Methods for developing GLP-1 related polyagonists

Abstract

Embodiments herein relate generally to peptides with agonistic or antagonistic activity to one or more human receptors selected from glucagon receptor, amylin receptor, calcitonin receptor, gastic inhibitory peptide (GIP) receptor and GLP-1 receptor. In some embodiments, the peptides disclosed herein are polyagonists and target two or more receptors. In some embodiments, one or both peptides comprise partial agonistic or antagonistic activity. Other embodiments herein relate to methods for screening and / or identifying peptides that have biological activity and / or agonistic activity to a receptor disclosed herein.

Description

Attorney Docket No: 01123-0022-00PCTMETHODS FOR DEVELOPING GLP-1 RELATED POLYAGONISTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 689,046, filed August 30, 2024, the disclosure of which is hereby incorporated by reference in its entirety.REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0002] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on August 19, 2025, is named “01123-0022-00PCT-ST26.xml” and is 26,442 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.GOVERNMENT SUPPORT

[0003] This invention was made with government support under National Institutes of Health (NIH) grants DK031036, DK082659, R01AG045215, R21AG071436, and R35GM122610. The government has certain rights in the invention.FIELD

[0004] Embodiments herein relate generally to peptides with agonistic or antagonistic activity to one or more human receptors selected from glucagon receptor, amylin receptor, calcitonin receptor, gastric inhibitory peptide (GIP) receptor and GLP-1 receptor. In some embodiments, the peptides disclosed herein are polyagonists and target two or more receptors. In some embodiments, one or both peptides comprise partial agonistic or antagonistic activity. Other embodiments herein relate to methods for screening and / or identifying peptides that have biological activity and / or agonistic activity to a receptor disclosed herein.BACKGROUND

[0005] Incretin peptides are hormones and peptides that, among other things, help regulate glycemic homeostasis and often regulate blood glucose levels in mammals, sometimes by increasing the amount of insulin released in response to glucose. Incretin peptides may also reduce glucagon production, increase satiety, delay gastric emptying, regulate white andAttorney Docket No: 01123-0022-00PCT brown adipose tissue and elicit weight loss. In addition, they can sometimes improve insulin sensitivity, and may increase islet cell neogenesis (the formation of new islets). Notable mammalian incretin peptides include glucose-dependent insulinotropic polypeptide, also known as gastric inhibitory polypeptide (GIP), glucagon, glucagon-like peptide- 1 (GLP-1), glucagon-like peptide-2 (GLP-2), oxyntomodulin, glicentin, glicentin-related pancreatic polypeptide (GRPP), amylin, GIP receptor and calcitonin. GLP-1, GLP-2, GRPP, oxyntomodulin and glicentin are produced by enzymatic digestion of proglucagon in a celltype and / or tissue-specific manner, often by digestion with dipeptidyl peptidase IV.

[0006] Mammalian incretins have been shown to induce their biological activity by binding to and / or signaling through their cognate receptors, which often times are somewhat promiscuous in their binding specificity. For example, GLP-1, glucagon and oxyntomodulin can bind to and signal through the GLP-1 receptor (GLP-1R), while glucagon can also bind and signal through the glucagon receptor. Some incretins are poly-active and may exert agonistic activity when binding to one receptor and antagonistic activity when binding to a second receptor.

[0007] Incretin peptide mimetics and synthetic analogues of incretins are useful for modulating a number of metabolic responses in mammals and some have proved useful for treating certain metabolic diseases and / or disorders.SUMMARY

[0008] Disclosed herein, in some aspects, are incretin peptides derived from, in part, one or more of four C.elegans peptides termed PDF-la, PDF-lb, PDF-2 and NLP-49.

[0009] Disclosed herein, in some aspects, are methods of making, testing, screening and / or selecting peptides that can induce a desired biological activity similar to human incretin peptides in a mammal, or in a mammalian cell. In some embodiments, the peptides presented herein are derived from, or are variants of, any one of SEQ ID NOs: 1-14, or portions thereof. In some embodiments, a peptide has at least 70% identity to an amino acid sequence selected from any one of SEQ ID NOs: 1-14, or a portion thereof. In some embodiments, a peptide having at least 70% identity to an amino acid sequence selected from any one of SEQ ID NOs: 1-14, or a portion thereof, further comprises about 20% or more identity to a human incretin sequence of any one of SEQ ID NOs: 15-26.

[0010] In some aspects, presented herein is a method of determining whether a peptide activates a human receptor, the method comprising (i) contacting a mammalian cell with aAttorney Docket No: 01123-0022-00PCT peptide having at least 70% identity to an amino acid sequence selected from any one of SEQ ID NOs: l-14, and (ii) determining if the peptide induces an increase in intracellular cyclic AMP (cAMP) production, wherein the cell expresses at least one human receptor selected from a glucagon receptor, GLP-1 receptor, GLP-2 receptor, Amylin receptor, GIP receptor and calcitonin receptor, and wherein an increase in intracellular cAMP as determined in (ii) indicates the peptide activates the receptor. In some embodiments, the mammalian cell is a human cell. In some embodiments, the cell overexpresses the one or more receptors. In some embodiments, an increase in cAMP is determined by comparison of cAMP levels determined in (ii) with a standard curve, or by comparison to cAMP levels produced in a cell that is contacted with a control peptide or vehicle.

[0011] In some aspects, presented herein is a method of determining whether a peptide antagonizes a human receptor, the method comprising (i) contacting a mammalian cell with a peptide having at least 70% identity to an amino acid sequence selected from any one of SEQ ID NOs: l-14, and (ii) determining if the peptide inhibits intracellular cyclic AMP (cAMP) production, wherein the cell expresses at least one human receptor selected from a glucagon receptor, GLP-1 receptor, GLP-2 receptor, Amylin receptor, GIP receptor and calcitonin receptor, and wherein an inhibition of intracellular cAMP production as determined in (ii) indicates the peptide antagonizes the receptor. In some embodiments, the mammalian cell is a human cell. In some embodiments, the cell overexpresses the one or more receptors.

[0012] In some aspects, presented herein is a method of determining whether a peptide affects food intake of a mammal, the method comprising (i) administering a peptide having at least 70% identity to an amino acid sequence selected from any one of SEQ ID NOs: 1-14 to a mammal, and (ii) determining an amount of food intake by the mammal over a period of at least 48 hours, wherein a change in the amount of food intake following administration of the peptide indicates the peptide affects food intake of the mammal. In some embodiments, a change in the amount of food intake is determined by (a) comparing the amount of food intake before administration of the peptide with the amount of food intake after administration of the peptide; or (b) comparing a change in the amount of food intake of the mammal after administration of the peptide, to a change in the amount of food intake of a control mammal after administration of a control peptide or control vehicle to the control mammal, wherein the control mammal is of the same species.

[0013] In some aspects, presented herein is a method of determining the effect of a peptide on blood glucose or blood insulin level in a mammal, the method comprising (i)Attorney Docket No: 01123-0022-00PCT administering a peptide having at least 70% identity to an amino acid sequence selected from any one of SEQ ID NOs: 1-14 to a mammal, and (ii) determining an amount of glucose or insulin in the blood of the mammal before and after administration of the peptide, wherein a change in the amount of glucose or insulin in the blood as determined in (ii) indicates the peptide affects blood glucose and / or blood insulin level, optionally wherein the effect of the peptide on the blood glucose or the blood insulin level is determined during a fasting state of the mammal, during a fed state of the mammal, or after the mammal is glucose challenged. In some embodiments, the amount of food intake is determined by providing an excess amount of food to the mammal and determining the amount of food that is uneaten by the mammal after a period of, for example, 30 minutes to 24 hours following the providing of the food.

