID NO: 105

[0059] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 83, wherein X33 is I or G; X47 is L or P; and X57 is T or K.

[0060] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 2, 7, 32, and 40.

[0061] In certain embodiments, the disclosure provides the synthetic GLP-1R binding protein further comprising a C-terminal extension of SEQ ID NO: 165. wherein X63 is R, G, D, or P.

[0062] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4-L4-D5 (Formula IB), wherein DI, D2, D3, D4 and D5 are domains 1, 2, 3, 4 and 5 respectively, and LI, L2, L3, and L4 are loops 1, 2, 3, and 4 respectively; and (ii) an amino acid of SEQ ID NO: 156, wherein DI, D2, D3, D4, and D5 independently comprise any of the following combinations: (a) DI comprises an amino acid sequence of SEQ ID NO: 158, wherein X2 is G or S; X3 is E or Q; XI 0 is V or Y; X14 is L, A, or R; XI 6 is R or Y; XI 8 is A, K, R, or F; X21 is S, I, F, Y, V, R, Q, L, or W; X24 is T, L, R, or K; X25 is I or F; X28 is L, R. N, or Q; X29 is R. E, G. Q, or H; (b) D2 comprises an amino acid sequence of SEQ ID NO: 160, wherein: X36 is F or Y; X41 is D, K, or R; (c) D3 comprises an amino acid sequence of SEQ ID NO: 100; (d) D4 comprises an amino acid sequence of SEQ ID NO: 161; and (e) D5 comprises an amino acid sequence of SEQ ID NO: 162. In certain embodiments, the synthetic GLP-1R binding protein comprises LI, which comprises an amino acid sequence of DGIX34X35, wherein X34 is T, K, R; X35 is D or Q; and / or L2, which comprises an amino acid sequence of SEQ ID NO: 104; and / or L3, which comprises an amino acid sequence of SEQ ID NO: 105; and / or L4, which comprises an amino acid sequence of LX63TG, wherein X63 is R, G. D, P.

[0063] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 156, wherein X2 is G or S; X3 is E or Q; XI 0 is V or Y; X14 is L, A, or R; XI 6 is R or Y; XI 8 is A, K, R, or F; X21 is S, I, F, Y, V, R, Q, L, or W; X24 is T, L, R, or K; X25 is I or F; X28 is L, R, N, or Q; X29 is R, E, G, Q, or H; X36 is F or Y; X41 is D, K. or R; X34 is T, K, or R; X35 is D or Q; and X63 is R, G, D. or P.

[0064] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 108-151.

[0065] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an ammo acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4-L4-D5 (Formula IB), wherein DI, D2, D3, D4 and D5 are domains 1, 2, 3, 4 and 5 respectively, and LI, L2, L3 and L4 are loops 1, 2, 3, and 4 respectively; and (ii) an amino acid of SEQ ID NO: 157, wherein DI, D2, D3, D4, and D5 independently comprise any of the following combinations: (a) DI comprises an amino acid sequence of SEQ ID NO: 159, wherein X21 is W or L; X24 is T, R, or K; X28 is L, N, or Q; and X29 is R, E, G, Q, or H; (b) D2 comprises an amino acid sequence of SEQ ID NO: 160, wherein X36 is F or Y ; and X41 is D, K, or R; (c) D3 comprises an amino acid sequence of SEQ ID NO:100; (d) D4 comprises an amino acid sequence of SEQ ID NO: 161; and (e) D5 comprises an amino acid sequence of SEQ ID NO: 162. In certain embodiments, the synthetic GLP- 1R binding protein comprises LI, which comprises an amino acid sequence of DGIX34X35, wherein X34 is T, K, or R; and X35 is D or Q; and / or L2, which comprises an amino acid sequence of SEQ ID NO: 104; and / or L3, which comprises an amino acid sequence of SEQ ID NO: 105, wherein X63 is R, G, D, or P; and / or L4 comprises an amino acid sequence of LX63TG.

[0066] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 157, wherein X21 is L or W; X24 is T, R, or K; X28 is L, N, or Q; X29 is R, E, G, Q, or H; X34 is T, K, or R; X35 is D or Q; X36 is F or Y; X41 is D, K, or R; and X63 is R, G, D, or P.

[0067] In another aspect, the disclosure provides a synthetic GLP-1R binding protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 130-151.

[0068] In certain embodiments, a synthetic GLP-1R binding protein of the disclosure further comprises an effector. The effector can comprise a protein that extends serum halflife. By way of non-limiting example, the effector can comprise a synthetic serum albumin binding protein. In some embodiments, the effector can comprise a protein that has an amino acid sequence comprising SEQ ID NO: 163. In certain embodiments, the effector can be attached to the GLP-1R binding protein by an amino acid linker. For example, the effector can comprise an amino acid linker having an amino acid sequence comprising SEQ ID NO: 164. To give but one example, a synthetic GLP-1R binding protein comprising an effector can have an amino acid sequence comprising SEQ ID NO: 155.

[0069] Serum half-life of a synthetic GLP-1R binding protein can be increased with an effector (e.g, that binds to albumin, e.g., human serum albumin). In certain embodiments the half-life of a synthetic GLP-1R binding protein comprising an effector is greater than the half-life of a synthetic GLP-1R binding protein without the effector. In certain embodiments, the synthetic GLP-1R binding protein without an effector has an amino acid sequence comprising any one of SEQ ID NOs: 2-83 and 108-154. In certain embodiments, the synthetic GLP-1R binding protein without the effector is a reference GLP-1R agonist. For example, in some embodiments, the reference GLP-1R agonist has an amino acid sequence comprising SEQ ID NO: 106.

[0070] Depending upon the circumstances, it is understood that in a GLP-1R binding protein containing DI and D4 domains, the DI domain can be flanked by one or more N- terminal amino acids and / or the D4 can be flanked by one or more C-terminal amino acids.

[0071] In another aspect, the disclosure provides synthetic GLP-1R binding protein comprising an amino acid sequence according to any of SEQ ID NOs: 2 to 83 and 108-154.

[0072] In certain embodiments, the synthetic GLP-1R binding proteins provided herein have a binding affinity for GLP-1R stronger than 10 pM. The synthetic GLP-1R binding protein can have a binding affinity between about 10 pM to about 0.001 nM; about 7.5 pM to about 0.01 nM; about 7.5 pM to about 0.75 nM; about 5 pM to about 0.5 nM; about 2.5 pM to about 0.25 nM; about 1 pM to about 1 nM; about 0.5 pM to about 1 nM; about 0.25 pM to about 1 nM; about 0. 10 pM to about 1 nM; about 75 nM to about 1 nM; about 50 nM to about 1 nM; about 25 nM to about 1 nM; about 10 nM to about 1 nM; or about 5 nM to about 1 nM.

[0073] Depending upon the circumstances, the synthetic GLP-1R binding protein has a binding affinity stronger than about 10 pM, about 7.5 pM, about 5 pM. about 2.5 pM, about 1 pM, about 0.75 pM, about 0.5 pM, about 0.25 pM, about 0.1 pM, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, about 0. 1 nM, about 0.01 nM, or about 0.001 nM.

[0074] In certain embodiments, the amino acid sequence of the synthetic GLP-1R binding protein has at least 85 (e.g, 90, 95, 96, 97, 98, 99, 99.5) percent identity to that of SEQ ID NO: 2.

[0075] In certain embodiments, the amino acid sequence of the synthetic GLP-1R binding protein has an amino acid sequence comprising or according to one or more sequences set forth in Table 8.

[0076] Also provided herein is a pharmaceutical composition comprising a synthetic GLP-1R binding protein disclosed herein, and a pharmaceutically acceptable carrier. The synthetic GLP-1R binding protein can further comprise an effector. In certain embodiments, the effector can extend serum half-life. By way of non-limiting example, the effector can comprise a synthetic serum albumin binding protein. Further, the effector can comprise a protein (e.g., a half-life extending protein, e.g., a synthetic albumin bindingprotein, e.g., a human serum albumin binding protein) that has an amino acid sequence comprising SEQ ID NO: 163. In certain embodiments, the effector can be attached to the GLP-1R binding protein by an amino acid linker. In some such embodiments, the linker can have an amino acid sequence comprising SEQ ID NO: 164. In some embodiments, the pharmaceutical composition comprises a synthetic GLP-1R binding protein comprising an effector and has an amino acid sequence comprising SEQ ID NO: 155. In certain embodiments, the serum half-life of the synthetic GLP-1R binding protein comprising an effector (e.g., that extends serum half-life) is greater than the serum half-life of the synthetic GLP-1R binding protein without the effector. In some embodiments, the synthetic GLP-1R binding protein has an amino acid sequence according to any one of SEQ ID NOs: 2-83 and 108-154. In certain embodiments, the synthetic GLP-1R binding protein without the effector (e.g.. that extends serum half-life) is a GLP-1R agonist. For example, in some embodimnets, the GLP-1R agonist has an amino acid sequence comprising SEQ ID NO: 106.

[0077] In another aspect, the disclosure provides a method of targeting GLP-1R. The method comprises contacting a cell that expresses GLP-1R on its cell surface with a composition comprising a synthetic GLP-1R binding protein or pharmaceutical composition as provided herein. In addition, the disclosure provides a method of modulating GLP-1R activity. The method comprises contacting a cell that expresses GLP- 1R on its cell surface with a composition comprising a synthetic GLP-1R binding protein or pharmaceutical composition provided herein. In each of the methods, the synthetic GLP-1R binding protein can further comprise an effector. Furthermore, depending upon the circumstances, the synthetic GLP-1R binding protein or the pharmaceutical composition agonizes (with at least equivalent therapeutic effect), promotes, or increases GLP-1R activity (e.g., GLP-lR-mediated) in the presence of a GLP-1R ligand (e.g., GLP- 1) or a reference GLP-1R agonist ( e.g., semaglutide, liraglutide, dulaglutide. lixisenatide, exenatide, etc.) relative to GLP-1R activity (e.g., GLP-lR-mediated) in the absence of the GLP-1R ligand (e.g., GLP-1) or reference GLP-1R agonist ( e.g., semaglutide, liraglutide, dulaglutide, lixisenatide, exenatide, etc.) .

[0078] In yet another aspect, the disclosure provides a method of biasing signaling through a G protein coupled receptor (GPCR), where the method involves modulating activity of a GPCR that is capable of activating one or more downstream pathways. In certainembodiments, the method comprises modulating one or more activities of a GPCR (e.g., GLP-1R) that is capable of activating a first downstream signaling pathway in a cell and a second, different downstream signaling pathway in the cell, wherein the GPCR (e.g., GLP- 1R) comprises an agonist binding site such that when an agonist binds to the agonist binding site, the agonist activates the first and the second downstream signaling pathways in the cell. The method comprises: contacting the cell with a miniprotein comprising: (i) an agonist that binds to the agonist binding site of the GPCR. and (ii) a stabilization domain that interacts with a surface region of the GPCR adjacent to the agonist binding site, wherein the stabilization domain stabilizes the interaction of the agonist in the agonist binding site of the GPCR and causes the GPCR to activate the first downstream signaling pathw ay preferentially over the second downstream signaling pathway relative the same or a similar agonist without the stabilization domain. Signaling pathways can be selected from, among others, cAMP -mediated signaling pathways, P-arrestin signaling mediated pathways, PI3K- mediated signaling pathways, PKCb-mediated signaling pathways, etc. In certain embodiments, a first signaling pathway is a cAMP-mediated signaling pathway, and a second signaling pathway is a P-arrestin-mediated signaling pathway. Synthetic GLP-1R binding proteins can be as provided by the disclosure and reference GLP-1R agonists can include, for example, commercially available agonists such as liraglutide, dulaglutide, lixisenatide. or exenatide. In certain embodiments, the miniprotein comprises a synthetic GLP-1R binding protein as provided herein.

[0079] In another aspect, the disclosure provides a method of biasing signaling of cAMP- mediated pathways over p-arrestin-mediated pathways. In certain embodiments, when a synthetic GLP-1R binding protein (e.g., a synthetic GLP-1R binding protein disclosed herein) interacts with a surface region of a GLP-1R. such as in a cell expressing a GLP-1R, downstream signaling is biased to a cAMP signaling pathway over a arrestin-mediated signaling pathway in the cell. For example, when such a cell is contacted with a synthetic GLP-1R binding protein disclosed herein, the synthetic GLP-1R binding protein preferentially activates the cAMP-mediated signaling pathway over a p arrestin-mediated signaling pathway in the cell.

[0080] In another aspect, the present disclosure provides a method of treatment wherein a subject in need thereof is administered a synthetic GLP-1R binding protein to bias a cAMP signaling-mediated pathway over a P-arrestin-mediated signaling pathway, each of which ismodulated by a GLP-1R, wherein when a synthetic GLP-1R binding protein is administered to the subject it preferentially activates the c AMP-mediated signaling pathway over a P-arrestin-mediated signaling pathway in the subject. Depending upon the circumstances, the administration of an effective amount of a synthetic GLP-1R binding protein provided herein reduces a side effect mediated by a GLP-1R activated P-arrestin- mediated signaling pathway such that the side effect is reduced relative to administration of a reference GLP-1R agonist that lacks a protein stabilizing domain capable of stabilizing the interaction of the GLP-1R agonist in an agonist binding site of the GLP-1R. In certain embodiments, a reference GLP-1R agonist comprises only a GLP-1R agonist portion that contacts all or part of a GLP-1 binding site on a GLP-1R, whereas synthetic GLP-1R binding proteins provided herein interacts or otherwise interfaces with a larger surface area of the GLP-1 R. Such differences in the interactions between the agonist and the receptor can modulate downstream activity of GLP-1 R, for example, biasing signaling towards one pathway (e.g., cAMP mediated signaling pathway) over another pathways (e.g., a -arrestin mediated signaling pathway).

[0081] In another aspect, the disclosure provides a method of promoting or increasing GLP-1 R-mediated activity in a subject in need thereof. The method comprises administering to the subject an effective amount of a synthetic GLP-1R binding protein or a pharmaceutical composition as provided herein.

[0082] In another aspect, the disclosure provides a method of treating one or more endocrine and / or metabolic disorders (e.g., type 2 diabetes mellitus, obesity, pancreatic dysfunction including uncontrolled blood glucose levels), gastrointestinal disorders (e.g. gastric motility disorders, e.g., irritable bowel syndrome, e.g., irritable bowel disease), renal disorders (e.g., reduced kidney function), hepatic disorders (e.g., non-alcoholic steatohepatitis), neurodegenerative / neurological disorders (e.g., Alzheimer’s disease, e.g., Parkinson's disease) and / or cardiovascular disorders (e.g., heart attack, and stroke) in a subject in need thereof. The method comprises administering to the subject an effective amount of a synthetic GLP-1 R binding protein or a pharmaceutical composition as provided herein. In certain embodiments, the subject is diagnosed as having or at risk of having endocrine and / or metabolic disorders (e.g., ty pe 2 diabetes mellitus, obesity, pancreatic dysfunction including uncontrolled blood glucose levels), gastrointestinal disorders (e.g., gastric motility disorders, e.g., irritable bowel syndrome, e.g., irritablebowel disease), renal disorders (e.g., reduced kidney function), hepatic disorders (e.g., nonalcoholic steatohepatitis), neurodegenerative / neurological diseases (e.g, Alzheimer’s, Parkinson’s) and / or cardiovascular disorders (e.g, heart attack, and stroke).

[0083] In certain embodiments, the subject is administered a synthetic GLP-1R binding protein or a pharmaceutical composition as provided herein before, during, or after administration or use of one or more other agents (e.g., as provided herein). For example, the one or more agents can be selected from: insulin, an agent for decreasing amount of blood glucose (e.g., produced by liver), an agent for decreasing amount of blood sugar absorbed by gastrointestinal tract (e.g, stomach and / or intestines), metformin, a reduced calorie diet, increased physical activity, and combinations thereof.

[0084] In another aspect, the disclosure provides a method of increasing half-life of a GLP-1R binding protein comprising linking an effector that extends half-life to the C- terminus of a synthetic GLP-1R binding protein disclosed herein. In certain embodiments, the effector comprises a protein that extends serum half-life. By way of non-limiting example, the effector can comprise an albumin binding protein (e.g., a synthetic albumin binding protein). In certain embodiments, the synthetic albumin binding protein has an amino acid sequence comprising SEQ ID NO: 163. Half-life of a synthetic GLP-1R binding protein comprising an effector that is or comprises an albumin binding protein is increased as compared to a GLP-1R ligand (e.g., GLP-1) or a GLP-1R agonist without an effector (e.g., a synthetic GLP-1R binding protein without the effector, e.g., a reference GLP-1R agonist, e.g., semaglutide, etc.). In some embodiments, the reference GLP-1R agonist has an amino acid sequence comprising SEQ ID NO: 106.

[0085] In another aspect, the disclosure provides a method of reducing blood glucose concentration in a subject in need thereof comprising administering to the subject an effective amount of a synthetic GLP-1 R binding protein or a pharmaceutical composition as disclosed herein, thereby to reduce blood glucose concentration relative to blood glucose concentration prior to administration. In certain embodiments, the synthetic GLP-1R binding protein has an amino acid sequence selected from any of those set forth in SEQ ID NOs: 108-154 or 156-157.

[0086] These and other aspects and features of the disclosure are described in the following detailed description and claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0087] FIG. 1A is a schematic illustration of a GLP-1 receptor (GLP-1R) before and after activation. Upon activation after GLP-1 binding, the GLP-1R mediates downstream signaling through several pathways.

[0088] FIG. IB is a schematic illustration of a GLP-1 receptor (GLP-1 R) before and after activation by a synthetic GLP-1R binding protein. Upon activation after binding an exemplary synthetic GLP-1R binding protein, the GLP-1R mediates downstream signaling through several pathways.

[0089] FIG. 1C is a schematic illustration of a space-filling model showing in 90° rotated views a GLP-1 receptor (GLP-1R, grey) bound to GLP-1 (black).

[0090] FIG. ID is a schematic illustration of a space-filling model show ing in 90° rotated views a GLP-1 receptor (GLP-1R, grey) bound to an exemplary synthetic GLP-1R binding protein (black).

[0091] FIG. 2 is a line graph of circular dichroism (CD) spectra showing the stability profile of Reference Miniprotein 1 (SEQ ID NO: 1) over a wavelength range of 200-260 nm during heating and cooling measured at 5°C intervals between temperatures of 25°C - 95 °C (not all data points shown for clarity ).

[0092] FIG. 3 is a line graph show ing levels of GLP-1 R agonism of Reference Miniprotein 1 (SEQ ID NO: 1) as compared to a control peptide (exendin-4) over a concentration of about 1 O’6- 10'11M as measured by normalized cAMP accumulation as determined in a luciferase-based cyclic AMP assay designed to detect intracellular signaling mediated by a G-protein coupled receptor, namely downstream signaling from GLP-1 R.

[0093] FIG. 4A and FIG. 4B are line graphs of circular dichroism (CD) spectra showing the stability profiles of exemplary GLP-1 R binding proteins disclosed herein over a wavelength range of 200-260 nm during heating and cooling measured at 5°C intervals between temperatures of 25°C - 95°C (not all data points shown for clarity). FIG. 4A show s CD spectra of the synthetic GLP-1 R binding protein of Reference Miniprotein 2 (SEQ ID NO: 2) and FIG. 4B show s CD spectra of the GLP-1R binding protein of Reference Miniprotein 11 (SEQ ID NO: 11).

[0094] FIG. 5 is a line graph showing levels of GLP-1R agonism of Reference Miniprotein 1 (SEQ ID NO: 1), Reference Miniprotein 2 (SEQ ID NO: 2) and Reference Miniprotein 11 (SEQ ID NO: 11) as compared to a control reference product (an exemplary reference GLP-1R agonist, semaglutide, SEQ ID NO: 106) over a concentration of about I O'6- 1 O'" M as measured by normalized cAMP accumulation in a luciferase-based cyclic AMP assay designed to detect intracellular, downstream signaling from GLP-1R.

[0095] FIG. 6 is a line graph showing levels of cAMP-mediated or p-arrestin-mediated signaling via GLP-1R agonism by Reference Miniprotein 46 (SEQ ID NO: 46) or a control reference product (an exemplary GLP-1R agonist, semaglutide, SEQ ID NO: 106) over a concentration of about 10'4-10'12M as measured by normalized luminescence levels in fluorescence-emitting assays that detect intracellular, downstream signaling of cAMP or p- arrestin from GLP-1R.

[0096] FIG. 7 is a line graph showing blood glucose concentration (mg / dL. y-axis) over time (x-axis, mins) after glucose injection in db / db mice previously administered Reference Miniprotein 120 (SEQ ID NO: 120), semaglutide (SEQ ID NO: 106), or a phosphate- buffered saline (PBS) vehicle.

[0097] FIG. 8A (cAMP signaling). FIG. 8B (P-arrestin signaling), and FIG. 8C (receptor internalization) are graphs showing activity characteristics of an exemplary synthetic GLP- 1R binding protein as compared to an exemplary reference GLP-1R agonist, semaglutide (SEQ ID NO: 106). FIG. 8A is a line graph showing GLP-1R agonist activity (luminescence signal, y-axis) of Reference Miniprotein 120 (SEQ ID NO: 120) as compared to an exemplary reference GLP-1R agonist, semaglutide (SEQ ID NO: 106), over a concentration of about 10'6- 1 O'" M (x-axis, log M) as measured by normalized cyclic AMP (cAMP) accumulation in a luciferase-based cAMP assay designed to detect intracellular, downstream signaling from GLP-1R. FIG. 8B is a line graph showing levels of P-arrestin-2 recruitment (luminescence signal, y-axis) to GLP-1R over a concentration of about 10'4-10'12M (x-axis, log M) of Reference Miniprotein 120 (SEQ ID NO: 120) or semaglutide (SEQ ID NO: 106) as measured by a luminescent reporter assay that detects intracellular, downstream signaling of P-arrestin-2 from GLP-1R. FIG. 8C is a line graph that show s levels of GLP-1R internalization Reference Miniprotein 120 (SEQ ID NO: 120) or semaglutide (SEQ ID NO: 106), which w ere tested over a concentration of about 10'4- 10'12M (x-axis, log M) and receptor internalization w as measured by luminescent signal(luminescence signal, y-axis) generated by colocalization of internalized GLP-1R and FYVE in the endosome.

[0098] FIG. 9 is a line graph showing serum protein concentration ( .M. x-axis) over time (y-axis) of an exemplary GLP-1R binding protein linked to an exemplary synthetic albumin binding protein (Reference Miniprotein 155 (SEQ ID NO: 155) after intravenous injection in mice.DETAILED DESCRIPTION

[0099] The disclosure is based, in part, upon the discovery of synthetic GLP-1R binding proteins that specifically bind GLP-1R and modulate the GLP-1R axis, for example, by agonizing downstream GLP-lR-signaling activity. The GLP-1R binding proteins described herein have specific binding activity for GLP-1R. have the ability' to agonize downstream GLP-lR-mediated signaling activity, and are, among other things, thermally and chemically stable, resistant to oxidation, resistant to protease degradation, deamination, and glycosylation, and have mM-level solubility. The binding proteins can be monovalent, bivalent, or multivalent and can also be conjugated (e.g., via chemical conjugation or as a fusion protein) to an effector.

[0100] Accordingly, the present disclosure provides, among other things, synthetic GLP- 1R binding proteins, methods of making such binding proteins, and methods of using such proteins to treat a disorder mediated by the GLP-1R axis.I. DEFINITIONS

[0101] Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. For example, nomenclatures utilized in connection with, and techniques of, e.g, polypeptide and polynucleotide chemistry' and synthesis, molecular and cellular biology, protein biology and biochemistry, immunology, etc. as described herein are those well-known and commonly used in the art.

[0102] As used herein, the singular forms "a." "an" and "the" include plural referents unless context clearly dictates otherwise. Thus, for example, in some embodiments, reference to, e.g., a synthetic GLP-1R binding protein includes a single binding protein, a plurality' of synthetic binding proteins, etc.

[0103] As used herein, the expression “and / or” in connection with two or more recited objects includes individually each of the recited objects and the various combinations of two or more of the recited objects, unless otherwise understood from the context and use.

[0104] Where the use of the term “about” is before a quantitative value, the present disclosure also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred. Ranges can be expressed in this disclosure as from “about” one particular value, and / or to “about” another particular value. When values are expressed as approximations by use of the antecedent “about,” it is understood that the disclosure also contemplates embodiments that specify the particular values and ranges of values without the approximations.

[0105] As used herein, the phrases “solvent accessible residue” and “solvent accessible amino acid” refer to an amino acid that, when disposed in a folded molecule (e.g., in its a tertiary conformation) and in a solvent, is characterized in that the amino acid is at least partially accessible or exposed to the solvent. Solvent accessible amino acids can be determined using a variety of approaches including, e.g, Rosetta software suite, Neighbor Count, and Neighbor vector algorithms (Durham et. al. (2009) J. MOL. MODEL. 15(9): 1093-108).

[0106] As used herein, the phrase “conservative substitution” refers to a substitution with a structurally and / or functionally similar amino acid. The following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S) and Threonine (T); 2) Aspartic Acid (D) and Glutamic Acid (E); 3) Asparagine (N) and Glutamine (Q); 4) Arginine (R) and Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W). Conservative substitutions may also be defined by the BLAST (Basic Local Alignment Search Tool) algorithm, the BLOSUM substitution matrix (e.g, BLOSUM 62 matrix), or the PAM substitution^ matrix (e.g, the PAM 250 matrix). In certain embodiments, a binding protein of the disclosure comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. 11. 12, 13, 14, or 15 conservative substitutions relative to a reference amino acid sequence.

[0107] As used herein, the phrase “corresponding to” designates a position / identity of an amino acid or a nucleic acid in a polymeric molecule such as an amino acid in an amino acid sequence or a nucleic acid in a nucleic acid sequence. It is understood by the skilledartisan that such amino acids or nucleic acids in such a polymer are often designated using a canonical numbering system based on a reference related polymer, so that, for example, an ammo acid in a first polymer '‘corresponding to” position seven in the reference amino acid, for example, need not actually be the seventh amino acid in the first polymer. Those of ordinary skill in the art are aware of methodology to identify “corresponding” amino acids or nucleic acids between two molecules (e.g, a polymer and a reference polymer), including, such as. commercially available algorithms, databases, or other information given context regarding particular polymers.

[0108] As used herein, the term “domain” refers to a region or segment of a given synthetic binding protein disclosed herein, and can include one or more structural features (e.g, amino acid, primary7, structure or secondary7structure features) and / or one or more functional features (e.g., binding properties). In the context of secondary structure, a “structural domain” can be an uninterrupted linear sequence that adopts a single type of secondary structure, for example, ten continuous amino acid residues that are all part of the same alpha helix structure or beta sheet. In the context of binding, a “binding domain” can be a discontinuous portion of the overall amino acid sequence that facilitates chemical interactions with a target molecule or indirectly stabilizes such interactions.

[0109] As used herein, the phrase “effective amount” refers to the amount of an active agent (e.g.. a synthetic GLP-1R binding protein disclosed herein) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or administration route.

[0110] As used herein, the term “effector” refers to a molecule or molecular entity7that confers one or more particular characteristics on itself or another molecule or molecular entity with or to which it is associated. For example, an effector may include a synthetic binding protein, e.g., a miniprotein, or something other than a miniprotein that is associated with a synthetic GLP-1R binding protein disclosed herein (e.g., via a covalent linkage), such as a detectable label (e.g., visualizable or otherw ise measurable such as by fluorescence or radiolabel detection), small molecule, nanoparticle (e.g., a lipid nanoparticle, a polymer nanoparticle, etc.), polynucleotide (e.g., an aptamer, an siRNA, an shRNA, an oligonucleotide, etc.), a radionuclide, etc. An effector may be a synthetic binding protein (e.g, a monovalent synthetic binding protein linked to a GLP-1R binding protein disclosedherein to create a bivalent synthetic protein where one or both of the proteins causes a change, e.g., in a cellular function, e.g., in a disease state, etc.).

[0111] As used herein, the term "loop" refers to (i) a structure (e.g., polypeptide) that connects two structural domains (e.g. a loop may be disposed between two alpha helices, between an alpha helix and a beta sheet, or between two beta sheets in a given synthetic GLP-1R binding protein) and / or (ii) a structure (e.g., peptide) present at the N- and / or C- terminal end of a given monovalent synthetic binding protein.

[0112] As used herein, the term “linker” refers to a structure (e.g., a polypeptide linker, or a chemical crosslinker, e.g., a homobifunctional or a heterobifunctional cross linking agent) between two molecules (e.g., two synthetic GLP-1R binding proteins disclosed herein) or between, e.g., a synthetic GLP-1R binding protein and an effector, wherein each of the entities that are linked is covalently linked to one another.

