GIPR inhibitors and methods of use

Abstract

This disclosure provides variant GIP peptides and polypeptides capable of inhibiting GIPR, and methods and uses thereof.

Description

GIPR INHIBITORS AND METHODS OF USE

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority from US Provisional Application 63 / 561,960 filed March 6, 2024, the contents of which are incorporated herein by reference.

[0003] FIELD

[0004] The present invention relates to polypeptides with GIPR inhibitory activity.

[0005] BACKGROUND

[0006] Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone that coordinates nutrient intake with systemic metabolism. It is secreted from endocrine K cells primarily located in the proximal part of the small intestine. GIP is a stimulatory ligand for the GIP receptor (GIPR). Upon stimulation by nutrients, GIP is secreted to the blood stream and activates GIPR which results in insulin secretion, lipid deposition in adipose tissue, reduction in bone resorption, increased bone formation, and increased gastrointestinal blood flow. GIPR, which is expressed in pancreatic beta cells, adipose tissue, the central nervous system, and on leukocyte subsets has a principal role in maintaining glucose homeostasis and controlling metabolism. For this reason GIPR is of significant interest as a target for metabolic disorders such as type 2 diabetes and obesity, although its presence on certain endocrine tumors make it a potential target for tumor therapy. In addition to full-length GIP(l-42), truncated endogenous variants of wild-type GIP with inhibitory activity have been identified, namely GIP(l-30)NH2, GIP(3-42), and GIP(3-30)NH2. Nevertheless, there remains a need for new GIPR inhibitors with improved properties such as increased inhibitory activity.

[0007] SUMMARY

[0008] The present invention relates to polypeptides with GIPR inhibitory activity.

[0009] In an embodiment, there is provided, a polypeptide comprising an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises an amino acid sequence with 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions relative to any one of SEQ ID NO: 9- 16, 25-48, and 73-114, and wherein the C-terminal portion comprises an amino acid sequence at least 70% identical to any one of SEQ ID NO: 2 and 166-168.

[0010] In an embodiment, there is provided, a peptide comprising an amino acid sequence with 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions relative to any one of SEQ ID NO: 9- 16, 25-48, and 73-114.

[0011] In an embodiment, there is provided, a nucleic acid molecule encoding the polypeptide or the peptide as described herein.

[0012] In an embodiment, there is provided, a vector comprising the nucleic acid molecule as described herein.

[0013] In an embodiment, there is provided a host cell comprising the nucleic acid molecule or the vector as described herein.

[0014] In an embodiment, there is provided a pharmaceutical composition comprising the polypeptide, the peptide, the nucleic acid molecule, or the vector as described herein, and a pharmaceutically acceptable carrier, excipient, and / or stabilizer.

[0015] In an embodiment, there is provided the polypeptide, the peptide, the nucleic acid molecule, the vector, or the pharmaceutical composition as described herein for use in inhibiting GIPR signaling in a cell.

[0016] In an embodiment, there is provided a method for inhibiting GIPR signaling in a cell, comprising contacting the cell with the polypeptide, the peptide, the nucleic acid molecule, the vector, or the pharmaceutical composition as described herein.

[0017] In an embodiment, there is provided a method of treating or preventing a disease or disorder associated with GIPR signaling in a subject, comprising administering to the subject the polypeptide, the peptide, the nucleic acid molecule, the vector, or the pharmaceutical composition as described herein.

[0018] In an embodiment, there is provided a use of the polypeptide, the peptide, the nucleic acid molecule, the vector, or the pharmaceutical composition as described herein for inhibiting GIPR signaling in a cell.

[0019] In an embodiment, there is provided a use of the polypeptide, the peptide, the nucleic acid molecule, the vector, or the pharmaceutical composition as described herein for treating or preventing a disease or disorder associated with GIPR signaling in a subject.

[0020] DETAILED DESCRIPTION

[0021] The present inventors have discovered peptides that can be used to create N- terminal variants of GIP with GIPR-inhibitory activity. The peptides may be used to replace the N-terminal portion of GIP or a GIP analog to produce a GIP variant polypeptide with GIPR- inhibitor activity. As demonstrated herein by way of example, GIP variant polypeptides were generated by peptide synthesis. As shown in Example 1, the GIP variant polypeptides are approximately 100-fold more potent than the previously reported truncation variants of GIP.

[0022] Sequences, compositions, and methods for carrying out the invention are presented in terms of examples and embodiments in the present disclosure. However, the invention is not limited to the described examples and embodiments, and a person skilled in the art will appreciate that many other embodiments of the invention are possible without deviating from the basic concept of the invention, and that any such work around will also fall under scope of this invention. It is envisioned that other styles and configurations of the present invention can be easily incorporated into the teachings of the present invention, and the configurations shall be shown and described for purposes of clarity and disclosure and not by way of limitation of scope.

[0023] Molecules of the Invention

[0024] Peptides providing GIP-inhibitory activity and which may be incorporated into polypeptides, such as GIP variants are provided.

[0025] In some embodiments, there is provided a polypeptide comprising an N-terminal portion and a C-terminal portion, wherein the polypeptide is a GIP variant and is a GIPR inhibitor. As used herein, a “GIP variant”, “GIP variant polypeptide”, “GIP derivative”, or “GIP derivative polypeptide” according to the present invention refers to a polypeptide derived from GIP, for example full-length human GIP (SEQ ID NO: 1) or truncated 1-30 human GIP, in which the N-terminal portion comprises a peptide of the invention as described herein and the C- terminal portion comprises an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% identical to any one of SEQ ID NO: 2 and 166-168.

[0026] The GIP variants of the present invention are modified GIP polypeptides containing one or more structural differences compared to the wild type GIP polypeptide. As used herein, the term “wild type GIP” refers to the naturally occurring structure of GIP as found in an organism. As used herein, a “wild type GIP” is a GIP polypeptide that is isolated from an organism or a GIP polypeptide that is produced synthetically and has the structure and / or function of GIP as found in an organism. The terms “naturally occurring GIP”, “native GIP”, and “wild type GIP” may be used interchangeably.