[0014] In some aspects, presented herein is a method of determining the effect of a peptide on body weight in a mammal, the method comprising (i) administering a peptide having at least 70% identity to an amino acid sequence selected from any one of SEQ ID NOs: 1-14 to a mammal, and (ii) determining a body weight of the mammal before and after administration of the peptide, wherein a change in body weight (e.g., a decrease in body weight) as determined in (ii) indicates the peptide affects body. In some embodiments, body weight is determined 24 hours, 48 hours, 2 days, 3 days, 1 week, 2 weeks, or 1 month after administration of the peptide. In some embodiments, a change in body weight is determined by comparing the body weight after administration of the peptide to a body weight of the mammal prior to the administration.

[0015] In some embodiments, a mammal is a rodent, (e.g., a mouse or a rat). In some embodiments, a mammal is a human.

[0016] In some embodiments, a peptide herein is conjugated to a lipid, lipid nanoparticle, albumin, or PEG.

[0017] In some aspects, presented herein is a method of making or screening a peptide library or a member of a peptide library for biological activity. In some embodiments, a method of screening a peptide library or a member of a peptide library comprises contacting a mammalian cell with one or more peptides of the library. In some embodiments, constructing a peptide library comprises modifying a peptide sequence comprising any one of SEQ ID NOs:l-14.

[0018] In some aspects, presented herein is a peptide found to activate one or more of a human glucagon receptor, GLP-1 receptor, GLP-2 receptor, amylin receptor, GIP receptor or a calcitonin receptor according to a method disclosed herein. In some aspects, presentedAttorney Docket No: 01123-0022-00PCT herein is a peptide found to antagonize one or more of a human glucagon receptor, GLP-1 receptor, GLP-2 receptor, amylin receptor, GIP receptor or a calcitonin receptor according to a method disclosed herein. In some embodiments, presented herein is a peptide having biological activity. In some embodiments, presented herein is a peptide that affects food intake, blood glucose level, blood insulin level, or body weight of a mammal according to a method disclosed herein.

[0019] Additional objects and advantages will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0020] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

[0021] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiment(s) and together with the description, serve to explain the principles described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 shows a consensus sequence (SEQ ID NO: 15) of the human proglucagon polypeptide showing peptide precursor peptides of glucagon, GLP-1 and GLP-2.

[0023] Figures 2 A and 2B show embodiments of protease processing of a human proglucagon polypeptide (SEQ ID NO: 15), and potential resulting peptides and potential tissue-specific associations.

[0024] Figure 3 shows an alignment of human calcitonin (CAL) (SEQ ID NO: 25) and C.elegans PDF- la peptide (SEQ ID NO: 1) (Top) and an alignment of human proglucagon (Glc) (SEQ ID NO: 15) and C.elegans PDF-la peptide (SEQ ID NO: 1) (Bottom).

[0025] Figure 4 shows an alignment of human proglucagon (SEQ ID NO: 15) and C.elegans PDF-la peptide (SEQ ID NO: 1) with references to the processed peptide portions of proglucagon. Sequence alignment of PDF-la with proglucagon is greatest in the region of, GRRP, glucagon, and the last few amino acids of GLP-1.

[0026] Figure 5 shows an alignment of C.elegans PDF-la peptide (SEQ ID NO: 1), C.elegans PDF-lb peptide (SEQ ID NO: 6), Tirzepatide (SEQ ID NO: 27) and a portion of human proglucagon (SEQ ID NO: 15).Attorney Docket No: 01123-0022-00PCT

[0027] Figure 6 shows an alignment of Human proglucagon (Uniprot Ref. P01275) (SEQ ID NO: 15), C.elegans NLP-49 peptide (SEQ ID NO: 13), C.elegans PDF-2 peptide (SEQ ID NO: 9), C.elegans PDF-la peptide (SEQ ID NO: 1), C.elegans PDF-lb peptide (SEQ ID NO: 6).

[0028] Figure 7 shows an alignment of Human amylin (IAPP; Uniprot Ref. Pl 0997) (SEQ ID NO: 24) and C.elegans PDF-la peptide (SEQ ID NO: 1).

[0029] Figure 8 shows an alignment of Human calcitonin (Uniprot Ref. P01258) (SEQ ID NO: 25), C.elegans PDF-la peptide (SEQ ID NO: 1), Human proglucagon (Uniprot Ref. P01275) (SEQ ID NO: 15), and Human amylin (IAPP; Uniprot Ref. Pl 0997) (SEQ ID NO: 24).

[0030] Figure 9 shows an alignment of Human gastric inhibitory peptide (GIP; Uniprot Ref. P09681) (SEQ ID NO: 26), and C.elegans PDF-la peptide (SEQ ID NO: 1).DESCRIPTION OF THE SEQUENCES

[0031] Table 1 provides a listing of certain sequences referenced herein.Table 1: Description of the SequencesAttorney Docket No: 01123-0022-00PCTAttorney Docket No: 01123-0022-00PCTDETAILED DESCRIPTION

[0032] As used herein, the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term about generally refers to a range of numerical values (e.g., + / -5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some instances, the term about may include numerical values that are rounded to the nearest significant figure.

[0033] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0034] In this application, the use of “or” means “and / or” unless stated otherwise. In the context of a multiple dependent claim, the use of “or” refers back to more than one preceding independent or dependent claim in the alternative only. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.

[0035] As used herein, the transition term “consisting essentially of’ or grammatical variations thereof, when referring to steps of a claimed process signifies that the process comprises no additional steps beyond those specified that would materially affect the basic and characteristics of the process. As used herein, the transition term “consisting essentially of’ or grammatical variations thereof, when referring to a composition or product, such as a kit, signifies that it comprises no additional components beyond those specified that would materially affect its basic and characteristics. As used herein, the transition term “consisting essentially of’ or grammatical variations thereof, when referring to a peptide having a percent identity to another peptide means that the peptide consists of an amino acid sequence having the indicated identity, where the peptide may comprise other non-amino acid moieties or nonprotein based molecules conjugated (e.g., by covalent linkage) to the indicated peptide, nonlimiting examples of which include lipids, PEG, toxins, carriers, nucleotides, nucleic acids, and the like.Attorney Docket No: 01123-0022-00PCT

[0036] As used herein, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise.

[0037] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0038] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0039] The term “percent identical” or “percent identity” refers to sequence identity between two amino acid sequences. Identity can be determined by aligning two amino acid sequences using a suitable method and comparing the amino acids of the two peptides at each position of the alignment. When an equivalent position in the compared sequences is occupied by the same amino acid, then the molecules are identical at that position. For any particular alignment, percent identity is often calculated as the number of amino acids that are the same divided by the arithmetic mean sequence length that is compared. Various alignment algorithms and / or programs may be used to determine percent identity, including FASTA, BLAST, or ENTREZ. FASTA and BLAST are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default settings. ENTREZ is available through the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md. In one embodiment, the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, e.g., each amino acid gap is weighted as if it were a single amino acid or mismatch between the two sequences.

[0040] In some embodiments, the amino acid sequences of two or more peptides are compared and the two or more peptides are determined to share conserved amino acids (e.g., conserved amino acid residues). The term, “conserved amino acids” and “conserved amino acid residues” are used interchangeably and said terms refer to those amino acids that are the same between two or more peptide sequences that are compared by an alignment. The term, “non-conserved amino acids” refers to those amino acids that are not the same between two or more peptide sequences that are compared by an alignment.Attorney Docket No: 01123-0022-00PCT

[0041] In some embodiments a peptide is modified. In some embodiments, a peptide can be modified by deletion of one or more amino acids, addition of one or more amino acids or substitution of one or more amino acids. A peptide can be modified using any suitable method.