[0113] As used herein, the terms / phrases “synthetic binding protein,” “synthetic miniprotein,” and “miniprotein” are used interchangeably, and refer to a polypeptide between about 25 to about 100 amino acids in length, e.g., from about 30 to about 90 amino acids, from about 45 to about 70 amino acids in length, from about 35 to about 65 amino acids, from about 50 to about 70 amino acids in length, from about 40 to about 65 amino acids, from about 50 to about 65 amino acids in length, or 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 62, 63, 64, or 65 amino acids in length, that are capable of binding to a given target, e.g. GLP-1R with a desired binding affinity (e.g., stronger than 1 pM).

[0114] As used herein, the phrase “percent identity” and “% identity” refers to the extent to which two sequences (e.g., a polypeptide) have the same amino acid or nucleotide at the same positions in an alignment. The percent identity between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. It is contemplated that a reference sequence can be an amino acid sequence corresponding to an entire GLP-1R binding protein or a portion thereof. A reference sequence may be an amino acid sequence that corresponds to a particular domain or domains (e.g. , an alpha helix, a loop region) or a combination of domains (e.g., a combination of a loop and an alpha helix). Alignment for purposes of determining percent sequence identity (e.g., amino acid sequence identity) can be achieved in various ways thatare within the skill in the art. for instance, using publicly available computer software such as BLAST (Basic Local Alignment Search Tool), BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, MUSCLE, or BioPython software. For a discussion of basic issues in searching sequence databases, see Altschul et al. (1994) NATURE GENETICS 6: 119-129, which is incorporated by reference herein. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0115] As used herein, the term “synthetic” refers to a molecule that is (i) not naturally occurring, (ii) not present in nature, (iii) does not comprise entirely natural components, or (iv) a combination of any one of (i), (ii) and (iii). For example, a synthetic peptide does not exist naturally, is produced or otherwise modified by human intervention, such as techniques including recombinant or cell-free synthesis, and / or the peptide may comprise one or more non-naturally occurring amino acids.

[0116] As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0117] As used herein, the phrase “pharmaceutically acceptable carrier” as used herein refers to an agent (e.g, excipient, carrier, buffer, etc.) suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Standard pharmaceutical carriers may include, for example a phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see e.g., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rded. 2020).

[0118] As used herein, “stabilization domain” refers to a portion of a synthetic binding protein (e.g. a miniprotein) that interfits with a receptor surface at one or more regions adjacent a native ligand / agonist binding site on the receptor. A stabilization domain can, in some embodiments, increase the surface area of contact between a synthetic binding protein (e.g, an agonist) as compared to the surface area of contact between a native ligand or receptor agonist without a stabilization domain. Such increases can be measured using methods known to those of skill in the art (e.g, Rosetta, e.g, solvent-accessible surface area (“SAS A”)), and, depending on circumstances, will be based on modeled and / or solved structures.

[0119] As used herein, the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g, murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably includes humans.

[0120] As used herein, “treat”, “treating”, and “treatment” refer to the treatment of a disease, disorder, or symptom or manifestation of such in a subj ect, e.g. , in a human. This includes: (a) preventing a disease or disorder, (b) inhibiting the disease, disorder, etc., i.e., slowing or arresting its progress or development; and (c) relieving the disease, disorder, etc., i.e., causing regression of the disease state. As used herein, “prevent”, “preventing” and “prevention” refer to causing a disease, disorder, or symptom or manifestation of such not to occur for at least a period of time in at least some subjects.

[0121] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps. Similarly, throughout the description, where compositions are described as consisting essentially of specific components, or where processes and methods are described as consisting essentially of specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist of the recited components, and that there are processes and methods according to the present disclosure that consist of the recited processing steps.

[0122] Throughout the text, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0123] Further, it should be understood that elements and / or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present disclosure, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present disclosure and / or in methods of the present disclosure, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and any invention provided herein. For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of any invention described and depicted herein.

[0124] The use of any and all examples, or exemplary language herein, for example, "‘such as” or “including,” is intended merely to illustrate better the present disclosure and does not pose a limitation on the scope of any invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of any invention disclosed herein.

[0125] It should be understood that the expression “at least one of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use.

[0126] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0127] It should be understood that the order of steps or order for performing certain actions is immaterial so long as disclosed invention(s) remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0128] As used herein, all numerical values or numerical ranges include whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, for example, reference to a range of 90-100%, includes 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc.II. GLUCAGON LIKE PEPTIDE-1 RECEPTOR (GLP-1R)

[0129] Provided herein are synthetic binding proteins (also referred to as miniproteins), that bind the glucagon-like peptide-1 receptor (GLP-1R). GLP-1R binds the peptide hormone glucagon-like peptide-1 (GLP-1), a peptide hormone of 30 or 31 amino acids in length that is A pically secreted by enteroendocrine L cells in the intestine and alpha cells in the pancreas. See, e.g., Zhao et al. (2021) supra; see also Underwood et al. (2010) J BIOL. CHEM. 285:723. GLP-1, among other things, can regulate glucose homeostasis, as can synthetic GLP-1R agonists. See. e.g., Zhao et al. (2021) supra. Although certain agonists of GLP-1R (e.g., semaglutide) have been found to be effective in treating disorders including type 2 diabetes mellitus, obesity, and liver disease (e.g, improvement of nonalcoholic fatty liver disease / non-alcoholic steatohepatitis) and reduction in cardiac inflammation, the doses needed, especially the higher doses used in effective treatment of obesity, can result in increase in adverse effects such as diarrhea, nausea, and vomiting. See. e.g., Gorgojo- Martinez (2023) J. CLIN. MED. 12: 145.

[0130] GLP-1 R is a seven helix transmembrane G protein-coupled receptor (GPCR) with three intracellular and three extracellular loops. See, e.g., Mayendraraj et al. (2022) PEPTIDES. 151 :170749. GLP-1R has a molecular weight of approximately 53 kilodaltons (kDa), (Zhang et al. (2020) DIABETES. 68:34) and is expressed in various tissues throughout the body including pancreas, lungs, kidneys, liver, the central nervous system, the peripheral nervous system (e.g, vagus nen e, skin nerves), the cardiovascular system, and the gastrointestinal tract (e.g, intestines). See, e.g., Zhao et al. (2021) supra; Yokomori et al. supra, and Zhang et al. supra. GLP-1 and exendin-4 (a 39 amino acid peptide with 50% identity to GLP-1 that is not metabolized by DPP-4) bind an extracellular domain of GLP- 1R. which then mediates G-protein or beta-arrestin binding to an intracellular domain, resulting in intracellular signaling. See, e.g., Gayatri et al. (2023) How SYNTHETIC DRUGSWORK, (2023) Eds. Kazmi et al. Academic Press, pp. 95-122. See also, Underwood et al. supra; Sonoda el a / . (2008) PROC. NATL. ACAD. SCI. 105:6614; and Mayendraraj et al. (2022), supra.

[0131] As depicted schematically in FIG. 1A, soluble GLP-1 is believed to bind to GLP- 1R and cause downstream signaling including activation of cyclic adenosine monophosphate (cAMP), protein kinase C delta (PKC8), and phosphoinositide 3-kinase (PI3K). See, e.g., Mayendraraj et al. (2022), supra; Sonoma et al. (2008), supra; and Fleming et al. (2017) CELL SIGNAL. 40: 1. Without being limited by theory, it is believed that these messengers can, among other things, modulate insulin release. Thus, it follows that if GLP-1 R axis dysregulation occurs, insulin and / or other metabolic dysregulation can also occur. FIG. IB depicts schematically a miniprotein agonist that binds to and agonizes the activity' of the GLP-1 R.

[0132] GLP-1 and other peptide ligands are believed to be unstructured or disordered in solution, but become structured (or ordered) upon binding to GLP-1 R. GLP-1 and the GLP- 1 peptide analogs (e.g, semaglutide), given their size, have limited ability to be stabilized in the binding cleft of the GLP-1 R. FIG. 1C depicts two views of a space filling model showing the GLP-1 peptide (shown in black) disposed within the binding cleft of GLP-1 R. where views are rotated by 90°. Exogenous GLP-1 R agonists (e.g.. semaglutide. liraglutide. dulaglutide, lixisenatide, exenatide etc.) are small and have a relatively small binding interface to bind with, and be stabilized by the GLP-1 R ectodomain outside of the binding cleft.

[0133] The synthetic GLP-1R binding proteins disclosed herein are designed to have a larger binding surface area to interface with GLP-1 R than currently available GLP-1 R peptide agonists (e.g, semaglutide, liraglutide, dulaglutide, lixisenatide, exenatide, etc). thereby facilitating stronger binding to the GLP-1 R ectodomain than GLP-1 or approved GLP-1R peptide agonists. See, e.g., Runge (2008) J. BIOL. CHEM. 287: 17; see also Zhang (2020) MOL. CELL 80: 3. FIG. ID depicts two views rotated by 90° of a space filling model showing a miniprotein (shown in black) interacting with a larger three-dimensional surface area of the GLP-1 R than the GLP-1 peptide in FIG. 1C. The additional binding domains of the miniprotein interfit with regions of the GLP-1 R adjacent to the GLP-1 binding cleft thereby stabilizing the interaction betw een the GLP-1 mimetic moiety of miniprotein and the binding cleft of GLP-1 R.III. GLP-1R BINDING PROTEINS

[0134] The disclosure provides synthetic GLP-1R binding proteins, and methods of identifying, making, characterizing, formulating, and using such GLP-1R binding proteins. The synthetic GLP-1R binding proteins have several advantages over small molecule and large molecule (e.g., biologies) based therapeutics, as well as over approved GLP-1R agonists including at least decreased side effects (e.g., relative to other approved GLP-1R agonists) and improved half-life (as compared to, e.g.. exendin-4, liraglutide, etc.). For example, small molecule therapeutics may suffer from off-target activities and / or may have long half-lives, which can risk negative impact on one more organs or organ systems, such as kidneys. Similarly, large molecules such as biologies are expensive to produce, are challenging to produce in uniform batches of drug substance (including, especially, e.g., at commercially-scalable amounts), and can be challenging to formulate, transport, store, and administer to subjects.

[0135] Furthermore, in some instances, GLP-1R agonists (e.g., semaglutide, liraglutide, dulaglutide, lixisenatide, exenatide, etc.) can have a variety of undesirable side effects, which can intensify at higher doses (e.g., such as used to treat obesity) and can also be burdened by short half-lives (e.g., exendin-4, liraglutide, etc.).

[0136] The synthetic GLP-1R binding proteins disclosed herein avoid certain such disadvantages as they have high binding specificity to GLP-1R. can be engineered to have desired pharmacodynamic and pharmacokinetic properties (e.g., a desirable circulating halflife in plasma), reduced immunogenicity (e.g, do not elicit an immune response against a therapeutic drug, e.g., a therapeutic agonist, e.g., a GLP-1R agonist, e.g., semaglutide, liraglutide, dulaglutide, lixisenatide, exenatide, etc.), are chemically and thermally stable, and are resistant to protease degradation, deamination and post translational modification (e.g.. glycosylation), and are stable in different redox environments.

[0137] A synthetic GLP-1R binding protein described herein can have mM-level solubility7. In some embodiments, the GLP-1R binding protein can have a solubility greater than 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, or 80 mg / mL in an aqueous solution.

[0138] The GLP-1R binding proteins described herein designed to specifically bind GLP- 1R. and preferably bind GLP-1R over GLP-1. Preferably, the binding proteins do not bindGLP-1. Depending upon the circumstances, the synthetic GLP-1R binding proteins bind GLP-1R and stabilize the GLP-1 R ectodomain and receptor at a level greater than that with GLP-1 or a reference GLP-1R agonist (e.g., a commercially-available GLP-1R agonist, e.g., semaglutide, liraglutide, dulaglutide, lixisenatide, exenatide, etc.). Synthetic GLP-1R miniproteins as provided herein can, in some embodiments, bind with an affinity at least at a level of a reference GLP-1R agonist (e.g., a commercially-available GLP-1R agonist, e.g., semaglutide. liraglutide, dulaglutide, lixisenatide. exenatide, etc.).

[0139] The synthetic GLP-1R binding proteins disclosed herein can be conjugated (e.g., chemically conjugated to or are fusion proteins) to one or more effectors. For the example, the GLP-1 R binding proteins described herein can be bound to one or more other synthetic binding proteins, which can be the same or different. The resulting proteins can be monovalent, bivalent, or multivalent. For example, a first GLP-1R binding protein described herein can be conjugated to (e.g., by a linker) and / or produced as a fusion (e.g., a genetic fusion) that binds to a second binding protein that binds a different target of interest such as, e.g., serum albumin (e.g., to extend serum half-life), etc. The resulting molecule is bivalent and can bind GLP-1R as well as the second target molecule (e.g., albumin).

[0140] In one aspect, the disclosure provides a synthetic GLP-1R binding protein that comprises:(a) an amino acid sequence from 30 amino acids to 95 amino acids in length:(b) a net negative charge in phosphate buffered saline (PBS);(c) a binding affinity for GLP-1 R stronger than 1 pM: and(d) a stability profile such that the protein (i) retains at least 90% binding affinity' to GLP-1 R upon cooling to room temperature after thermal denaturation at 95°C in PBS for at least about five minutes relative to the protein prior to thermal denaturation; (ii) retains at least 90% binding affinity to GLP-1 R after incubation for 16 hours at 37°C of incubation in PBS relative to the protein under the same conditions prior to incubating; and / or (iii) retains at least 90% binding affinity to GLP-1R in PBS following chemical denaturation in 4 M urea for 1 hour at room temperature relative to the protein prior to chemical denaturation.

[0141] In another aspect, the disclosure provides a synthetic GLP-1R binding protein, the binding protein comprising:(a) an amino acid sequence from 30 amino acids to 95 amino acids in length;(b) a net negative charge in PBS;(c) a binding affinity for GLP-1R stronger than 1 pM;(d) at least one alpha helix;(e) at least three beta sheets;(f) at least three amino acid loops, where a first loop having a first amino acid sequence connects a terminal amino acid (e.g. , a C-terminal amino acid) of a first alpha helix to a terminal amino acid (e.g., an N-terminal amino acid) of a first beta sheet, and a second loop having a second amino acid sequence connects a second, terminal amino acid (e.g., a C-terminal amino acid) of the first beta sheet to a terminal amino acid (e.g., an N-terminal amino acid) of a second beta sheet, and a third loop having a third amino acid sequence connects a third, terminal amino acid (e.g, a C-terminal amino acid) of the second beta sheet to a terminal amino acid (e.g, an N- terminal amino acid) of a third beta sheet; and (g) a hydrophobic core defined by at least two hydrophobic amino acids present in at least one of the alpha helices and / or at least one of the beta sheets.

[0142] In another aspect, the disclosure provides a synthetic GLP-1R binding protein, the binding protein comprising:(a) the first alpha helix contains at least one hydrophobic amino acid, wherein, optionally, one or more of the at least one hydrophobic amino acids is not solvent accessible;(b) the first, second, and / or third beta sheet each contains at least two or three hydrophobic amino acids, wherein, optionally, one or more of the at least two or three hydrophobic amino acids is not solvent accessible;(c) the first alpha helix contains at least one or two solvent accessible amino acids;(d) the first, second, and / or third beta sheet contains at least one or two solvent accessible amino acids;(e) the first alpha helix contains at least one or two solvent accessible amino acids;(f) the first and / or second and / or third loop contains at least one hydrophobic amino acid; or (g) the binding protein comprises any combination of elements selected from (a), (b), (c). (d), (e), and (I .

[0143] In another aspect, the disclosure provides a synthetic GLP-1R binding protein, the binding protein comprising:(a) an amino acid sequence from 30 amino acids to 95 amino acids in length;(b) a net negative charge in PBS;(c) a binding affinity for GLP-1R stronger than 1 pM;(d) at least one alpha helix;(e) at least four beta sheets;(f) at least four amino acid loops, where a first loop having a first amino acid sequence connects a terminal amino acid (e.g. , a C-terminal amino acid) of a first alpha helix to a terminal amino acid (e.g., an N-terminal amino acid) of a first beta sheet, and a second loop having a second amino acid sequence connects a second, terminal amino acid (e.g., a C-terminal amino acid) of the first beta sheet to a terminal amino acid (e.g., an N-terminal amino acid) of a second beta sheet; a third loop having a third amino acid sequence connects a third, terminal amino acid (e.g. , a C-terminal amino acid) of the second beta sheet to a terminal amino acid (e.g. , an N- terminal amino acid) of a third beta sheet; and a fourth loop having a fourth amino acid sequence connects a fourth, terminal amino acid (e.g., a C-terminal amino acid) of the third beta sheet to a terminal amino acid (e.g., an N-terminal amino acid) of a fourth beta sheet; and (g) a hydrophobic core defined by at least two hydrophobic amino acids present in at least one of the alpha helices and / or at least one of the beta sheets.

[0144] In another aspect, the disclosure provides a synthetic GLP-1R binding protein, the binding protein comprising:(a) the first alpha helix contains at least one hydrophobic amino acid, wherein, optionally, one or more of the at least one hydrophobic amino acids is not solvent accessible;(b) the first, second, third and / or fourth beta sheet each contains at least two or three hydrophobic amino acids, wherein, optionally, one or more of the at least two or three hydrophobic amino acids is not solvent accessible;(c) the first alpha helix contains at least one or two solvent accessible amino acids;(d) the first, second, third and / or fourth beta sheet contains at least one or two solvent accessible amino acids;(e) the first alpha helix contains at least one or two solvent accessible amino acids;(f) the first and / or second and / or third and / or fourth loop contains at least one hydrophobic amino acid; or (g) the binding protein comprises any combination of elements selected from (a), (b), (c), (d), (e), and (I).

[0145] Synthetic GLP-1R binding proteins of the present disclosure may include naturally-occurring or non-naturally occurring amino acids. It is understood that certain amino acids may have and / or take on different characteristics (e.g., hydrophobic, hydrophilic, neutral, etc.) depending upon the context (e.g., macro and / or micro-environment including, but not limited to surrounding amino acids, environmental conditions such as solvent type, pH, etc.). Various terms and phrases may be used herein to describe, identify, and / or characterize ammo acids. In addition, a single amino acid, at any given time, may have more than one characteristic or identity. Depending upon the context, a hydrophobic amino acid may be selected from alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine, try ptophan, tyrosine, lysine, and arginine. A hydrophilic amino acid may be an amino acid selected from cysteine, aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine, arginine, serine, threonine, tryptophan, and tyrosine. A charged amino acid may be an amino acid selected from arginine, histidine, lysine, aspartic acid (aspartate), and glutamic acid (glutamate). A positively-charged amino acid may be an amino acid selected from arginine, histidine, and lysine. A negatively-charged amino acid may be an amino acid selected from aspartic acid (aspartate) and glutamic acid (glutamate). An uncharged or neutral amino acid may be selected from alanine, cysteine, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan, and tyrosine. Sy nthetic GLP-1R binding proteins of present disclosure have a primary structure comprising certain key features. For example, synthetic GLP-1 R binding proteins can have amino acid sequences that include various combinations of hydrophobic and solvent accessible amino acids organized into certain domains. Primary structures (i.e., amino acid sequences) of the synthetic GLP-1R binding proteins will have a combination of one or more types (e.g., hydrophobic, e.g, solvent accessible) amino acids.

[0146] In each of the foregoing aspects, the synthetic GLP-1R binding protein comprises one or more of the following features: (a) free of tryptophan amino acids; (b) free of methionine amino acids; (c) free of lysine amino acids; (d) does not comprise an unpaired cysteine amino acid when cysteine amino acids are present in the protein; (e) free of glycosylation sites; (f) free of protease cleavage sites; and (g) soluble up to at least 1 mM in PBS at 4°C for one month.

[0147] Preferably, the GLP-1R binding sites are designed to be free of glycosylation sites, for example, free of peptide sequences that are substrates for glycosylation (e.g., N-X-S / T.which can be a substrate for an oligosaccharyltransferase (OST) complex). Alternatively or in addition, the GLP-1R binding sites are designed to be free of protease cleavage sites, for example, free of peptide sequences that are substrates for proteases (e.g., L-X-R-R, which can be a substrate for a Kexin / KEX2 protease). Similarly, the GLP-1R binding proteins may also be designed to be free of other protease cleavage sites for other proteolytic enzymes such as trypsin, chymotrypsin, elastase, subtilisin. etc.

[0148] Some synthetic GLP-1R binding proteins may be designed to avoid cleavage by certain other enzymes, such as dipeptidy 1 peptidase 4 (DPP-4), which is associated with the rapid degradation of GLP-1. As is known to those of skill in the art, DPP-4 cleaves substrate peptides, more preferably those containing proline or alanine amino acids at position two of a given peptide with reference to its amino terminus (amino acid number two of the amino region of a given substrate peptide, for example, depicted as NH2 - Amino Acid (Position 1) - Amino Acid (Position 2). . . .COOH). See, e.g., Mulvihill et al. (2014) ENDOCR REV. 35(6): 992-1019. Thus, for example, the present disclosure contemplates that synthetic GLP-1R binding proteins with an alanine at amino acid position two can be more vulnerable to proteolytic degradation (e.g., via DPP-4, such as indicated by measurements taken using a commercially-available signaling assay) than synthetic GLP-1R binding proteins not having an alanine at position two (e.g.. and, instead, having, for example, a glycine or other amino acid).

[0149] Depending upon the circumstances, the N-terminus of the first alpha helix may be preceded by one or more N-terminal amino acids and / or the C-terminus of the third or, if present, the fourth beta sheet may be followed by one or more C-terminal amino acids. In certain embodiments, the third beta sheet is followed by a C-terminal extension comprising a fourth loop region and a fourth beta sheet. In some embodiments, the fourth loop and fourth beta sheet include a total of at least 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. In certain embodiments, the fourth loop and fourth beta sheet include a total of at least 8 amino acids in length. By way of non-limiting example, a C-terminal extension can have an amino acid comprising SEQ ID NO: 165, where X63 is selected from R, G, D, or P.

[0150] Under certain circumstances, it is contemplated that in certain synthetic GLP-1R binding proteins with additional amino acids at the N-terminal position (e.g., N-terminal to the first alpha helix, e.g., alpha helices with amino acid sequences as set forth herein) may bind to the GLP-1 R but may fail to act as an agonist (i.e., the sy nthetic GLP-1 R bindingprotein binds but does not activate / signal through GLP-1R). As a result in certain embodiments, such GLP-1R binding proteins lack an additional N-terminal sequence that would otherwise result in loss or reduction of receptor agonist activity.

[0151] A synthetic GLP-1R binding protein of the disclosure may have a structural arrangement comprising combinations of one or more alpha helices and / or beta sheets, with each helix and / or sheet attaching to one or more loop regions. Certain non-limiting examples are provided herein.

[0152] For example, a structural arrangement in a synthetic GLP-1R binding protein may be depicted as N-HniLn2En3Ln4En5Ln6En7-C, where H is an alpha helix, E is a beta sheet, L is a loop, each of m-n? represents an integer indicating the number of amino acids in that structural domain (e.g., nl is 30, n2 is 5, n3 is 6, n4 is 4, n5 is 6, n6 is 5, and n7 is 6), N and C represent N-terminal and C-terminal domains, respectively. As disclosed herein, certain GLP-1R binding proteins can be represented according to a formula (Formula IA): D1-L2- D2-L2-D3-L3-D4, wherein DI corresponds to an alpha helical domain, and D2, D3, and D4 each correspond to a beta sheet domain; LI, L2, and L3 are each, independently, loops. The components of Formula IA can correspond to the aforementioned structural arrangement as follows: DI to Hni and each of D2, D3, and D4 to each of En3, Ens, and En7. The number of E amino acids does not have to be the same across sheets, for example, ns, ns, and n? may be, but do not have to be, the same numbers. Similarly, for example, the number of L amino acids does not have to be the same across loops; m and may be, but do not have to be, the same number. For example, an exemplary' formula of a synthetic GLP-1R binding protein may comprise N-H30L5E6L4E6L5E6-C as depicted pictorially below denoting the amino acids in a helix domain (H) or a loop domain (L):N-HHHHHHHHHHHHHHHHHHHHHHHHHHHHHLLLLLEEEEEELLLLEEEEEELLLLLEEEEEE-C.A person skilled in the art can determine which amino acids of a given sequence constitute a loop, sheet, or a helix. See, e.g., Mirdita, et al. (2022) NAT. METHODS(19): 679-682.

[0153] In some embodiments, Formula IA may optionally include a C-terminal extension comprising at least one additional loop and one additional beta sheet, represented as L4-D5, wherein the N-terminal side of the fourth loop (L4) is connected to the C-terminal side of the third beta sheet (D4) of Formula IA and the N-terminal side of the fourth beta sheet (D5) is connected to the C-terminal side of the fourth loop (L4).

[0154] In one aspect, a structural arrangement in a synthetic GLP-1R binding protein may be depicted as N-HniLn2En3Ln4En5Ln6En7Ln8En9-C, where H is an alpha helix, E is a beta sheet, L is a loop, each of m-ns represents an integer indicating the number of amino acids in that structural domain (e.g., nl is 30, n2 is 5, n3 is 6, n4 is 4, n5 is 6, n6 is 5, n7 is 6, n8 is 4, and n9 is 4), N and C represent N-terminal and C-terminal domains, respectively. As disclosed herein, certain GLP-1R binding proteins can be represented according to a formula (Formula IB): D1-L1-D2-L2-D3-L3-D4-L4-D5, wherein DI corresponds to an alpha helical domain, and D2, D3, D4 and D5 each correspond to a beta sheet domain; LI , L2, L3 and L4 are each, independently, loops. The components of Formula 1 can correspond to the aforementioned structural arrangement as follows: DI to H and each of D2. D3, D4 and D5 to each of En3, Ens, En7 and En4. The number of E amino acids does not have to be the same across sheets, for example, , ns, and may be, but do not have to be, the same numbers. Similarly, for example, the number of L amino acids does not have to be the same across loops; m and may be, but do not have to be, the same number. For example, an exemplary formula of a synthetic GLP-1R binding protein may comprise N-H30L5E6L4E6L5E6L4E4-C as depicted pictorially below denoting the amino acids in a helix domain (H) or a loop domain (L):N-HHHHHHHHHHHHHHHHHHHHHHHHHHHHHLLLLLEEEEEELLLLEEEEEELLLLLEEEEEELLLLE EEE-C.A person skilled in the art can determine which amino acids of a given sequence constitute a loop, sheet, or a helix. See, e.g., Mirdita, el al. (2022), supra .

[0155] Any given H domain (e.g., Hni) in an alpha helix that is part of a synthetic GLP- 1R binding protein may independently contain between about 4 amino acids and about 20 amino acids in length. An H domain may independently comprise 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more amino acids in length. Any given E domain (e.g, En3) in a beta sheet that is part of a synthetic GLP-1R binding protein may independently contain between about 4 and about 15 amino acids in length. An E domain may independently comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids in length.

[0156] Loops disposed between alpha helices and / or beta sheets may also be of the same or different lengths. Each loop may independently comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 amino acids in length.

[0157] In a protein with more than one alpha helical structure, each alpha helix may comprise the same number of amino acids in each of its H domains or different numbers of amino acids in length in reference to the primary structure of each helical region. That is, in some synthetic GLP-1R binding proteins having more than one alpha helix, each H domain in the binding protein is the same length. In some synthetic GLP-1R binding proteins having one or more alpha helix, one or more H domains has a different length relative to other H domains in the binding protein. In some embodiments, an H domain has zero, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty -two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine or more conserved amino acids (e.g, relative to other GLP-1R binding proteins).

[0158] In a protein with more than one beta sheet, each beta sheet may comprise the same number of amino acids in each of its E domains or different numbers of amino acids in length in reference to the primary structure of each sheet region. That is, in some synthetic GLP-1R binding proteins having more than one beta sheet, each E domain in the binding protein is the same length. In some synthetic GLP-1R binding proteins having one or more beta sheets, one or more E domains has a different length relative to other E domains in the binding protein. In some embodiments, an E domain has zero, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more conserved amino acids (e.g., relative to other GLP-1R binding proteins).

[0159] In an exemplary' GLP-1R binding protein disclosed herein, the binding protein comprises at least one alpha helix, at least three beta sheets, and at least three loops (a first loop, a second loop, and a third), wherein the first loop has a first amino acid sequence that connects a terminal amino acid (e.g. , a C-terminal amino acid) of a first alpha helix to a terminal amino acid (e.g., an N-terminal amino acid) of a first alpha helix, a second loop having a second amino acid sequence connects a second, terminal amino acid (e.g., a C- terminal amino acid) of the first beta sheet to a terminal amino acid (e.g, an N-terminal amino acid) of a second beta sheet; and a third loop having a third amino acid sequence connects a third, terminal amino acid (e.g., a C-terminal amino acid) of the second beta sheet to a terminal amino acid (e.g, an N-terminal amino acid) of a third beta sheet.