[0027] The GIP variants contain mutations, substitutions, additions, deletions, truncations, or other structural variations compared to wild type GIP. In some embodiments, the GIP variants contain mutations or other structural variations to the receptor-binding region of wild type GIP. In some embodiments, the GIP variants contain truncations of the C-terminal portion of wild type GIP.

[0028] As used herein, the term “ligand” refers to a molecule that specifically binds to one or more receptors. As used herein, an “antagonist” is a polypeptide, peptide, small molecule, or other compound (a ligand) that inhibits one or more biological activities induced by the agonism, activation, or signaling of a receptor. As used herein, an “agonist” is a polypeptide, peptide, small molecule, or other compound (a ligand) that induces one or more biological activities induced by the agonism, activation, or signaling of a receptor. As used herein, “antagonism” refers to the activity of antagonist and “agonism” refers to the activity of an agonist. As used herein, the term “GIPR inhibitor” and “GIPR antagonist” may be used interchangeably to mean a polypeptide, peptide, small molecule, or other compound that inhibits one or more biological activities induced by the agonism, activation, or signaling of GIPR. As used herein, the term “GIPR agonist” refers to a polypeptide, peptide, small molecule, or other compound that induces one or more biological activities induced by the agonism, activation, or signaling of GIPR.

[0029] GIPR inhibitors may prevent the binding of ligands by achieving partial or complete occupation of the site or sites on GIPR that they require for interaction (orthosteric inhibitors). For example, a GIPR inhibitor may block the binding of native GIP ligand to GIPR, preventing normal activation of GIPR, or block the binding of a synthetic GIPR agonist to GIPR. Alternatively, GIPR inhibitors may prevent the binding of ligands by engaging sites on GIPR and inducing GIPR to adopt a conformation or conformations that cannot be recognized by the ligands (allosteric inhibitors). GIPR inhibitors may inhibit the entire repertoire or only a subset of GIPR intracellular signaling pathways.

[0030] The GIP variants of the present invention are antagonists of GIPR. In some embodiments, the GIP variant may exhibit at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 15-fold, 20-fold, 50-fold, or 100-fold higher antagonism of GIPR compared to a truncated wild-type GIP (e.g. GIP(l-30)NH2, GIP(3-42), GIP(3-30)NH2). In some embodiments, the GIP variant is an antagonist that inhibits one or more biological activities of GIPR as described herein. The amount of agonism provided by a ligand is typically measured by its EC50 value. The EC50 can be measured for a given agonist ligand by determining theconcentration of agonist ligand needed to elicit half of the maximum biological response of an agonist ligand. Smaller EC50 values, as measured in concentration of agonist ligand, indicate increased agonism because a lower concentration of the agonist ligand is required to elicit the maximum biological response. By contrast, the activity of an antagonist may be measured in the terms of IC50 values obtained from an inhibitory potency assay. The IC50 is typically defined as the concentration of the antagonist at which 50 % of a signal elicited via the receptor by a reference agonist is inhibited. IC50 may sometimes be reported as pIC50, which is the negative log of the IC50 value in moles per liter (molar or M). In some embodiments, GIP variants of the present invention inhibit GIPR with a pIC50 of greater than greater than 7.00, greater than 7.25, greater than 7.50, greater than 7.75, greater than 8.00, greater than 8.25, or greater than 8.50. The pIC50 may be measured by techniques known in the art, for example by the inhibition of the calcium flux induced by GIP agonism of GIPR expressed on HEK cells.

[0031] Receptors may exhibit more than one biological response when activated by an agonist ligand. The magnitude of the agonism or antagonism of a ligand may be measured with respect to one or more of the biological responses exhibited by the activated receptor. Agonism may be measured by one or more of increased phosphorylation of the receptor when bound by the ligand; increased G protein signaling through the receptor when bound by the ligand; increased arrestin recruitment to the receptor when bound by the ligand; increased induction of intracellular calcium flux when the receptor is bound by the ligand; increased production of intracellular cyclic AMP when the receptor is bound by the ligand; and / or increased receptor internalization when the receptor is bound by the ligand. Antagonism may be measured by inhibition of one or more of the above receptor responses. Measurements of agonism or antagonism may be determined using routine methods known in the art.

[0032] A calcium flux assay measures the calcium flux in a cell induced by agonism of a cell receptor when bound by an agonist, such as when GIPR is agonized by GIP. Calcium flux assays are known in the art, and may be performed as disclosed in WO 2008 / 012689 or as disclosed in Gaertner et al., 2008, PNAS, 105(46): 17706-17711. In an exemplary calcium flux assay, Chinese Hamster Ovary (CHO) cells or Human Embryonic Kidney (HEK) cells engineered to express GIPR are seeded 20000 cells / well in wells of black- walled clear-bottom 384-well plates. Test samples of variant ligands are diluted in PBS supplemented with 1% BSA and 25 mM HEPES to generate dilution series for dose-response experiments: 12-point doseresponse starting at 688 nM with a 2.5-fold dilution interval for each treatment. Cells are loaded with a calcium-sensitive fluorescent dye (Screen Quest™ Fluo-8 No Wash Calcium Assay Kit, AAT Bioquest) according to the manufacturer’s instructions, then a first addition of either testsample dilutions or vehicle alone were added. 5 minutes later, cells are stimulated with 100 nM test or reference samples and fluorescence signals (ex. 490 nm, em. 525 nm) are recorded.

[0033] A sequestration assay measures the internalization and / or down-regulation of a surface receptor on a cell in response to exposure of the cell to a ligand of the receptor. Sequestration assays are known in the art, and may be performed as disclosed in WO 2008 / 012689 or as disclosed in Gaertner et al., 2008, PNAS, 105(46): 17706-17711. In an exemplary sequestration assay, CHO cells expressing the receptor of interest are seeded at a density of 80,000 cells / well. After overnight incubation, medium is removed and replaced with medium containing ligands, and cells are incubated for 1 h at 37°C. Medium is then removed and cells are fixed with 4% paraformaldehyde and washed twice with PBS. Cells are then labeled with fluorescent label-conjugated anti-ligand antibody in PBS supplemented with 1% BSA (PBS-1% BSA) on ice for 1 h. Plates are washed three times with PBS-1% BSA and fluorescence values for each well are determined using a FLEXstation fluorimeter (Molecular Devices). Results are expressed as percentage control level of surface receptor: 100 (mean fluorescence [ligand added, anti-ligand] - mean negative control fluorescence [anti- ligand]) / (mean positive control fluorescence [no ligand added, anti-ligand] - mean fluorescence [anti-ligand]).