[0042] In some embodiments, a peptide comprises a conservative substitution of one or more amino acids. A “conservative substitution” refers to the substitution of an amino acid with a another chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Non-limiting examples of conservative substitutions include amino acids listed in one of the following eight groups, each of which list examples of amino acids that are conservative substitutions for one another: Group 1 : Alanine (A), Glycine (G); Group 2: Aspartic acid (D), Glutamic acid (E); Group 3: Asparagine (N), Glutamine (Q); Group 4: Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); Group 6: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Group 7: Serine (S), Threonine (T); and Group 8: Cysteine (C), Methionine (M). In some embodiments, a peptide comprising one or more conservative substitutions retains a desired function described herein (e.g., receptor binding or biological activity).

[0043] In some embodiments a peptide comprises a modification. In certain embodiments, one or more amino acids in a peptide are modified. Non-limiting examples of a modified amino acid or peptide include Phosphorylation; De-phosphorylation; Oxidization; Reduction; Citrullination; lipidation (including but not limited to: prenylation, myristylation, palmitoylation; isoprenylation; famesylation; geranylgeranylation; and the like); Acylation; Alkylation; Amidation; Arginylation; Polyglutamylation; Polyglycylation; Adenylylation; Sulfation; Carbamylation; Carbonylation; Glutathionylation; Succynlation; Nitration; Nitrosylation; Di-sulfide bonding (Cysteine); Transglutamination; Deamidation; Misfolding; Aggregation; Ubiquitination; Sumoylation; and Racemization. The aforementioned modifications are not intended to be limited by scope or category but rather an illustration of how peptides or amino acids may be modified. In some embodiments, a peptide comprising a modification (e.g., a modification of an amino acid) retains a desired function described herein. A peptide disclosed herein can be made and / or modified using a suitable method known in the art and any amino acid of a peptide disclosed herein can substituted with another amino acid using a suitable method. In some embodiments, a peptide or one or more amino acids of a peptide are modified to increase or decrease in vivo half-life of a peptide, improve solubility, increase or decrease receptor binding (e.g., binding affinity and / or on-offAttorney Docket No: 01123-0022-00PCT rate), change receptor binding specificity, increase or decrease signaling through a receptor, and / or the like.

[0044] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. In some embodiments, a peptide comprises at least 5, at least 10, at least 15, at least 20 or at least 25 amino acids in length. In some embodiments, a peptide comprises less than 250, less than 200, less than 150, less than 100, less than 75 or less than 50 amino acids in length. In some embodiments, a peptide comprises or consists of 5 to 250 amino acids, 5 to 200 amino acids, 5 to 150 amino acids, 5 to 100 amino acids, 10 to 100 amino acids, or 20 to 50 amino acids in length.

[0045] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.

[0046] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0047] As used herein “subsequence” refers to a portion of a protein or peptide that is at least one amino acid less in length than the full-length protein or peptide.

[0048] As used herein “variant” and grammatical variations thereof, refers to a protein or peptide comprising an amino acid sequence that deviates from a reference protein or peptide sequence. Modified and variant proteins or peptides may therefore have greater or less activity or function than a reference protein or peptide but at least retain partial activity or function of a reference protein or peptide.

[0049] The term “subject” refers to a mammal. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some embodiments a mammal is a human. In some embodiments a mammal is a rodent. In some embodiments, a mammal is a mouse or a rat. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female.

[0050] In some embodiments, peptides disclosed herein are modified and / or mutated. In some embodiments, a peptide can be modified or mutated by deleting, substituting and / orAttorney Docket No: 01123-0022-00PCT adding one or more amino acids, using a suitable method. In some embodiments, peptides disclosed herein (e.g., mutated and / or variant peptides) are generated and screened for biological activity. In some embodiments, peptides disclosed herein (e.g., mutated and / or variant peptides) are generated by modifying or mutating amino acids in any one of SEQ ID NOs: 1-14. In some embodiments, mutated and / or variant peptides are generated by modifying or substituting one or more non-conserved amino acids of any one of SEQ ID NOs: 1-14. Non-conserved amino acids are identified in SEQ ID NOs: 1-14 by aligning one or more of SEQ ID NOs: 1-14 (C.elegans peptides) with one or more of SEQ ID NOs: 15-26 (Human peptides). In some embodiments, amino acids in SEQ ID NOs: 1-14 that are conserved between one or more of SEQ ID NOs: 1-14 (C.elegans peptides) and one or more of SEQ ID NOs: 15-26 (Human peptides) are not mutated, substituted, deleted, or modified. In some embodiments, a peptide is mutated by substitution of one or more non-conserved amino acids with alanine. In some embodiments, a peptide is mutated using a suitable high throughput method, non-limiting examples of which include alanine-scanning mutagenesis, or a variation thereof, or a method disclosed in: Heydenreich, el al. (2017) Scientific Reports 7 (6787); Strain-Damerell C, Burgess-Brown NA. (2019) High-Throughput Site-Directed Mutagenesis. Methods Mol Biol. 2025:281-296; Heydenreich FM, Miljus T, Milic D, Veprintsev DB. (2020) High-throughput Site-directed Scanning Mutagenesis Using a Two- fragment PCR Approach. Bio Protoc. 10(l):e3484; Han Xiao, Zehua Bao, and Huimin Zhao (2015) Industrial & Engineering Chemistry Research 54 (16), 4011-4020; or the like. In some embodiments, a library of mutated peptides is generated using a suitable method, for example one of the methods disclosed above.

[0051] In some embodiments, disclosed herein is a method of determining whether a peptide activates a human receptor selected from a glucagon receptor, GLP-1 receptor, GLP-2 receptor, Amylin receptor, GIP receptor and a calcitonin receptor on a cell. In some embodiments, the peptides are derived from one of SEQ ID NOs: 1-14. In some embodiments, the peptides are variants of one of SEQ ID NOs: 1-14. In some embodiments a peptide selected from one of SEQ ID NOs: 1-14 is mutated and or modified. In certain embodiments, the modified and / or mutated peptides are tested to determine if they bind to, and / or activate one or more human receptors selected from a glucagon receptor, GLP-1 receptor, GLP-2 receptor, Amylin receptor, GIP receptor and calcitonin receptor by a suitable method. In some embodiments, methods disclosed herein provide a process to select peptides that are agonist or polyagonist to one or more receptors selected from a glucagon receptor,Attorney Docket No: 01123-0022-00PCTGLP-1 receptor, GLP-2 receptor, Amylin receptor, GIP receptor and a calcitonin receptor. In some embodiments, peptide disclosed herein are tested for biological activity in vitro and / or in vitro. For example, in some embodiments a peptide is contacted with a cell to determine if it induces a desired biological activity (e.g., receptor agonist activity) and in other embodiments, a peptide is administered to a mammal to determine if the peptide induces a desired biological activity.

[0052] Disclosed herein, in some embodiments, are method a making, testing, screening and / or selecting peptides that can induce a desired biological activity. In some embodiments, a peptide has at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% identity to an amino acid sequence selected from any one of SEQ ID NOs: 1- 14, or a portion thereof. In some embodiments, a peptide comprises, consists of, or consists essentially of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to an amino acid sequence selected from any one of SEQ ID NOs: 1-14, or to a portion thereof. In some embodiments, a peptide comprises, consists of, or consists essentially of an amino acid sequence having 80% to 99%, 80% to 95%, 80% to 90%, 85% to 99%, or 85% to 95% identity to a peptide selected from SEQ ID NOs: 1-14, or to a portion thereof. In certain embodiments, a peptide comprises, consists of, or consists essentially of an amino acid sequence having about 80%, 85%, 90%, 95%, 97%, 99% or 100% identity to a peptide selected from SEQ ID NOs: 1-14, or to a portion thereof.