[0160] In an exemplary' GLP-1R binding protein disclosed herein, the binding protein can comprise at least one alpha helix, at least four beta sheets, and at least four loops (a first loop,a second loop, a third loop, and a fourth loop), wherein the first loop has a first amino acid sequence that connects a terminal amino acid (e.g, a C-terminal amino acid) of a first alpha helix to a terminal amino acid (e.g, an N-terminal amino acid) of a first beta sheet, a second loop having a second amino acid sequence connects a second, terminal amino acid (e.g., a C- terminal amino acid) of the first beta sheet to a terminal amino acid (e.g, an N-terminal amino acid) of a second beta sheet; a third loop having a third amino acid sequence connects a third, terminal amino acid (e.g, a C-terminal amino acid) of the second beta sheet to a terminal amino acid (e g, an N-terminal amino acid) of a third beta sheet; and a fourth loop having a fourth amino acid sequence connects a fourth, terminal amino acid (e.g, a C- terminal amino acid) of the third beta sheet to a terminal amino acid (e.g., an N-terminal amino acid) of a fourth beta sheet.

[0161] At least a portion of the beta sheets in a synthetic GLP-1R binding protein described herein can constitute a stabilization domain. A stabilization domain can comprise one or more portions of a protein sequence that, when folded, interfits with one or more regions of the receptor adjacent a native ligand (e.g., GLP-1) or agonist (e.g, GLP-1R agonist, e.g, binding site on the receptor). A stabilization domain can, in some embodiments, increase the surface area of contact between a synthetic binding protein (e.g, an agonist, e.g. a GLP-1R agonist, e.g, a synthetic GLP-1R agonist in accordance with the present disclosure) as compared to the surface area of contact between a native ligand (e.g, GLP-1) or receptor agonist (e.g., a reference receptor agonist, e.g, semaglutide, etc.) without a stabilization domain. Increase in surface area can, in some embodiments, be calculated by using a program (e.g, Rosetta) to calculate solvent-accessible surface area (“SASA”). It will be understood to those of skill in the art that, depending on circumstances, such measurements can be based on models or on solved structures. GLP-1R binding agonists as provided herein can be modeled and measured using Rosetta and SASA and compared to, e.g., reference GLP-1R agonists (e.g, semaglutide).

[0162] Depending upon the circumstances (e.g., GLP-1R activity), cAMP-mediated pathway signaling is thought to be an important component of therapeutic effect / relevance of GLP-1R agonists (e.g., reference GLP-1R agonists, e.g., semaglutide, liraglutide, dulaglutide, lixisenatide, exenatide, etc.). Accordingly, having a molecule that can bias cAMP signaling over other signaling pathways can provide benefits over currently available GLP-1 R therapeutics. Without wishing to be bound by theory, engagement of a synthetic GLP-1Rbinding protein ectodomain via an agonist-site binding and protein stabilization domain (e.g., alpha helical and beta sheet portions, respectively) can result in biased agonism, preferentially activating a certain signaling pathway (e.g., cAMP) over another (e.g., |3- arrestin).

[0163] In some embodiments, a synthetic GLP-1R binding protein may have (a) a first alpha helix containing at least one hydrophobic amino acid, wherein, optionally, one or more of the at least one hydrophobic amino acids is not solvent accessible; (b) a first, second, third and / or fourth beta sheet each containing at least two or three hydrophobic amino acids, wherein, optionally, one or more of the at least two or three hydrophobic amino acids is not solvent accessible; (c) a first alpha helix containing at least one or two solvent accessible amino acids; (d) a first, second, third and / or fourth beta sheet containing at least one or two solvent accessible amino acids; (e) a first alpha helix containing at least one or two solvent accessible amino acids; (f) a first and / or second and / or third loop and / or fourth containing at least one hydrophobic amino acid; or (g) the binding protein comprises any combination of elements selected from (a), (b), (c), (d), (e), and (f).

[0164] In some embodiments, a synthetic GLP-1R binding protein may have (a) a first, second, third, and, optionally, a fourth beta sheets each containing at least two hydrophobic amino acids; (b) a first, second, third and. optionally, a fourth beta sheet each containing at least one solvent accessible amino acid; (c) a first, second, third, and, optionally, fourth beta sheet each containing at least two hydrophobic and one solvent accessible amino acids; (d) a first alpha helix contains at least four solvent accessible amino acids; and / or (e) a first, second, and / or third loop, and / or optionally a fourth loop contains at least one hydrophobic amino acid.

[0165] A synthetic GLP-1R binding protein may have one of several consensus sequence structures. Consensus sequences will generally have certain ‘'fixed’’ amino acid positions as well as those that can be varied, such as by changing to another amino acid. Sometimes changing amino acids at certain positions can alter the function of the synthetic binding protein by increasing or decreasing affinity for the target (i.e., GLP-1R). However, all the binding proteins disclosed herein, although having different primary structures have a minimal “threshold” binding affinity to GLP-1R. In some embodiments, a threshold binding affinity may be greater than about 10 pM, about 1 pM, about 100 nM, about 10 nM, or about 1 nM.

[0166] In certain embodiments, a synthetic GLP-1R binding protein disclosure may be represented according to a formula shown as one or more domains, wherein each domain optionally has one or more conserved amino acid residues and / or a particular structure (e.g, loop, e.g., helix). For example, an exemplary synthetic GLP-1R binding protein comprises an amino acid sequence arranged in a primary structure of:D1-L1-D2-L2-D3-L3-D4 (Formula IA) where DI represents an alpha helix, D2, D3, and D4 represent beta sheets, and LI, L2, and L3 represent loops connecting the alpha helix and / or beta sheets.

[0167] In certain embodiments, a synthetic GLP-1R binding protein disclosure may be represented according to a formula shown as one or more domains, wherein each domain optionally has one or more conserved amino acid residues and / or a particular structure (e.g, loop, e.g., helix). For example, an exemplary synthetic GLP-1R binding protein comprises an amino acid sequence arranged in a primary structure of:D 1 -L 1 -D2-L2-D3-L3-D4-L4-D5 (Formula IB) where DI represents an alpha helix; D2, D3, D4, and optionally D5 represent beta sheets; and LI, L2, L3, and optionally L4 represent loops connecting the alpha helix and / or beta sheets.

[0168] The amino acid sequence of the starting “parental” protein and exemplary consensus sequences for various miniproteins developed in Examples 1-4 and 7 are set forth in TABLE 1. Bold, underlined, italicized residues represent helices and correspond to DI, and bold, underlined residues represent sheets and correspond to D2, D3, and D4 in order along a given consensus sequence. Exemplary consensus sequences for DI, D2, D3, D4, LI, L2, and L3 for each miniprotein are set forth in TABLE 2A. Positions for each variable amino acid along the length of a consensus sequence as set forth in TABLE 1 are set forth in TABLE 2B

[0169] As demonstrated in the Examples that follow, a synthetic GLP-1R binding protein having the amino acid sequence of a first miniprotein (referred to a Reference Miniprotein 1) HAEGTFTSDVSSYLERLASRSFLTILQLRADGITDFSLDLDLLTGELTVTDNNTGETTT VQL (SEQ ID NO: 1) was developed, characterized, and optimized.

[0170] It is contemplated that a synthetic GLP-1R binding protein can comprise from about 30 amino acids to about 90 amino acids in length, from about 35 amino acids to about85 amino acids in length, from about 35 amino acids to about 75 amino acids in length, from about 35 amino acids to about 65 amino acids in length, from about 35 amino acids to about 55 amino acids in length, from about 35 amino acids to about 50 amino acids in length, from about 40 amino acids to about 85 amino acids in length, from about 40 amino acids to about 75 amino acids in length, from about 40 amino acids to about 65 amino acids in length, from about 45 amino acids to about 70 amino acids in length, from about 45 amino acids to about 65. from about 45 amino acids to about 60 amino acids in length, from about 50 ammo acids to about 75 amino acids, from about 50 amino acids to about 70 amino acids in length, from about 50 amino acids to about 65 amino acids in length, from about 60 amino acids to about 70 amino acids in length, or from about 60 amino acids to about 65 amino acids in length. In certain embodiments, the synthetic GLP-1R binding protein comprises 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 ammo acids.

[0171] A synthetic GLP-1R binding protein may have an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more or 100% identity with or to a reference sequence or component thereof, wherein the reference sequence is selected from any of SEQ ID NOs: 1- 105 and 108-166, and portions (e.g, domains) thereof. In some embodiments, a synthetic GLP-1R binding protein may have an amino acid sequence comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%. 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more or 100% identity with or to a reference sequence or component thereof selected from any of SEQ ID NOs: 1-70 and 108- 155, and portions thereof. In some embodiments, a synthetic GLP-1R binding protein may have an amino acid sequence comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%. 99%. 99.5%, 99.6%, 99.7%, 99.8%. 99.9% or more or 100% identity with or to a reference sequence or component thereof selected from TABLE 8. In certain embodiments, a synthetic GLP-1R binding protein has an amino acid sequence at least 60 (e.g., 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5) percent identical to SEQ ID NO: 1. In certain embodiments, a synthetic GLP-1R binding protein has an amino acid sequence at least 85 (e.g., 90, 95, 96. 97. 98. 99. 99.5) percent identical to SEQ ID NO: 2.TABLE 1. Exemplary Miniprotein Consensus Sequences*Bolded and italicized text indicates amino acids present in an alpha helix (positions 1-30); and bolded, underlined text indicates amino acids present in a beta sheet (positions 36-41 ; 46-51 ; 57-61, and 66-70).TABLE 2A. Exemplary Miniprotein Domain Consensus SequencesTABLE 2B. Exemplary Miniprotein Variable Substitutions

[0172] In one aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI. L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 71, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 84, wherein XI is H, L, Y, I, a peptide bond, or is absent; X2 is A, G. S, T, D, E, P, H, V, N, a peptide bond, or is absent; X3is E, a peptide bond, or is absent; X4 is G, A, a peptide bond, or is absent; X5 is T, P, V, H, G, Y, a peptide bond, or is absent; X6 is F, A, a peptide bond, or is absent; XI 1 is S or R; X13 is Y or I; X16 is R or V; X18 is A, D, or L; X19 is S, I, E, V, or L; X20 is R or I; X21 is S or F; X23 is L, I, or V; X25 is I, L, Y, or A; X27 is Q or L; X28 is L, G, N, or Q; and X29 is R or G;(b) D2 comprises an amino acid sequence of X36X37X38X39LD, wherein X36 is F or T; X37 is S, V. or L; X38 is L. F, Y. or R; and X39 is D or V;(c) D3 comprises an amino acid sequence of X46X47TX49X50D, wherein X46 is E, V. or D; X47 is L or P; X49 is V or F; and X50 is T or I; and(d) D4 comprises an amino acid sequence of X57X58X59VX61, wherein X57 is T, K, or R; X58 is T, F, K, L, or R; X59 is T. V, F, R, or H; and X61 is Q, S, T, H.

[0173] Furthermore, LI can comprises an amino acid sequence of DGX33TX35, wherein X33 is I, G, V, K, or F; and X35 is D or G; and / or L2 can comprise an amino acid sequence of X42X43X44X45. wherein X42 is L, D. or E; X43 is L. Y, or E; X44 is T or F; and X45 is G or F; and / or L3 can comprise an amino acid sequence of NX53X54X55E, wherein X53 is N or T; X54 is T or F; and X55 is G or L.

[0174] In addition, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 71 may have a C-terminus of X62, wherein X is L or S. In some embodiments, the synthetic GLP-1R binding protein comprises an amino acid, X62, which may or may not be a C-terminal amino acid and may be L or S. That is, in some embodiments, X62 is not a C- terminal amino acid. That is, in certain embodiments, one or more additional amino acids can be present following X62 e.g., bound, e.g, via a peptide bond) to amino acid X62.

[0175] In certain embodiments, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 71, where X62 is a C-terminal amino acid selected from L or S, further comprises a C-terminal extension comprising an additional loop (L4) and beta sheet (D5), wherein the amino acid sequence of L4-D5 is set forth in SEQ ID NO: 165 (X63TGEFSED). In some embodiments, X63 is R, G, D, or P.

[0176] In certain embodiments, the synthetic GLP-1R binding protein comprises an amino acid sequence of SEQ ID NO: 71, wherein XI is H, L, Y, I, a peptide bond, or is absent; X2 is A, G. S, T, D, E, P, H, V, N, a peptide bond, or is absent; X3 is E, a peptide bond, or is absent; X4 is G, A, a peptide bond, or is absent; X5 is T, P, V, H, G, Y, a peptide bond, or is absent; X6 is F, A. a peptide bond, or is absent; XI 1 is S or R; X13 is Y or I; XI 6 is R or V; X18 is A, D, or L; X19 is S, I, E, V, or L; X20 is R or I; X21 is S or F; X23 is L, I,or V; X25 is I, L, Y, or A; X27 is Q or L; X28 is L, G, N, or Q; X29 is R or G; X33 is I, G, V, K. or F; X35 is D or G; X36 is F or T; X37 is S, V, or L; X38 is L, F, Y, or R; X39 is D or V; X42 is L, D, or E; X43 is L, Y, or E; X44 is T or F; X45 is G or F; X46 is E, V, or D; X47 is L or P; X49 is V or F; X50 is T or I; X53 is N or T; X54 is T or F; X55 is G or L; X57 is T, K, or R; X58 is T, F, K, L, or R; X59 is T, V, F, R, or H; X61 is Q, S, T, or H; and X62 is L or S. In some embodiments, the synthetic GLP-1R binding protein optionally comprises a C-terminal extension of SEQ ID NO: 165. wherein X63 is R. G, D. or P.

[0177] In certain embodiments, the GLP-1R binding protein comprises an amino acid sequence selected from any of SEQ ID NOs: 1-29 and 31-70.

[0178] In one aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 72, wherein DI, D2, D3. and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 85, wherein XI is H, L, Y, I, a peptide bond, or is absent; X2 is A, G, S, T, D, E, P, H, V, N, a peptide bond, or is absent; X3 is E, a peptide bond, or is absent; X4 is G, a peptide bond, or is absent; X5 is T, P, V, H. G, Y, a peptide bond, or is absent; X6 is F, A. a peptide bone, or is absent; X13 is Y or I; XI 8 is A, D, or L; XI 9 is I, S, or E; X20 is R or I; X23 is I, L, or V; X28 is L or G; and X29 is R or G;(b) D2 comprises an amino acid sequence of SEQ ID NO: 97, wherein X38 is L or Y;(c) D3 comprises an amino acid sequence of SEQ ID NO: 99, wherein X47 is P or L; and(d) D4 comprises an amino acid sequence of X57X58X59VX61, wherein X57 is T, K, or R; X58 is F, T, or L; X59 is T. H, V, or R; and X61 is Q, S, or T.

[0179] Furthermore, LI can comprise an amino acid sequence of DGX33TX35. wherein X33 is I, G, V. K, or F; and X35 is D or G; and / or L2 can comprise an amino acid sequence of X42X43TG, wherein X42 is D, L, or E; and X43 is L or Y; and / or L3 can comprise an amino acid sequence of SEQ ID NO: 105.

[0180] In certain embodiments, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 72, further comprises a C-terminal extension comprising an additional loop (L4) and beta sheet (D5), wherein the amino acid sequence of L4-D5 is set forth in SEQ ID NO: 165 (X63TGEFSED). In some embodiments, X63 is R, G, D, or P.

[0181] In certain embodiments, the synthetic GLP-1R binding protein comprises an amino acid sequence of SEQ ID NO: 72, wherein XI is H, L, Y, I, a peptide bond, or is absent; X2 is A, G, S, T, D, E, P, H, V, N, a peptide bond, or is absent; X3 is E, a peptide bond, or is absent; X4 is G, a peptide bond, or is absent; X5 is T, P. V, H, G, Y, a peptide bond, or is absent; X6 is F, A, a peptide bond, or is absent; XI 3 is Y or I; XI 8 is A, D, or L; XI 9 is I, S, or E; X20 is R or I; X23 is I, L, or V; X28 is L or G; X29 is R or G; X33 is I, G, V, K, or F; X35 is D or G; X38 is L or Y; X42 is D, L, or E; X43 is L or Y; X47 is P or L; X57 is T. K, or R; X58 is F, T. or L; X59 is T, H, V, or R; and X61 is Q, S. or T. In some embodiments, the synthetic GLP-1R binding protein optionally comprises a C-terminal extension of SEQ ID NO: 165, wherein X63 is R, G, D, or P.

[0182] In certain embodiments, the GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 2, 6-9,11-17, 20, 23, 28-32, 37-40, 42, 44, and 46-70.

[0183] In one aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI. D2, D3, and D4 are domains 1, 2, 3. and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 73, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 86, wherein XI is H. L, or Y;X2 is A, G. S, T. D, E. P, H. V, or N; X5 is T. P, V. H, G. or Y; X6 is F or A; X18 is A or D;XI 9 is I, S, or E; X20 is R or I; X23 is I or L; X28 is L or G; X29 is R or G;(b) D2 comprises an amino acid sequence of SEQ ID NO: 97, wherein X38 is L or Y;(c) D3 comprises an amino acid sequence of SEQ ID NO: 100; and(d) D4 comprises an amino acid sequence of X57X58X59VX61, wherein X57 is T, K. or R; X58 is F or T; X59 is T or H; and X61 is Q, S, or T.

[0184] Furthermore, LI can comprise an amino acid sequence of DGX33TX35. wherein X33 is I or V; X35 is D or G; and / or L2 can comprise an amino acid sequence ofX42X43TG, wherein X42 is D, L, or E; and X43 is L or Y; and / or L3 can comprise an amino acid sequence of SEQ ID NO: 105.

[0185] In certain embodiments, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 73, further comprises a C-terminal extension with an amino acid sequence of SEQ ID NO: 165 (X63TGEFSED). In some embodiments, X63 is R, G, D or P.

[0186] In certain embodiments, the synthetic GLP-1R binding protein comprises an amino acid sequence of SEQ ID NO: 73, wherein XI is H, L, or Y; X2 is A, G, S, T, D, E, P, H, V, or N; X5 is T, P, V, H, G, or Y; X6 is F or A; X18 is A or D; X19 is I, S, or E; X20 is R or I; X23 is I or L; X28 is L or G; X29 is R or G; X33 is I or V; X35 is D or G; X38 is L or Y; X42 is D. L, or E; X43 is L or Y; X57 is T, K, or R; X58 is F or T; X59 is T or H; and X61 is Q, S, or T. In some embodiments, the synthetic GLP-1R binding protein optionally comprises a C-terminal extension of SEQ ID NO: 165, wherein X63 is R, G, D or P.

[0187] In certain embodiments, the GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 2, 6, 8, 9, 11-14, 20, 28, 37, and 46-59.

[0188] In one aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI. D2, D3, and D4 are domains 1, 2, 3. and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 74, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 87, wherein XI is H, I, a peptide bond, or is absent; XI 3 is Y or I; XI 8 is D, A, or L; XI 9 is S or I; X23 is V, I, or L; X28 is L or G:(b) D2 comprises an amino acid sequence of SEQ ID NO: 98;(c) D3 comprises an amino acid sequence of SEQ ID NO: 100; and(d) D4 comprises an amino acid sequence of X57X58X59VQ, wherein X57 is T or K; X58 is T or F; and X59 is T, V, or R.

[0189] Furthermore, LI can comprise an amino acid sequence of SEQ ID NO: 103. wherein X33 is K, I, F, or G; and / or L2 can comprise an amino acid sequence of SEQ ID NO: 104; and / or L3 can comprise an amino acid sequence of SEQ ID NO: 105.

[0190] In certain embodiments, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 74, further comprises a C-terminal extension comprising an additional loop (L4) and beta sheet (D5), wherein the amino acid sequence of L4-D5 is set forth in SEQ ID NO: 165 (X63TGEFSED). In some embodiments, X63 is R, G, D, or P.

[0191] In certain embodiments, the synthetic GLP-1R binding protein comprises an amino acid sequence of SEQ ID NO: 74, wherein XI is H, I, a peptide bond, or is absent; X13 is Y or I; X18 is D, A, or L; X19 is S or I; X23 is V, I, or L; X28 is L or G; X33 is K, I, F, or G; X57 is T or K; X58 is T or F; and X59 is T, V, or R.

[0192] In certain embodiments, the GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 15-17. 23. 29. 31. 38, 40, 42, and 60.

[0193] In one aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 75, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 88, wherein XI is H, a peptide bond, or is absent; X2 is A, a peptide bond, or is absent; X3 is E, a peptide bond, or is absent; X4 is G, a peptide bond, or is absent; X5 is T, a peptide bond, or is absent; X6 is F, a peptide bond, or is absent; X13 is Y or I; X18 is A or L; X19 is S or I; X23 is L or I; and X28 is L or G;(b) D2 comprises an amino acid sequence of SEQ ID NO: 98;(c) D3 comprises an amino acid sequence of SEQ ID NO: 100; and(d) D4 comprises an amino acid sequence of X57X58TVQ, wherein X57 is T or K; and X58 is T, F, or L.

[0194] Furthermore, LI can comprise an amino acid sequence of SEQ ID NO: 103, wherein X33 is I or G; and / or L2 can comprise an amino acid sequence of X42LTG, wherein X42 is L or D; and / or L3 can comprise an amino acid sequence of SEQ ID NO: 105.

[0195] In certain embodiments, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 75, further comprises a C-terminal extension comprising an additional loop (L4) and beta sheet (D5), wherein the amino acid sequence of L4-D5 is set forth in SEQ ID NO: 165 (X63TGEFSED). In some embodiments, X63 is R, G, D, or P.

[0196] In certain embodiments, the synthetic GLP-1R binding protein comprises an amino acid sequence of SEQ ID NO: 75, wherein XI is H, a peptide bond, or is absent; X2 is A, a peptide bond, or is absent; X3 is E, a peptide bond, or is absent; X4 is G, a peptide bond, or is absent; X5 is T, a peptide bond, or is absent; X6 is F, a peptide bond, or is absent; XI 3 is Y or I; X18 is A or L; X19 is S or I; X23 is L or I; X28 is L or G; X33 is I or G; X42 is L or D; X57 is T or K; and X58 is T. F, or L.

[0197] In certain embodiments, the GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 30, 39, 44, and 61-70.

[0198] In one aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4 respectively, and LI, L2, and L3 are loops 1, 2, and 3 respectively; and (ii) an amino acid of SEQ ID NO: 76, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 89 wherein XI is H, L, Y, a peptide bond, or is absent; X2 is A, G. S, T. D, E. P, H. V, or N; and XI 3 is Y or I;(b) D2 comprises an amino acid sequence of SEQ ID NO: 98;(c) D3 comprises an amino acid sequence of SEQ ID NO: 99, wherein X47 is P or L; and(d) D4 comprises an amino acid sequence of SEQ ID NO: 102, wherein X57 is T or K.

[0199] Furthermore, LI can comprise an amino acid sequence of SEQ ID NO: 103, wherein X33 is I or G; and / or L2 can comprise an amino acid sequence of SEQ ID NO: 104; and / or L3 can comprise an amino acid sequence of SEQ ID NO: 105.

[0200] In certain embodiments, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 76, further comprises a C-terminal extension comprising an additional loop (L4) and beta sheet (D5), wherein the amino acid sequence of L4-D5 is set forth in SEQ ID NO: 165 (X63TGEFSED). In some embodiments, X63 is R, G, D, or P.

[0201] In certain embodiments, the synthetic GLP-1R binding protein comprises an amino acid sequence of SEQ ID NO: 76, wherein XI is H, L, Y, a peptide bond, or absent; X2 is A, G, S, T, D, E, P, H, V, or N; XI 3 is Y or I; X33 is I or G; X47 is P or L; and X57 is T or K.

[0202] In certain embodiments, the GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 2, 7-9. 29. 32, 40, 46-54, and 60.

[0203] In one aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1 , 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 77, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 90, wherein XI is H, L, Y, a peptide bond, or is absent; X2 is A, G. S, T. D, E. P, H, V, or N; and XI 3 is Y or I;(b) D2 comprises an amino acid sequence of SEQ ID NO: 98;(c) D3 comprises an amino acid sequence of SEQ ID NO: 99, wherein X47 is L or P; and(d) D4 comprises an amino acid sequence of SEQ ID NO: 102, wherein X57 is T or K.

[0204] Furthermore, LI can comprise an amino acid sequence of SEQ ID NO: 103, wherein X33 is I or G; and / or L2 can comprise an amino acid sequence of SEQ ID NO: 104; and / or L3 can comprise an amino acid sequence of SEQ ID NO: 105.

[0205] In certain embodiments, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 77, further comprises a C-terminal extension comprising an additional loop (L4) and beta sheet (D5), wherein the amino acid sequence of L4-D5 is set forth in SEQ ID NO: 165 (X63TGEFSED). In some embodiments, X63 is R, G, D, or P.

[0206] In certain embodiments, the synthetic GLP-1R binding protein comprises an amino acid sequence of SEQ ID NO: 77, wherein XI is H, L, Y, a peptide bond, or is absent; X2 is A, G, S, T, D, E, P, H, V, or N; X13 is Y or I; X33 is I or G; X47 is L or P; and X57 is T or K.

[0207] In certain embodiments, the GLP-1 R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 2, 7-9, 29, 32, 40, 46-54, and 60.

[0208] In one aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI. L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid ofSEQ ID NO: 78, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:

[0209] (a) DI comprises an amino acid sequence of SEQ ID NO: 91, wherein XI is H or Y; X2 is A, G, S, P, or V; X4 is G or A; XI 1 is S or R; X13 is Y or I: X16 is R or V; X18 is A or L; XI 9 is S, I, E, V, or L; X20 is R or I; X21 is S or F; X23 is L or E X25 is I, L, Y, or A; X27 is Q or L; X28 is L, G, N, or Q; and X29 is R or G;(b) D2 comprises an amino acid sequence of X36X37X38X39LD, wherein X36 is F or T; X37 is S, V, or L; X38 is L, F, Y, or R; and X39 is D or V;(c) D3 comprises an amino acid sequence of X46X47TX49X50D, wherein X46 is E, D, or V; X47 is L or P; X49 is V or F; and X50 is T or I; and(d) D4 comprises an amino acid sequence of X57X58X59VX61, wherein X57 is T, K, or R; X58 is T, F, L, K, or R; X59 is T, H, or F; and X61 is Q, T, or H.

[0210] Furthermore, LI can comprise an amino acid sequence of SEQ ID NO: 103, wherein X33 is I or G; and / or L2 can comprise an amino acid sequence of X42X43X44X45, wherein X42 is L or D; X43 is L or E; X44 is T or F; and X45 is G or F; and / or L3 can comprise an amino acid sequence of NX53X54X55E. wherein X53 is N or T; X54 is T or F; and X55 is G or L.

[0211] In addition, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 78 may have a C-terminus of X62, wherein X is L or S. In some embodiments, the synthetic GLP-1R binding protein comprises an amino acid, X62, which may or may not be a C-terminal amino acid and may be L or S. That is, in some embodiments, X62 is not a C- terminal amino acid. That is, in certain embodiments, one or more additional amino acids can be present following X62 (e.g., bound, e.g, via a peptide bond) to amino acid X62.

[0212] In certain embodiments, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 78, where X62 is a C-terminal amino acid selected from L or S, further comprises a C-terminal extension comprising an additional loop (L4) and beta sheet (D5), wherein the amino acid sequence of L4-D5 is set forth in SEQ ID NO: 165 (X63TGEFSED). In some embodiments. X63 is R, G, D, or P.

[0213] In certain embodiments, the synthetic GLP-1R binding protein comprises an amino acid sequence of SEQ ID NO: 78, wherein XI is H or Y; X2 is A, G, S, P, or V: X4 is G or A; Xl l is S or R; X13 is Y or I; X16 is R or V; X18 is A or L; X19 is S, I, E, V, or L;X20 is R or I; X21 is S or F; X23 is L or I; X25 is I, L, Y, or A; X27 is Q or L; X28 is L. G, N, or Q; X29 is R or G; X33 is I or G; X36 is F or T; X37 is S, V, or L; X38 is L, F, Y, or R; X39 is D or V; X42 is L or D; X43 is L or E; X44 is T or F; X45 is G or F; X46 is E, D, or V; X47 is L or P; X49 is V or F; X50 is T or I; X53 is N or T; X54 is T or F; X55 is G or L; X57 is T, K, or R; X58 is T, F, L, K, or R; X59 is T, H, or F; X61 is Q, T, or H; and X62 is L or S.

[0214] In certain embodiments, the GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 1-7, 9-11, 18-22, 24-27. 30-47, 51, and 53.

[0215] In one aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 79, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 92, wherein XI is H or Y; X2 is A, G, S, P. or V; X23 is I or L; X28 is L or G; and X29 is R or G;(b) D2 comprises an amino acid sequence of SEQ ID NO: 98;(c) D3 comprises an amino acid sequence of SEQ ID NO: 101, wherein X46 is E or D; and X47 is L or P; and(d) D4 comprises an amino acid sequence of X57X58X59VQ, wherein X57 is T, K. or R; X58 is F or T; and X59 is T or H.