[0034] In an embodiment, the polypeptide of the present invention comprises an amino acid sequence with 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions relative to any one of SEQ ID NO: 9-16, 25-48, and 73-114. In some embodiments, this amino acid sequence is located near the N terminus of the polypeptide. In some embodiments, this amino acid sequence is located such that the beginning of the amino acid sequence lies within 15 residues of the N terminus of the polypeptide, such as within 15, 12, 10, 8, 6, 5, 4, 3, 2, 1 residues of the N terminus, or consists of the N terminus of the polypeptide.

[0035] In an embodiment, the polypeptide of the present invention comprises an amino acid sequence with 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions relative to any one of SEQ ID NO: 17-24, 49-72, and 115-165. In an embodiment, the polypeptide of the present invention comprises or consists of an amino acid sequence of any one of SEQ ID NO: 17-24, 49-72, and 115-165.

[0036] In some embodiments, the polypeptide according to the present invention comprises an N-terminal portion and a C-terminal portion, wherein said N-terminal portion comprises an amino acid sequence with 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions relative to any one of SEQ ID NO: 9-16, 25-48, and 73-114, and the C-terminal portion comprises an aminoacid sequence at least 70% identical to any one of SEQ ID NO: 2 and 166-168. In some embodiments, the C-terminal portion is truncated relative to wild type GIP, such as a truncation of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or more C-terminal amino acids relative to wild type GIP. In some embodiments, the truncated C-terminal portion of GIP is SEQ ID NO: 166 or 168 containing a truncation of the 13 C-terminal amino acids relative to human GIP.

[0037] In some embodiments, the polypeptides or peptides according to the present invention comprise the amino acid sequence of any one of SEQ ID NO: 9-165 or a functional fragment of any thereof that retains GIPR inhibitory activity.

[0038] In some embodiments, the N-terminal portion comprises the amino acid sequence XXXXXXXISXY (SEQ ID NO: 3), wherein X is any amino acid. In some embodiments, the N- terminal portion comprises the amino acid sequence [D or W or S or I][S or G or W or L][F or W or G or N or C][F or H or W or L][R or E or I or L or A or P or G][N or R or P or C or T][W or F]IS[Y or D]Y (SEQ ID NO: 4). In some embodiments, the N-terminal portion comprises the amino acid sequence XXXXXXFISDY (SEQ ID NO: 5), wherein X is any amino acid. In some embodiments, the N-terminal portion comprises the amino acid sequence [D or W or S or I][S or G or W or L][F or W or G or N or C][F or H or W or L][R or E or I or L or A or P or G][N or R or P or C or T]FISDY (SEQ ID NO 6). In some embodiments, the N-terminal portion comprises the amino acid sequence [D or I][L or G][C or W][F or W][E or G][T or R]FISDY (SEQ ID NO: 7). In some embodiments, the N-terminal portion comprises the amino acid sequence DGW[F or W][E or G][T or R]FISDY (SEQ ID NO: 8). In such embodiments, the C-terminal portion comprises an amino acid sequence at least 70% identical to any one of SEQ ID NO: 2 and 166- 168.

[0039] In some embodiments, the N-terminal portion consists of no more than 11 amino acids, such as no more than 11 or 10 amino acids. In some embodiments, the N-terminal portion consists of 11 amino acids or consists of 10 amino acids. In an embodiment, the N-terminus of the C-terminal portion adjoins directly to the C-terminus of the N-terminal portion, i.e. the N- terminal portion and the C terminal portion are directly adjoined. In some embodiments, the N- terminal portion is adjoined to the C-terminal portion via a peptide linker. In some embodiments, the N-terminal portion is located at the extreme N terminus of the polypeptide.

[0040] In some embodiments, there is provided a peptide comprising an amino acid sequence with 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions relative to any one of SEQ ID NO: 9- 16, 25-48, and 73-114. In some embodiments, the amino acid sequence XXXXXXXISXY (SEQ ID NO: 3), wherein X is any amino acid. In some embodiments, the amino acid sequence [D orW or S or I] [S or G or W or L][F or W or G or N or C][F or H or W or L][R or E or I or L or A or P or G][N or R or P or C or T][W or F]IS[Y or D]Y (SEQ ID NO: 4). In some embodiments, the amino acid sequence XXXXXXFISDY (SEQ ID NO: 5), wherein X is any amino acid. In some embodiments, the amino acid sequence [D or W or S or I] [S or G or W or L][F or W or G or N or C][F or H or W or L][R or E or I or L or A or P or G][N or R or P or C or T]FISDY (SEQ ID NO 6). In some embodiments, the peptide comprises the amino acid sequence [D or I][L or G][C or W][F or W][E or G][T or R]FISDY (SEQ ID NO: 7). In some embodiments, the peptide comprises the amino acid sequence DGW[F or W][E or G][T or R]FISDY (SEQ ID NO: 8).

[0041] Polypeptides and peptides are polymers that comprise amino acids linked by peptide bonds. As used herein, the term “amino acid” is used to describe any amino acid, natural or otherwise, that can be incorporated into a polypeptide or a peptide. Amino acids are small molecules comprising an amine (-NH2) group, a carboxyl (-COOH), and a variable side chain (R-group) specific to each amino acid. Amino acids are covalently linked by peptide bonds between the amine group of one amino acid to the carboxyl group of another amino acid to form polypeptides. Amino acids within a polypeptide are often referred to in the art as “residues”.