[0053] In some embodiments, a peptide comprising, consisting of, or consisting essentially of an amino acid sequence having identity to one of SEQ ID NOs: 1-14 further comprises identity to one or more of SEQ ID NOs: 15-26. In some embodiments, a peptide has at least 40%, at least 50%, at least 60%, at least 70% identity, at least 80%, at least 90% or at least 95% to an amino acid sequence selected from any one of SEQ ID NOs: 15-26, or a portion thereof. In some embodiments, a peptide disclosed herein has about 20% to 90%, 20% to 80%, 20% to 60%, 20% to 40%, 5% to 40%, 5% to 20%, 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to an amino acid sequence selected from any one of SEQ ID NOs: 15-26. In certain embodiments, a peptide comprises, consists of, or consists essentially of an amino acid sequence having about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or 100% identity to a peptide selected from SEQ ID NOs: 15-26, or to a portion thereof. In certain embodiments, a peptide comprises, consists of, or consists essentially of an amino acid sequence having less than 60%, less than 50%, less than 40%,Attorney Docket No: 01123-0022-00PCT less than 30%, or less than 20% identity to a peptide selected from SEQ ID NOs: 15-26, or to a portion thereof.

[0054] In some embodiments, a peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO:2and / or 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 16. In some embodiments, a peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:2 and SEQ ID NO: 16.

[0055] In some embodiments, a peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO:7and / or 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 16. In some embodiments, a peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:7 and SEQ ID NO: 16.

[0056] In some embodiments, a peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: lOand / or 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 16. In some embodiments, a peptide comprises all of the amino acid residues that are conserved between SEQ ID NO: 10 and SEQ ID NO: 16.

[0057] In some embodiments, a peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 14. In some embodiments, a peptide comprises all of the amino acid residues that are conserved between SEQ ID NO: 14 and SEQ ID NO: 16.

[0058] In some embodiments, a peptide comprises an amino acid sequence of: PXnFXnSX; PXnYXnPXnFXnSX; PXnYRXPXnDXnFXnKRXn, or PXnYRXnPXnYXnDXnFXnKRXn, where X is any amino acid and n is a number between 0 and 15, or between 1 and 10 .

[0059] In some embodiments, a peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 3and / or SEQ ID NO: 18. In some embodiments, a peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:3 and SEQ ID N0: 18.

[0060] In some embodiments, a peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 8 and / or SEQ ID NO: 18. In some embodiments, a peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:8 and SEQ ID N0: 18.

[0061] In some embodiments, a peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 1 land / or SEQ ID NO: 18. In some embodiments, a peptide comprises all of the amino acid residues that are conserved between SEQ ID NO: 11 and SEQ ID N0: 18.Attorney Docket No: 01123-0022-00PCT

[0062] In some embodiments, a peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 14 and / or SEQ ID NO: 18. In some embodiments, a peptide comprises all of the amino acid residues that are conserved between SEQ ID NO: 14 and SEQ ID NO: 18.

[0063] In some embodiments, a peptide comprises an amino acid sequence of PXnFXnSXnMDXnAXnLXnLXn;XnSXnAXnPLLXnRAPQXnMXnDXnAXnLXnGXnMDXnKLXnRXnGLXn; or PXnYRXPXnDXnFXnKRXnSXnLINGLIGXnKLXnKRSNXnLINXn, where X is any amino acid and n is a number between 0 and 15, or between 1 and 10.

[0064] In some embodiments, a peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 4 and / or SEQ ID NO: 17. In some embodiments, a peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:4 and SEQ ID NO: 17.

[0065] In some embodiments, a peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 12 and / or SEQ ID NO: 17. In some embodiments, a peptide comprises all of the amino acid residues that are conserved between SEQ ID NO: 12 and SEQ ID NO: 17.

[0066] In some embodiments, a peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO:4 and / or SEQ ID NO: 19. In some embodiments, a peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:4 and SEQ ID NO: 19.

[0067] In some embodiments, a peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 12 and / or SEQ ID NO: 19. In some embodiments, a peptide comprises all of the amino acid residues that are conserved between SEQ ID NO: 12 and SEQ ID NO: 19.

[0068] In some embodiments, a peptide comprises an amino acid sequence of GXnDXnKXnRXnL; XnGXnMDXnKLXnRXnGLXn; or LINGLIGXnKLXnKRSNXnLINXn, where X is any amino acid and n is a number between 0 and 15, or between 1 and 10.

[0069] In some embodiments, a peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO:5 and / or SEQ ID NO:21. In some embodiments, a peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:5 and SEQ ID NO:21.Attorney Docket No: 01123-0022-00PCT

[0070] In some embodiments, a peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO:5 and / or SEQ ID NO:22. In some embodiments, a peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:5 and SEQ ID NO:22.

[0071] In some embodiments, a peptide comprises an amino acid sequence of XnGXnGRRXn; XnGAGRRXn; XnKXnGAGRRXn; XnKXnSGAGRRXn; XnKLSGAGRRXn; or XnNKLSGAGRRXn, where X is any amino acid and n is a number between 0 and 15, or between 1 and 10.

[0072] In some embodiments, a peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 1 and / or SEQ ID NO:24. In some embodiments, a peptide comprises all of the amino acid residues that are conserved between SEQ ID NO: 1 and SEQ ID NO:24.

[0073] In some embodiments, a peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 1 and / or SEQ ID NO:25. In some embodiments, a peptide comprises all of the amino acid residues that are conserved between SEQ ID NO: 1 and SEQ ID NO:25.

[0074] In some embodiments, a peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 1 and / or SEQ ID NO:25. In some embodiments, a peptide comprises all of the amino acid residues that are conserved between SEQ ID NO: 1 and SEQ ID NO:26.

[0075] In some embodiments, a peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO:6 and / or SEQ ID NO:24. In some embodiments, a peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:6 and SEQ ID NO:24.

[0076] In some embodiments, a peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO:6 and / or SEQ ID NO:25. In some embodiments, a peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:6 and SEQ ID NO:25.

[0077] In some embodiments, a peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO:9 and / or SEQ ID NO:24. In some embodiments, a peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:9 and SEQ ID NO:24.Attorney Docket No: 01123-0022-00PCT

[0078] In some embodiments, a peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO:9 and / or SEQ ID NO:25. In some embodiments, a peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:9 and SEQ ID NO:25.

[0079] In some embodiments, a peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 13 and / or SEQ ID NO:24. In some embodiments, a peptide comprises all of the amino acid residues that are conserved between SEQ ID NO: 13 and SEQ ID NO:24.

[0080] In some embodiments, a peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 13 and / or SEQ ID NO:25. In some embodiments, a peptide comprises all of the amino acid residues that are conserved between SEQ ID NO: 13 and SEQ ID NO:25.

[0081] In some embodiments, a peptide comprises an amino acid sequence of 3 to 9 amino acids having 80% to 99%, 85% to 99%, 90% identity to SEQ ID NO:5 or wherein the peptide comprises the amino acid sequence of SEQ ID NO: 5.

[0082] In some embodiments, a peptide is conjugated to a suitable moiety that may extend in vivo half-life and / or direct in vivo biological activity of a peptide, non-limiting examples of which include a lipid, lipid nanoparticle, albumin, and PEG.

[0083] In some embodiments, a library of peptides is generated where the library comprises a plurality of mutated and / or modified peptides derived from one or more of SEQ ID NOs: 1- 14. In some embodiments, a peptide is a member of a peptide library. In some embodiments, a method comprises contacting a mammalian cell with the peptide library and / or identifying a peptide of the library that has biological activity.

[0084] In some embodiments, a method comprises contacting a cell (e.g., a mammalian cell) with a peptide disclosed herein and determining if the peptide induces an increase in intracellular cyclic AMP (cAMP) production in the cell using a suitable method. In some embodiments, the cell expresses at least one mammalian receptor selected from a glucagon receptor, GLP-1 receptor, GLP-2 receptor, Amylin receptor, GIP receptor and calcitonin receptor. In some embodiments, a method comprises contacting a cell (e.g., a mammalian cell) with a peptide disclosed herein, and determining if the peptide induces an increase in intracellular cyclic AMP (cAMP) production in the cell; where an increase in intracellular cAMP indicates the peptide activates one or more of a receptor selected from a glucagon receptor, GLP-1 receptor, GLP-2 receptor, Amylin receptor, GIP receptor and calcitoninAttorney Docket No: 01123-0022-00PCT receptor. In some embodiments, an increase in intracellular cAMP indicates the peptide is an agonist of a receptor expressed on the mammalian cell.