[0216] Furthermore, LI can comprise an amino acid sequence of SEQ ID NO: 103. wherein X33 is I or G; and / or L2 can comprise an amino acid sequence of SEQ ID NO: 104; and / or L3 can comprise an amino acid sequence of SEQ ID NO: 105.

[0217] In certain embodiments, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 79, further comprises a C-terminal extension comprising an additional loop (L4) and beta sheet (D5), wherein the amino acid sequence of L4-D5 is set forth in SEQ ID NO: 165 (X63TGEFSED). In some embodiments, X63 is R, G, D. or P.

[0218] In certain embodiments, the synthetic GLP-1R binding protein comprises an amino acid sequence of SEQ ID NO: 79, wherein XI is H or Y; X2 is A, G, S, P, or V; X23is I or L; X28 is L or G; X29 is R or G; X33 is I or G; X46 is E or D; X47 is L or P; X57 is T, K, or R; X58 is F or T; and X59 is T or H.

[0219] In certain embodiments, the GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 2, 5-7. 9, 11, 32, 37. 40. 42. 46. 47. 51, and 53.

[0220] In one aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 80, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 93, wherein XI is H or Y; X2 is A, G, S, P, or V;(b) D2 comprises an amino acid sequence of SEQ ID NO: 98;(c) D3 comprises an amino acid sequence of SEQ ID NO: 99, wherein X47 is L or P; and(d) D4 comprises an amino acid sequence of SEQ ID NO: 102, wherein X57 is T or K.

[0221] Furthermore, LI can comprise an amino acid sequence of SEQ ID NO: 103. wherein X33 is I or G; and / or L2 can comprise an amino acid sequence of SEQ ID NO: 104; and / or L3 can comprise an amino acid sequence of SEQ ID NO: 105.

[0222] In certain embodiments, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 80, further comprises a C-terminal extension comprising an additional loop (L4) and beta sheet (D5), wherein the amino acid sequence of L4-D5 is set forth in SEQ ID NO: 165 (X63TGEFSED). In some embodiments, X63 is R, G, D, or P.

[0223] In certain embodiments, the synthetic GLP-1R binding protein comprises an amino acid sequence of SEQ ID NO: 80, wherein XI is H or Y; X2 is A, G, S, P. or V: X33 is I or G; X47 is L or P; and X57 is T or K.

[0224] In certain embodiments, the GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 2, 7, 9, 32 ,40, 46, 47, 51, and 53

[0225] In one aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4,respectively, and LI, L2. and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 81, wherein DI, D2, D3. and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 94, wherein XI 3 is Y or I;XI 6 is R or V; XI 8 is A or L; X 19 is S, I, E, V, or L; X20 is R or T; X23 is L or I; X25 is I, L, Y, or A; X27 is Q or L; X28 is L, G, or Q; and X29 is R or G;(b) D2 comprises an amino acid sequence of X36X37X38X39LD, wherein X36 is F or T; X37 is S, V. or L; X38 is L. F, Y. or R; X39 is D or V;(c) D3 comprises an amino acid sequence of X46X47TVX50D, wherein X46 is E, D, or V; X47 is L or P; X50 is T or I; and(d) D4 comprises an amino acid sequence of X57X58X59VX61, wherein X57 is T, K, or R; X58 is T, F, L, K, or R; X59 is T, H, or F; and X61 is Q or T.

[0226] Furthermore, LI can comprise an amino acid sequence of SEQ ID NO: 103, wherein X33 is I or G; and / or L2 can comprise an amino acid sequence of X42X43X44X45. wherein X42 is L or D; X43 is L or E; X44 is T or F; and X45 is G or F; and / or L3 can comprise an amino acid sequence of NX53X54X55E, wherein X53 is N or T; X54 is T or F; and X55 is G or L.

[0227] In addition, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 81 may have a C-terminus of X62, wherein X is L or S. In some embodiments, the synthetic GLP-1R binding protein comprises an amino acid, X62, which may or may not be a C-terminal amino acid and may be L or S. That is, in some embodiments, X62 is not a C- terminal amino acid. That is, in certain embodiments, one or more additional amino acids can be present following X62 (e.g., bound, e.g, via a peptide bond) to amino acid X62.

[0228] In certain embodiments, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 81, where X62 is a C-terminal amino acid selected from L or S, further comprises a C-terminal extension comprising an additional loop (L4) and beta sheet (D5), wherein the amino acid sequence of L4-D5 is set forth in SEQ ID NO: 165 (X63TGEFSED). In some embodiments, X63 is R, G, D, or P.

[0229] In certain embodiments, the synthetic GLP-1R binding protein comprises an amino acid sequence of SEQ ID NO: 81, wherein XI 3 is Y or I; XI 6 is R or V; XI 8 is A or L; XI 9 is S, I, E, V, or L; X20 is R or I; X23 is L or I; X25 is I, L, Y, or A; X27 is Q or L; X28 is L. G, or Q; X29 is R or G; X36 is F or T; X37 is S, V. or L; X38 is L ,F, Y, or R; X39is D or V; X46 is E, D. or V: X47 is L or P; X50 is T or I; X57 is T, K, or R; X58 is T, F, L, K, or R; X59 is T, H, or F; X61 is Q or T; X33 is I or G; X42 is L or D; X43 is L or E; X44 is T or F; X45 is G or F; X53 is N or T; X54 is T or F; X55 is G or L; and X62 is L or S.

[0230] In certain embodiments, the GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 1 -7, 10, 1 1, 18-22, 24, 27, and 30-45.

[0231] In one aspect, the disclosure provides a synthetic GLP- 1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI. L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 82. wherein DI, D2, D3. and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 95, wherein X23 is I or L; X28 is L or G; and X29 is R or G;(b) D2 comprises an amino acid sequence of SEQ ID NO: 98;(c) D3 comprises an amino acid sequence of SEQ ID NO: 101. wherein X46 is E or D; and X47 is L or P; and(d) D4 comprises an amino acid sequence of X57X58X59VQ, wherein X57 is T, K, or R; X58 is F or T; and X59 is T or H.

[0232] Furthermore, LI can comprise an amino acid sequence of SEQ ID NO: 103, wherein X33 is I or G; and / or L2 can comprise an amino acid sequence of SEQ ID NO: 104; and / or L3 can comprise an amino acid sequence of SEQ ID NO: 105.

[0233] In certain embodiments, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 82, further comprises a C-terminal extension comprising an additional loop (L4) and beta sheet (D5), wherein the amino acid sequence of L4-D5 is set forth in SEQ ID NO: 165 (X63TGEFSED). In some embodiments, X63 is R, G, D, or P.

[0234] In certain embodiments, the synthetic GLP-1R binding protein comprises an amino acid sequence of SEQ ID NO: 82, wherein X23 is I or L; X28 is L or G; X29 is R or G; X33 is I or G; X46 is E or D; X47 is L or P; X57 is T, K, or R; X58 is F or T; and X59 is T or H.

[0235] In certain embodiments, the GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 2, 5-7. 11. 32. 37. 40, and 42.

[0236] In one aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary’ structure of D1-L1-D2-L2- D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 83, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 96;(b) D2 comprises an amino acid sequence of SEQ ID NO: 98;(c) D3 comprises an amino acid sequence of SEQ ID NO: 99, wherein X47 is L or P; and(d) D4 comprises an amino acid sequence of SEQ ID NO: 102, wherein X57 is T or K.

[0237] Furthermore, LI can comprise an amino acid sequence of SEQ ID NO: 103, wherein X33 is I or G; and / or L2 can comprise an amino acid sequence of SEQ ID NO: 104; and / or L3 can comprise an amino acid sequence of SEQ ID NO: 105.

[0238] In certain embodiments, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 83, further comprises a C-terminal extension comprising an additional loop (L4) and beta sheet (D5), wherein the amino acid sequence of L4-D5 is set forth in SEQ ID NO: 165 (X63TGEFSED). In some embodiments, X63 is R, G, D, or P.

[0239] In certain embodiments, the synthetic GLP-1R binding protein comprises an amino acid sequence of SEQ ID NO: 83, wherein X33 is I or G; X47 is L or P; and X57 is T or K.

[0240] In certain embodiments, the GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 2, 7. 32, and 40.

[0241] A synthetic GLP-1R binding protein can further comprises a C-terminal extension (e.g., C-terminal to position 62, or a position corresponding to 62 with reference to the sequences set forth in Table 1, e.g., linked to the C-terminal amino acid of a given GLP-1R binding protein, etc. ) of SEQ ID NO: 165, wherein X63 is R, G, D, or P.

[0242] In one aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4-L4-D5 (Formula IB), wherein DI, D2, D3, D4 and D5 are domains 1. 2, 3, 4 and 5 respectively, and LL L2. L3, and L4 are loops 1, 2, 3. and 4 respectively: and (ii) anamino acid of SEQ ID NO: 156, wherein DI, D2, D3, D4 and D5 independently comprise any of the following combinations: a) DI comprises an amino acid sequence of SEQ ID NO: 158, wherein X2 is G or S; X3 is E or Q; X10 is V or Y; X14 is L, A, or R; X16 is R or Y; X18 is A, K, R, or F; X21 is S, I, F, Y, V, R, Q, L, or W; X24 is T, L, R, or K; X25 is I or F; X28 is L, R, N, or Q; X29 is R, E, G, Q, or H; b) D2 comprises an amino acid sequence of SEQ ID NO: 160, wherein: X36 is F or Y; X41 is D, K, R; c) D3 comprises an amino acid sequence of SEQ ID NO: 100; d) D4 comprises an amino acid sequence of SEQ ID NO: 161; and e) D5 comprises an amino acid sequence of SEQ ID NO: 162.

[0243] Furthermore, LI can comprise an amino acid sequence of DG1X34X35. wherein X34 is T, K, or R; X35 is D or Q; and / or L2 can comprise an amino acid sequence of SEQ ID NO: 104; and / or L3 comprises an amino acid sequence of SEQ ID NO: 105, wherein: X63 is R, G, D, or P; and / or L4 can comprise an amino acid sequence of LX63TG, wherein X63 is R, G, D, or P.

[0244] In certain embodiments, the synthetic GLP-1R binding protein comprises an amino acid sequence of SEQ ID NO: 156. wherein X2 is G or S; X3 is E or Q: X10 is V or Y; X14 is L, A, R; X16 is R or Y; XI 8 is A, K, R, or F; X21 is S, I, F, Y, V, R, Q, L, or W; X24 is T, L, R, or K; X25 is I or F; X28 is L, R, N, or Q; X29 is R, E, G, Q, or H; X36 is F or Y; X41 is D, Kor R; X34 is T, K, or R; X35 is D or Q; X63 is R, G, D, or P.

[0245] In certain embodiments, the GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 108-151.

[0246] In one aspect, the disclosure provides a synthetic GLP-1R binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2- D3-L3-D4-[L4-D5] (Formula IB), wherein DI, D2, D3, D4 and D5 are domains 1, 2, 3, 4 and 5 respectively, and LI, L2, L3 and L4 are loops 1, 2, 3, and 4 respectively; and (ii) an amino acid of SEQ ID NO: 157. wherein DI, D2, D3. D4 and D5 independently comprise any of the follow ing combinations: a) DI comprises an amino acid sequence of SEQ ID NO: 159, wherein: X21 is W or L; X24 is T, R, or K; X28 is L, N, Q: X29 is R, E, G, Q, or H;b) D2 comprises an amino acid sequence of SEQ ID NO: 160, wherein: X36 is F or Y; X41 is D. K, R; c) D3 comprises an amino acid sequence of SEQ ID NO: 100; d) D4 comprises an amino acid sequence of SEQ ID NO: 161; and e) D5 comprises an amino acid sequence of SEQ ID NO: 162.

[0247] Furthermore, LI can comprise an amino acid sequence of DGIX34X35. wherein: X34 is T. K, R; X35 is D or Q; and / or L2 can comprise an amino acid sequence of SEQ ID NO: 104; and / or L3 comprises an amino acid sequence of SEQ ID NO: 105, wherein: X63 is R, G, D, or P; and / or L4 can comprise an amino acid sequence of LX63TG, wherein X63 is R, G, D, or P.

[0248] In certain embodiments, the synthetic GLP-1R binding protein comprises an amino acid sequence of SEQ ID NO: 157, wherein X21 is L, W; X24 is T, R, K; X28 is L, N, Q: X29 is R, E, G, Q. or H; X34 is T, K. or R; X35 is D or Q; X36 is F or Y; X41 is D, K, or R; and X63 is R, G, D, or P.

[0249] In certain embodiments, the GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 130-151.

[0250] The synthetic GLP-1R binding proteins disclosed herein can have a binding affinity’ from between about 10 pM to about 0.001 nM; about 7.5 pM to about 0.01 nM; about 7.5 pM to about 0.75 nM; about 5 pM to about 0.5 nM; about 2.5 pM to about 0.25 nM; about 1 pM to about 1 nM; about 0.5 pM to about 1 nM; about 0.25 pM to about 1 nM; about 0. 10 pM to about 1 nM; about 75 nM to about 1 nM; about 50 nM to about 1 nM; about 25 nM to about 1 nM; about 10 nM to about 1 nM; or about 5 nM to about 1 nM. Furthermore, the synthetic GLP-1R binding protein can have a binding affinity stronger than about 10 pM, 7.5 pM, 5 pM, about 2.5 pM, about 1 pM, about 0.75 pM, about 0.5 pM, about 0.25 pM, about 0. 1 pM, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, about 0.1 nM, about 0.01 nM, or about 0.001 nM.

[0251] In some embodiments, a threshold binding affinity’ may be greater than about 10 pM, about 1 pM, about 100 nM. about 10 nM, about 1 nM, about 100 pM, about 10 pM, or about 1 pM.

[0252] It is contemplated that optimization of synthetic binding proteins may be achieved using optimized designs, such as, for example, modifications of one or more amino acids by substitution at one or more positions with a different amino acid. Optimization, such as by amino acid modifications, allows tunability of certain characteristics such as changes to (e.g, increases in) binding affinity and / or avidity7.

[0253] Synthetic GLP-1R binding proteins can be optimized by affinity maturation techniques. For example, affinity maturation may be used on a sequence of a binding protein to create another synthetic GLP-1R binding protein with at least the same or better selectivity and / or affinity for GLP-1R as compared to the starting sequence. Affinity maturation can be accomplished using techniques known to those of ordinary skill in the art, including, for example, generating libraries using error prone PCR, degenerate codons, synthetic oligonucleotide pools, or a combination thereof. These libraries can then be transformed into yeast and improved variants may be isolated by methods such as magnetic, flow cytometric, and / or FACS-based approaches. Computational design / redesign strategies may also be used when affinity7maturing proteins and computer programs for implementing such approaches are known in the art. Prior to affinity maturation, synthetic binding proteins may be characterized to determine functional and structural features, such as binding affinity (e.g, for GLP-1R) and conformation.

[0254] Synthetic GLP-1R binding proteins provided herein are engineered to have certain characteristics (e.g, binding affinity / avidity, e.g., for a target, e.g, for GLP-1R). Various in silico, in vitro, and in vivo characterization assays may be used to evaluate these GLP-1R binding proteins. For example, binding assays can be used to determine binding specificity7to a target, e.g. GLP-1R, as compared to binding to another molecule such as, e.g.. another receptor or a ligand such as GLP-1 receptor. Other assays can be used to determine binding affinity7of a binding protein, e.g, a GLP-1 R binding protein for its target, which can include for example, surface plasmon resonance (SPR), and flow cytometry.

[0255] The synthetic GLP-1 R binding proteins of the present disclosure are designed to have certain stability characteristics. For example, a binding protein is stable in that it may retain at least 10%. 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 9%, 94%, 95%, 96%, 97%, 98%, 99% or substantially all of its binding affinity to GLP-1R upon cooling to room temperature after thermal denaturation at 95°C in a solution (e.g, phosphatebuffered saline (PBS)) for at least about 5. 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more minutes relative to the synthetic GLP-1R binding protein prior to thermal denaturation.

[0256] In some embodiments, a synthetic GLP-1R binding protein is stable in that it may retain at least 10%. 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 9%, 94%, 95%, 96%, 97%, 98%, 99% or substantially all of its binding affinity to GLP-1 R after incubation at 37°C (e.g., for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more hours) relative to the synthetic GLP-1R binding protein prior to incubation.

[0257] Synthetic GLP-1R binding proteins of the present disclosure may also display stability’ in resistance to chemical denaturation and / or retention of stability after exposure to chemical denaturants. For example, a synthetic GLP-1R binding protein may be stable in that it may retain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 9%, 94%, 95%, 96%, 97%, 98%, 99% or substantially all of its binding affinity to GLP-1R in PBS following exposure to a denaturing chemical (e.g., 4M urea) at room temperature for about 15 minutes, 30 minutes. 45 minutes, 1, 2. 3, 4, 5, 6, 7, 8 or more hours relative to the binding affinity of the synthetic GLP-1R binding protein prior to exposure to the chemical denaturant.

[0258] Synthetic GLP-1R binding proteins engineered, developed, and produced herein are selected and / or specific for GLP-1R. That is, in some embodiments, a synthetic GLP-1R binding proteins does not bind to a non-GLP-lR. In some embodiments, a synthetic GLP-1R binding protein binds to another receptor, but binds to GLP-1R with a much greater affinity. For example, the binding affinity of a synthetic GLP-1R binding protein may be between 1 and 1000-fold greater than that for any other binding partner (e.g., a non-GLP-lR, e.g., an ectodomain of glucagon, e.g., an ectodomain of GIP).

[0259] Affinity of a synthetic GLP-1R binding protein may be modified and may vary depending on modifications made to, for example, its primary sequence. A binding affinity' may be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70- fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, at least 250-fold greater, at least 500-fold greater, or at least 1000-fold greater than the affinity of thesynthetic GLP-1R binding protein for an unrelated [different] target (e.g., a non-GLP-lR, e.g., an ectodomain of glucagon, e.g, an ectodomain of GIP).IV. SYNTHESIS OF GLP-1R BINDING PROTEINS

[0260] The synthetic GLP-1R binding proteins described herein may be produced bymethods known to those of ordinary skill in the art. Methods may include, for example, biological approaches, such as recombinant approaches and / or chemical approaches, such as solid phase and / or liquid phase chemical synthesis, etc., or combinations thereof.

[0261] With regard to recombinant approaches, a variety of methodologies can be implemented to produce the binding proteins disclosed herein. For example, DNA molecules encoding the binding proteins can be synthesized chemically and / or cloned / produced using recombinant DNA methodologies. The resulting DNA molecules encoding binding proteins of interest can be ligated to other nucleotide sequences, including, for example, expression control sequences, to produce a gene expression construct (i.e., expression vector). Thereafter, the resulting expression vectors are introduced into host cells using conventional transfection or transformation techniques. Exemplary host cells include E. coll cells. Pichia Pastoris cells, Saccharomyces cerevisiae cells. Kluyveromyces lactis cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), and human hepatocellular carcinoma cells (e.g., Hep G2). The transformed host cells can be grown under conditions that permit the host cells to express the genes that encode the binding proteins.

[0262] Specific expression and purification conditions will vary depending upon the expression system employed. For example, if a gene is expressed in E. coli, it is first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, e.g., T7, lac, Trp or Tac, and, in some contexts, a prokaryotic signal sequence or fusion to a protein such as, e.g., Trx, MBP, SUMO, or OsmY. The expressed protein may be secreted. The expressed protein can be harvested after disruption of the cells by French press or sonication (e.g.. in the presence of 4-6 M urea). Alternatively, or in addition, the binding proteins can be harvested and purified or isolated from cell extracts using techniques known in the art, e.g., affinity- tags such as glutathione-S -transferase (GST) or histidine tags. Protease cleavage with SUMO Protease (Ulp), thrombin, enterokinase. TEV protease, 3C protease may be used to cleave affinity tags and fusion proteins from the miniprotein binder. The protein may be further purified with reverse phase HPLC using a C-18 column and eluted in a solvent gradient (e.g, gradient of acetonitrile). The protein may then be lyophilized to remove solvent and may be resuspended in phosphate buffered saline. Purification by reverse phase HPLC may be used to remove endotoxin from samples expressed in E. coli.

[0263] If the engineered gene is expressed in eukaryotic host cells, e.g., CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, a poly A sequence, and a stop codon. Optionally, the vector or gene construct may contain enhancers. The vector may also optionally contain fusion domains which can be used to facilitate expression and secretion. Vectors may also optionally contain enzyme (e.g., protease) cleavage sites. The gene construct can be introduced into eukaryotic host cells using conventional transfection (e.g., for mammalian) and transformation (e.g., for yeast).

[0264] In addition, the synthetic binding proteins may be produced in cell-free systems. For example, chemical synthesis such as organic chemical synthesis using liquid and / or solid phase chemical processes may be used. Such processes and tools for performing such processes, such as various automatic synthesizers, are well known to those of ordinary skill in the art and such tools are widely commercially available. More specifically, methods of chemically synthesizing polypeptides are well known in the art and include, but are not limited to, sohd-phase peptide synthesis, liquid-phase peptide synthesis, and organic synthesis methods. In some synthetic approaches, an amino group of one amino acid (or amino acid derivative) is linked to a carboxyl group of another amino acid (or amino acid derivative) that has been activated by reacting it w ith a reagent such as dicyclohexylcarbodiimide (DCC). When the free amino group attacks the activated carboxyl group, a peptide bond is formed and dicyclohexylurea is released. In such methods, other potentially reactive groups (such as the a-amino group of the N-terminal amino acid or amino acid derivative and the carboxyl group of the C-terminal amino acid or amino acid derivative) may be blocked (“protected”) from participating in the chemical reaction. Thus, only particular active groups react such that the desired product is formed. Blocking groups useful for this purpose include, without limitation, tertbutoxy carbonyl groups (t-Boc) and benzoyloxy carbonyl groups to protect amine groups; and simple esters (such as methyl and ethyl groups) and benzyl esters to protect carboxyl groups. Blocking groups can typically be subsequently removed with a treatment that leaves peptide bonds intact (for example,treatment with dilute acid). This process of protecting reacting groups that should not react, coupling to form a peptide bond, and deprotecting reactive groups may be repeated. A peptide may be synthesized by sequentially adding amino acids to a growing peptide chain.

[0265] Both liquid-phase and solid phase peptide synthesis methods can be used to make the binding proteins described herein. Tn solid-phase peptide synthesis, the growing peptide chain is typically linked to an insoluble matrix (such as, for example, polystyrene beads) by linking the carboxy terminal amino acid to the matrix. At the end of synthesis, the peptide can be released from the matrix using a cleaving reagent that does not disrupt peptide bonds, such as hydrofluoric acid (HF). Protecting groups are also typically removed at this time. Automated, high throughput, and / or parallel peptide synthesis methods may also be used in accordance with the disclosure. For more information about peptide synthesis methods, see, e.g, Merrifield (1969) ADV. ENZYMOL. RELAT. AREAS MOL. BIOL.. 32: 221-96; Fridkin et al. (1974) ANN. REV. BIOCHEM. 43(0): 419-43; Merrifield (1997) METH. ENZYMOL. 289: 3-13;Sabatino et al. (2009) CURR. OPIN. DRUG DISCOV. DEVEL., 11(6): 762-70.

[0266] Once synthesized, the binding proteins can be purified using standard approaches including, for example, chromatographic (e.g.. reverse phase HPLC) and affinity binding approaches. The resulting binding proteins can then be characterized using a variety of chemical, biological and biophysical approaches.V. CHARACTERIZATION OF GLP-1R BINDING PROTEINSA. Biophysical Characterization

[0267] The synthetic binding proteins described herein may be characterized using a variety of approaches to determine, e.g., secondary' and tertiary conformation, binding affinity, binding selectivity, stability (e.g, thermostability, chemical stability, propensity to degrade, etc.), solubility, etc.

[0268] For example, protein conformation may be measured via circular dichroism spectroscopy, infrared spectroscopy, NMR, X-ray crystallography, cryo-electron microscopy and AlphaFold (alphafold.ebi.ac.uk / ). Binding affinity and / or selectivity may be determined using assays such as flow' cytometric analyses using, e.g., yeast or mammalian cells, biolayer interferometry and / or surface plasmon resonance measurements, each of which will be able to determine different types and specificities of binding.

[0269] Binding affinity and / or avidity can be determined by measuring the equilibrium dissociation constant (KD) of a synthetic GLP-1R binding protein to a target. In some embodiments, the binding affinity (KD) of synthetic GLP-1R binding proteins in the range of 10'5M or less, or ranging down to IO'10M or lower, (e.g., about 1 O'6, 1 O'7, 10'8, 10'9, IO'10M or less).

[0270] In some embodiments, the synthetic GLP-1R binding protein comprises a binding affinity characterized by a dissociation constant ranging from about 1 pM to 10 pM. In some embodiments, the binding affinity is between about 0.001 nM to about 10 pM; about 0.75 nM to about 7.5 pM; about 0.5 nM to about 5 pM; about 0.25 nM to about 2.5 pM; about 1 nM to about 1 pM; about 1 nM to about 0.5 pM; about 1 nM to about 0.25 pM; about 1 nM to about 0. 10 pM; about 1 nM to about 75 nM; about 1 nM to about 50 nM; about 1 nM to about 25 nM; about 1 nM to about 10 nM; or about 1 nM to about 5 nM. In some embodiment, the binding affinity is stronger than about 10 pM, 7.5 pM, 5 pM, about 2.5 pM, about 1 pM, about 0.75 pM, about 0.5 pM, about 0.25 pM, about 0.1 pM, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM. about 3 nM, about 2 nM, about 1 nM, about 0.75 nM. about 0.5 nM, about 0.25 nM, about 0.1 nM to about 0.01 nM, or about 0.01 nM to about 0.001 nM. One of ordinary skill in the art will readily know how to calculate equilibrium dissociation constants using measured Ka (1 / sec) and Kd (1 / secM) of the synthetic GLP-1R binding proteins.

[0271] Other analytical techniques (some of which are also used for synthesis and purification) include, without limitation. HPLC, LCMS, quantitative thin layer chromatography and others known to those of skill in the art. Stability can be measured using assays that expose binding proteins to elevated temperatures (e.g.. 37 °C, e.g.. 95 °C, etc.) and / or chemical denaturants (e.g., urea, and guanidine hydrochloride) and then observe whether the protein refolds into its pre-exposure structure / conformation and / or regains binding activity to a given target molecule. Degradation can be evaluated using techniques such as reverse phase HPLC or gel electrophoresis to monitor resistance of a synthetic binding protein to degradation.B. Biochemical Characterization

[0272] The biological activity of the binding proteins can be determined via in vitro and in vivo assays (see, e.g., Examples 4, 5, and 6). Such assays can be used to determine whether a binding protein has agonistic (or antagonistic) properties. For example, the suitable assays can be performed to determine whether a synthetic GLP-1R binding protein disclosed herein can, e.g., partially or completely inhibit downstream GLP-1R- mediated signaling.

[0273] Various assays may be used to evaluate efficacy of synthetic GLP-1R binding proteins to determine their ability to agonize GLP-lR-mediated signaling. Certain assays can measure the agonism of GLP-1R signaling by its natural ligands (e.g., GLP-1). In the assay, a synthetic GLP-1 R binding protein binds to GLP-1 R, agonizing its downstream signaling activity (e.g., cAMP activity).

[0274] Assays conducted with cells, such as mouse (e.g., L929), hamster (e.g., BHK), or human (e.g, HEK293, e.g, islet cells, e.g., primary islet cells such as from pancreatic islet cell isolation procedures) cells, may be used to evaluate and characterize synthetic GLP-1R binding proteins. For example, the cells may have a visualizable reporter that is detectable upon GLP-1 R-mediated signaling. In some embodiments, GLP-1R signaling is measured by contacting a population of cells with a GLP-1R ligand (e.g., GLP-1, e.g., a GLP-1R agonist, e.g., semaglutide) and measuring cellular activity (e.g., cAMP activity). Such a measurement may be compared to a measurement made after contacting a population of cells with a synthetic GLP-1 R binding protein as provided in accordance with the present disclosure. The amount of signaling activity can be induced or increased contacting the population of cells (before, concomitant with, or after exposure to a ligand) with a sy nthetic GLP-1 R binding protein (including, e.g, as compared to binding with a different GLP-1R agonist such as, e.g., GLP-1. e.g., a GLP-1R agonist, e.g.. a reference GLP-1R agonist, e.g., semaglutide).

[0275] Cellular signaling, e.g., through a GPCR such as GLP-1 R, can be measured using any number of commercial assays. For example, an assay may be used that can measure normalized cAMP accumulation via luciferase activity, wherein luciferase activity only occurs downstream of cAMP activity triggered by binding and signaling through GLP-1 R (see, e.g., GloSensor™ from Promega).

[0276] In some embodiments, a concentration of the synthetic GLP-1R binding protein of about 105- 10"12M (e.g, 10’5, 10’6, 10’7. 10’8, 10’9, IO-10, IO’11,1012) may be enough to cause cAMP activation (as measured, e.g., by detectable reporter) through binding of a synthetic GLP-1R binding protein to a GLP-1R receptor in accordance with the present disclosure.