[0042] Methionine residues can be conservatively substituted with non-oxidizable amino acid analogs or amino acid derivatives to reduce complications from methionine oxidation during synthesis. In some embodiments, one or more methionine residues in the polypeptides or peptides of the present invention, including SEQ ID NOs: 17-24, 49-72, and 115-165, are conservatively substituted with an amino acid analog or an amino acid derivative such as, but not limited to, norleucine (Nle). The C-terminal residue of a polypeptide may be amidated. In some embodiments, the polypeptide of any one of SEQ ID NOs: 2, 17-24, 49-72, and 115-168 comprises a C-terminal amidation. It is known in the art the N-terminal glutamine or glutamic acids can spontaneously convert to pyroglutamate in various in vitro and in vivo conditions (Cao et al., 2022, J. Pharm. Sci., I l l :335-344). In some embodiments, an N-terminal residue designated herein as glutamine (Q) is present as a pyroglutamate.

[0043] Polypeptides and peptides, including GIP variants of the present invention, may comprise modifications such as, for example, phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, acylation, prenylation, alkylation, oxidation, or other modifications known in the art. In some embodiments, the GIP variants described herein comprise in their N-terminal portion and / or in their C-terminal portion one or more modifications selected from phosphorylation, glycosylation, ubiquitination, nitrosylation,methylation, acetylation, lipidation, acylation, prenylation, alkylation, oxidation, or other modifications known in the art.

[0044] Polypeptides and peptides, including GIP variants of the present invention, may comprise non-proteinogenic amino acids and / or amino acid analogs including artificial, synthetic, modified, or unnatural amino acids known in the art beyond the 20 genetically- encoded amino acids. Examples of non-proteinogenic amino acids or amino acid analogs that can be incorporated into polypeptides or peptides of the present invention include, but are not limited to, norleucine, P-amino acids, homo-amino acids, synthetic proline and pyruvic acid derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, substituted leucine derivatives, ornithine, linear core amino acids, N- methyl amino acids, N-acetyl amino acids, and amino acids with synthetic R-groups. Polypeptides and peptides may also comprise amino acid derivatives. As used herein, the term “amino acid derivatives” describes amino acids that have been derived from the modification of one of the 20 genetically-encoded amino acids. Amino acid derivatives can be synthetic, such as by chemical reaction, or they can be naturally occurring in organisms, such as in vivo metabolites. An example of an amino acid derivative is pyroglutamate / pyroglutamic acid, a cyclized derivative of glutamine in which the free amino group of glutamic acid cyclizes to form a lactam. In some embodiments, GIP variants described herein comprise one or more substitutions with a non-proteinogenic amino acid and / or an amino acid analog.

[0045] As used herein, the term "sequence identity" refers to the percentage of sequence identity between two nucleic acid (polynucleotide) or two amino acid (polypeptide) sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions / total number of positions multiplied by 100%). The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin, S. and Altschul, S., 1990, PNAS, 87(6):2264-2268, modified in Karlin, S. and Altschul, S., 1993, PNAS,90(12):5873-5877. Such an algorithm is incorporated into the BLAST programs. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a given nucleic acid molecule. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score=50, wordlength=3 to obtain amino acid sequences homologous to a given polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res., 25(17):3389-3402. Alternatively, PSLBLAST can be used to perform an iterated search which detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSLBlast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers, E. and Miller, W., 1988, Bioinformatics, 4(1): 11-17. Such an algorithm is incorporated in the ALIGN program which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0046] Conservative substitutions are substitutions in the sequence of a peptide or polypeptide that do not lead to a significant loss of function or which lead only to a small loss of function. Such a loss of function due to one or more conservative substitutions may be considered not to be significant if said loss amounts to less than 20 %, less than 15 %, less than 10 %, less than 6 %, or less than 4 % with respect to the function of the polypeptide having the unsubstituted sequence. Conservative substitutions are often substitutions wherein an amino acid side chain is replaced by an amino acid side chain that is related, or similar in physicochemical properties, to the replaced residue. Such conservative substitutions may be made, for example, using one of the 20 natural amino acids according to Table 1 wherein amino acids in the same block in the middle column and preferably in the same line in the right-hand column may be substituted for each other. Conservative substitutions may also be made using amino acidanalogs or amino acid derivatives such as, for example, a substitution of methionine with norleucine.Table 1

[0047] Polypeptides and peptides of the present invention can be prepared in many ways, for example, using known techniques of protein chemistry (for example, chemical peptide synthesis) or molecular biology (i.e. genetic engineering and fermentation — in general, biotechnology).

[0048] The polypeptides or peptides of the present invention may be prepared using the known techniques of protein chemistry as described, for example, in Gaertner et al., 2008, PNAS, 105(46): 17706-17711 or in Akondi et al., 2021, Chimia, 75(6):489-494.

[0049] A method of preparing polypeptides or peptides of the present invention involves in vitro chemical synthesis. The polypeptides or peptides may be synthesized in part or in whole using chemical means. For example, solid-phase peptide synthesis, such as methods based on tBoc or Fmoc chemistry, may be used. Enzymatic synthesis may also be used in part or in full.

[0050] In addition, the polypeptides or peptides of the present invention can be prepared using genetic engineering. The polypeptides or peptides of the present invention may be produced by culturing a host cell comprising a nucleic acid molecule expressing the polypeptides or peptides of the present invention under conditions which induce expression of the polypeptides or peptides. In some embodiments, the host cell is a bacterial cell (e.g. E.coh), a yeast cell (e.g. Saccharomyces cerevisiae), or a mammalian cell (e.g. a human cell, a mouse cell, a CHO cell, a HEK cell, a HeLa cell).

[0051] Biological synthesis other than by expression in a host cell may be used, e.g. the polypeptides or peptides of the present invention may be produced by translation from RNA invitro. Polypeptides or peptides of the present invention can, for example, also be prepared by digesting longer polypeptides using proteases.

[0052] Biological methods, including genetic engineering, fermentation, and expression are in general restricted to the production of polypeptides based on L-amino acids, but manipulation of translation machinery in vivo or in vitro (e.g. of aminoacyl tRNA molecules) can be used to allow the introduction of D-amino acids (or of other non-natural amino acids, such as iodotyrosine or methylphenylalanine, azidohomoalanine, etc.). Where D-amino acids are included, however, it is preferred to use chemical synthesis. Polypeptides or peptides of the present invention may have covalent modifications at the C-terminus and / or N-terminus.