[0085] In some embodiments, a method comprises contacting a cell (e.g., a mammalian cell) with a peptide disclosed herein and determining if the peptide antagonizes a human incretin receptor disclosed herein. In some embodiments, a method comprises contacting a cell (e.g., a mammalian cell) with a peptide disclosed herein and determining if the peptide blocks or inhibits binding of a human ligand to the incretin receptor. In some embodiments, a method comprises contacting a cell (e.g., a mammalian cell) with a peptide disclosed herein and determining if the peptide inhibits an increase in intracellular cyclic AMP (cAMP) production in the cell using a suitable method, where the increase in cAMP is induced by a known agonistic of the receptor. In some embodiments, the cell expresses at least one mammalian receptor selected from a glucagon receptor, GLP-1 receptor, GLP-2 receptor, Amylin receptor, GIP receptor and calcitonin receptor. In some embodiments, a method comprises contacting a cell (e.g., a mammalian cell) with a peptide disclosed herein, and determining if the peptide induces an increase in intracellular cyclic AMP (cAMP) production in the cell; where an increase in intracellular cAMP indicates the peptide activates one or more of a receptor selected from a glucagon receptor, GLP-1 receptor, GLP-2 receptor, Amylin receptor, GIP receptor and calcitonin receptor. In some embodiments, an increase in intracellular cAMP indicates the peptide is an agonist of a receptor expressed on the mammalian cell.

[0086] Any suitable cell can be used for a method described herein. In some embodiments, a cell is an oocyte or a red blood cell. In some embodiments, a cell is a mammalian cell. In some embodiments, a mammalian cell is a human cell. In some embodiments a mammalian cell is known to express one, two or three receptors selected from a glucagon receptor, GLP-1 receptor, GLP-2 receptor, Amylin receptor, GIP receptor and calcitonin receptor. In certain embodiments, a mammalian cell is derived from a suitable mammalian tissue or organ (e.g., pancreas, liver, kidney, liver, intestine, brain and the like). In some embodiments, a mammalian cell is engineered to express one or more receptors using a suitable method. In some embodiments, a mammalian cell expresses or overexpresses one or more receptors selected from a glucagon receptor, GLP-1 receptor, GLP-2 receptor, Amylin receptor, GIP receptor and calcitonin receptor.

[0087] In some embodiments, a method comprises (i) contacting a mammalian cell with a peptide having at least 40%, at least 50%, at least 60%, at least 70% identity, at least 80%, atAttorney Docket No: 01123-0022-00PCT least 90% or at least 95% to an amino acid sequence selected from any one of SEQ ID NOs: l-14, and (ii) determining if the peptide induces an increase in intracellular cyclic AMP (cAMP) production, wherein the cell expresses one, or at least one human receptor, selected from a glucagon receptor, GLP-1 receptor, GLP-2 receptor, Amylin receptor, GIP receptor and calcitonin receptor, and if an increase in intracellular cAMP is detected in (ii), the peptide activates the receptor and / or is an agonist of the one or at least one receptor. In some embodiments, an increase in cAMP is determined by comparison of the cAMP levels determined in (ii) with a standard curve, or by comparison to cAMP levels produced in a cell that is contacted with a control peptide or vehicle. In some embodiments, a control peptide comprises a peptide that does not bind to one or more receptors of interest.

[0088] In some embodiments, a method comprises determining whether a peptide affects food intake of a mammal, the method comprising (i) administering a peptide (e.g., a peptide having at least 70% identity to an amino acid sequence selected from any one of SEQ ID NOs: 1-14 to a mammal), and (ii) determining an amount of food intake by the mammal over a period of at least 48 hours, wherein a change in the amount of food intake following administration of the peptide indicates the peptide affects food intake of the mammal. In certain embodiments, an amount of food intake is determined over a suitable period of time, non-limiting examples of which include a period of 24 hours, 48 hours, 72 hours or longer. In some embodiments, a change in the amount of food intake is determined by (a) comparing the amount of food intake before administration of the peptide with the amount of food intake after administration of the peptide; or (b) comparing a change in the amount of food intake of the mammal after administration of the peptide, to a change in the amount of food intake of a control mammal after administration of a control peptide or control vehicle to the control mammal, wherein the control mammal is of the same species. A suitable control animal can be used for a method described herein. In some embodiments, a control animal is the same species, the same or comparable body weight and / or the same or comparable age as an experimental animal. In some embodiments, an amount of food intake is determined using a suitable method, for example by providing an excess amount of food to the mammal and determining the amount of food that is uneaten by the mammal after a period of, for example, 30 minutes to 24 hours following the providing of the food.

[0089] In some embodiments, a method comprises determining the effect of a peptide on blood glucose or blood insulin level in a mammal, the method comprising (i) administering a peptide (e.g., a peptide having at least 70% identity to an amino acid sequence selected fromAttorney Docket No: 01123-0022-00PCT any one of SEQ ID NOs: 1-14) to a mammal, and (ii) determining an amount of glucose or insulin in the blood of the mammal before and after administration of the peptide, wherein a change in the amount of glucose or insulin in the blood as determined in (ii) indicates the peptide affects blood glucose and / or blood insulin level. Blood glucose levels and / or blood insulin levels can be measured or determined using a suitable method. In some embodiments, blood glucose levels, blood insulin levels or changes thereof are measured or determined using a suitable mammal (e.g., a rodent) that is in a fasting state, in a fed state, or when the mammal is glucose challenged.

[0090] In some embodiments, a method disclosed herein comprises determining body weight, or a change in body weight of a mammal. Body weight, and / or changes in body weight of a mammal can be determined using a suitable method. In some embodiments, a method disclosed herein comprises determining body weight of the mammal before and after administration of a peptide or control peptide, such as 24 hours, 48 hours, 2 days, 3 days, 1 week, 2 weeks, or 1 month after administration of a peptide. In some embodiments, a method of determining the effect of a peptide on body weight of a mammal comprising (i) administering a peptide (e.g., a peptide having at least 70% identity to an amino acid sequence selected from any one of SEQ ID NOs: 1-14) to a mammal, and (ii) determining a body weight of the mammal before and after the administering of (i), wherein a change in the body weight of the mammal indicates the peptide comprises biological activity. In some embodiments, a decrease in body weight indicates a peptide has biological activity. In some embodiments, a decrease in body weight indicates a peptide has agonistic activity or is an agonist to a receptor of interest. In some embodiments, body weight is determined 24 hours, 48 hours, 2 days, 3 days, 1 week, 2 weeks, or 1 month after administration of a peptide. In some embodiments, body weight of the mammal is measured prior to the administration.

[0091] In some embodiments, a mammal is a rodent. In some embodiments, a rodent is a rat or a mouse. In some embodiments, a mammal is a primate. In some embodiments, a mammal is a human.

[0092] In some embodiments, a method comprises constructing the peptide library using a suitable method. In some embodiments, a peptide library is constructed using a starting sequence comprising any one of SEQ ID NOs: 1-14.