[0277] Characterization assays may also be conducted in vivo. For example, synthetic GLP-1R binding proteins may be tested for efficacy in agonizing GLP-lR-mediated signaling by comparing downstream activity (e.g., in mice) treated with a control GLP-1R ligand / agonist e.g., GLP-1, e.g., semaglutide) as compared to downstream activity in mice treated with a GLP-1 R binding protein as provided in accordance with the present disclosure. In certain embodiments, synthetic GLP-1 R binding proteins have at least about the same ability7as a GLP-1R ligand / agonist (e.g, GLP-1, e.g., a GLP-1R agonist, e.g., a reference GLP-1R agonist, e.g., semaglutide) to agonize downstream activity, and, in some instances, synthetic GLP-1R binding proteins result in more sensitive and / or sustained signaling as compared to a control ligand / agonist (e.g., GLP-1, e.g., semaglutide).VI. GLP-1R-BINDING PROTEIN CONJUGATES

[0278] It is contemplated that the synthetic binding proteins may be engineered to modily certain desired properties (e.g., binding affinity, binding avidity7, or pharmacokinetic or pharmacodynamic properties). That is, a GLP-1R binding protein may also include a GLP- 1R binding protein conjugate. Production of such conjugates can be achieved by conjugating (e.g, chemical conjugation or via a fusion protein) a synthetic binding protein to an effector. For example, the synthetic binding protein can be conjugated to a second binding molecule, e.g., a second synthetic binding molecule, which can be the same or different from the first synthetic binding protein, or an antibody or antibody fragment) or a molecule that directly e.g., bovine serum albumin (BSA), murine serum albumin (MSA), or human serum albumin (HSA)) or indirectly (e.g, an engineered binding site for BSA, MSA. or HSA) enhances the PK or PD properties of the binding molecule. By way of non-limiting example, half-life can be extended by a variety7of approaches know n to those of skill in the art including use of polyethylene glycol (PEG), fusion proteins (e.g., Fc fusions, albumin fusions), engineered Fc binding such as engineered binding to neonatal Fc receptor (FcRn). antibody conjugation (e.g., to an antibody or fragment thereof, e.g., other protein engineering approaches that change the stability7and / or clearance of a protein from an organism. It is contemplated that certain half-life extension approaches such as PEGylation may reduce clearance and prolongcirculation. Fusions, such as to Fc or albumin, and / or addition of binding domains such as engineered HSA domains may prolong half-life due to circulatory half-life properties of the molecules (e.g., longer circulatory half-life via binding to albumin in the serum). For instance, a serum albumin binding domain may facilitate longer half-life via binding to albumin in circulating blood. One mechanism that extension could occur may include via recycling of a synthetic GLP-1R binding protein by binding to neonatal Fc receptor (FcRn), which could thereby extend the serum half-life of the synthetic GLP-1R binding protein.

[0279] By way of non-limiting example, a binding protein that binds to serum albumin (e.g, human serum albumin) has an amino acid sequence comprising SEQ ID NO: 163 (LEEAIEEAIERLREAGITSEYFFRLIEKGKTVEGVRALTEEILKS).

[0280] It is contemplated that a variety of effectors can be used to modify the properties of the synthetic GLP-1R binding protein disclosed herein. Furthermore, it is contemplated that the effector may be a cytotoxic molecule or detectable label (e.g., radiolabel or fluorescent tag) which can be used in a detection assay, e.g, a diagnostic assay.

[0281] It certain embodiments the effector can be chemically conjugated to the synthetic binding protein or can be incorporated into the synthetic binding protein as a fusion protein. The chemical conjugation can be accomplished by including a conjugation site into the synthetic binding protein, e.g., via inclusion of a derivatizable amino acid (e.g., a lysine or cysteine amino acid). The conjugation site can then be used to link the effector to the binding protein, either directly or indirectly (e.g, via a linker, such as homobifunctional or a heterobifunctional cross-linking agent). By way of non-limiting example, linkers may be or include hydrazone, PEG, bifunctional 4-(4-acetylphenoxy) butanoic acid moiety, maleimidocaproyl, maleimidomethyl cyclohexane- 1 -carboxylate, maleimidocaproyl group with a tetrapeptide portion consisting of the amino acid sequence, glycine-glycine- phenylalanine-glycine. and / or maleimidocaproyl group with PEG. Exemplary homo- and heterobifunctional cross-linking agents can also include, for example, EDC (l-ethyl-3-(3- dimethyl aminopropyl) carbodiimide hydrochloride), sulfo-NHS (N-hydroxysulfo succinimide), NHS (N-hydroxysuccinimide), dimethyl pimelimidate dihy drochloride, suberic acid bis(N-hydroxysuccinimide ester). DSG (disuccinimidyl glutarate). DSS (disuccinimidyl suberate), DSP (dithiobis(succinimidyl propionate)), sulfo-SMCC (sulfosuccinimidyl-4-(N- maleimidomethyl)cyclohexane-l -carboxylate), SPDP ((succinimidyl 3-(2- pyridyldithiojpropionate), N-succinimdyl oxy carbony lethyl methanethio sulfonate. 6-Maleimidohexanoic acid N-hydroxysuccinimide ester, 6-Maleimidocaproic acid sulfo-N- succinimidyl ester, maleimidoacetic acid N-hydroxysuccinimide ester, N-succinimidyl iodoacetate, and 4-(4-maleimidophynyl) butyric acid N-hydroxysuccinimide ester.(00282] Linkers may be used to link two or more synthetic GLP-1R binding proteins to one another, and may also be used to link one or more synthetic GLP-1 R binding proteins to one or more effectors (e.g., another synthetic binding protein, e.g., a synthetic albumin binding protein). A linker may be a peptide linker or a chemical linker. Linkers may be covalently bound (e.g.. to an amino acid) to a synthetic GLP-1R binding protein and covalently bound to a second agent (e.g. an effector, e.g., a second binding protein, e.g., a serum binding protein, e.g., a human serum albumin binding protein). The composition and / or length of the linker may be designed with a particular functionality' in mind, but preferably is non-immunogenic. A linker may contain one or more glycine amino acids and / or one or more serine amino acids. Exemplary linkers can comprise one or multiples of (Gly2Ser)n, (GlysSer)n, or (Gly4Ser)n, where n can be 1, 2, 3, etc. In some embodiments, a linker has an amino acid sequence comprising SEQ ID NO: 164 (GGDKTVERKGGDKTVERKGG).(00283] A synthetic GLP-1 R binding protein as provided herein can further comprise an effector. Serum half-life of the GLP-1 R binding protein provided herein can be extended, such as with an effector, e.g., an effector comprising a synthetic binding protein (e.g, a synthetic albumin binding protein, e.g.. that binds to human serum albumin).

[0284] It is contemplated that the synthetic GLP-1 R binding proteins disclosed herein can monovalent or multivalent. Multivalent proteins may include, but are not limited to bivalent and trivalent formats. A multivalent molecule may include two, three, four, or more monovalent synthetic GLP-1 R binding proteins or, for example, one synthetic GLP-1 R binding protein and another binding protein that binds another target (e.g, serum albumin). In any of these formats, at least one linker can connect a C-terminal amino acid of a first monovalent binding protein to an N-terminal amino acid of a second monovalent synthetic binding protein, such that the first and the second monovalent synthetic GLP-1 R binding proteins are linked together. For example, in some embodiments, a multivalent (e.g.. bivalent) miniprotein may comprise two miniproteins, each independently between about 35 and 85 amino acids and associated (e.g., linked, conjugated) with one another. In some embodiments, multivalent molecules may be fused and / or combined to another molecule,such as an effector, e.g., a half-life extender (e.g., a site or a protein that binds to serum albumin to extend serum half-life, etc.) or a multivalent protein may comprise a miniprotein (e.g., a synthetic GLP-1R binding protein), and a miniprotein engineered to bind to serum albumin (e.g., to extend serum half-life). In some such embodiments, a half-life extension molecule may be fused or conjugated to either a C-terminal amino acid or an N-terminal amino acid (or to both the N- and C-terminal amino acids) of a synthetic GLP-1R binding protein disclosed herein.

[0285] In certain embodiments, a total length, from N-to-C-terminus of a synthetic GLP- IR binding protein of the disclosure comprising an effector can be greater than 100 amino acids. For example, in some embodiments, the disclosure contemplates that a synthetic GLP- 1R binding protein comprising an effector can be between about 35 to 85 amino acids in length (e.g., 35, 40, 45, 50, 51. 52. 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67. 68. 69. 70, 71, 72, 73, 74, or 75. 80, 85, etc.) and that a synthetic albumin binding protein can be between about 35 to 85 amino acids in length (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, etc.). Such a binding protein can further comprise a linker (e.g., an amino acid linker). Such a linker can have, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. 17. 18. 19, 20, 21, 22, 23, 24, or 25 amino acids. In certain embodiments, a linker has or comprises about 20 amino acids (e.g., as in SEQ ID NO: 164). By way of non-limiting example, a multivalent binding protein, including, a synthetic GLP- 1R binding protein further comprising a linker (e.g., that binds to albumin, e.g., that extends half-life, e.g.. as in SEQ ID NO: 163) can have a total of over 100 amino acids in length (e.g, 105, 110. 120, 130, 140. 150, e.g, 130, 131. 132, 133, 134. 135, 136. 137, 138. 139. 140). In certain embodiments, a synthetic GLP-1R binding protein conjugate comprises about 137 amino acids (e.g., as in SEQ ID NO: 155).

[0286] For example, serum half-life of a synthetic GLP-1R binding protein provided herein can be extended with an effector comprising a human serum albumin binding protein. A synthetic albumin binding protein can be engineered, such as provided herein (e.g, with an amino acid sequence comprising SEQ ID NO: 163). Synthetic albumin binding proteins can bind to albumin from more than one species (e.g., human, mouse, canine, cynomolgus, bovine, etc.). Synthetic albumin binding proteins can be developed and produced using methods such as disclosed herein (e.g, de novo protein design, affinity maturation, rational selection / mutation. etc.).

[0287] In certain embodiments, conjugates comprising a GLP-1R binding protein and an effector that can extend serum half-life (e.g., serum half-life, e.g.. via an albumin binding protein) can increase half-life substantially. For example, native GLP-1 exhibits a short serum half-life ranging from approximately 1.5 to 5 minutes. See, e.g., Lau et cd. (2015) J. MED. CHEM. 58: 7370-7380; see also Lau et al. (2022) EXPERT REVIEW OF CLINICAL PHARMACOLOGY 15(3): 251-268. In certain embodiments, incorporation of an effector, such as an albumin binding protein (e.g, with an amino acid sequence comprising SEQ ID NO: 163) can extend half-life of a synthetic GLP-1R binding protein. For example, in some embodiments, serum half-life can be extended by at least 15 minutes, 30 minutes, 60 minutes, 1 hour, 2 hours. 3 hours, 4 hours, 5 hours, or 6 hours post-administration (e.g., postinjection. e.g., to a subject, e.g, to a subject in need thereof). In some embodiments, the serum half-life extension is relative to a native GLP-1 ligand or existing GLP-1R agonist (e.g., semaglutide). In some embodiments, the half-life extension is relative to the same or similar synthetic GLP-1R binding protein without an effector (e.g., that extends serum half- life). In certain embodiments, the serum half-life of a GLP-1R binding protein comprising an effector that extends half-life (e.g, an effector that is or comprises an albumin binding protein, e.g., that binds to serum albumin such as human serum albumin) is extended relative to the same or similar synthetic GLP-1 R binding protein without the effector (e.g., without being linked to a synthetic binding protein that binds to albumin).

[0288] By way of non-limiting example, a sy nthetic GLP-1 R-albumin binding protein conjugate can have an amino acid sequence as set forth in SEQ ID NO: 155 (HGEGTFTSDVSSYLERLAIRLFITILQLRADGITDFSLDLDDLTGELTVTDNNTGETFT VQLRTGEFSEDGGDKTVERKGGDKTVERKGGLEEAIEEAIERLREAGITSEYFFRLIE KGKTVEGVRALTEEILKSGS), which comprises a synthetic albumin binding protein of SEQ ID NO: 163 and an amino acid linker of SEQ ID NO: 164.

[0289] It is contemplated that a synthetic GLP-1 R binding protein comprising an effector that binds to albumin (e.g., serum albumin, e.g., human serum albumin) has a longer half-life than the half-life of a GLP-1R binding protein without the effector (e.g., same or similar synthetic GLP-1 R binding protein, e.g., without an effector that extends half-life such as by binding to serum albumin). In certain embodiments, the synthetic GLP-1R binding protein without the effector has an amino acid sequence that is or comprises a sequence set forth in any one of SEQ ID NOs: 2-105 and 108-159. In some embodiments, the GLP-1R bindingprotein comprising the effector has a longer half-life than as compared to a reference GLP- 1R agonist (e.g., semaglutide). In some embodiments, the reference GLP-1R agonist has an amino acid sequence comprising SEQ ID NO: 106.

[0290] In certain embodiments, the disclosure provides a multivalent (e.g., bivalent) protein comprising multiple synthetic GLP-1R binding proteins disclosed herein. The multivalent protein can comprise a first synthetic GLP-1R binding protein and a second synthetic GLP-1R binding protein linked together through at least one linker. A linker (e.g., a glycine and serine containing linker (e.g., GGS), can connect a C-terminal amino acid of the first synthetic GLP-1R binding protein to an N-terminal amino acid of the second synthetic GLP-1R binding protein. Depending upon the circumstances, the multivalent binding protein can have a binding affinity stronger than the binding affinity of each synthetic GLP-1R binding protein alone. In certain embodiments, the multivalent protein comprises a synthetic GLP-1R binding protein having an amino acid sequence, wherein the amino acid sequence comprises an amino acid sequence set forth in TABLE 8.VII. PHARMACEUTICAL COMPOSITIONS

[0291] Once produced, a synthetic GLP-1R binding protein disclosed herein can be formulated into a pharmaceutical composition.

[0292] For therapeutic use, a synthetic GLP-1R binding protein disclosed herein is combined with a pharmaceutically acceptable carrier. Various carriers (e.g., diluents, excipients, etc.) used in formulating and preparing pharmaceutical compositions are known and / or readily accessible to those of skill in the art. Depending upon the circumstances, a carrier can include a liquid (e.g. a sterile liquid) or a solid. A carrier may be selected from or comprise water, aqueous solvents, non-aqueous solvents, dispersion media, surfactants, antioxidants, buffers, adjuvants, tonicity agents, stabilizers, bulking agents, lyoprotectants, metal ions, chelating agents, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art. Typically a carrier is approved by United States Food and Drug Administration and meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or other International Pharmacopoeia. Suitable formulations for use in the present disclosure are found in see e.g.. Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rded. 2020). For a brief review of methods for drug delivery, see, e.g.,Langer (1990) SCIENCE 249: 1527-1533. The resulting pharmaceutical compositions are suitable for administration to a subject (e.g, an animal, e.g. a mammal, e.g., a human).

[0293] A pharmaceutical composition may contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stabi 1 i ty, rate of dissolution or release, adsorption, or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogensulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, polyethylene glycol (PEG), sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants (see e.g., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rded. 2020)).

[0294] In certain embodiments, a pharmaceutical composition may contain a sustained- or controlled-dehvery formulation. Techniques for formulating sustained- or controlled- delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. Sustained-release preparations may include, e.g., porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices may includepolyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L- glutamate, poly (2-hydroxyethyl-inethacrylate), ethylene vinyl acetate, or poly-D(-)-3- hydroxybutyric acid. Sustained release compositions may also include liposomes that can be prepared by any of several methods know n in the art.

[0295] Depending upon the circumstances, a pharmaceutical composition may contain nanoparticles, or lipid droplets, e.g., polymeric nanoparticles, liposomes, or micelles (see Anselmo et al. (2016) BIOENG. TRANSL. MED. 1: 10-29).

[0296] Pharmaceutical compositions containing a synthetic GLP-1R binding protein can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intraperitoneal, intradermal, inhalation, transdermal, topical, transmucosal, intrathecal and rectal administration. In certain embodiments, the synthetic peptide is administered by subcutaneous administration.

[0297] Useful formulations can be prepared by methods known in the pharmaceutical art. For example, see e.g., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rded. 2020). Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzy l alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity' such as sodium chloride or dextrose.

[0298] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, polyethoxylated castor oil or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0299] Pharmaceutical formulations preferably are sterile. Formulations can be sterilized, for example, by methods appropriate to retain activity' and stability of the synthetic GLP-1R binding protein included therein. Sterilization can be accomplished by any suitable method,e.g., filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.

[0300] Depending upon the drug substance and formulation, the resulting dosage forms can be stable for extended periods of time, such as 1 month, 3 months, 6 months, 1 year, 2 years, 3 years, or more, when the dosage form is a liquid or solid. The formulations can be stable at room temperature or higher. It is contemplated that the dosage form is stable at ambient conditions in PBS. Alternatively the dosage form is frozen (e.g., a liquid or a lyophilizate) and stable under appropriate temperatures such as, e.g, -20°C, -80°C).

[0301] Depending upon the circumstances, the dosage forms can be formulated as a unit dose, which can include, for example, about 0.25 mg. 0.5 mg. 0.75 mg. 1 mg, 1.25 mg. 1.5 mg, 1.75 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 25 mg, 50 mg, 100 mg, 250 mg, 500 mg, 1 g, 1.5 g, 2.5 g, 5 g, or 10 g of the drug substance.

[0302] The compositions described herein may be administered locally or systemically. It is contemplated that the compositions described herein are generally administered by parenteral administration. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. In certain embodiments, the pharmaceutical composition is administered subcutaneously or may be administered intravenously, e.g., via intravenous infusion. In certain embodiments, it is contemplated that the synthetic constructs disclosed herein can be administered by systemic administration.

[0303] Generally, a therapeutically effective amount of active component, for example, a synthetic GLP-1R binding protein disclosed herein, is in the range of 0.1 mg / kg to 1000 mg / kg. e.g., 1 mg / kg to 100 mg / kg, e.g.. 10 mg / kg to 500 mg / kg, e.g., 500 mg / kg to 1000 mg / kg. In certain embodiments, the effective amount is in the range of 15 to 50 mg / kg. In certain embodiments, the effective amount is 15 mg / kg. In certain embodiments, the effective amount is 30 mg / kg. In certain embodiments, the effective amount is 50 mg / kg. A dose may also be a flat dose, for example. 0.25 mg to 25 mg, and depending on context (e.g.. administration route such as subcutaneous vs oral), dose can change. In certain embodiments (e.g., subcutaneous administration) a dose may be about 0.25 mg to about 2.5 mg (e.g., 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.25 mg, 1.5 mg, 2 mg, or 2.5 mg. In other embodiments, (e.g, oral administration) a dose may be about 5 mg to about 25 mg (e.g., 5 mg, 7.5 mg, 10 mg, 12.5 mg. 15 mg, 20 mg, or 25 mg). The amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the patient,the in vivo potency of the active component, the pharmaceutical formulation, and the route of administration. The initial dosage can be increased beyond the upper level in order to rapidly achieve the desired blood-level or tissue-level. Alternatively, the initial dosage can be smaller than the optimum, and the daily dosage may be progressively increased during the course of treatment. Human dosage can be optimized, e.g, in a conventional Phase I dose escalation study. Dosing frequency can vary, depending on factors such as route of administration, dosage amount, serum half-life of the synthetic peptide, and the disease, disorder, or condition being treated. Exemplary dosing frequencies are once per day, once per week and once every' two weeks.

[0304] Among other things, the disclosure provides a pharmaceutical composition comprising a synthetic GLP-1R binding protein as provided herein and a pharmaceutically acceptable carrier. It is contemplated that a synthetic GLP-1R binding protein further comprises an effector. In certain embodiments, the effector is or comprises a protein that extends serum half-life (e.g., of the GLP-1R binding protein). In some embodiments, the effector is or comprises a synthetic serum albumin binding protein (e.g., as set forth in SEQ ID NO: 163). The effector (e.g, synthetic serum albumin binding protein, e.g., as set forth in SEQ ID NO: 163) can be linked to the synthetic GLP-1R binding protein through a linker (e.g, an amino acid linker, e.g.. having an amino acid sequence comprising SEQ ID NO: 164). In certain embodiments, an exemplary synthetic GLP-1R binding protein comprising an effector that binds to serum albumin has an amino acid sequence comprising SEQ ID NO: 155.

[0305] It is contemplated that a pharmaceutical composition comprising a GLP-1R binding protein (e.g.. as provided herein, e.g., as set forth in any one of SEQ ID NOs: 1-105 and 108-151, e.g.. including GLP-1R binding proteins and domains thereof) comprising an effector that extends serum half-life (e.g., a GLP-1R binding protein conjugate) have a longer half-life as compared to the GLP-1R binding protein without the effector and / or as compared to a reference GLP-1R agonist (e.g., semaglutide, e.g., as set forth in SEQ ID NO: 106).VIII. METHODS OF USE AND TREATMENT

[0306] The GLP-1R binding proteins described herein can be used in a variety of different approaches.

[0307] For example, the binding proteins can be used in a method of targeting GLP-1R. The method comprises contacting a cell that expresses GLP-1R on its cell surface with a composition comprising the synthetic GLP-1R binding protein disclosed herein. In addition the GLP-1R binding proteins described herein can be used to modulate GLP-1R activity. The method comprises contacting a cell that expresses GLP-1R on its cell surface with a composition comprising the synthetic GLP-1R binding protein disclosed herein. In each method, the GLP-1 R binding protein or the pharmaceutical composition comprising a GLP- 1R binding protein further comprises an effector. In each of the foregoing methods, the synthetic GLP-1R binding protein or the pharmaceutical composition can agonize or increase GLP-1R activity at least equivalent or better to that of a GLP-1R ligand (e.g., GLP-1) relative to GLP-1 R activity in the presence of the GLP-1 R ligand or agonist (e.g., GLP-1, e.g.. a GLP-1R agonist, e.g., a reference GLP-1R agonist, e.g.. semaglutide, liraglutide, dulaglutide, lixisenatide, exenatide, etc.).

[0308] The GLP-1R binding proteins may be used in treatment of a disease, disorder, or condition mediated by GLP-1 R. GLP-1 R-signaling mediates various intracellular signaling activities downstream of GLP-1R binding a ligand (e.g., GLP-1), including those related to cell signaling via cyclic adenosine monophosphate (cAMP), protein kinase C delta (PKC5), and phosphoinositide 3-kinase (PI3K). See, e.g., Zhao et al. (2021), supra.; see also, Mayendraraj et al. (2022) supra; Sonoda et al., (2008) supra; and Fleming et al. (2017) CE L SIGNAL. 40: 1.

[0309] In certain embodiments, the disclosure provides a method of increasing GLP-1R- mediated activity in a subject in need thereof. The method comprises administering to the subject an effective amount of the pharmaceutical composition comprising a synthetic GLP- 1R binding protein (e.g., including a binding protein conjugate) disclosed herein. As such, the compositions described herein can be used in treating one or more endocrine and / or metabolic disorders (e.g., type 2 diabetes mellitus, obesity, pancreatic dysfunction including uncontrolled blood glucose levels), gastrointestinal disorders (e.g., gastric motility disorders, e.g., irritable bowel syndrome, e.g., irritable bowel disease), renal disorders (e.g, reduced kidney function), hepatic disorders (e.g., non-alcoholic steatohepatitis), neurodegenerative / neurological diseases (e.g., Alzheimer’s. Parkinson's) and / or cardiovascular disorders (e.g., heart attack, and stroke) in a subject in need thereof.

[0310] Exemplar^' diseases, disorders, or conditions that may be treated with the GLP-1R binding proteins disclosed herein include those such as type 2 diabetes mellitus, obesity, pancreatic dysfunction including uncontrolled blood glucose levels, gastric motility disorders including, e.g., irritable bowel syndrome and / or irritable bowel disease, renal disorders including reduced kidney function, hepatic disorders including non-alcoholic steatohepatitis and / or nonalcoholic fatty liver disease, cardiovascular disorders including heart attack and stroke, and neurodegenerative disorders including Alzheimer's disease and Parkinson’s Disease, and any other disease, disorder, or condition thought be mediated or impacted by the GLP-1R axis. For example, the synthetic GLP-1R binding proteins disclosed herein may be used to treat type 2 diabetes mellitus, obesity', or nonalcoholic fatty liver disease. In some embodiments, a disease that can be treated using a synthetic GLP-1R binding protein is selected from pancreatic dysfunction including uncontrolled blood glucose levels, gastric motility disorders including, e.g., irritable bowel syndrome and / or irritable bowel disease, renal disorders including reduced kidney function, hepatic disorders including non-alcoholic steatohepatitis and / or nonalcoholic fatty liver disease, cardiovascular disorders including heart attack and stroke, and neurodegenerative disorders including Alzheimer’s disease and Parkinson’s Disease.

[0311] In certain circumstances, the compositions described herein can be used to treat a subject diagnosed as having one or more endocrine and / or metabolic disorders (e.g., ty pe 2 diabetes mellitus, obesity', pancreatic dysfunction including uncontrolled blood glucose levels, gastrointestinal disorders (e.g., gastric motility disorders, e.g., irritable bowel syndrome, e.g.. irritable bowel disease), renal disorders (e.g., reduced kidney function), hepatic disorders (e.g., non-alcoholic steatohepatitis), neurodegenerative / neurological diseases (e.g., Alzheimer’s, Parkinson’s) and / or cardiovascular disorders (e.g., heart attack, and stroke).

[0312] Compositions of the present disclosure may be used to treat a subject in need of one or more of the following: (i) to reduce the risk of adverse cardiovascular events in presence of type II diabetes and cardiovascular disease; (ii) to improve HbAlc in presence of increased risk of cardiovascular events; (iii) to manage weight in those 12 and older with a body mass index (BMI) of 30 or greater; (iv) for combination with long acting insulin; and (v) for weight loss / weigh management.

[0313] It is contemplated that therapy can be accomplished using a synthetic GLP-1R binding protein alone, as a monotherapy, or as part of a combination therapy. The combination therapy may include one or more additional agents or therapeutic approaches know n to those of skill in the art for treating one or more endocrine and / or metabolic disorders (e.g., type 2 diabetes mellitus, obesity, pancreatic dysfunction including uncontrolled blood glucose levels), gastrointestinal disorders (e.g, gastric motility disorders, e.g., irritable bowel syndrome, e.g., irritable bowel disease), renal disorders (e.g.. reduced kidney function), hepatic disorders (e.g, non-alcoholic steatohepatitis), neurodegenerative / neurological diseases (e.g., Alzheimer’s, Parkinson’s) and / or cardiovascular disorders (e.g., heart attack, and stroke), and may have been previously used, be already ongoing, or added to a treatment for a subject in need thereof.

[0314] A subject may be evaluated, e.g, by a healthcare provider, before, during, and / or after treatment with a composition provided herein. Depending on the outcome of the evaluation, a treatment may be continued or ceased, treatment frequency or dosage may change, or the patient may be treated with a different synthetic GLP-1R binding protein. Subjects may be administered a composition comprising the synthetic GLP-1R binding protein for a discrete period of time according to dosage paradigms described herein, including, optionally, until the disease, disorder, or condition is treated.

[0315] Subjects that can be treated include those suspected as having, having, or at risk of having a GLP-1R dysfunction and / or a GLP-1R mediated disease, disorder, or condition. The methods described herein may include a step of selecting a treatment for a subject in need thereof. The method includes (a) identifying (e.g, diagnosing) the subject with GLP- 1R dysfunction and / or a GLP-1R mediated disease, disorder, or condition, and (b) selecting a synthetic GLP-1R binding protein as described herein, to treat the subject.

[0316] Synthetic GLP-1R binding proteins administered in an effective amount to a subject in need thereof may result in (a) reduced blood glucose levels, (b) reduced HbAlc levels, (c) reduced insulin dose or elimination of need for insulin (d) reduced w eight, (e) reduced risk or incidence of heart attack or stroke, (f) improved organ function (g) reduced pain, (h) improved neurological condition, (h) increased quality of life, (i) increased life span, and (j) any combinations of any of the above.

[0317] Certain GLP-1R dysfunction and / or a GLP-1R mediated disease, disorder, or conditions can be treated by modulating, e.g., increasing GLP-lR-mediated signaling. Forexample, GLP-1 can act by reducing blood glucose concentration by promoting synthesis and release of insulin. See. e.g, Smith et al. (2019) NEUROCHEM INT. 128: 94-105. In certain diseases, a level of meal-stimulated GLP-1 (a GLP-1R ligand) is decreased (see. e.g., Toft- Nielsen et al. (2001) JCEM 86(8): 3717-23). As is known to those of skill in the art, however, GLP-1 is considered to have a short half-life in vivo (e.g., ~ < 5 minutes; see Lau et al. (2015), supra), thus, extension and / or activation of GLP-1R is at least one potential benefit of synthetic GLP-1 R agonist therapeutics.