[0053] The present invention provides polypeptides and peptides as disclosed above and, moreover, nucleic acid molecules encoding said polypeptides and peptides. In some embodiments, the nucleic acid molecules encoding polypeptides and peptides of the present invention are RNA or DNA. The skilled person is able to design or identify nucleic acid molecules encoding said polypeptides or peptides of the present invention using methods known in the art. In some embodiments, the nucleic acid molecules encoding polypeptides or peptides of the present invention are incorporated into a vector, such as a plasmid, episome, artificial chromosome, virus, or a viral vector. In some embodiments, the nucleic acid molecules encoding polypeptides or peptides of the present invention or the vector comprising said nucleic acid molecule are comprised within a host cell to enable expression of the polypeptides or peptides of the present invention. In some embodiments, the host cell is a bacterial cell, a yeast cell, a vertebrate cell, a mammalian cell, a human cell, or a cell of an immortalized cell line such as a CHO cell, a HEK cell, or a HeLa cell.

[0054] Pharmaceutical Compositions

[0055] The peptides or polypeptides described herein may be incorporated into pharmaceutical compositions for administration to a subject in need thereof. Said pharmaceutical compositions may comprise a pharmaceutically acceptable carrier, excipient, and / or stabilizer. The pharmaceutical compositions may be provided for use as a medicament. The preparation of pharmaceutical compositions is well known to the person skilled in the art.

[0056] A pharmaceutical composition according to the present invention may be administered to a subject in a therapeutically effective amount. As used herein, a "therapeutically effective amount" means an amount of the pharmaceutical composition or polypeptide or peptide therein effective to provide a therapeutic, prophylactic or diagnostic benefit to a subject. In some embodiments, a therapeutically effective amount of the pharmaceutical composition is anamount capable of inducing a clinical response in a subject in the treatment of a particular disease or disorder. Determination of a therapeutically effective amount of the pharmaceutical composition is well within the capability of those skilled in the art. The therapeutically effective amount may vary according to a variety of factors such as the subject’s condition, weight, sex, and age.

[0057] Pharmaceutical compositions provided herein may be prepared in various pharmaceutical dosage forms, such as an instant release, controlled release, sustained release, or target drug-delivery system. Commonly used dosage forms include, for example, solutions and suspensions, (micro-) emulsions, ointments, gels, creams, pastes, foams, suppositories, ovules, implants, patches, liposomes, tablets, dragees, lozenges, soft or hard shell capsules, amorphous or crystalline powders, effervescent powders or tablets, aerosols, and lyophilized formulations. Depending on the route of administration used, special devices may be required for application or administration of a dosage form, such as syringes and needles, inhalers, pumps, injection pens, applicators, special flasks, or other devices for administration, which may also be implanted within a body. Pharmaceutical dosage forms provided herein may be manufactured by any of the methods known in the art, such as, for example, by conventional mixing, sieving, dissolving, melting, granulating, dragee-making, tabletting, suspending, extruding, spray-drying, levigating, emulsifying, (nano / micro-) encapsulating, entrapping, or lyophilization processes.

[0058] In an embodiment, a pharmaceutical composition of the present invention may comprise more than one polypeptide or peptide, or a nucleic acid or vector encoding said polypeptide or peptide of the present invention. In an embodiment, the pharmaceutical composition of the present invention may comprise (a) at least one polypeptide or peptide, or a nucleic acid or vector encoding said polypeptide or peptide of the present invention; and (b) at least one further pharmaceutical agent or therapeutic agent. In an embodiment, the further pharmaceutical agent or therapeutic agent may be, for example, an anti-inflammatory drug, an immunosuppressant, an antibiotic, an antiviral agent, a small molecule drug, a cytotoxic agent, or an antibody. In some embodiments, the further pharmaceutical agent or therapeutic agent is formulated in admixture with, or in a separate pharmaceutical composition from, the at least one polypeptide or peptide, or a nucleic acid or vector encoding said polypeptide or peptide, e.g. for simultaneous or for sequential administration. In some embodiments, the further pharmaceutical agent or therapeutic agent is conjugated to a polypeptide or peptide of the present invention.

[0059] Pharmaceutical compositions provided herein may further comprise a pharmaceutically acceptable carrier, excipient, and / or stabilizer as known in the art. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations, and may comprise, for example but not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (such as Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0060] Methods and Uses

[0061] In some embodiments, the polypeptide or peptide of the present invention is used for inhibiting GIPR signaling in a cell. Inhibiting GIPR signaling comprises antagonism of GIPR as described herein. In some embodiments, the cell is, but not limited to, a pancreatic beta islet cell, a pancreatic alpha cell, a pancreatic upsilon cell, an adipocyte, a pericyte, a mesothelial cell, an endothelial cell, an osteocyte, an osteoblast, an osteoclast, a cardiomyocyte, a neuron, an oligodendrocyte, a gastric mucus-secreting cell, a ciliated cell, a T cell, a myeloid cell, a GIPR- expressing immortalized cell, or a GIPR-expressing cancer cell. In some embodiments, the cell is a primary cell or transformed cell that natively expresses GIPR or is genetically modified to express GIPR. In some embodiments, the cell is contacted with a peptide or polypeptide of the present invention in vitro. In some embodiments, the cell is contacted with a polypeptide or peptide of the present invention in vivo or ex vivo.

[0062] In some embodiments, the polypeptide or peptide of the present invention is used for treating or preventing a disease or disorder associated with GIPR signaling in a subject. As used herein, a “a disease or disorder associated with GIPR signaling” refers to a disease or disorder for which inhibition of GIPR signaling (e.g. antagonism) in the subject leads to complete or partial treatment or prevention of the disease or disorder. In some embodiments, the disease or disorder associated with GIPR signaling is a metabolic disorder, a GIPR-mediated cancer, or a GIPR-expressing cancer. In some embodiments, the disease or disorder associated with GIPR signaling is obesity, hypertension, cardiovascular disease, non-alcoholic fatty liverdisease, cushing’s syndrome, Alzheimer’s disease, Parkinson’s disease, metabolic syndrome, or diabetes (e.g. Type I or Type II). In some embodiments, the subject is human.