[0093] In some embodiments, presented herein is a peptide found to activate one or more of a human glucagon receptor, GLP-1 receptor, GLP-2 receptor, Amylin receptor, GIP receptor or a calcitonin receptor according to a method disclosed herein. In some embodiments, aAttorney Docket No: 01123-0022-00PCT peptide disclosed herein has biological activity or is determined to have biological activity. Non-limiting examples of biological activity include an ability to increase or decrease food intake in a mammal, an ability to increase or decrease body weight in a mammal, an ability to increase or decrease blood glucose levels in a mammal, an ability to increase or decrease blood insulin levels in a mammal, and an ability to induce cAMP production in a cell (e.g., a mammalian cell). In some embodiments, presented herein is a peptide that is determined to have biological activity according to a method disclosed herein.EXAMPLESExample 1 - Peptide Synthesis

[0094] Peptides are synthesized on Gyros Protein Technologies Symphony X synthesizers via solid phase peptide chemistry (SPPS) / Fmoc chemistry using polystyrene or PEG based resins. Fmoc chemistry employs Fmoc (fluorenylmethyloxycarbonyl) protecting group at the N-terminus of each amino acids, which is removed by a base (usually 20% piperidine in DMF) before the next coupling cycle. As an example, SEQ ID NO: 1 is made using TentaGel RAM resin (with a loading of 0.18 mmol / g) and sequential coupling and deprotection of Fmoc-protected amino acid derivatives. A lipidated extended Lysine residue is introduced as a single, preformed Fmoc derivative at position 20 of the sequence. Upon completion of SPPS, TFA mediated cleavage from the resin along with concomitant protecting group removal is performed, followed by isolation via precipitation and filtration with diethyl ether to yield 920 mg of crude material. Disulfide bond formation is affected using H2O2 in an aqueous solution at pH 8.6. Preparative reverse-phase HPLC is performed on a Teledyne Isco ACCQPrep HP 150 System with a variable wavelength UV absorbance detector, and the material is lyophilized to provide 126 mg of purified peptide. Purity is determined by analytical reverse-phase HPLC performed on an Dionex UltiMate 3000 system and mass identity is confirmed on a Kratos Axima CFR Plus MALDI-MS. The measured mass is compared with the calculated mass, and the product is further characterized by amino acid analysis that determines a percent peptide content.Example 2 - Biological Activity of Peptides in Cell-Based Cyclic AMP Activity Assay

[0095] Peptide are tested in cAMP cell-based assays to determine their relative activity against a selected receptor (e.g., GLP-1, glucagon, GIP, amylin and / or calcitonin receptors).Attorney Docket No: 01123-0022-00PCTPeptide activation of the various receptors often results in downstream production of cAMP second messenger which can be measured in a functional activity assay. Evaluation of the potency (EC50) of peptides for the different human receptors is determined using stably transfected CH0-K1 cells overexpressing a non-tagged receptor in a GPCR cell-based cAMP assay.

[0096] Experimental protocol: Cells are seeded in a total volume of 20 pL into white walled, 384-well microplates and are incubated at 37° C overnight prior to testing. Prior to testing cell plating media is exchanged with 10 pL of Assay buffer (HBSS+10 mM HEPES). All peptides are run in assay buffer containing 0.1% casein. Briefly, intermediate dilution of sample stocks is performed to generate 4x sample in assay buffer. 5 pL of 4x sample is added to cells and incubated at 37° C for 30 minutes. Final assay vehicle concentration is adjusted to about 1%. After appropriate peptide incubation, assay signal is generated through incubation with 5 ul of cAMP-specific antibody and 20 ul cAMP XS+ED / CL lysis cocktail for one hour followed by incubation with 20 ul cAMP XS+EA reagent for 2 hours at room temperature. Microplates are read following signal generation with PerkinElmer Envision instrument for chemiluminescent signal detection. Data is normalized to the maximal and minimal response observed in the presence of control ligand and vehicle. Results are expressed as a percent efficacy relative to the maximum response of the control ligand.Example 3 - Acute Food Intake Studies in Lean Mice

[0097] To assess the acute effect of the peptides in vivo, studies are performed in lean mice to determine the effect of the peptides on acute food intake.

[0098] C57BL / 6j male mice at 6 weeks of age are purchased from Charles River (Spain) and housed individually. Mice are kept in 12 hr light / dark cycles (lights on at 8 am) in temperature — (22° C.) and humidity (45-55%) — controlled rooms. Mice are fed ad libitum a standard rodent chow diet (CHD) (A04, U8220G10R, SAFE). At eight weeks of age, C57BL / 6j mice are acclimatized to reverse light / dark cycles for at least 7 days prior to peptide administration.

[0099] Mice are randomized in the experimental groups according to body weight. Twelve- hour fasted mice receive a single subcutaneous (s.c.) injection 2 hr before the lights are turned off at doses of 3, 10 and 50 nmol / kg (mols peptide / body weight of mouse; n=6 per group). As control, mice receive a single injection of equivalent volume of the corresponding vehicle. Food is replaced immediately after the lights are turned off. Food intake is measuredAttorney Docket No: 01123-0022-00PCT at 8, 24, 48 and 72 hours post-injection using a precision balance. Cumulative food intake is calculated for the indicated time periods.

[0100] Peptides are reconstituted in vehicle containing 20 mM Tris-HCl, 7.5 mg / mL mannitol, pH=7 or in 5 mM NaAc, 2.5 mg / mL mannitol, pH=5 at 1 mg / ml. All peptide solutions are aliquoted and stored at -20° C until use. Dilutions for injection are performed in saline buffer.

[0101] Statistical evaluation of the data is carried out using one-way ANOVA, followed by Bonferroni post-hoc test to determine where statistically significant differences exist between vehicle and peptide-treated groups. Differences are considered statistically significant at p<0.05. Data analysis is carried out with GraphPad software (GraphPad Prism).Example 4 - Effect on Blood Glucose after Single Administration of Peptides

[0102] For glucose-lowering analysis of the peptides and to determine if the incretin portion of the peptides have an effect in vivo, 4 hr fasted mice (n=6 per group) receive a single s.c. injection of peptide, all at 10 nmol / kg or an equivalent volume of the respective vehicle. Blood samples are collected from tail clips at 0, 120 and 240 minutes after injection for glucose measurements. Glucose is measured using a glucometer (Accu-chek Aviva, Roche Diagnostics). Calculations of glucose lowering at 120 and 240 min is compared to time 0 minutes.

[0103] Data are provided as the mean±standard error (SEM). Statistical evaluation of the data is carried out using one-way ANOVA, followed by Bonferroni post-hoc test to determine where statistically significant differences exist between vehicle and peptide- treated groups. Differences are considered statistically significant at p<0.05. Data analysis is carried out with GraphPad software (GraphPad Prism).Example 5 - Acute Food Intake and Body Weight Reduction in Lean Rats

[0104] Sprague Dawley male rats (9 weeks of age) are purchased from Janvier Labs (France). The rats are maintained in temperature-controlled conditions (22° C) and in humidity-controlled rooms (45-55%), and rats are acclimatized to a single housing at least 5 days prior to testing for individual registration of food consumption. Rats are fed ad libitum a standard rodent chow diet (CHD) (A04, U8220G10R, SAFE).

[0105] Sprague Dawley rats (350-400 g) are randomized in the different experimental groups by body weight and are fasted 8 hr prior to turning the lights off. Two hours prior toAttorney Docket No: 01123-0022-00PCT the onset of a dark cycle, the rats are injected (s.c.) with peptides or the corresponding vehicle at doses of 3, 10 or 50 nmol / kg (n=5 per group) for each experiment. For all experimental groups, food is replaced immediately after the lights are turned off.

[0106] Food intake is measured at different time-intervals of 4 hr, 12 hr, 24 hr, 48 hr and 72 hr post-injection using a precision balance. Body weight and cumulative food intake are also calculated for the indicated time periods. Mean of cumulative food intake in each dose group is compared to vehicle and is reported as a percentage of mean reduction relative to vehicle.