[0318] In one aspect, the disclosure provides a method of increasing serum half-life of a synthetic GLP-1R binding protein, comprising adding an effector that extends serum half-life to the C-terminus of a synthetic GLP-1R binding protein (e.g., that does not comprise an effector, e.g., that does not comprise a protein that binds to serum albumin). In certain embodiments, the effector binds to serum albumin. For example, in some embodiments, the effector is or compnses a synthetic albumin binding protein. By way of non-hmiting example, one such synthetic albumin binding protein has an amino acid sequence comprising or consisting of SEQ ID NO: 163. It is contemplated that a synthetic GLP-1R binding protein comprising an effector (e.g, that binds to albumin) has a longer half-life as compared to the half-life in the absence of the effector (e.g., a GLP-1 R binding protein without an effector) and / or a reference GLP-1R agonist (e.g., semaglutide, etc.). An exemplary reference GLP-1R agonist has an amino acid sequence comprising that set forth in SEQ ID NO: 106.

[0319] In one aspect, the disclosure provides a method of reducing blood glucose concentration in a subject in need thereof, comprising administering to the subject an effective amount of a synthetic GLP-1 R binding protein as provided herein, thereby to reduce blood glucose concentration relative to blood glucose concentration prior to administration. In certain embodiments, a GLP-1R binding protein has an amino acid sequence comprising any one of SEQ ID NOs: 108-154. In some embodiments, the GLP-1R binding protein has an amino acid sequence comprising SEQ ID NO: 120.

[0320] GLP-1 R-mediated signaling may be evaluated by detecting levels of one or more downstream molecules such as, for example, blood glucose levels, levels of cAMP, markers of cardiovascular health (e.g., C-reactive protein (CRP), an accepted and established biomarker of inflammation that is often elevated in people that are overweight or obese (see, e.g., Verma et al., (2023) ECLINICALMEDICINE 55: 101737; Lingvay et al., (2023) OBESITY31(1): 111-122; and Aronson et al., (2004) INT J OBES RELAT METAB DISCORD 28(5): 674-9 )). In certain embodiments, for example, CRP is expressed at > about 1 mg / dL in a subject having or being treated for obesity prior to treatment with a GLP- 1R agonist such as a synthetic GLP-1R agonist (e.g, a synthetic GLP-1R binding protein, e.g., that binds and catalyzes downstream signaling) as provided herein. During and / or after treatment with a GLP-1R agonist therapeutic (e.g., a synthetic GLP-1R binding protein, e.g., that binds and catalyzes signaling), a level of CRP may be lower (e.g.. equal or less than about 1 mg / dL).

[0321] The present disclosure provides methods of treating a subject in need thereof by administering an effective amount of the synthetic GLP-1R binding protein to the subject. The methods and compositions described herein can be used alone or in combination with other therapeutic agents and / or modalities. The phrase administered "in combination,” as used herein, is understood to mean that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, such that the effects of the treatments on the patient overlap at a point in time. In certain embodiments, the delivery7of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In certain embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what w ould be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0322] In certain embodiments, a method or composition described herein, is administered in combination with one or more additional therapies, e.g, surgery, radiation therapy, or administration of another therapeutic preparation. In certain embodiments, the additional therapy may include at least one other agent. Exemplary such agents include, for example, small molecules and biologies. Exemplary small molecules can include, e.g..metformin, sodium-glucose cotransporter-2 (SGLT2) inhibitors and DPP-4 inhibitors. Exemplary biologies can include, for example, insulin, apelin receptor agonists (see, e.g, Yamamoto et al. (2011) BIOCHIM BIOPHYS ACTA 1810(9): 853-862; Yue et al. (2011) ENDOCRINOLOGY 152(1): 59-68), myostatin antagonists (see, e.g., Latres et al. (2015) SKELET. MUSCLE 5:34), and molecules to counteract muscle loss associated with GLP- 1 / GLP-lR-related therapies such as receptor-based traps for activin TGFp family ligands (e.g., a heterodimeric Fc-fusion having ECDs from ActRIIB and Alk4), which can. among other things, inhibit signaling of activin class ligands such as ActA, ActB, GDF8, and GDF11 (see, e.g., Gos X etal. (2022) ISCIENCE 25(1): 103590). Exemplary other treatments include, but are not limited to, an agent for decreasing amount of blood glucose (e.g, produced by liver), an agent for decreasing amount of blood sugar absorbed by gastrointestinal tract (e.g., stomach and / or intestines), a reduced calorie diet, increased physical activity, and combinations thereof. In certain embodiments, the additional therapy may include a combination of therapeutics of different classes.

[0323] Without wishing to be bound by theory, the present disclosure contemplates that synthetic binding proteins provided herein can be used to bias signaling downstream of G- protein coupled receptors (GPCRs). For example, as shown in FIGs. 1A and IB. there are various signaling pathways downstream of GLP-1R, including cAMP and P-arrestin. Binding of a molecule such GLP-1 or a reference GLP-1R agonist (e.g., semaglutide, liraglutide, dulaglutide, lixisenatide, exenatide, etc.).

[0324] cAMP signaling through GLP-1 R is thought to be associated with responses such as e.g, glucose control and insulin secretion (see, e.g, Kuna et al. (2013) AM J PHYSIOL ENDOCRINOL METAB. 305(2): E161-70), whereas P-arrestin-2-mediated signaling is thought to be involved in receptor internalization, desensitization, and downregulation. See, e.g., Jones et al., (2018) NAT COMMUN. 9(1): 1602. It is contemplated that avoidance of GLP-1R desensitization and downregulation (such as via reduced P-arrestin recruitment) may be involved in efficacy of GLP-1R agonist therapeutics. See, e.g., Jones, B. (2021) BJP 179(4): 492-510. Interplay between p-arrestin and intemalization / recy cling machinery and processing is still not fully understood; however, evidence supports that less P-arrestin involvement might result in prolonged cAMP signaling and thus, improvement or enhancement of certain desired physiological responses. For example, in an experiment using double-knockout p-arrestin 1 / 2 HEK293 cells, receptor internalization was decreasedwhile GLP-1R agonist-induced cAMP (and PKA) signaling is prolonged. See, e.g., Bitsi, et al. (2023) SCIENCE ADVANCES 9(18): eadf7737. Accordingly, it is contemplated that biasing signaling to increase cAMP signaling over P-arrestin signaling (or, said differently, enhancing cAMP signaling while also repressing p-arrestin signaling) can, in some contexts, improve or enhance positive effects (e.g, blood sugar regulation, weight maintenance or weight loss, improved cardiovascular, hepatic, and / or renal outcomes etc.) of GLP-1R agonists, including synthetic GLP-1R binding proteins of the present disclosure, at lower concentrations / doses and / or with fewer side effects (e.g., less nausea) as compared to currently used GLP-1R agonist therapeutics.

[0325] Stabilization of the GLP-1R ectodomain may also be involved in biased signaling. It is contemplated that binding of a synthetic GLP-1R binding protein (such as those disclosed herein), can stabilize GLP-1R as compared to binding of, e.g., GLP-1 or a reference GLP-1 R agonist (e.g., semaglutide), which may, in some instances, change (e.g., bias, e.g, prolong) signaling towards one pathway (e.g., cAMP) over another (e.g., p- arrestin). Thus, in some aspects, the present disclosure provides methods of modulating activity of a GPCR, where the GPCR is capable of modulating (e.g., activating) one or more downstream pathways. In certain embodiments, the present disclosure provides methods of modulating activity of a GPCR such as GLP-1R, which receptor is capable of activating a first downstream signaling pathway in a cell and a second, different downstream signaling pathway in the cell, and wherein the GPCR comprises an agonist binding site such that when an agonist binds to the agonist binding site, the agonist activates the first and the second downstream signaling pathways in the cell. The method comprises: contacting the cell with a miniprotein comprising: (i) an agonist that binds to the agonist binding site of the GPCR, and (ii) a stabilization domain that interacts with a surface region of the GPCR adjacent to the agonist binding site, wherein the stabilization domain stabilizes the interaction of the agonist in the agonist binding site of the GPCR and causes the GPCR to activate the first downstream signaling pathway preferentially over the second downstream signaling pathway relative the same or a similar agonist without the stabilization domain. Signaling pathways can be selected from, among others, cAMP-mediated signaling pathways. P-arrestin signaling mediated pathways, PI3K-mediated signaling pathways, PKCS-mediated signaling pathways, etc. In certain embodiments, a first signaling pathway is a cAMP-mediated signaling pathway, and a second signaling pathway is a arrestin-mediated signalingpathway. A synthetic GLP-1R binding protein can be as provided by the present disclosure and a reference GLP-1R agonist may include, for example, liraglutide, dulaglutide, lixisenatide, or exenatide.

[0326] It has been discovered that, when a synthetic GLP-1R binding protein disclosed herein interacts with a surface region of a GLP-1 R, such as in a cell expressing a GLP-1 R, downstream signaling can be biased in favor of a cAMP signaling pathway over a P arrestin- mediated signaling pathway in the cell. Accordingly, the disclosure provides a method of biasing GLP-!R receptor activity in a subject in need thereof. The method comprises administering a synthetic GLP-1 R binding protein to the subject, wherein the GLP-1 R binding protein biases a cAMP signaling-mediated pathw ay preferentially over a -arrestin- mediated signaling pathw ay where each pathw ay is modulated by the GLP-1 R. As a result, administration of the GLP-1R binding protein preferentially activates the cAMP -mediated signaling pathway over a P-arrestin-mediated signaling pathway in the subject. It is contemplated that such an approach can reduce a side effect mediated by a GLP-1R activated P-arrestin-mediated signaling pathway such that the side effect is reduced relative to administration of a reference GLP-1 R agonist that lacks a protein stabilizing domain capable of stabilizing the interaction of the GLP-1R agonist in an agonist binding site of the GLP-1R. In some embodiments, the reference GLP-1R agonist comprises only a GLP-1R agonist portion that contacts all or part of a GLP-1 binding site on a GLP-1 R, whereas synthetic GLP-1 R binding proteins provided herein interacts or otherwise interfaces with a larger surface area of the GLP-1R. It is contemplated that, in some embodiments, these structural modifications change downstream activity of GLP-1R, which can bias signaling towards one pathway (e.g., a cAMP mediated signaling pathway) over another pathway (e.g., a P-arrestin mediated signaling pathway).IX. KITS

[0327] Synthetic GLP-1R binding proteins of the present disclosure may be included as part of a kit. A kit may comprise a container comprising or consisting essentially of a unit of a pharmaceutical composition comprising a synthetic GLP-1R binding protein, instructions for use, and optionally, one or more agents (e.g, a buffer or diluent, if appropriate, to dissolve the binding protein or dilute a solution containing the binding protein), and a dispenser. A kit may include a label indicating the intended use of the contents of the kit.The contents of the kit may be used for treating, monitoring, and / or diagnosing a subject in need thereof.

[0328] The present disclosure is further illustrated by the following examples which should not be construed as further limiting.EXAMPLESEXAMPLE 1: INITIAL SCREENING

[0329] This Example describes an initial in vitro screen of synthetic proteins using a functional readout of GLP-1R activation in cells as well as biophysical measurements of the interaction with the ectodomain of GLP-1R to identify synthetic proteins capable of binding and activating human GLP-1R. The synthetic proteins were designed in silico as depicted in FIG. ID and w ere screened for members having certain characteristics, such as binding to the target (GLP-1R) at particular concentrations of target (e.g., 10 pM, e.g., 1 pM, etc. ).Once identified, certain synthetic binding proteins were synthesized for use in downstream screening and discovery processes.

[0330] A set of synthetic binding proteins containing 96 unique proteins were individually tested for GLP-1R binding by surface plasmon resonance (SPR). Initially, miniproteins encoded by each of these sequences w ere expressed and purified from E. coli to use in SPR testing. For all proteins that showed specific binding to GLP-1R (as determined by measurable binding to the GLP-1R ectodomain protein and not to a streptavidin protein chip surface), binding affinities were determined. In parallel with binding affinity testing, each of the 96 proteins was individually tested for the ability to activate GLP-1 R in cells using a cAMP reporter cell line. (GloSensor™, which is engineered to detect cAMP activity) For all proteins that showed signaling activity EC50 values w ere determined.

[0331] Six proteins were further characterized using affinity' maturation, binding assays, and activity assays, and a reference miniprotein ("Reference Miniprotein 1”; SEQ ID NO: 1) as selected for further optimization.

[0332] Reference Miniprotein 1 was confirmed to bind to the GLP-1R ectodomain by SPR (kon 3.8 e7s^M’1; koff 7.81 s’1; KD 2.54e’7M) and folded and thermostable as shown by circular dichroism spectroscopy from about 25°C to about 95°C (FIG. 2). In addition to binding, Reference Miniprotein 1 show ed a concentration-dependent GLP-1R agonism as compared to a control (exendin-4) on a luciferase-based cyclic AMP assay designed to detectintracellular signaling through G-protein coupled receptors. As shown in FIG. 3, the line graph indicates levels of GLP-1R agonism of Reference Miniprotein 1 (SEQ ID NO: 1) as compared to a control peptide (exendin-4) over a concentration of about 10‘6- I O'" M as measured by normalized cAMP accumulation as determined by luciferase activity and intensity as measured using an exemplary fluorescent cyclic adenosine monophosphate (cAMP) assay (GloSensor™), which used cAMP levels as an indicator of GLP-1R activity, because cAMP activity is downstream of GLP-1R signaling.EXAMPLE 2: OPTIMIZATION OF GLP-1R MINIPROTEINS FOR GLP-1R ECTODOMAIN

[0333] This Example describes the optimization of GLP-1R miniproteins developed from Reference Miniprotein 1 for binding to and signaling through the GLP-1R ectodomain.

[0334] At the outset, a synthetic miniprotein library of greater than 1 billion variants of Reference Miniprotein 1 (SEQ ID NO. 1) was screened for binding to human GLP-1R ectodomain using standard yeast surface display techniques. Miniprotein variants that bound to the GLP-1R ectodomain were isolated from non-binding miniproteins through iterative rounds of magnetic and fluorescent selection, using standard labeling and selection techniques. Thousands of unique miniprotein sequences were identified. As a result, miniproteins of SEQ ID NOs: 2 - 28 were generated via affinity’ maturation of Reference Miniprotein 1, and then selected for further validation. After testing Reference Miniproteins 2-28 (SEQ ID NOs: 2-28), a rationally-selected set of mutations and combinations of mutations was generated to engineer synthetic binding proteins with improved binding, signaling, and / or stability relative to Reference Miniproteins 2-28. Rational selection included introducing variations or mutations known to those of skill in the art and / or combining with those engineered and discovered using Reference Miniproteins 2-28. Miniproteins encoded by each of these sequences were expressed and purified from E. coli. to increase thermal stability and resulted in 69 new proteins (some of which were made through further modifications such as truncations (see Example 5), and a subset of which were further characterized as described in Examples 3, 4 and 5).

[0335] Each of Reference Miniproteins 2-70 (SEQ ID NOs: 2-70) had the same secondary structural arrangement of loop amino acids, alpha helix amino acids, and beta sheet amino acids as its parent, Reference Miniprotein 1 (SEQ ID NO: 1). Miniproteins encoded by each of these sequences were expressed and purified from E. coli.

[0336] The binding characteristics of exemplary Reference Miniproteins (1-18, 20-25, 27- 28, 32-39, 46, and 60-66; corresponding to SEQ ID NOs: 1-18, 20-25, 27-28, 32-39, 46, and 60-66) to human GLP-1R ectodomain were measured and binding activities of each miniprotein were determined using SPR (see data in TABLE 3A).

[0337] Thermostability of the GLP-1R binding miniproteins was also analyzed. GLP-1R binding miniproteins were exposed to 4M urea at temperatures of at least 50°C. These proteins retained their properly folded three-dimensional structure as confirmed by circular dichroism spectroscopy. FIG. 4A (Reference Miniprotein 2, SEQ ID NO: 2) and FIG. 4B (Reference Miniprotein 11, SEQ ID NO: 11) show exemplary CD spectra of exemplary GLP-1R binding proteins in 5°C intervals between 25°C - 95°C, demonstrating that GLP-1R binding proteins with improved affinity7for the GLP-1R ectodomain retain their folding (not all data points between 25°C - 95°C are shown in FIGS. 4A and 4B for clarity, but all were measured). In addition, each of these miniproteins also retained their properly folded three- dimensional structure after heating to 95°C (see Example 3) as confirmed by circular dichroism spectroscopy (but without urea exposure).

[0338] The amino acid sequences of Reference Miniproteins 2-70 share about 61.0%- 98.4% identity with one another, and about 72.2 - 90.8 % identity with that of Reference Miniprotein 1 (SEQ ID NO: 1).TABLE 3A. Binding Characteristics of Exemplary Reference Miniproteins

[0339] Reference Miniprotein 2 (SEQ ID NO: 2) was also used to develop a consensus sequence of SEQ ID NO: 77, to assay which subset of proteins was most similar in sequence to Reference Miniprotein 2 (SEQ ID NO: 2) and also displayed binding activity to the GLP- 1R ectodomain. Reference Miniproteins 2, 7-9, 29, 32, 40, 46-54, and 60 (SEQ ID NOS: 2, 7-9, 29, 32, 40. 46-54, and 60) share about 93.8 % - 98.4 % identity with one another and about 96.8% - 98.4 % identity with that of Reference Miniprotein 2 (SEQ ID NO: 2) and about 93.8% - 95.2% identity with that of Reference Miniprotein 1 (SEQ ID NO: 1).

[0340] Measurements for binding characteristics of proteins not shown in TABLE 3A are shown in TABLE 3B (Reference Miniproteins 40, and 47-54; corresponding to SEQ ID NOS: 40, and 47-54, respectively).TABLE 3B. Binding Characteristics Of Exemplary Reference MiniproteinsEXAMPLE 3: VARIANT MINIPROTEIN STABILITY

[0341] This Example describes in vitro chemical stability of various Reference Miniproteins 2-70 (SEQ ID NOs: 2-70) using biophysical characterization after exposure to exemplary chemical denaturants.

[0342] Each of these GLP-1R miniproteins developed and characterized in Example 2 were exposed to 4M urea. Measurements of whether these proteins had properly folded three-dimensional structure after heating to 95°C and then returning to 25°C as measured by circular dichroism (CD) spectroscopy were determined. Examples are shown in Example 2, FIGS. 4A and 4B, demonstrating exemplar}' Reference Miniproteins 2 (SEQ ID NO: 2) and 11 (SEQ ID NO: 11) were thermally-stable from 25°C - 95°C. In addition, of the miniproteins tested, Reference Miniproteins 1-13. 16-22, 24-35, 37, 39. 42. 46-54, and 60-70 (corresponding to SEQ ID NOs: 1-13, 16-22, 24-35, 37, 39, 42, 46-54, and 60-70, respectively) were thermally stable, retaining their conformations across a variety of conditions and maintaining binding across a range of temperatures (as shown by proper refolding after heating to 95°C and then cooling to 25°C). Reference Miniproteins 1-13, 16- 22, 24-35, 37-70 (SEQ ID NOs: 1-13, 16-22, 24-35, and 37-70, respectively) all showed chemical stability in 4M urea because after exposure to urea and subsequent dilution into PBS, the Reference Miniproteins each folded into their expected, respective, molecular structures (as prior to urea exposure) at 25°C as measured by CD spectroscopy.EXAMPLE 4: VALIDATION OF SIGNALING ACTIVITY OF HUMAN GLP-1R MINIPROTEINS

[0343] This Example describes assays for signaling activity by human GLP-1R miniproteins.

[0344] In addition to Reference Miniprotein 1 (SEQ ID NO: 1), all other synthesized GLP-1R binding Reference Miniproteins 2-70 (SEQ ID NOs: 2-70) were tested for cAMP signaling activity’ based on GLP-1R agonism using a binary' signaling assay (GloSensor™ assay, cAMP-Glo™; Promega, using a reduced number of protein concentrations rather than a full titration series as set forth in the manufacturer’s instructions) (see TABLE 4).

[0345] Signaling activity was measured using the GloSensor™ assay (cAMP-Glo™; Promega) according to the manufacturer’s instructions.

[0346] Of the 70 tested Reference Miniproteins (SEQ ID NOs: 2-70), a subset of Reference Miniproteins (Reference Miniproteins 1-7, 9-11. 18-22, 24-27. 30-47, 51, and 53; SEQ ID NOs: 1-7, 9-1 1, 18-22, 24-27, 30-47, 51, and 53) showed that in addition to binding to GLP-1R ectodomain with stronger affinity’ than Reference Miniprotein 1 (SEQ ID NO: 1) as shown in Example 2, they also were able to agonize and signal (as shown by cAMP signaling measured using the GloSensor™ assay) through GLP-1R.

[0347] The proteins that signaled were characterized as Series 2 (Series 2.0, 2.1, and 2.2) miniproteins. Exemplary- Series 2 Reference Miniproteins were compared to a reference GLP-1 agonist, semaglutide (purchased from Peptide Sciences; Molecular Formula C187H291N45O59; PubChem CID: 56843331; CAS Number 910463-68-2; SEQ ID NO: 106). As shown in FIG. 5 the line graph indicates levels of GLP-1R agonism of Reference Miniprotein 2 (SEQ ID NO: 2) and Reference Miniprotein 11 (SEQ ID NO: 11) as compared to a control reference product (semaglutide. SEQ ID NO: 106) and to the parental Reference Miniprotein 1 (SEQ ID NO: 1) over a concentration of about as measured by normalized cAMP accumulation in luciferase / cAMP assay that could measure downstream signaling from GLP-1R. The data demonstrate that the exemplary- Reference Miniproteins w ere able to signal in a concentration-dependent manner, and with greater potency- than Reference Miniprotein 1 (SEQ ID NO: 1) as determined by the GloSensor™ assay.

[0348] The 39 Series 2 miniproteins (Reference Miniproteins 1-7. 9-11. 18-22, 24-27, 30- 47, 51, and 53; corresponding to SEQ ID NOs: 1-7, 9-11, 18-22, 24-27, 30-47, 51, and 53) were similar to one another. The amino acid sequences of Reference Miniproteins 1-7, 9-11, 18-22, 24-27, 30-47, 51, and 53 shared about 66.2%-98.4% identity with one another and approximately about 96.8%-98.4% identity with that of Reference Miniprotein 2 (SEQ ID NO: 2).

[0349] The amino acid sequences of Series 2 proteins corresponding to each consensus sequence (SEQ ID NOs: 78, 79, and 80) were also highly similar w-ithin the entire Series and within each subgroup of Series 2 proteins corresponding to a Series 1.0 - 1.6 consensus sequence (SEQ ID NOs: 71-77).

[0350] Reference Miniprotein 2 (SEQ ID NO: 2) w as also used to develop a consensus sequence of SEQ ID NO: 80 to assay which subset of proteins was most similar in sequence to Reference Mimprotein 2 (SEQ ID NO: 2) and also displayed signaling activity greater than that of SEQ ID NO: 1. Reference Miniproteins 2, 7, 9, 32, 40, 46, 47, 51 and 53 having SEQ ID NOS: 2, 7, 9, 32, 40, 46, 47, 51 and 53 share about 93.8%-98.4% identity7with one another and about 96.8-98.4% identity w ith that of Reference Miniprotein 2 (SEQ ID NO: 2) and about 86.46%-89.0% identity with that of Reference Miniprotein 1 (SEQ ID NO: 1).EXAMPLE 5: MODIFICATIONS AND INTERROGATIONS OF GLP-1RMINIPROTEINS

[0351] This Example describes the design, production, and binding characterization of: (i) fixed N-terminus binding proteins and (ii) truncated GLP-1R binding proteins.Fixed N-Terminus GLP-1R Binding, Proteins

[0352] Series 3.0 (consensus sequence of SEQ ID NO: 81) and Series 3.1 (consensus sequence of SEQ ID NO: 82) Reference Miniproteins were developed by using a fixed N- terminal segment. The first twelve amino acids of Reference Miniproteins 1-7, 10, 1 1, 18- 22, 24, 27, and 30-45 (SEQ ID NOs: 1-7, 10, 11, 18-22, 24, 27, and 30-45) were fixed to comprise the sequence of HAEGTFTSDVSS (SEQ ID NO: 107), which aligns with the sequence of human GLP-1 bound by GLP-1R.

[0353] Consensus sequences of SEQ ID NOs: 80 (Series 3.0) and 81 (Series 3.1) each included a sequence in which amino acid positions 1-12 were fixed (positions 1-12 are with reference to a 62 amino acid miniprotein having a formula D1-L1-D2-L2-D3-L3-D4, wherein DI is an alpha helix, D2-D4 are beta sheets, and L1-L3 are loops). Reference Miniproteins 2, 5-7, 11, 32, 37, 40, and 42 having SEQ ID NOs: 2, 5-7, 11, 32, 37, 40, and 42, respectively share about 86.4%-98.4% identity’ with one another and about 89.2% - 96.8% identity with that of Reference Miniprotein 2 (SEQ ID NO: 2) and about 85.1% - 89.2% % identity with that of Reference Miniprotein 1 (SEQ ID NO: 1) and show signaling activity greater than that of SEQ ID NO: 1.

[0354] Reference Miniprotein 2 (SEQ ID NO: 2) was also used to develop a consensus sequence of SEQ ID NO: 83 (Series 3.2) to assay which subset of proteins was most similar in sequence to Reference Miniprotein 2 and displayed signaling activity’ greater than that of SEQ ID NO: 1. Reference Miniproteins 2, 7, 32, and 40, having SEQ ID NOS: 2, 7, 32, and 40, respectively, share about 93.8% - 96.8% identify with one another and about 96.8% - 96.8% identify with that of Reference Miniprotein 2 (SEQ ID NO: 2) and about 86.4% - 89.2 % identify with that of Reference Miniprotein 1 (SEQ ID NO: 1).Truncated GLP-1 R Binding Proteins

[0355] Exemplar^’ Reference Miniprotein amino acid sequences were systematically truncated at the N-terminus beginning with the removal of amino acid 1 (Reference Miniprotein 60; SEQ ID NO: 60), then removing amino acids 1 and 2 (ReferenceMiniproteins 61 and 62; SEQ ID NOs: 61 and 62); 1, 2, and 3 (Reference Miniproteins 63 and 64; SEQ ID NOs: 63 and 64); 1. 2, 3, and 4 (Reference Miniproteins 65 and 66; SEQ ID NOs: 65 and 66); 1, 2, 3, 4, and 5 (Reference Miniproteins 67 and 68; SEQ ID NOs: 67 and 68); and 1, 2, 3, 4, 5, and 6 (SEQ ID NOs: 69 and 70).

[0356] Miniproteins with these truncations did not signal, thus, no signaling data are available. Binding characteristics were determined including binding affinities (KD) for the human GLP-1R ectodomain using SPR (TABLE 4). These data indicate that N-terminal truncations can affect functional signaling without affecting receptor binding (to the human GLP-1R ectodomain) of synthetic GLP-1R miniproteins. That is, the synthetic GLP-1R miniproteins can still bind, but, without the N-terminal portions set forth herein, signaling downstream of GLP-1R does not occur.TABLE 4. Binding Characteristics To GLP-1REXAMPLE 6: BIASED SIGNALING USING SYNTHETIC GLP-1R BINDINGPROTEINS

[0357] This Example describes evaluation of exemplary synthetic GLP-1R binding proteins and signaling bias of cAMP-mediated signaling over P-arrestin-mediated signaling in vitro.

[0358] In this example, Reference Miniprotein 46 (SEQ ID NO: 46) was compared to a reference GLP-1R agonist (semaglutide, SEQ ID NO: 106) on a cell-based assay that monitors downstream signaling via protein: protein interactions (NanoBit™; Promega). To perform the assay, an engineered HEK293 cell line was constructed. The cell line stably overexpresses GLP-1R that has been fused on its C-terminus to a small “Bit” containing one half of the split luciferase system that produces signaling in the NanoBit assay (see. e.g, Promega catalog, N2014-2016; see also Reyes-Acaraz et al. (2022) COMMUNICATIONS BIOLOGY 5:212). Cells were transiently transfected with a fusion construct containing |3- arrestin 2 linked to large “Bit” containing the other half of split luciferase system. Reference Miniprotein 46 or semaglutide were added to cells and luminescence readout was measured. Each assay was conducted in triplicate. Luminescence was normalized and plotted against protein concentrations (10'4-1012M) and EC50s were calculated. cAMP signaling was measured using GloSensor™ as previously described (see Example 4), using the same plate on the same day. That is, both -arrestin 2 and cAMP signaling levels were measured in the same cells, but at different times, using different assays.

