GIPR antibody and its fusion protein with GLP-1, as well as its pharmaceutical composition and application

Abstract

The present invention provides a GIPR antibody and a fusion protein thereof with GLP-1, and a pharmaceutical composition thereof. Also provided is a method for treating, preventing or ameliorating one or more symptoms of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, type 2 diabetes, or obesity using a GIPR antibody and a fusion protein thereof with GLP-1.

Description

Technical Field

[0001] The present invention provides antibodies that specifically bind to GIPR, fusion proteins thereof with GLP-1, and pharmaceutical compositions thereof. The present invention also provides methods for using the GIPR antibodies and fusion proteins thereof with GLP-1 for treating, preventing or ameliorating one or more symptoms of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, type 2 diabetes, or obesity. Background of the Invention

[0003] Gastric inhibitory polypeptide (GIP) is a polypeptide hormone secreted by intestinal K cells in the human body after eating, and its forms include 42-peptide and 30-peptide. GIP participates in the physiological process of stimulating insulin secretion by activating the gastric inhibitory polypeptide receptor (GIPR) on the surface of pancreatic β cells (Tseng et al., 1996, J. Clin. Invest. 98: 2440-2445; Ravn et al., 2013, J. Biol. Chem. 288: 19760-72). Therefore, the classical biological function of GIP is similar to that of GLP-1, so the two are collectively referred to as enterokinin. However, GIPR is widely distributed in addition to the pancreas, including bone, heart, stomach, intestine and adipose tissue, etc. (Peter et al., 2013, J. Biol. Chem. 288: 19760-72). The diverse distribution indicates that the GIP / GIPR pathway has more biological functions beyond blood glucose regulation. Experimental evidence shows that the GIP / GIPR signaling pathway is at least closely related to lipid metabolism in these tissues (Yip and Wolfe, 2000, Life Sci. 66: 91-103). Experimental data also show that the blood circulation GIP concentration in obese or diabetic patients will increase (Creutzfeldt et al., 1978, Diabetologia 14: 15-24; Flatt et al., 1984, J. Endocrinol. 101: 249-256; Salera et al., 1982, J. Clin. Endocrinol. Metab. 55: 329-336; et al., 2003, J. Clin. Endocrinol. Metab. 88: 2706-2713). After blocking the GIPR signal with a GIPR inhibitor, a significant decrease in body weight and alleviation of insulin resistance symptoms can be observed in high-fat diet-induced obese mice, and even type 2 diabetes caused by high-fat diet can be reversed (Peter et al., 2013, J. Biol. Chem. 288: 19760-72).

[0004] The long-acting glucagon-like peptide-1 analogue (GLP-1 analogue) is a new generation and currently the most effective type 2 diabetes drug (Tomlinson et al., 2015, Expert Opin. Investig. Drugs 25: 1744 - 7658; Gallwitz, 2015, Eur. Endocr. 11: 21 - 25). Long-acting GLP-1 drugs are also being tried in clinical trials for the treatment of non-alcoholic fatty liver disease (NAFLD). The research results show that it has significant effects on improving the hepatic tissue morphology, reducing the ratio of alanine aminotransferase / aspartate aminotransferase, and decreasing hepatic fat content in NAFLD patients (Samson et al., 2013, J. Diabetes Complications 27: 401 - 6; Portillo-Sanchez and Cusi, 2016, Clin. Diabetes Endocrinol. 2: 9).

[0005] If GLP-1 drugs and GIPR inhibitors can be used in combination, including co-administration or fusion, it may achieve the effect of simultaneously improving insulin resistance and excessive fat accumulation (i.e., obesity). Thus, while reducing blood sugar, it also interferes with fat metabolism, that is, the GLP-1 part improves glucose metabolism, reduces appetite, and lowers blood sugar and body weight; the GIPR antibody part reduces further fat accumulation and improves liver function. The fat-reducing effect of the GIPR antibody part and the weight-reducing effect of the GLP-1 part are superimposed to treat non-alcoholic fatty liver disease / non-alcoholic steatohepatitis through dual actions. This article provides a fusion protein drug, which will be beneficial for treating patients suffering from one or more of non-alcoholic fatty liver disease / non-alcoholic steatohepatitis, type 2 diabetes, and obesity. Summary of the Invention

[0006] This article provides an antibody that can specifically bind to GIPR, and this antibody is an antagonist of GIPR.

[0007] This article also provides an antibody that can specifically bind to GIPR, and the antibody contains one, two, three, four, five, or six amino acid sequences, where each amino acid sequence is independently selected from the following amino acid sequences:

[0008] a. Light chain CDR1 amino acid sequences: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 15;

[0009] b. Amino acid sequences of light chain CDR2: SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, and SEQ ID NO: 16;

[0010] c. Amino acid sequences of light chain CDR3: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 17;

[0011] d. Amino acid sequences of heavy chain CDR1: SEQ ID NO: 18, SEQ ID NO: 23, and SEQ ID NO: 26;

[0012] e. Amino acid sequences of heavy chain CDR2: SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, and SEQ ID NO: 29; and

[0013] f. Amino acid sequences of heavy chain CDR3: SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, and SEQ ID NO: 30.