[0107] Data are provided as the mean±standard error (SEM).Example 6 - Effect in Food Intake and Body Weight after Repeated Dosing in DIO Mice

[0108] C57BL / 6j male mice (8 weeks of age) are fed a HFD (60% kcal from fat, D12492 Research Diets) for 14 weeks. Mice are individualized in separated cages (1 mice per cage) 21 days before the peptide treatment and acclimated to daily s.c. injections with saline during the last 10 days during which body weight loss is not observed.

[0109] Two days before peptide treatment mice are fasted for 4 hr, after which blood glucose is measured. Mice are randomized in the experimental groups (n=6 mice per group) according to body weight and blood glucose levels. Mean body weight is typically about 50 g at the start of the study. Mice receive a daily injection of vehicle or peptides (10 nmol / kg) for 20 days. Body weight and cumulative food intake are recorded daily.

[0110] After the treatment, mice are fasted for 4 hr and blood glucose is measured using a glucometer (Accu-chek Aviva, Roche Diagnostics). Plasma insulin is measured by ELISA (Mercodia).

[0111] Body composition is assessed by quantitative nuclear magnetic resonance at the end of the study. For total fat and lean mass analysis, images are acquired in a Bruker Biospec 47 / 40 spectrometer (Bruker Biospin GmbH). The MRI system works at 4.7 Teslas and is equipped with a 12 cm shielded gradient coil with a maximum gradient strength of 450 mT / m. For these experiments, a 70-mm volume radiofrequency (RF) coil, acting as a transmitter and receiver, is used. Mice are anaesthetized with a mixture of oxygen and isoflurane and are placed in prone position inside the RF coil. Animal respiration and temperature are monitored throughout the experiment using a 1035 MR-compatible monitor system (SA Instruments Inc., Stony Brook). First, 2D spin-echo T1 weighted (T1WI) imagesAttorney Docket No: 01123-0022-00PCT covering the whole body of the mouse are acquired. The parameters for this sequence are: repetition time (TR)=1 s; echo time (TE)=10 ms; slice thickness=1.0 mm; field of view (FOV)=10><5 cm2; matrix size=256* 128: number of averaged experiments (NA)=2. Thirty coronal slices are acquired in 4 min. Then, the spectrometer frequency is centered on the fat signal and the same images as above are acquired, but a water suppression sequence, consisted of three narrow-band saturation pulses each followed by a spoiling gradient, is applied on the water signal, so only the fat signal is visible in the images. Images are processed using the ImageJ 1.52 package (NIH).

[0112] All data shown are provided as the mean±standard error (SEM). Statistical evaluation of the data is carried out using one-way ANOVA, followed by Bonferroni post- hoc test to determine where statistically significant differences exist between vehicle and peptide-treated groups. Differences are considered statistically significant at p<0.05. Data analysis is carried out with GraphPad software (GraphPad Prism).EQUIVALENTS

[0113] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and Examples detail certain embodiments and describe the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the embodiment may be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

Claims

Attorney Docket No: 01123-0022-00PCTWhat is Claimed is:

1. A method of determining whether a peptide activates a human receptor selected from a glucagon receptor, GLP-1 receptor, GLP-2 receptor, Amylin receptor, calcitonin receptor and gastric inhibitory peptide (GIP) receptor on a cell, the method comprising (i) contacting a mammalian cell with a peptide having at least 70% identity to an amino acid sequence selected from any one of SEQ ID NOs: l-14, and (ii) determining if the peptide induces an increase in intracellular cyclic AMP (cAMP) production, wherein the cell expresses at least one human receptor selected from a glucagon receptor, GLP-1 receptor, GLP-2 receptor, Amylin receptor, GIP receptor and calcitonin receptor, and wherein an increase in intracellular cAMP as determined in (ii) indicates the peptide activates the receptor.

2. The method of claim 1, wherein an increase in cAMP is determined by comparison of the cAMP levels determined in (ii) with a standard curve, or by comparison to cAMP levels produced in a cell that is contacted with a control peptide or vehicle.

3. The method of any one of claims 1-2, wherein the control peptide does not bind to the receptor.

4. A method of determining whether a peptide antagonizes a human receptor selected from a glucagon receptor, GLP-1 receptor, GLP-2 receptor, Amylin receptor, calcitonin receptor and gastric inhibitory peptide (GIP) receptor on a cell, the method comprising (i) contacting a mammalian cell with a peptide having at least 70% identity to an amino acid sequence selected from any one of SEQ ID NOs: 1-14, and (ii) determining if the peptide inhibits intracellular cyclic AMP (cAMP) production, wherein the cell expresses at least one human receptor selected from a glucagon receptor, GLP-1 receptor, GLP-2 receptor, Amylin receptor, calcitonin receptor and gastric inhibitory peptide (GIP) receptor, and wherein an inhibition of intracellular cAMP production as determined in (ii) indicates the peptide antagonizes the receptor.

5. The method of any one of claims 1-4, wherein the mammalian cell is a human cell.

6. The method of any one of claims 1-5, wherein the cell overexpresses the receptor.

7. A method of determining whether a peptide affects food intake of a mammal, the method comprising (i) administering a peptide having at least 70% identity to an amino acid sequence selected from any one of SEQ ID NOs: 1-14 to a mammal, and (ii) determining an amount of food intake by the mammal over a period of at least 48 hours, wherein a change inAttorney Docket No: 01123-0022-00PCT the amount of food intake following administration of the peptide indicates the peptide affects food intake of the mammal.

8. The method of claim 7, wherein a change in the amount of food intake is determined by (a) comparing the amount of food intake before administration of the peptide with the amount of food intake after administration of the peptide; or (b) comparing a change in the amount of food intake of the mammal after administration of the peptide, to a change in the amount of food intake of a control mammal after administration of a control peptide or control vehicle to the control mammal, wherein the control mammal is of the same species.

9. A method of determining the effect of a peptide on blood glucose or blood insulin level in a mammal, the method comprising (i) administering a peptide having at least 70% identity to an amino acid sequence selected from any one of SEQ ID NOs: 1-14 to a mammal, and (ii) determining an amount of glucose or insulin in the blood of the mammal before and after administration of the peptide, wherein a change in the amount of glucose or insulin in the blood as determined in (ii) indicates the peptide affects blood glucose and / or blood insulin level, optionally wherein the effect of the peptide on the blood glucose or the blood insulin level is determined during a fasting state of the mammal, during a fed state of the mammal, or after the mammal is glucose challenged.

10. The method of any one of claims 7-8, wherein the amount of food intake is determined by providing an excess amount of food to the mammal and determining the amount of food that is uneaten by the mammal after a period of, for example, 30 minutes to 24 hours following the providing of the food.

11. The method of any one of claims 7-10, wherein the method further comprises determining body weight of the mammal before and after administration of the peptide or control peptide, such as 24 hours, 48 hours, 2 days, 3 days, 1 week, 2 weeks, or 1 month after administration of the peptide.

12. A method of determining the effect of a peptide on body weight of a mammal, the method comprising (i) administering a peptide having at least 70% identity to an amino acid sequence selected from any one of SEQ ID NOs: 1-14 to a mammal, and (ii) determining a body weight of the mammal before and after the administering of (i), wherein a change in the body weight of the mammal indicates the peptide comprises biological activity.

13. The method of claim 12, wherein the body weight is determined 24 hours, 48 hours, 2 days, 3 days, 1 week, 2 weeks, or 1 month after administration of the peptide and compared to body weight of the mammal prior to the administration.Attorney Docket No: 01123-0022-00PCT14. The method of any one of claims 1-13, wherein the mammal is a rodent.

15. The method of claim 14, wherein the rodent is a rat or a mouse.

16. The method of any one of claims 1-15, wherein the peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to an amino acid sequence selected from any one of SEQ ID NOs: l-14.

17. The method of claim 16, wherein the peptide further comprises an amino acid sequence having 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to an amino acid sequence selected from any one of SEQ ID NOs: 15-26.