[0063] “Treating” or “treatment”, or “preventing” or “prevention”, as used herein, refers to an approach for obtaining beneficial or desired results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, dimini shment of extent of disease, stabilisation of the state of disease, prevention of development of disease, prevention of spread of disease, delay or slowing of disease progression, suppression of disease, delay or slowing of disease onset, and amelioration or palliation of the disease state. “Treating” or “preventing” can also mean prolonging survival of a patient beyond that expected in the absence of treatment and can also mean inhibiting the progression of disease temporarily or preventing the occurrence of disease. “Treating” may be distinguished from “preventing” in that “treating” typically occurs in a subject who already has a disease or disorder, whereas “preventing” typically occurs in a subject who does not have a disease or disorder. As will be appreciated, there may be overlap in treatment and prevention. For example, it is possible to be “treating” a disease in a subject, while at same time “preventing” symptoms or progression of the disease.

[0064] Particular embodiments of the disclosure include, without limitation, the following:1. A polypeptide comprising an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises an amino acid sequence with 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions relative to any one of SEQ ID NO: 9-16, 25-48, and 73-114, and wherein the C-terminal portion comprises an amino acid sequence at least 70% identical to any one of SEQ ID NO: 2 and 166-168.2. The polypeptide of embodiment 1, wherein the amino acid substitution is a conservative substitution.3. The polypeptide of embodiment 1, wherein the N-terminal portion comprises the amino acid sequence of any one of SEQ ID NO: 9-16, 25-48, and 73-114.4. The polypeptide of embodiment 1, wherein the N-terminal portion comprises the amino acid sequence XXXXXXXISXY (SEQ ID NO: 3), wherein X is any amino acid.5. The polypeptide of embodiment 4, wherein the N-terminal portion comprises the amino acid sequence [D or W or S or I] [S or G or W or L][F or W or G or N or C][F or H or W or L][R or E or I or L or A or P or G][N or R or P or C or T][W or F]IS[Y or D]Y (SEQ ID NO: 4).6. The polypeptide of embodiment 1, wherein the N-terminal portion comprises the amino acid sequence XXXXXXFISDY (SEQ ID NO: 5), wherein X is any amino acid.7. The polypeptide of embodiment 6, wherein the N-terminal portion comprises the amino acid sequence [D or W or S or I] [S or G or W or L][F or W or G or N or C][F or H or W or L][R or E or I or L or A or P or G][N or R or P or C or T]FISDY (SEQ ID NO 6).8. The polypeptide of embodiment 6, wherein the N-terminal portion comprises the amino acid sequence [D or I][L or G][C or W][F or W][E or G][T or R]FISDY (SEQ ID NO: 7).9. The polypeptide of embodiment 6, wherein the N-terminal portion comprises the amino acid sequence DGW[F or W][E or G][T or R]FISDY (SEQ ID NO: 8).10. The polypeptide of any one of embodiments 1-9, wherein the N-terminal portion consists of 10-11 amino acids.11. The polypeptide of any one of embodiments 1-10, wherein the C-terminal portion comprises the amino acid sequence of any one of SEQ ID NO: 2 and 166-168.12. The polypeptide of any one of embodiments 1-11, wherein the polypeptide inhibits GIPR with a pIC50 of greater than 7.00, greater than 7.25, greater than 7.50, greater than 7.75, greater than 8.00, greater than 8.25, or greater than 8.50.13. A peptide comprising an amino acid sequence with 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions relative to any one of SEQ ID NO: 9-16, 25-48, and 73-114.14. The peptide of embodiment 13, wherein the amino acid substitution is a conservative substitution.15. The peptide of embodiment 13, comprising the amino acid sequence of any one of SEQ ID NO: 9-16, 25-48, and 73-114.16. The peptide of embodiment 13, comprising the amino acid sequence XXXXXXXISXY (SEQ ID NO: 3), wherein X is any amino acid.17. The peptide of embodiment 16, comprising the amino acid sequence [D or W or S or I] [S or G or W or L][F or W or G or N or C][F or H or W or L][R or E or I or L or A or P or G][N or R or P or C or T][W or F]IS[Y or D]Y (SEQ ID NO: 4).18. The peptide of embodiment 13, comprising the amino acid sequence XXXXXXFISDY (SEQ ID NO: 5), wherein X is any amino acid.19. The peptide of embodiment 18, comprising the amino acid sequence [D or W or S or I] [S or G or W or L][F or W or G or N or C][F or H or W or L][R or E or I or L or A or P or G][N or R or P or C or T]FISDY (SEQ ID NO 6).20. The peptide of embodiment 18, comprising the amino acid sequence [D or I] [L or G][C or W][F or W][E or G][T or R]FISDY (SEQ ID NO: 7).21. The peptide of embodiment 18, comprising the amino acid sequence DGW[F or W][E or G][T or R]FISDY (SEQ ID NO: 8).22. A nucleic acid molecule encoding the polypeptide of any one of embodiments 1-12 or the peptide of any one of embodiments 13-21.23. A vector comprising the nucleic acid molecule of embodiment 22.24. A host cell comprising the nucleic acid molecule of embodiment 22 or the vector of embodiment 23.25. A pharmaceutical composition comprising the polypeptide of any one of embodiments 1- 12, the peptide of any one of embodiments 13-21, the nucleic acid molecule of embodiment 22, or the vector of embodiment 23, and a pharmaceutically acceptable carrier, excipient, and / or stabilizer.26. The pharmaceutical composition of embodiment 25, further comprising a therapeutic agent.27. The polypeptide of any one of embodiments 1-12, the peptide of any one of embodiments 13-21, the nucleic acid molecule of embodiment 22, the vector of embodiment 23, or the pharmaceutical composition of embodiment 25 or 26 for use in inhibiting GIPR signaling in a cell.28. The polypeptide, peptide, nucleic acid molecule, vector, or pharmaceutical composition for use of embodiment 27, wherein the cell is a pancreatic beta islet cell, a pancreatic alpha cell, a pancreatic upsilon cell, an adipocyte, a pericyte, a mesothelial cell, an endothelial cell, an osteocyte, an osteoblast, an osteoclast, a cardiomyocyte, a neuron, an oligodendrocyte, a gastric mucus-secreting cell, a ciliated cell, a T cell, a myeloid cell, a GIPR-expressing immortalized cell, or a GIPR-expressing cancer cell.29. The polypeptide, peptide, nucleic acid molecule, vector, or pharmaceutical composition for use of embodiment 27 or 28, wherein the cell is in vitro.30. The polypeptide, peptide, nucleic acid molecule, vector, or pharmaceutical composition for use of embodiment 27 or 28, wherein the cell is in a subject.31. The polypeptide of any one of embodiments 1-12, the peptide of any one of embodiments 13-21, the nucleic acid molecule of embodiment 22, the vector of embodiment 23, or the pharmaceutical composition of embodiment 25 or 26 for use in treating or preventing a disease or disorder associated with GIPR signaling in a subject.32. The polypeptide, peptide, nucleic acid molecule, vector, or pharmaceutical composition for use of embodiment 31, wherein the disease or disorder is obesity, hypertension, cardiovascular disease, non-alcoholic fatty liver disease, cushing’s syndrome, Alzheimer’s disease, Parkinson’s disease, metabolic syndrome, or diabetes.33. A method for inhibiting GIPR signaling in a cell, comprising contacting the cell with the polypeptide of any one of embodiments 1-12, the peptide of any one of embodiments 13-21, the nucleic acid molecule of embodiment 22, the vector of embodiment 23, or the pharmaceutical composition of embodiment 25 or 26.34. The method of embodiment 33, wherein the cell is a pancreatic beta islet cell, a pancreatic alpha cell, a pancreatic upsilon cell, an adipocyte, a pericyte, a mesothelial cell, an endothelial cell, an osteocyte, an osteoblast, an osteoclast, a cardiomyocyte, a neuron, an oligodendrocyte, a gastric mucus-secreting cell, a ciliated cell, a T cell, a myeloid cell, a GIPR- expressing immortalized cell, or a GIPR-expressing cancer cell, a GIPR-expressing immortalized cell, or a GIPR-expressing cancer cell.35. The method of embodiment 33 or 34, wherein the cell is in vitro.36. The method of embodiment 33 or 34, wherein the cell is in a subject.37. A method of treating or preventing a disease or disorder associated with GIPR signaling in a subject, comprising administering to the subject the polypeptide of any one of embodiments 1-12, the peptide of any one of embodiments 13-21, the nucleic acid molecule of embodiment 22, the vector of embodiment 23, or the pharmaceutical composition of embodiment 25 or 26.38. The method of embodiment 37, wherein the disease or disorder is obesity, hypertension, cardiovascular disease, non-alcoholic fatty liver disease, cushing’s syndrome, Alzheimer’s disease, Parkinson’s disease, metabolic syndrome, or diabetes.39. Use of the polypeptide of any one of embodiments 1-12, the peptide of any one of embodiments 13-21, the nucleic acid molecule of embodiment 22, the vector of embodiment 23, or the pharmaceutical composition of embodiment 25 or 26 for inhibiting GIPR signaling in a cell.40. The use of embodiment 39, wherein the cell is a pancreatic beta islet cell, a pancreatic alpha cell, a pancreatic upsilon cell, an adipocyte, a pericyte, a mesothelial cell, an endothelial cell, an osteocyte, an osteoblast, an osteoclast, a cardiomyocyte, a neuron, an oligodendrocyte, a gastric mucus-secreting cell, a ciliated cell, a T cell, a myeloid cell, a GIPR-expressing immortalized cell, or a GIPR-expressing cancer cell.41. The use of embodiment 39 or 40, wherein the cell is in vitro.42. The method of embodiment 39 or 40, wherein the cell is in a subject.43. Use of the polypeptide of any one of embodiments 1-12, the peptide of any one of embodiments 13-21, the nucleic acid molecule of embodiment 22, the vector of embodiment 23, or the pharmaceutical composition of embodiment 25 or 26 for treating or preventing a disease or disorder associated with GIPR signaling in a subject.44. The method of embodiment 43, wherein the disease or disorder is obesity, hypertension, cardiovascular disease, non-alcoholic fatty liver disease, cushing’s syndrome, Alzheimer’s disease, Parkinson’s disease, metabolic syndrome, or diabetes.