[0359] As shown in FIG. 6, the delta between the EC50 for semaglutide cAMP signaling and semaglutide p-arrestin signaling (as represented by normalized luminescence levels) was about 1-log difference. In contrast to the difference in signaling pathway activation / activity for semaglutide, Reference Miniprotein 46 showed about a 3-log difference in the EC50 for cAMP-mediated vs. P-arrestin-mediated signaling, supporting that synthetic GLP-1R binding proteins (which each contain stabilization domains) demonstrate signaling bias for cAMP signaling over -arrestin signaling. Furthermore, using modeled structures, SASA of GLP-1R miniproteins of the present disclosure showed binding with -700 A2more interface area than that of a reference GLP-1R agonist (e.g., semaglutide).EXAMPLE 7: C-TERMINAL EXTENSION OF SYNTHETIC GLP-1R BINDING PROTEINS

[0360] This Example describes the design, production, and binding characterization of exemplary synthetic GLP-1R binding proteins that were modified to add a C-terminal extension including a loop and p sheet.

[0361] Reference Miniprotein 46 (SEQ ID NO: 46) was used as a starting point and modified by computational protein design to attach a loop and fourth P-sheet having an amino acid sequence of SEQ ID NO: 165 to the C-terminal side of the C-terminal amino acid at position 62. This resulted in Reference Miniprotein 109 (SEQ ID NO: 109, HGEGTFTSDVSSYLERLAIRSFITILQLRADGITDFSLDLDDLTGELTVTDNNTGETFT VQLRTGEFSED), which can be represented according to Formula IB, D1-L1-D2-L2-D3- L3-D4-L4-D5, where DI is an alpha helix, D2-D5 are beta sheets and L1-L5 are loops. This miniprotein showed improved signaling activity as measured by activation of GLP-1R in the GloSensor™ assay (as described in Example 4). Reference Miniprotein 109 has an EC50 of 1.89 nM (also shown in TABLE 6, below), which is a 2-fold improvement over SEQ ID NO: 46.EXAMPLE 8: OPTIMIZATION OF GLP-1R MINIPROTEINS WITH C-TERMINALEXTENSIONS

[0362] This Example describes optimization of the GLP-1R binding miniproteins developed from Reference Miniprotein 109. Each of these GLP-1R binding proteins has a C- terminal extension relative to Reference Miniproteins 1-69 (SEQ ID NOs: 1-69).

[0363] In this Example, Reference Miniprotein 109 (SEQ ID NO: 109) was used as a starting point for further optimization. To begin, mutations in the amino acid sequence were rationally selected and introduced to improve binding affinity for GLP-1R and to improve efficacy as a GLP-1R agonist with the improvement characterized as relative to Reference Miniprotein 109 (SEQ ID NO: 109). Reference Miniproteins 110-132 (SEQ ID NOs: 110- 132) were generated and characterized for binding and signaling. Binding affinity to the human GLP-1R ectodomain was measured by SPR see TABLE 5A. KD (M)), and ka and kd were also determined. EC50 was determined by measuring GLP-1R mediated signaling in the GloSensor™ assay for Reference Miniproteins 110-132 (SEQ ID NOs: 110-132;TABLE 5A)TABLE 5A. Binding Characteristics Of Exemplary GLP-1R Binding Proteins With C-Terminal Extensions

[0364] Reference Miniprotein 120 (SEQ ID NO: 120;HGEGTFTSDVSSYLERLAIRLFITILQLRADGITDFSLDLDDLTGELTVTDNNTGETFTVQLRTGEFSED) was then selected for further optimization by using its amino acid sequence as a starting point to generate a saturation mutagenesis library. The library was screened for binding to the human GLP-1R ectodomain using standard yeast surface display techniques. Miniprotein variants that bound to the GLP-1R ectodomain were isolated from non-binding miniproteins through iterative rounds of magnetic and fluorescent selection, using standard labeling and selection techniques. Thousands of unique miniprotein sequences were identified and Reference Miniproteins 133-154 (SEQ ID NOs: 133-154) were selected for further binding and activity characterization. Miniproteins encoded by each of these sequences were expressed and purified from E. coli. Binding affinity to the human GLP-1R ectodomain for Reference Miniproteins 133-154 was measured by surface plasmon resonance (see TABLE 5B, SPR, KD (M)), and ka and kd were also determined. EC50s of Reference Mini proteins 133-154 were determined by measuring GLP-lR-mediated signaling in the GloSensor™ assay as described in Example 4 (TABLE 5B, EC50).TABLE 5B. Binding Characteristics Of Exemplary GLP-1R Binding Proteins With C-Terminal Extensions

[0365] Overall, each of the Reference Miniproteins 109-155 (SEQ ID NOs: 109-105) had the same secondary structural arrangement of loop amino acids, alpha helix amino acids, and beta sheet amino acids as its parent, Reference Miniprotein 109 (SEQ ID NO: 109), according to Formula IB. Each of Reference Miniproteins 109-155 (SEQ ID NOs: 109-155) also showed chemical stability in 4M urea as determined by folding into their expected, respective, molecular structures (as prior to urea exposure) after exposure to urea and subsequent dilution into PBS, at 25°C as measured by CD spectroscopy (data not shown). Furthermore, thermal stability was measured for Reference Miniproteins 109 and 120 (SEQ ID NOs: 109 and 120) and each was thermally stable, retaining their conformations across a variety of conditions and maintaining binding across a range of temperatures (as shown by proper refolding after heating to 95°C and then cooling to 25°C) (data not shown ).EXAMPLE 9: IN VIVO BINDING AND SIGNALING ACTIVITY OF SYNTHETIC GLP-1R BINDING PROTEINS

[0366] This Example describes evaluation of exemplary synthetic GLP-1R binding proteins in vivo.

[0367] In this example, a mouse model of type II diabetes and obesity, db / db mice, was used (Coleman, D.L. (1978) DIABETOLOGIA 14(3): 141-8; db / db mice from Jackson Laboratory, Bar Harbor, ME (B6.BKS(D)-Leprdb / J; JAX stock # 00697)). db / db mice have a homozygous mutation. Leprdb, which is a spontaneous mutation that results in morbid obesity, chronic hyperglycemia, pancreatic beta cell atrophy, and hypoinsulinemia. db / db mice are often used to model phase I to III diabetes type II and obesity.

[0368] The efficacy of SEQ ID NO: 120 (Reference Miniprotein 120) on glucose tolerance was tested in db / db mice. Male db / db mice (10 to 12 weeks old; n=5 for each group) were selected and split into three groups: (i) Reference Miniprotein 120 (SEQ ID NO: 120); (ii) semaglutide (positive control; SEQ ID NO: 106); and (iii) vehicle (negative control).

[0369] Mice were fasted for 16 hours. Fifteen hours into the fasting period, Reference Miniprotein 120 (SEQ ID NO: 120) was administered subcutaneously to the first group at a dosage of 791.65 pg / kg body weight, semaglutide was administered to the second group at a dosage of 41 1.4 pg / kg body weight (see, e.g., Lau et al. (2015) J. MED. CHEM. 58(18):7370- 7380), and phosphate-buffered saline (PBS) vehicle was administered to the third group. After completion of the fasting period, glucose (2 g / kg body weight of 20% glucose solution) was administered via intraperitoneal injection. Blood glucose levels (mg / dL) were measured at baseline (0 minutes) and at 15, 30, 60, 90, and 120 minutes post-glucose administration using tail vein blood samples and a calibrated glucometer (FIG. 7). The results demonstrate that mice treated with Reference Miniprotein 120 (SEQ ID NO: 120) had significantly lower blood glucose levels at all measured time points compared to the vehicle control, with a marked reduction in the area under the glucose concentration-time curve (AUC). These results indicate improved glucose tolerance and enhancement of glucose metabolism in a model of Type II diabetes after administration of Reference Miniprotein 120 (SEQ ID NO: 120).EXAMPLE 10: BIASED SIGNALING USING SYNTHETIC GLP-1R BINDING PROTEINS DELAYS RECEPTOR INTERNALIZATION

[0370] In this Example, Reference Miniprotein 120 (SEQ ID NO: 120) was compared to a reference GLP-1R agonist (semaglutide, SEQ ID NO: 106) using a cell-based assay that monitors downstream signaling via protein: protein interactions (NanoBit™; Promega). To perform the assay, an engineered HEK293 cell line was constructed. The cell line stably overexpresses GLP-1R that has been fused, on its C-terminus, to a small “Bit” containing one half of the split luciferase system that produces signaling in the NanoBit assay (see, e.g., Promega Catalog, N2014-2016; see also Reyes-Acaraz et al. (2022) COMMUNICATIONS BIOLOGY 5:212). Cells were transiently transfected with a fusion construct containing the endosomal marker protein FYVE linked to a large “Bit” containing the other half of the split luciferase system. The activity of Reference Miniprotein 120 (SEQ ID NO: 120) andsemaglutide (SEQ ID NO: 106) was measured in three experiments: (i) cAMP signaling; (ii) P-arrestin signaling; and (iii) receptor internalization. Receptor internalization of GLP-1R is P-arrestin-signaling-mediated.

[0371] Reference Miniprotein 120 or semaglutide were added to cells and luminescence readout was measured. Each assay was conducted in triplicate. Luminescence was normalized and plotted against EC50 protein concentrations (10'4-10’12M). Measurements are summarized in TABLE 6. As shown in FIG. 8A (cAMP signaling), FIG. 8B (P-arrestin signaling), and FIG. 8C (receptor internalization), the difference between the EC50 for semaglutide cAMP signaling and semaglutide P-arrestin signaling (as represented bynormalized luminescence levels) is about 1-log, showing that semaglutide has cAMP signaling, P-arrestin signaling, and exhibits receptor internalization. In contrast to semaglutide, Reference Miniprotein 120 showed cAMP-mediated signaling, but P-arrestin- mediated signaling and receptor internalization were undetectable, indicating that synthetic GLP-1R binding proteins (e.g., Reference Miniprotein 120; SEQ ID NO: 120) have a signaling bias for cAMP over P-arrestin. Furthermore, using modeled structures, SASA of GLP-1R miniproteins of the present disclosure showed binding with -700 A2more interface area than that of a reference GLP-1R agonist (e.g., semaglutide).TABLE 6. Binding And Activity Characteristics Of GLP-1R Binding ProteinsEXAMPLE 11: MINIPROTEIN HALF-LIFE EXTENSION WITH AN ALBUMINBINDING DOMAIN

[0372] This Example describes extending the serum half-life of an exemplary synthetic GLP- 1R binding protein using an exemplary synthetic albumin binding protein.

[0373] A synthetic albumin binding miniprotein (SEQ ID NO: 163;LEEAIEEAIERLREAGITSEYFFRLIEKGKTVEGVRALTEEILKS) was developed. Briefly, the synthetic albumin-binding protein of SEQ ID NO: 163 was engineered through de novo protein design to specifically target human serum albumin (HSA). Utilizing the Protein Data Bank (PDB) structure 1TF0, the protein structure was computationally relaxed, and novel sequences were generated using the ProteinMPNN model. The new sequences were validated using Alphafold2. The resultant synthetic albumin-binding protein comprises three-helix bundle structure, optimized for stability and binding affinity to human serum albumin (HSA). The synthetic albumin-binding protein of SEQ ID NO: 163 also binds to serum albumin from mouse, canine, cynomolgus, and bovine as assessed by SPR.

[0374] The albumin binding miniprotein (SEQ ID NO: 163) has nanomolar binding affinity to albumin from several species (human, mouse, canine, cynomolgus, and bovine), as measured by surface plasmon resonance (SPR) analysis (TABLE 7).TABLE 7. Binding Characteristics Of An Exemplary Synthetic Albumin-Binding Protein

[0375] The synthetic albumin binding protein was then fused to the C-terminus of an exemplary GLP-1R miniprotein (Reference Miniprotein 120; SEQ ID NO: 120) using an amino acid linker (SEQ ID NO: 164; GGDKTVERKGGDKTVERKGG) to connect the GLP-1Rminiprotein (Reference Miniprotein 120; SEQ ID NO: 120) to the albumin binding miniprotein (SEQ ID NO: 163) to produce Reference Miniprotein 155 (SEQ ID NO: 155).[003761 To determine extent of half-life extension after attachment to the albumin binding domain, the pharmacokinetic half-life of Reference Miniprotein 155 (SEQ ID NO: 155) was measured in mice. Reference Miniprotein 155, with a purity exceeding 99% as confirmed by high-performance liquid chromatography, was dissolved in sterile phosphate-buffered saline and administered to male C57BL / 6J mice (8-12 weeks old; 18-22 g; n= 5 mice) via tail vein injection at a dose of 3.96 mg / kg to 7.56 mg / kg body weight. Blood samples were collected from the submandibular vein in EDTA coated tubes at 0 (pre-dose), 0.08, 0.25, 0.50, 1, 2, 4, 6, 8, 12, 24, 36 and 48 hours after initial injection. Plasma was separated by centrifugation and stored at - 80°C until analysis.

[0377] Concentrations of Reference Miniprotein 155 in the plasma samples were quantified by SPR, using a calibration curve prepared with known concentrations of Reference Miniprotein 155 in the assay buffer and establishing a lower limit of quantification at 13 nM. The half-life of GLP-1 is known to have a short half-life in vivo (~ < 5 mins). In contrast, Reference Miniprotein 155 showed approximately half of its maximal concentration of injected protein was still present at least six hours after injection (FIG. 9), which falls within suitable ranges for therapeutic applications, including daily dosing.TABLE S. Table of SequencesINCORPORATION BY REFERENCE

[0378] All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc. , whether supra or infra, are hereby incorporated by reference in their entirety for all purposes. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.EQUIVALENTS

[0379] Any invention provided may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on any invention provided herein. Scope of any invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

CLAIMSWhat is Claimed is:

1. A synthetic GLP-1R binding protein, the binding protein comprising:(a) an amino acid sequence from 30 amino acids to 95 amino acids in length;(b) a net negative charge in phosphate buffered saline (PBS);(c) a binding affinity for GLP-1R stronger than 1 pM; and(d) a stability profile such that the protein (i) retains at least 90% binding affinity to GLP-1R upon cooling to room temperature after thermal denaturation at 95°C in PBS for at least about five minutes relative to the protein prior to thermal denaturation; (ii) retains at least 90% binding affinity to GLP-1R after incubation for 16 hours at 37°C of incubation in PBS relative to the protein under the same conditions prior to incubating; and / or (iii) retains at least 90% binding affinity to GLP-1R in PBS following chemical denaturation in 4 M urea for 1 hour at room temperature relative to the protein prior to chemical denaturation.

2. A synthetic GLP-1R binding protein, the binding protein comprising:(a) an amino acid sequence from 30 amino acids to 95 amino acids in length;(b) a net negative charge in PBS;(c) a binding affinity for GLP-1R stronger than 1 pM;(d) at least one alpha helix;(e) at least three beta sheets;(f) at least three amino acid loops, where a first loop having a first amino acid sequence connects a terminal amino acid (e.g., a C-terminal amino acid) of a first alpha helix to a terminal amino acid (e.g., an N-terminal amino acid) of a first beta sheet, and a second loop having a second amino acid sequence connects a second, terminal amino acid (e.g., a C-terminal amino acid) of the first beta sheet to a terminal amino acid (e.g., an N-terminal amino acid) of a second beta sheet, and a third loop having a third amino acid sequence connects a third, terminal amino acid (e.g., a C- terminal amino acid) of the second beta sheet to a terminal amino acid (e.g., an N-terminal amino acid) of a third beta sheet; and(g) a hydrophobic core defined by at least two hydrophobic amino acids present in at least one of the alpha helices and / or at least one of the beta sheets.

3. The synthetic GLP-1R binding protein of claim 1 or 2, further comprising an optional fourth amino acid loop and / or an optional fourth beta sheet, wherein the fourth loop has a fourth amino acid sequence and connects a fourth, terminal amino acid (e.g., a C-terminal amino acid) of the fourth loop to a terminal amino acid (e.g., an N-terminal amino acid) of the fourth beta sheet.

4. The synthetic GLP-1R binding protein of any of claims 1-3, wherein the binding protein comprises one or more of the following features:(a) free of tryptophan amino acids;(b) free of methionine amino acids;(c) free of lysine amino acids;(d) does not comprise an unpaired cysteine amino acid when cysteine amino acids are present in the protein;(e) free of glycosylation sites;(f) free of protease cleavage sites; and(g) soluble up to at least 1 mM in PBS at 4°C for one month.

5. The synthetic GLP-1R binding protein of any of claims 1-4, wherein the binding affinity is between about 10 pM to about 0.001 nM; about 10 pM to about 0.01 nM, about 7.5 pM to about 0.75 nM; about 5 pM to about 0.5 nM; about 2.5 pM to about 0.25 nM; about 1 pM to about 1 nM; about 0.75 pM to about 1 nM, about 0.5 pM to about 1 nM; about 0.25 pM to about 1 nM; about 0.10 pM to about 1 nM; about 75 nM to about 1 nM; about 50 nM to about 1 nM; about 25 nM to about 1 nM; about 10 nM to about 1 nM; or about 5 nM to about 1 nM.

6. The synthetic GLP-1R binding protein of any of claims 1-5, wherein the binding affinity is stronger than about 10 pM, about 7.5 pM, about 5 pM, about 2.5 pM, about 1 pM, about 0.75 pM, about 0.5 pM, about 0.25 pM, about 0.1 pM, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, about 0.1 nM, about 0.01 nM, or about 0.001 nM.

7. The synthetic GLP-1R binding protein of any of claims 2-6, wherein the N-terminus of the first alpha helix is preceded by one or more N-terminal amino acids.

8. The synthetic GLP-1R binding protein of any of claims 2-7 wherein the C-terminus of the third beta sheet is followed by one or more C-tenninal amino acids.

9. The synthetic GLP-1R binding protein of any of claims 3-8 wherein the C-terminus of the fourth beta sheet is followed by one or more C-terminal amino acids.

10. The synthetic GLP-1R binding protein of any of the preceding claims, wherein the protein comprises from 35 amino acids to 85 amino acids in length, from 35 amino acids to 75 amino acids in length, from 35 amino acids to 65 amino acids in length, from 35 amino acids to 55 amino acids in length, from 35 amino acids to 50 amino acids in length, from 40 amino acids to 85 amino acids in length, from 40 amino acids to 75 amino acids in length, from 40 amino acids to 65 amino acids in length, from 45 amino acids to 70 amino acids in length, from 45 amino acids to 65 amino acids in length, from 45 amino acids to 60 amino acids in length, from about 50 amino acids to about 75 amino acids, from 50 amino acids to 70 amino acids in length, from 50 amino acids to 65 amino acids in length, or from 60 amino acids to 70 amino acids in length.

11. The synthetic GLP-1R binding protein of claim 10, wherein the protein comprises 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 amino acids.

12. The synthetic GLP-1R binding protein of any of claims 2-11, wherein(a) the first alpha helix contains at least one hydrophobic amino acid, wherein, optionally, one or more of the at least one hydrophobic amino acids is not solvent accessible;(b) the first, second, third and / or fourth beta sheet each contains at least two or three hydrophobic amino acids, wherein, optionally, one or more of the at least two or three hydrophobic amino acids is not solvent accessible;(c) the first alpha helix contains at least one or two solvent accessible amino acids;(d) the first, second, third and / or fourth beta sheet contains at least one or two solvent accessible amino acids;(e) the first alpha helix contains at least one or two solvent accessible amino acids;(f) the first and / or second and / or third and / or fourth loop contains at least one hydrophobic amino acid; or(g) the binding protein comprises any combination of elements selected from (a), (b), (c), (d), (e), and (f).

13. The synthetic GLP-1R binding protein of claim 12, wherein:(a) the first, second, third and / or fourth beta sheets each contains at least two hydrophobic amino acids;(b) the first, second, third and, when present, fourth beta sheet each contains at least one solvent accessible amino acid;(c) the first, second, third and, when present, fourth beta sheet each contains at least two hydrophobic and one solvent accessible amino acids;(d) the first alpha helix contains at least four solvent accessible amino acids; and / or(e) the first, second, third and / or fourth loop contains at least one hydrophobic amino acid.

14. A synthetic GLP-1R binding protein comprising:(i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI,L2, and L3 are loops 1, 2, and 3, respectively; and(ii) an amino acid of SEQ ID NO: 71, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 84, wherein XI is H, L, Y, I, a peptide bond, or is absent; X2 is A, G, S, T, D, E, P, H, V, N, a peptide bond, or is absent; X3 is E, a peptide bond, or is absent; X4 is G, A, a peptide bond, or is absent; X5 is T, P, V, H, G, Y, a peptide bond, or is absent; X6 is F, A, a peptide bond, or is absent; XI 1 is S or R; XI 3 is Y or I; XI 6 is R or V; XI 8 is A, D, or L; XI 9 is S, I, E, V, or L; X20 is R or I; X21 is S or F; X23 is L, I, or V; X25 is I, L, Y, or A; X27 is Q or L; X28 is L, G, N, or Q; and X29 is R or G;(b) D2 comprises an amino acid sequence of X36X37X38X39LD, wherein X36 is F or T; X37 is S, V, or L; X38 is L, F, Y, or R; and X39 is D or V;(c) D3 comprises an amino acid sequence of X46X47TX49X50D, wherein X46 is E, V, or D; X47 is L or P; X49 is V or F; and X50 is T or I; and(d) D4 comprises an amino acid sequence of X57X58X59VX61, wherein X57 is T, K, or R; X58 is T, F, K, L, or R; X59 is T, V, F, R, or H; and X61 is Q, S, T, or H.

15. The synthetic GLP-1R binding protein of claim 14, wherein LI comprises an amino acid sequence of DGX33TX35, wherein X33 is I, G, V, K, or F; and X35 is D or G; and / or L2 comprises an amino acid sequence of X42X43X44X45, wherein X42 is L, D, or E; X43 is L, Y, or E; X44 is T or F; and X45 is G or F; and / or L3 comprises an amino acid sequence of NX53X54X55E, wherein X53 is N or T; X54 is T or F; and X55 is G or L.

16. The synthetic GLP-1R binding protein of claim 12 or 13, comprising a C-terminal amino acid of X62, wherein X62 is L or S.

17. A synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 71, wherein XI is H, L, Y, I, a peptide bond, or is absent; X2 is A, G, S, T, D, E, P, H, V, N, a peptide bond, or is absent; X3 is E, a peptide bond, or is absent; X4 is G, A, a peptide bond, or is absent; X5 is T, P, V, H, G, Y, a peptide bond, or is absent; X6 is F, A, a peptide bond, or is absent; XI 1 is S or R; X13 is Y or I; X16 is R or V; X18 is A, D, or L; X19 is S, I, E, V, or L; X20 is R or I; X21 is S or F; X23 is L, I, or V; X25 is I, L, Y, or A; X27 is Q or L; X28 is L, G, N, or Q; X29 is R or G; X33 is I, G, V, K, or F; X35 is D or G; X36 is F or T; X37 is S, V, or L; X38 is L, F, Y, or R; X39 is D or V; X42 is L, D, or E; X43 is L, Y, or E; X44 is T or F; X45 is G or F; X46 is E, V, or D; X47 is L or P; X49 is V or F; X50 is T or I; X53 is N or T; X54 is T or F; X55 is G or L; X57 is T, K, or R; X58 is T, F, K, L, or R; X59 is T, V, F, R, or H; X61 is Q, S, T, or H; and X62 is L or S.

18. A synthetic GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 2-29 and 31-70.

19. A synthetic GLP-1R binding protein comprising:(i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and(ii) an amino acid of SEQ ID NO: 72, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 85, wherein XI is H, L, Y, I, a peptide bond, or is absent; X2 is A, G, S, T, D, E, P, H, V, N, a peptide bond, or is absent; X3 is E, a peptide bond, or is absent; X4 is G, a peptide bond, or is absent; X5 is T, P, V, H, G, Y, a peptide bond, or is absent; X6 is F, A, a peptide bone, or is absent; XI 3 is Y or I; XI 8 is A, D, or L; X19 is I, S, or E; X20 is R or I; X23 is I, L, or V; X28 is L or G; and X29 is R or G;(b) D2 comprises an amino acid sequence of SEQ ID NO: 97, wherein X38 is L or Y;(c) D3 comprises an amino acid sequence of SEQ ID NO: 99, wherein X47 is P or L; and(d) D4 comprises an amino acid sequence of X57X58X59VX61, wherein X57 is T, K, or R; X58 is F, T, or L; X59 is T, H, V, or R; and X61 is Q, S, or T.

20. The synthetic GLP-1R binding protein of claim 19, wherein LI comprises an amino acid sequence of DGX33TX35, wherein X33 is I, G, V, K, or F; and X35 is D or G; and / or L2 comprises an amino acid sequence of X42X43TG, wherein X42 is D, L, or E; and X43 is L or Y; and / or L3 comprises an amino acid sequence of SEQ ID NO: 105.

21. A synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 72, wherein XI is H, L, Y, I, a peptide bond, or is absent; X2 is A, G, S, T, D, E, P, H, V, N, a peptide bond, or is absent; X3 is E, a peptide bond, or is absent; X4 is G, a peptide bond, or is absent; X5 is T, P, V, H, G, Y, a peptide bond, or is absent; X6 is F, A, a peptide bond, or is absent; X13 is Y or I; X18 is A, D, or L; X19 is I, S, or E; X20 is R or I; X23 is I, L, or V; X28 is L or G; X29 is R or G; X33 is I, G, V, K, or F; X35 is D or G; X38 is L or Y; X42 is D, L, or E; X43 is L or Y; X47 is P or L; X57 is T, K, or R; X58 is F, T, or L; X59 is T, H, V, or R; and X61 is Q, S, or T.

22. A synthetic GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 2, 6-9,11-17, 20, 23, 28-32, 37-40, 42, 44, and 46-70.

23. A synthetic GLP-1R binding protein comprising:(i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and(ii) an amino acid of SEQ ID NO: 73, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 86, wherein XI is H, L, or Y; X2 is A, G, S, T, D, E, P, H, V, or N; X5 is T, P, V, H, G, or Y; X6 is F or A; X18 is A or D; X19 is I, S, or E; X20 is R or I; X23 is I or L; X28 is L or G; X29 is R or G;(b) D2 comprises an amino acid sequence of SEQ ID NO: 97, wherein X38 is L or Y;(c) D3 comprises an amino acid sequence of SEQ ID NO: 100; and(d) D4 comprises an amino acid sequence of X57X58X59VX61, wherein X57 is T, K, or R; X58 is F or T; X59 is T or H; and X61 is Q, S, or T.

24. The synthetic GLP-1R binding protein of claim 21, wherein LI comprises an amino acid sequence of DGX33TX35, wherein X33 is I or V; X35 is D or G; and / or L2 comprises an amino acid sequence of X42X43TG, wherein X42 is D, L, or E; and X43 is L or Y; and / or L3 comprises an amino acid sequence of SEQ ID NO: 105.

25. A synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 73, wherein XI is H, L, or Y; X2 is A, G, S, T, D, E, P, H, V, or N; X5 is T, P, V, H, G, or Y; X6 is F or A; X18 is A or D; X19 is I, S, or E; X20 is R or I; X23 is I or L; X28 is L or G; X29 is R or G; X33 is I or V; X35 is D or G; X38 is L or Y; X42 is D, L, or E; X43 is L or Y; X57 is T, K, or R; X58 is F or T; X59 is T or H; and X61 is Q, S, or T.

26. A synthetic GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 2, 6, 8, 9, 11-14, 20, 28, 37, and 46-59.

27. A synthetic GLP-1R binding protein comprising:(i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4 (Formula IA), wherein DI , D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and(ii) an amino acid of SEQ ID NO: 74, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 87, wherein XI is H, I, a peptide bond, or is absent; X13 is Y or I; X18 is D, A, or L; X19 is S or I; X23 is V, I, or L; X28 is L or G;(b) D2 comprises an amino acid sequence of SEQ ID NO: 98;(c) D3 comprises an amino acid sequence of SEQ ID NO: 100; and(d) D4 comprises an amino acid sequence of X57X58X59VQ, wherein X57 is T or K;X58 is T or F; and X59 is T, V, or R.

28. The synthetic GLP-1R binding protein of claim 25, wherein LI comprises an amino acid sequence of SEQ ID NO: 103, wherein X33 is K, I, F, or G; and / or L2 comprises an amino acid sequence of SEQ ID NO: 104; and / or L3 comprises an amino acid sequence of SEQ ID NO: 105.

29. A synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 74, wherein XI is H, I, a peptide bond, or is absent; X13 is Y or I; X18 is D, A, or L; X19 is S or I; X23 is V, I, or L; X28 is L or G; X33 is K, I, F, or G; X57 is T or K; X58 is T or F; and X59 is T, V, or R.

30. A synthetic GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 15-17, 23, 29, 31, 38, 40, 42, and 60.

31. A synthetic GLP-1R binding protein comprising:(i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and(ii) an amino acid of SEQ ID NO: 75, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 88, wherein XI is H, a peptide bond, or is absent; X2 is A, a peptide bond, or is absent; X3 is E, a peptide bond, or is absent; X4 is G, a peptide bond, or is absent; X5 is T, a peptide bond, or is absent; X6 is F, a peptide bond, or is absent; XI 3 is Y or I; XI 8 is A or L; XI 9 is S or I; X23 is L or I; and X28 is L or G;(b) D2 comprises an amino acid sequence of SEQ ID NO: 98;(c) D3 comprises an amino acid sequence of SEQ ID NO: 100; and(d) D4 comprises an amino acid sequence of X57X58TVQ, wherein X57 is T or K; and X58 is T, F, or L.