18. The method of any one of claims 1-17, comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 1.

19. The method of any one of claims 1-17, wherein the peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO:2 and / or 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 16.

20. The method of claim 19, wherein the peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:2 and SEQ ID NO: 16.

21. The method of claim 19, wherein the peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO:7 and / or 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 16.

22. The method of claim 21, wherein the peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:7 and SEQ ID NO: 16.

23. The method of any one of claims 1-17, wherein the peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 10 and / or 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 16.

24. The method of claim 23, wherein the peptide comprises all of the amino acid residues that are conserved between SEQ ID NO: 10 and SEQ ID NO: 16.

25. The method of claim 23, wherein the peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 14.

26. The method of claim 23, wherein the peptide comprises all of the amino acid residues that are conserved between SEQ ID NO: 14 and SEQ ID NO: 16.

27. The method of any one of claims 1-17, wherein the peptide comprises an amino acid sequence of:PXnFXnSX; PXnYXnPXnFXnSX; PXnYRXPXnDXnFXnKRXn, orPXnYRXnPXnYXnDXnFXnKRXn,Attorney Docket No: 01123-0022-00PCT where X is any amino acid and n is a number between 0 and 15, or between 1 and 10.

28. The method of any one of claims 1-17, wherein the peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 3 and / or SEQ ID NO: 18.

29. The method of claim 28, wherein the peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:3 and SEQ ID NO: 18.

30. The method of any one of claims 1-17, wherein the peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 8 and / or SEQ ID NO: 18.

31. The method of claim 30, wherein the peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:8 and SEQ ID NO: 18.

32. The method of any one of claims 1-17, wherein the peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 11 and / or SEQ ID NO: 18.

33. The method of claim 32, wherein the peptide comprises all of the amino acid residues that are conserved between SEQ ID NO: 11 and SEQ ID NO: 18.

34. The method of any one of claims 1-17, wherein the peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 14 and / or SEQ ID NO: 18.

35. The method of claim 34, wherein the peptide comprises all of the amino acid residues that are conserved between SEQ ID NO: 14 and SEQ ID NO: 18.

36. The method of any one of claims 1-17, wherein the peptide comprises an amino acid sequence of PXnFXnSXnMDXnAXnLXnLXn;XnSXnAXnPLLXnRAPQXnMXnDXnAXnLXnGXnMDXnKLXnRXnGLXn; orPXnYRXPXnDXnFXnKRXnSXnLINGLIGXnKLXnKRSNXnLINXn, where X is any amino acid and n is a number between 0 and 15, or between 1 and 10.

37. The method of any one of claims 1-17, wherein the peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 4 and / or SEQ ID NO: 17.

38. The method of claim 37, wherein the peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:4 and SEQ ID NO: 17.

39. The method of any one of claims 1-17, wherein the peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 12 and / or SEQ ID NO: 17.

40. The method of claim 39, wherein the peptide comprises all of the amino acid residues that are conserved between SEQ ID NO: 12 and SEQ ID NO: 17.

41. The method of any one of claims 1-17, wherein the peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO:4 and / or SEQ ID NO: 19.Attorney Docket No: 01123-0022-00PCT42. The method of claim 41, wherein the peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:4 and SEQ ID NO: 19.

43. The method of any one of claims 1-17, wherein the peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 12 and / or SEQ ID NO: 19.

44. The method of claim 43, wherein the peptide comprises all of the amino acid residues that are conserved between SEQ ID NO: 12 and SEQ ID NO: 19.

45. The method of any one of claims 1-17, wherein the peptide comprises an amino acid sequence of GXnDXnKXnRXnL; XnGXnMDXnKLXnRXnGLXn; or LINGLIGXnKLXnKRSNXnLINXn, where X is any amino acid and n is a number between 0 and 15, or between 1 and 10.

46. The method of any one of claims 1-17, wherein the peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO:5 and / or SEQ ID NO:21.

47. The method of claim 46, wherein the peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:5 and SEQ ID NO:21.

48. The method of any one of claims 1-17, wherein the peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO:5 and / or SEQ ID NO:22.

49. The method of claim 48, wherein the peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:5 and SEQ ID NO:22.

50. The method of any one of claims 1-17, wherein the peptide comprises an amino acid sequence of XnGXnGRRXn; XnGAGRRXn; XnKXnGAGRRXn; XnKXnSGAGRRXn;XnKLSGAGRRXn; or XnNKLSGAGRRXn, where X is any amino acid and n is a number between 0 and 15, or between 1 and 10.

51. The method of any one of claims 1-17, wherein the peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 1 and / or SEQ ID NO:24.

52. The method of claim 51, wherein the peptide comprises all of the amino acid residues that are conserved between SEQ ID NO: 1 and SEQ ID NO:24.

53. The method of any one of claims 1-17, wherein the peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 1 and / or SEQ ID NO:25 or to SEQ ID NO: 1 and / or SEQ ID NO:26.

54. The method of claim 53, wherein the peptide comprises all of the amino acid residues that are conserved between SEQ ID NO: 1 and SEQ ID NO:25 or to SEQ ID NO: 1 and / or SEQ ID NO:26.Attorney Docket No: 01123-0022-00PCT55. The method of any one of claims 1-17, wherein the peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO:6 and / or SEQ ID NO:24.

56. The method of claim 55, wherein the peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:6 and SEQ ID NO:24.

57. The method of any one of claims 1-17, wherein the peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO:6 and / or SEQ ID NO:25.

58. The method of claim 57, wherein the peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:6 and SEQ ID NO:25.

59. The method of any one of claims 1-17, wherein the peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO:9 and / or SEQ ID NO:24.

60. The method of claim 59, wherein the peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:9 and SEQ ID NO:24.

61. The method of any one of claims 1-17, wherein the peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO:9 and / or SEQ ID NO:25.

62. The method of claim 60, wherein the peptide comprises all of the amino acid residues that are conserved between SEQ ID NO:9 and SEQ ID NO:25.

63. The method of any one of claims 1-17, wherein the peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 13 and / or SEQ ID NO:24.

64. The method of claim 63, wherein the peptide comprises all of the amino acid residues that are conserved between SEQ ID NO: 13 and SEQ ID NO:24.

65. The method of any one of claims 1-17, wherein the peptide has 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% identity to SEQ ID NO: 13 and / or SEQ ID NO:2566. The method of claim 65, wherein the peptide comprises all of the amino acid residues that are conserved between SEQ ID NO: 13 and SEQ ID NO:25.

67. The method of any one of claim 1-17, wherein the peptide comprises an amino acid sequence of 3 to 9 amino acids having 80% to 99%, 85% to 99%, 90% identity to SEQ ID NO:5 or wherein the peptide comprises the amino acid sequence of SEQ ID NO: 5.

68. The method of any one of claims 1-67, wherein the peptide is conjugated to a lipid, lipid nanoparticle, albumin, or PEG.

69. The method of any one of claims 1-6 or 16-68, wherein the peptide is a member of a peptide library, and wherein the method comprises contacting the mammalian cell with the peptide library.Attorney Docket No: 01123-0022-00PCT70. The method of claim 69, wherein the method further comprises constructing the peptide library, optionally from a starting sequence comprising any one of SEQ ID NOs: 1- 14.

71. A peptide found to activate one or more of human glucagon receptor, GLP-1 receptor, GLP-2 receptor, Amylin receptor, GIP receptor or calcitonin receptor according to the method of any one of claims 1-6 or 16-70.

72. A peptide found to antagonize one or more of a human glucagon receptor, GLP-1 receptor, GLP-2 receptor, Amylin receptor, GIP receptor or calcitonin receptor according to the method of any one of claims 1-6 or 16-70.

73. A peptide found to peptide affects food intake, blood glucose level, blood insulin level, or body weight of a mammal according to the method of any one of claims 7-70.

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