[0065] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of’, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps.

[0066] As used herein, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. In embodiments comprising an “additional” or “second” component, the second component as used herein is different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.

[0067] All publications and patents cited herein are incorporated by reference in their entirety as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. In the case of any conflict between a definition of a term in the present disclosure and a definition in a cited publication or patent, the definition provided in the present disclosure is to be used in describing the present invention.

[0068] The present invention will now be described by way of non-limiting examples having regard to the appended drawings.

[0069] EXAMPLE 1

[0070] GIP variants with variant N-terminal portions (residues 0-10) and conserved GIP C-terminal portions (residues 11-42) were produced essentially using techniques described in Gaertner et al. (2008, PNAS, 105(46): 17706-17711) and in Akondi et al. (2021, Chimia, 75(6):489-494). The GIP variants were chemically synthesized and tested for pharmacological activity on CHO cells expressing GIPR.

[0071] The determined activities of the GIP variants are summarized in Table 2, showing sequences and inhibitory activities. GIP variants were synthesized with the modified positions of the protein (residues 0 through 10) as indicated (single-letter amino acid codes). The analogs were assessed for antagonist potency in a calcium flux assay using CHO-GIPR cells. “Av ANTAG pIC50” shows the average measured pIC50 for inhibiting calcium flux induced by GIP- mediated agonism of GIPR, averaged over 2-3 independent experimental replicates. As shown in Table 2, the GIP variant polypeptides are approximately 100-fold more potent than the previously reported truncation variants of GIP.

[0072] Table 2

[0073] EXAMPLE 2

[0074] Further GIP variants based on hGIP-lP25 (SEQ ID NO: 24) were produced and tested for their inhibitory activity on GIPR using HEK cells expressing GIPR. pIC50 was determined as explained in Example 1.