32. The synthetic GLP-1R binding protein of claim 29, wherein LI comprises an amino acid sequence of SEQ ID NO: 166, wherein X33 is I or G; and / or L2 comprises an amino acidsequence of X42LTG, wherein X42 is L or D; and / or L3 comprises an amino acid sequence of SEQ ID NO: 105.

33. A synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO:75, wherein XI is H, a peptide bond, or is absent; X2 is A, a peptide bond, or is absent; X3 is E, a peptide bond, or is absent; X4 is G, a peptide bond, or is absent; X5 is T, a peptide bond, or is absent; X6 is F, a peptide bond, or is absent; XI 3 is Y or I; XI 8 is A or L; XI 9 is S or I; X23 is L or I; X28 is L or G; X33 is I or G; X42 is L or D; X57 is T or K; and X58 is T, F, or L.

34. A synthetic GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 30, 39, 44, and 61-70.

35. A synthetic GLP-1R binding protein comprising:(i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and(ii) an amino acid of SEQ ID NO: 76, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 89, wherein XI is H, L, Y, a peptide bond, or is absent; X2 is A, G, S, T, D, E, P, H, V, or N; and X13 is Y or I;(b) D2 comprises an amino acid sequence of SEQ ID NO: 98;(c) D3 comprises an amino acid sequence of SEQ ID NO: 99, wherein X47 is P or L; and(d) D4 comprises an amino acid sequence of SEQ ID NO: 102, wherein X57 is T or K.

36. The synthetic GLP-1R binding protein of claim 33, wherein LI comprises an amino acid sequence of SEQ ID NO: 166, wherein X33 is I or G; and / or L2 comprises an amino acid sequence of SEQ ID NO: 104; and / or L3 comprises an amino acid sequence of SEQ ID NO: 105.

37. A synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO:76, wherein XI is H, L, Y, a peptide bond, or absent; X2 is A, G, S, T, D, E, P, H, V, or N; X13 is Y or I; X33 is I or G; X47 is P or L; and X57 is T or K.

38. A synthetic GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 2, 7-9, 29, 32, 40, 46-54, and 60.

39. A synthetic GLP-1R binding protein comprising:(i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4 respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and(ii) an amino acid of SEQ ID NO: 77, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 90, wherein XI is H, L, Y, a peptide bond, or is absent; X2 is A, G, S, T, D, E, P, H, V, or N; and X13 is Y or I;(b) D2 comprises an amino acid sequence of SEQ ID NO: 98;(c) D3 comprises an amino acid sequence of SEQ ID NO: 99, wherein X47 is L or P; and(d) D4 comprises an amino acid sequence of SEQ ID NO: 102, wherein X57 is T or K.

40. The synthetic GLP-1R binding protein of claim 37, wherein LI comprises an amino acid sequence of SEQ ID NO: 166, wherein X33 is I or G; and / or L2 comprises an amino acid sequence of SEQ ID NO: 104; and / or L3 comprises an amino acid sequence of SEQ ID NO: 105.

41. A synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 77, wherein XI is H, L, Y, a peptide bond, or is absent; X2 is A, G, S, T, D, E, P, H, V, or N; X13 is Y or I; X33 is I or G; X47 is L or P; and X57 is T or K.

42. A synthetic GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 2, 7-9, 29, 32, 40, 46-54, and 60.

43. A synthetic GLP-1R binding protein comprising:(i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and(ii) an amino acid of SEQ ID NO: 78, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 91, wherein XI is H or Y; X2 is A, G, S, P, or V; X4 is G or A; XI 1 is S or R; X13 is Y or I; X16 is R or V; X18 is A or L; X19 is S, I, E, V, or L; X20 is R or I; X21 is S or F; X23 is L or I; X25 is I, L, Y, or A; X27 is Q or L; X28 is L, G, N, or Q; and X29 is R or G;(b) D2 comprises an amino acid sequence of X36X37X38X39LD, wherein X36 is F or T; X37 is S, V, or L; X38 is L, F, Y, or R; and X39 is D or V;(c) D3 comprises an amino acid sequence of X46X47TX49X50D, wherein X46 is E, D, or V; X47 is L or P; X49 is V or F; and X50 is T or I; and(d) D4 comprises an amino acid sequence of X57X58X59VX61, wherein X57 is T, K, or R; X58 is T, F, L, K, or R; X59 is T, H, or F; and X61 is Q, T, or H.

44. The synthetic GLP-1R binding protein of claim 41, wherein LI comprises an amino acid sequence of SEQ ID NO: 166, wherein X33 is I or G; and / or L2 comprises an amino acid sequence of X42X43X44X45, wherein X42 is L or D; X43 is L or E; X44 is T or F; and X45 is G or F; and / or L3 comprises an amino acid sequence of NX53X54X55E, wherein X53 is N or T; X54 is T or F; and X55 is G or L.

45. The synthetic GLP-1R binding protein of claim 41 or 42, comprising a C-terminal amino acid of X62, wherein X is L or S.

46. A synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 78, wherein XI is H or Y; X2 is A, G, S, P, or V; X4 is G or A; XI 1 is S or R; X13 is Y or I; X16 is R or V; X18 is A or L; X19 is S, I, E, V, or L; X20 is R or I; X21 is S or F; X23 is L or I; X25 is I, L, Y, or A; X27 is Q or L; X28 is L, G, N, or Q; X29 is R or G; X33 is I or G; X36 is F or T; X37 is S, V, or L; X38 is L, F, Y, or R; X39 is D or V; X42 is L or D; X43 is L or E; X44 is T or F; X45 is G or F; X46 is E, D, or V; X47 is L or P; X49 is V or F; X50 is T or I; X53 is N or T; X54 is T or F; X55 is G or L; X57 is T, K, or R; X58 is T, F, L, K, or R; X59 is T, H, or F; X61 is Q, T, or H; and X62 is L or S.

47. A synthetic GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 2-7, 9-11, 18-22, 24-27, 30-47, 51, and 53.

48. A synthetic GLP-1R binding protein comprising:(i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4(Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and(ii) an amino acid of SEQ ID NO: 79, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 92, wherein XI is H or Y; X2 is A, G, S, P, or V; X23 is I or L; X28 is L or G; and X29 is R or G;(b) D2 comprises an amino acid sequence of SEQ ID NO: 98;(c) D3 comprises an amino acid sequence of SEQ ID NO: 101, wherein X46 is E or D; and X47 is L or P; and(d) D4 comprises an amino acid sequence of X57X58X59VQ, wherein X57 is T, K, or R; X58 is F or T; and X59 is T or H.

49. The synthetic GLP-1R binding protein of claim 46, wherein LI comprises an amino acid sequence of SEQ ID NO: 166, wherein X33 is I or G; and / or L2 comprises an amino acid sequence of SEQ ID NO: 104; and / or L3 comprises an amino acid sequence of SEQ ID NO: 105.

50. A synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 79, wherein XI is H or Y; X2 is A, G, S, P, or V; X23 is I or L; X28 is L or G; X29 is R or G; X33 is I or G; X46 is E or D; X47 is L or P; X57 is T, K, or R; X58 is F or T; and X59 is T or H.

51. A synthetic GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 2, 5-7, 9, 11, 32, 37, 40, 42, 46, 47, 51, and 53.

52. A synthetic GLP-1R binding protein comprising:(i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4(Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and(ii) an amino acid of SEQ ID NO: 80, wherein DI, D2, D3, and D4 independently comprise any of the following combinations: (a) DI comprises an amino acid sequence of SEQ ID NO: 93, wherein XI is H or Y; X2 isA, G, S, P, or V;(b) D2 comprises an amino acid sequence of SEQ ID NO: 98;(c) D3 comprises an amino acid sequence of SEQ ID NO: 99, wherein X47 is L or P; and(d) D4 comprises an amino acid sequence of SEQ ID NO: 102, wherein X57 is T or K.

53. The synthetic GLP-1R binding protein of claim 50, wherein LI comprises an amino acid sequence of SEQ ID NO: 166, wherein X33 is I or G; and / or L2 comprises an amino acid sequence of SEQ ID NO: 104; and / or L3 comprises an amino acid sequence of SEQ ID NO: 105.

54. A synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 80, wherein XI is H or Y; X2 is A, G, S, P, or V; X33 is I or G; X47 is L or P; and X57 is T or K.

55. A synthetic GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 2, 7, 9, 32 ,40, 46, 47, 51, and 53.

56. A synthetic GLP-1R binding protein comprising:(i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and(ii) an amino acid of SEQ ID NO: 81, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 94, wherein X13 is Y or I; X16 is R or V; XI 8 is A or L; X19 is S, I, E, V, or L; X20 is R or I; X23 is L or I; X25 is I, L, Y, or A; X27 is Q or L; X28 is L, G, or Q; and X29 is R or G;(b) D2 comprises an amino acid sequence of X36X37X38X39LD, wherein X36 is F or T; X37 is S, V, or L; X38 is L, F, Y, or R; X39 is D or V;(c) D3 comprises an amino acid sequence of X46X47TVX50D, wherein X46 is E, D, or V;X47 is L or P; X50 is T or I; and(d) D4 comprises an amino acid sequence of X57X58X59VX61, wherein X57 is T, K, or R; X58 is T, F, L, K, or R; X59 is T, H, or F; and X61 is Q or T.

57. The synthetic GLP-1R binding protein of claim 54, wherein LI comprises an amino acid sequence of SEQ ID NO: 166, wherein X33 is I or G; and / or L2 comprises an amino acidsequence of X42X43X44X45, wherein X42 is L or D; X43 is L or E; X44 is T or F; and X45 is G or F; and / or L3 comprises an amino acid sequence of NX53X54X55E, wherein X53 is N or T; X54 is T or F; and X55 is G or L.

58. The synthetic GLP-1R binding protein of claim 54 or 55, comprising a C-terminal amino acid of X62, wherein X is L or S.

59. A synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 81, wherein XI 3 is Y or I; XI 6 is R or V; XI 8 is A or L; XI 9 is S, I, E, V, or L; X20 is R or I; X23 is L or I; X25 is I, L, Y, or A; X27 is Q or L; X28 is L, G, or Q; X29 is R or G; X36 is F or T; X37 is S, V, or L; X38 is L ,F, Y, or R; X39 is D or V; X46 is E, D, or V; X47 is L or P; X50 is T or I; X57 is T, K, or R; X58 is T, F, L, K, or R; X59 is T, H, or F; X61 is Q or T; X33 is I or G; X42 is L or D; X43 is L or E; X44 is T or F; X45 is G or F; X53 is N or T; X54 is T or F; X55 is G or L; and X62 is L or S.

60. A synthetic GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 2-7, 10, 11, 18-22, 24, 27, and 30-45.

61. A synthetic GLP-1R binding protein comprising:(i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and(ii) an amino acid of SEQ ID NO: 82, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 95, wherein X23 is I or L; X28 is L or G; and X29 is R or G;(b) D2 comprises an amino acid sequence of SEQ ID NO: 98;(c) D3 comprises an amino acid sequence of SEQ ID NO: 101, wherein X46 is E or D; and X47 is L or P; and(d) D4 comprises an amino acid sequence of X57X58X59VQ, wherein X57 is T, K, or R; X58 is F or T; and X59 is T or H.

62. The synthetic GLP-1R binding protein of claim 59, wherein LI comprises an amino acid sequence of SEQ ID NO: 166, wherein X33 is I or G; and / or L2 comprises an amino acidsequence of SEQ ID NO: 104; and / or L3 comprises an amino acid sequence of SEQ ID NO: 105.

63. A synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO:82, wherein X23 is I or L; X28 is L or G; X29 is R or G; X33 is I or G; X46 is E or D; X47 is L or P; X57 is T, K, or R; X58 is F or T; and X59 is T or H.

64. A synthetic GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 2, 5-7, 11, 32, 37, 40, and 42.

65. A synthetic GLP-1R binding protein comprising:(i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4 (Formula IA), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, andLI, L2, and L3 are loops 1, 2, and 3, respectively; and(ii) an amino acid of SEQ ID NO: 83, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 96;(b) D2 comprises an amino acid sequence of SEQ ID NO: 98;(c) D3 comprises an amino acid sequence of SEQ ID NO: 99, wherein X47 is L or P; and(d) D4 comprises an amino acid sequence of SEQ ID NO: 102, wherein X57 is T or K.

66. The synthetic GLP-1R binding protein of claim 64, wherein LI comprises an amino acid sequence of SEQ ID NO: 166, wherein X33 is I or G; and / or L2 comprises an amino acid sequence of SEQ ID NO: 104; and / or L3 comprises an amino acid sequence of SEQ ID NO: 105.

67. A synthetic GLP-1R binding protein comprising, an amino acid sequence of SEQ ID NO:83, wherein X33 is I or G; X47 is L or P; and X57 is T or K.

68. A synthetic GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 2, 7, 32, and 40.

69. The synthetic GLP-1R binding protein of any one of claims 14-68 further comprising a C- terminal extension of SEQ ID NO: 165, wherein X63 is R, G, D, or P.

70. A synthetic GLP-1R binding protein comprising:(i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4- L4-D5 (Formula IB), wherein DI, D2, D3, D4 and D5 are domains 1, 2, 3, 4, and 5 respectively, and LI, L2, L3, and L4 are loops 1, 2, 3, and 4 respectively; and(ii) an amino acid of SEQ ID NO: 156, wherein DI, D2, D3, D4, and D5 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 158, wherein X2 is G or S; X3 is E or Q; X10 is V or Y; X14 is L, A, or R; X16 is R or Y; X18 is A, K, R, or F; X21 is S, I, F, Y, V, R, Q, L, or W; X24 is T, L, R, or K; X25 is I or F; X28 is L, R, N, or Q; X29 is R, E, G, Q, or H;(b) D2 comprises an amino acid sequence of SEQ ID NO: 160, wherein: X36 is F or Y; X41 is D, K, or R;(c) D3 comprises an amino acid sequence of SEQ ID NO: 100;(d) D4 comprises an amino acid sequence of SEQ ID NO: 161; and(e) D5 comprises an amino acid sequence of SEQ ID NO: 162.

71. The synthetic GLP-1R binding protein of claim 70, wherein LI comprises an amino acid sequence of DGIX34X35, wherein X34 is T, K, R; X35 is D or Q; and / or L2 comprises an amino acid sequence of SEQ ID NO: 104; and / or L3 comprises an amino acid sequence of SEQ ID NO: 105; and / or L4 comprises an amino acid sequence of LX63TG, wherein X63 is R, G, D, or P.

72. A synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 156, wherein X2 is G or S; X3 is E or Q; X10 is V or Y; X14 is L, A, or R; X16 is R or Y; XI 8 is A, K, R, or F; X21 is S, I, F, Y, V, R, Q, L, or W; X24 is T, L, R, or K; X25 is I or F; X28 is L, R, N, or Q; X29 is R, E, G, Q, or H; X36 is F or Y; X41 is D, K, or R; X34 is T, K, or R; X35 is D or Q; and X63 is R, G, D, or P.

73. A synthetic GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 108-151.

74. A synthetic GLP-1R binding protein comprising:(i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4- L4-D5 (Formula IB), wherein DI, D2, D3, D4 and D5 are domains 1, 2, 3, 4 and 5 respectively, and LI, L2, L3 and L4 are loops 1, 2, 3, and 4 respectively; and(ii) an amino acid of SEQ ID NO: 157, wherein DI, D2, D3, D4, and D5 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 159, wherein X21 is W or L; X24 is T, R, or K; X28 is L, N, or Q; and X29 is R, E, G, Q, or H;(b) D2 comprises an amino acid sequence of SEQ ID NO: 160, wherein X36 is F orY; and X41 is D, K, or R;(c) D3 comprises an amino acid sequence of SEQ ID NO: 100;(d) D4 comprises an amino acid sequence of SEQ ID NO: 161; and(e) D5 comprises an amino acid sequence of SEQ ID NO: 162.

75. The synthetic GLP-1R binding protein of claim 74, wherein LI comprises an amino acid sequence of DGIX34X35, wherein X34 is T, K, or R; and X35 is D or Q; and / or L2 comprises an amino acid sequence of SEQ ID NO: 104; and / or L3 comprises an amino acid sequence of SEQ ID NO: 105, wherein X63 is R, G, D, or P; and / or L4 comprises an amino acid sequence of LX63TG.

76. A synthetic GLP-1R binding protein comprising an amino acid sequence of SEQ ID NO: 157, wherein X21 is L or W; X24 is T, R, or K; X28 is L, N, or Q; X29 is R, E, G, Q, or H; X34 is T, K, or R; X35 is D or Q; X36 is F or Y; X41 is D, K, or R; and X63 is R, G, D, or P.

77. A synthetic GLP-1R binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 130-151.

78. The synthetic GLP-1R binding protein of any one of claims 70-77, further comprising an effector.

79. The synthetic GLP-1R binding protein of claim 78, wherein the effector comprises a protein that increases serum half-life.

80. The synthetic GLP-1R binding protein of claim 79, wherein the effector comprises a synthetic serum albumin binding protein.

81. The synthetic GLP-1R binding protein of any one of claims 78-80, wherein the effector has an amino acid sequence comprising SEQ ID NO: 163.

82. The synthetic GLP-1R binding protein of any one of claims 78-81, wherein the effector is linked to the GLP-1R binding protein by an amino acid linker.

83. The synthetic GLP-1R binding protein of claim 82, wherein the amino acid linker has an amino acid sequence comprising SEQ ID NO: 164.

84. The synthetic GLP-1R binding protein of any one of claims 78-83, wherein the amino acid sequence comprises SEQ ID NO: 155.

85. The synthetic GLP-1R binding protein of any one of claims 78-84, wherein the serum halflife of the GLP-1R binding protein with the effector is longer than the half-life of the same or similar GLP-1R binding protein without the effector.

86. The synthetic GLP-1R binding protein, wherein the GLP-1R binding protein without the effector has an amino acid sequence comprising any one of SEQ ID NOs: 2-70 or 108-154 or as set forth in any of SEQ ID NOs: 71-83 or 156 or 157.

87. The synthetic GLP-1R binding protein, wherein the GLP-1R binding protein without the effector is a reference GLP-1R agonist.

88. The synthetic GLP-1R binding protein of claim 87, wherein the reference GLP-1R agonist has an amino acid sequence comprising SEQ ID NO: 106.

89. The synthetic GLP-1R binding protein of any of claims 14-86, wherein the binding protein has a binding affinity for GLP-1R stronger than 10 pM.

90. The synthetic GLP-1R binding protein of any of claims 14-86 or 89, wherein the binding affinity is between about 10 pM to about 0.001 nM; about 7.5 pM to about 0.01 nM; about 7.5 pM to about 0.75 nM; about 5 pM to about 0.5 nM; about 2.5 pM to about 0.25 nM; about 1 pM to about 1 nM; about 0.5 pM to about 1 nM; about 0.25 pM to about 1 nM; about 0.10 pM to about 1 nM; about 75 nM to about 1 nM; about 50 nM to about 1 nM; about 25 nM to about 1 nM; about 10 nM to about 1 nM; or about 5 nM to about 1 nM.

91. The synthetic GLP-1R binding protein of any of claims 14-86 or 89-90, wherein the binding affinity is stronger than about 10 pM, about 7.5 pM, about 5 pM, about 2.5 pM, about 1 pM, about 0.75 pM, about 0.5 pM, about 0.25 pM, about 0.1 pM, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, about 0.1 nM, about 0.01 nM, or about 0.001 nM.

92. The synthetic GLP-1R binding protein of any of claims 14, 19, 23, 27, 31, 35, 39, 43, 48, 52, 56, 61, 65, 69, or 74 wherein DI is flanked by one or more N-terminal amino acids and / or D4 is flanked by one or more C-terminal amino acids.

93. The synthetic GLP-1R binding protein of claim any of claims 14-92, wherein the amino acid sequence of the binding protein has at least 85 (e.g., 90, 95, 96, 97, 98, 99, 99.5) percent identity to that of SEQ ID NO: 2.

94. The synthetic GLP-1R binding protein of any of claims 1-93, wherein the protein has an amino acid sequence comprising or according to one or more amino acid sequences set forth in Table 8.

95. A pharmaceutical composition comprising the synthetic GLP-1R binding protein of any one of claims 1-86 or 89-94; and a pharmaceutically acceptable carrier.

96. The pharmaceutical composition of claim 95, wherein the synthetic GLP-1R binding protein further comprises an effector.

97. The pharmaceutical composition of claim 96, wherein the effector comprises a protein that extends serum half-life of the GLP-1R binding protein.

98. The pharmaceutical composition of claim 97, wherein the effector comprises a synthetic serum albumin binding protein.

99. The pharmaceutical composition any one of claims 95-98, wherein the effector has an amino acid sequence comprising SEQ ID NO: 163.

100. The pharmaceutical composition any one of claims 95-98, wherein the effector is linked to the GLP-1R binding protein by an amino acid linker.

101. The pharmaceutical composition of claim 100, wherein the amino acid linker has an amino acid sequence comprising SEQ ID NO: 164.

102. The pharmaceutical composition any one of claims 95-101, wherein the amino acid sequence comprises SEQ ID NO: 155.

103. The pharmaceutical composition any one of claims 95-102, wherein the serum half-life of the GLP-1R binding protein is longer than the serum half-life of the same or similar GLP- 1R binding protein without the effector.

104. The pharmaceutical composition any one of claims 95-103, wherein the GLP-1R binding protein without the effector has an amino acid sequence comprising any one of SEQ ID NOs: 2-70 or 108-154 or as set forth in any of SEQ ID NOs: 71-83 or 156 or 157.

105. The pharmaceutical composition of claim 103 or 104, wherein the GLP-1R binding protein without the effector is a reference GLP-1R agonist.

106. The pharmaceutical composition of claim 105, wherein the reference GLP-1R agonist has an amino acid sequence comprising SEQ ID NO: 106.

107. A method of targeting GLP-1R, the method comprising contacting a cell that expresses GLP-1R on its cell surface with a composition comprising the synthetic GLP-1R binding protein of any of claims 1-86 or 89-94 or the pharmaceutical composition of any one of claims 95-104.

108. A method of modulating GLP-1R activity, the method comprising contacting a cell that expresses GLP-1R on its cell surface with a composition comprising the synthetic GLP-1R binding protein of any of claims 1-86 or 89-94 or the pharmaceutical composition of any one of claims 95-104.

109. The method of any of claim 107 or 108, wherein the synthetic GLP-1R binding protein promotes or increases GLP-1R activity in the presence of a GLP-1R ligand (e.g., GLP-1) or a reference GLP-1R agonist (e.g., semaglutide, liraglutide, dulaglutide, lixisenatide, exenatide, etc.) relative to GLP-1R activity in the absence of the GLP-1R ligand (e.g., GLP-1) or a reference GLP-1R agonist (e.g., semaglutide, liraglutide, dulaglutide, lixisenatide, exenatide, etc.).

110. A method of promoting or increasing GLP-lR-mediated activity in a subject in need thereof, the method comprising administering to the subject an effective amount of the synthetic GLP-1R binding protein of claims 1-86 or 89-94 or the pharmaceutical composition of any one of claims 95-104.

111. A method of treating one or more endocrine and / or metabolic disorders (e.g., type 2 diabetes mellitus, obesity, pancreatic dysfunction including uncontrolled blood glucose levels), gastrointestinal disorders (e.g., gastric motility disorders, e.g., irritable bowel syndrome, e.g., irritable bowel disease), renal disorders (e.g., reduced kidney function), hepatic disorders (e.g., nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD)), neurodegenerative / neurological disorders (e.g., Alzheimer’s disease, e.g., Parkinson’s disease) and / or cardiovascular disorders (e.g., heart attack, stroke), in a subject in need thereof, the method comprising administering to the subject an effective amount of the synthetic GLP-1R binding protein of claims 1-86 or 89-94 or the pharmaceutical composition of any one of claims 95-104.

112. The method of claim 111, wherein the administration is before, during, or after administration or use of one or more other agents.

113. The method of claim 112, wherein the one or more other agents is selected from: insulin, an agent for decreasing amount of blood glucose (e.g., produced by liver), an agent for decreasing amount of blood sugar absorbed by gastrointestinal tract (e.g., stomach and / or intestines), metformin, a reduced calorie diet, increased physical activity, and combinations thereof.

114. The method of any of claims 111-113, wherein the subject is diagnosed as having or at risk of having endocrine and / or metabolic disorders (e.g., type 2 diabetes mellitus, obesity, pancreatic dysfunction including uncontrolled blood glucose levels), gastrointestinal disorders (e.g., gastric motility disorders, e.g., irritable bowel syndrome, e.g., irritable bowel disease), renal disorders (e.g., reduced kidney function), hepatic disorders (e.g., nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD)), neurodegenerative / neurological diseases (e.g., Alzheimer’s, Parkinson’s) and / or cardiovascular disorders (e.g., heart attack, stroke).

115. A method of modulating one or more activities of a G-protein coupled receptor (GPCR) capable of activating a first downstream signaling pathway in a cell and a second, different downstream signaling pathway in the cell, wherein the GPCR comprises an agonist binding site such that when an agonist binds to the agonist binding site, the agonist activates the first and the second downstream signaling pathways in the cell, the method comprising: contacting the cell with a miniprotein comprising:(i) an agonist that binds to the agonist binding site of the GPCR, and(ii) a stabilization domain that interacts with a surface region of the GPCR adjacent to the agonist binding site, wherein the stabilization domain stabilizes the interaction of the agonist in the agonist binding site of the GPCR and causes the GPCR to activate the first downstream signaling pathway preferentially over the second downstream signaling pathway relative the same or a similar agonist without the stabilization domain.

116. The method of claim 115, wherein the GPCR is a GLP-1 receptor (GLP-1R).

117. The method of claim 115 or 116, wherein the first signaling pathway is a cAMP-mediated signaling pathway.

118. The method of any one of claims 115-117, wherein the second signaling pathway is a P arrestin-mediated signaling pathway.

119. The method of any one of claims 115-118, wherein the miniprotein comprises the synthetic GLP-1R binding protein of any one of claims 1-86 or 89-94.

120. A method of biasing a cAMP signaling pathway over a arrestin-mediated signaling pathway in a cell modulated by a GLP-1R, the method comprising: contacting the cell with the synthetic GLP-1 R binding protein of any one of claims 1-86 or 89-94 or the pharmaceutical composition of any one of claims 95-106, thereby to preferentially activate the cAMP-mediated signaling pathway over a P arrestin-mediated signaling pathway in the cell.

121. A method of biasing a cAMP signaling-mediated pathway over a P arrestin- mediated signaling pathway that are both modulated by a GLP-1R in a subject in need thereof, themethod comprising: administering to the subject an effective amount of the synthetic GLP- 1R binding protein of any one of claims 1-86 or 89-94 or the pharmaceutical composition of any one of claims 95-104, thereby to preferentially activate the cAMP-mediated signaling pathway over a P arrestin-mediated signaling pathway in the subject.

122. A method of reducing a side effect mediated by a GLP-1R activated arrestin-mediated signaling pathway in a subject in need thereof, the method comprising: administering to the subject an effective amount of the synthetic GLP-1R binding protein of any one of claims 1-86 or 89-94 or the pharmaceutical composition of claim of any one of claims 95-104, wherein the side effect is reduced relative to administration of a reference GLP-1R agonist that lacks a protein stabilizing domain capable of stabilizing the interaction of the GLP-1R agonist in an agonist binding site of the GLP-1R.

123. The method of claim 122, wherein the reference GLP-1R agonist just comprises the GLP- 1R agonist portion present in the synthetic GLP-1R binding protein of any one of claims 1- 86 or 89-94.

124. A method of increasing half-life of a synthetic GLP-1R binding protein, comprising adding an effector that extends serum half-life to the C-terminus of the synthetic GLP-1R binding protein of any one of claims 1-77.

125. The method of claim 124, wherein the effector is an albumin binding protein.

126. The method of claim 124 or 125, wherein the albumin binding protein is a synthetic albumin binding protein with an amino acid sequence comprising SEQ ID NO: 163.

127. The method of any one of claims 124-127, wherein the serum half-life of the GLP-1R binding protein is increased as compared to a GLP-1R agonist (e.g., semaglutide).

128. The method of claim 127, wherein the GLP-1R agonist has an amino acid sequence comprising SEQ ID NO: 106.

129. A method of reducing blood glucose concentration in a subject in need thereof, comprising administering to the subject an effective amount of the synthetic GLP-1R binding proteinof any one of claims 1-86 or 89-94, thereby to reduce blood glucose concentration relative to blood glucose concentration prior to administration.

130. The method of claim 129, wherein the synthetic GLP-1R binding protein is according to the synthetic GLP-1R binding protein of any one of claims 70-77.

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