[0075] In the Group 1 experiment (Table 3), single-substitution variants of hGIP-lP25 were generated and tested for inhibition of GIPR. Group 1 variants were compared to reference antagonist GIP(5-3i) palmitoylated analog ([N“-Ac, L14, R18, E21] hGIP(5-3i)-Kl l (yE-C16)) as disclosed in Yang B et al. (Mol Metab. 2022 Dec;66: 101638. doi:10.1016 / j.molmet.2022.101638). The results are disclosed in Table 3. “OAc” refers to N- acetylation of the N-terminal residue. All positions and substitutions are indicated relative to hGIP-lP25 (SEQ ID NO: 24).

[0076] Table 3

[0077] In the Group 2 experiment (Table 4), double-substitution variants of hGIP-lP25 were generated and tested for inhibition of GIPR. Group 2 variants were compared to reference antagonist GIP(5-3i) palmitoylated analog ([N“-Ac, L14, R18, E21] hGIP(5-3i)-Kl l (yE-C16)). The results are disclosed in Table 4. “OAc” refers to N-acetylation of the N-terminal residue. All positions and substitutions are indicated relative to hGIP-lP25 (SEQ ID NO: 24).

[0078] Table 4

[0079] In the Group 3 experiment (Table 5), truncated variants of hGIP-lP25 were generated and tested for inhibition of GIPR. Group 3 variants were truncated to comprise positions 1-30 of hGIP-lP25 and to include further substitutions or modifications. Group 3 variants were compared to reference antagonist GIP(5-3i) palmitoylated analog ([N“-Ac, L14, R18, E21] hGIP(5-3i)-Kl 1 (yE-C16)). The results are disclosed in Table 5. “OAc” refers to N- acetylation of the N-terminal residue. All positions and substitutions are indicated relative to hGIP-lP25 (SEQ ID NO: 24).

[0080] Table 5

[0081] The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching. The embodiments described were chosen and described in order to best explain the principles of the invention and its practical application, and to thereby enable others skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated. It isunderstood that various omissions or substitutions of equivalents are contemplated as circumstance may suggest or render expedient, but is intended to cover the application or implementation without departing from the spirit or scope of the claims of the present invention.

Claims

CLAIMS:

1. A polypeptide comprising an N-terminal portion and a C-terminal portion, wherein the N-terminal portion comprises an amino acid sequence with 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions relative to any one of SEQ ID NO: 16, 9-15, 25-48, and 73-114, and wherein the C-terminal portion comprises an amino acid sequence at least 70% identical to any one of SEQ ID NO: 2 and 166-168.

2. The polypeptide of claim 1, wherein the N-terminal portion comprises the amino acid sequence of any one of SEQ ID NO: 16, 9-15, 25-48, and 73-114.

3. The polypeptide of claim 1, wherein the N-terminal portion comprises the amino acid sequence XXXXXXFISDY (SEQ ID NO: 5), wherein X is any amino acid.

4. The polypeptide of claim 3, wherein the N-terminal portion comprises the amino acid sequence DGW[F or W][E or G][T or R]FISDY (SEQ ID NO: 8).

5. The polypeptide of any one of claims 1-4, wherein the C-terminal portion comprises the amino acid sequence of any one of SEQ ID NO: 2 and 166-168.

6. A peptide comprising an amino acid sequence with 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions relative to any one of SEQ ID NO: 16, 9-15, 25-48, and 73-114.

7. The peptide of claim 6, comprising the amino acid sequence of any one of SEQ ID NO: 16, 9-15, 25-48, and 73-114.

8. The peptide of claim 6, comprising the amino acid sequence XXXXXXFISDY (SEQ ID NO: 5), wherein X is any amino acid.

9. The peptide of claim 8, comprising the amino acid sequence DGW[F or W][E or G][T or R]FISDY (SEQ ID NO: 8).

10. A nucleic acid molecule encoding the polypeptide of any one of claims 1-5 or the peptide of any one of claims 6-9.

11. A vector comprising the nucleic acid molecule of claim 10.

12. A host cell comprising the nucleic acid molecule of claim 10 or the vector of claim 11.

13. A pharmaceutical composition comprising the polypeptide of any one of claims 1-5, the peptide of any one of claims 6-9, the nucleic acid molecule of claim 10, or the vector of claim 11, and a pharmaceutically acceptable carrier, excipient, and / or stabilizer.

14. The pharmaceutical composition of claim 13, further comprising a therapeutic agent.

15. The polypeptide of any one of claims 1-5, the peptide of any one of claims 6-9, the nucleic acid molecule of claim 10, the vector of claim 11, or the pharmaceutical composition of claim 13 or 14 for use in inhibiting GIPR signaling in a cell.

16. The polypeptide, peptide, nucleic acid molecule, vector, or pharmaceutical composition for use of claim 15, wherein the cell is a pancreatic beta islet cell, a pancreatic alpha cell, a pancreatic upsilon cell, an adipocyte, a pericyte, a mesothelial cell, an endothelial cell, an osteocyte, an osteoblast, an osteoclast, a cardiomyocyte, a neuron, an oligodendrocyte, a gastric mucus-secreting cell, a ciliated cell, a T cell, a myeloid cell, a GIPR-expressing immortalized cell, or a GIPR-expressing cancer cell.

17. The polypeptide, peptide, nucleic acid molecule, vector, or pharmaceutical composition for use of claim 15 or 16, wherein the cell is in vitro.

18. The polypeptide, peptide, nucleic acid molecule, vector, or pharmaceutical composition for use of claim 15 or 16, wherein the cell is in a subject.

19. The polypeptide of any one of claims 1-5, the peptide of any one of claims 6-9, the nucleic acid molecule of claim 10, the vector of claim 11, or the pharmaceutical composition of claim 13 or 14 for use in treating or preventing a disease or disorder associated with GIPR signaling in a subject.

20. The polypeptide, peptide, nucleic acid molecule, vector, or pharmaceutical composition for use of claim 19, wherein the disease or disorder is obesity, hypertension, cardiovascular disease, non-alcoholic fatty liver disease, cushing’s syndrome, Alzheimer’s disease, Parkinson’s disease, metabolic syndrome, or diabetes.