[0014] The present invention further provides a GLP-1 fusion protein, which comprises an antibody capable of specifically binding to GIPR, and one, two, three, four, five, six, seven, or eight GLP-1 fragments; the fusion protein connects the carboxyl terminus of a GLP-1 fragment to the amino terminus of a light chain or heavy chain of a GIPR antibody or connects the amino terminus of a GLP-1 fragment to the carboxyl terminus of a light chain or heavy chain of a GIPR antibody through a peptide linker sequence (Linker).

[0015] The present invention provides a GLP-1 fusion protein, which comprises a GIPR antibody and two GLP-1 fragments; the fusion protein connects the carboxyl terminus of a GLP-1 fragment to the amino terminus of a light chain of a GIPR antibody: N′-GLP-1-Linker-R-C′; or connects the carboxyl terminus of a GLP-1 fragment to the amino terminus of a heavy chain of a GIPR antibody: N′-GLP-1-Linker-R-C′; wherein: N′ represents the amino terminus of the polypeptide chain of the fusion protein, C′ represents the carboxyl terminus of the polypeptide chain of the fusion protein, GLP-1 represents a GLP-1 fragment, R is the amino acid sequence of the light chain or heavy chain of a GIPR antibody, and Linker represents a peptide linker sequence.

[0016] The present invention provides a polynucleotide encoding a GIPR antibody as described herein.

[0017] The present invention provides a polynucleotide encoding a fusion protein of a GIPR antibody and GLP-1 as described herein.

[0018] The present invention provides a vector comprising a polynucleotide encoding a GIPR antibody as described herein.

[0019] The present invention provides a vector comprising a polynucleotide encoding a fusion protein of a GIPR antibody and GLP-1 as described herein.

[0020] The present invention provides a host cell comprising a vector as described herein.

[0021] The present invention provides a pharmaceutical composition comprising a GIPR antibody as described herein and a pharmaceutically acceptable carrier.

[0022] The present invention provides a pharmaceutical composition comprising a fusion protein of a GIPR antibody and GLP-1 as described herein and a pharmaceutically acceptable carrier. The present invention provides the use of a GIPR antibody as described herein in the preparation of a medicament for treating, preventing or ameliorating non-alcoholic fatty liver disease.

[0023] The present invention provides the use of a fusion protein of a GIPR antibody and GLP-1 as described herein in the preparation of a medicament for treating, preventing or ameliorating non-alcoholic fatty liver disease.

[0024] The present invention provides the use of a GIPR antibody as described herein in the preparation of a medicament for treating, preventing or ameliorating type 2 diabetes.

[0025] The present invention provides the use of a fusion protein of a GIPR antibody and GLP-1 as described herein in the preparation of a medicament for treating, preventing or ameliorating type 2 diabetes.

[0026] The present invention provides the use of a GIPR antibody as described herein in the preparation of a medicament for weight loss or for treating, preventing or ameliorating obesity and obesity-related disorders.

[0027] The present invention provides the use of a fusion protein of a GIPR antibody and GLP-1 as described herein in the preparation of a medicament for weight loss or for treating, preventing or ameliorating obesity and obesity-related disorders.

[0028] The present invention provides the use of a GIPR antibody as described herein in the preparation of a medicament for simultaneously treating, preventing or ameliorating two or more of non-alcoholic fatty liver disease, obesity or type 2 diabetes.

[0029] The present invention provides the use of a fusion protein of a GIPR antibody described herein and GLP-1 in the preparation of a medicament for simultaneously treating, preventing or improving two or more diseases selected from non-alcoholic steatohepatitis, type 2 diabetes or obesity.

[0030] The present invention provides a method for treating, preventing or improving one or more symptoms of non-alcoholic steatohepatitis, which comprises administering to a subject a therapeutically effective amount of a GIPR antibody described herein.

[0031] The present invention provides a method for treating, preventing or improving one or more symptoms of non-alcoholic steatohepatitis, which comprises administering to a subject a therapeutically effective amount of a fusion protein of a GIPR antibody described herein and GLP-1.

[0032] The present invention provides a method for treating, preventing or improving one or more symptoms of type 2 diabetes, which comprises administering to a subject a therapeutically effective amount of a GIPR antibody described herein.

[0033] The present invention provides a method for treating, preventing or improving one or more symptoms of type 2 diabetes, which comprises administering to a subject a therapeutically effective amount of a fusion protein of a GIPR antibody described herein and GLP-1.

[0034] The present invention provides a method for treating, preventing or improving one or more symptoms of obesity, which comprises administering to a subject a therapeutically effective amount of a GIPR antibody described herein.

[0035] The present invention provides a method for treating, preventing or improving one or more symptoms of obesity, which comprises administering to a subject a therapeutically effective amount of a fusion protein of a GIPR antibody described herein and GLP-1. Brief Description of the Drawings

[0037] Figure 1 : shows the results of detecting the specific binding of the recombinantly expressed hGIPR antibody L10H8 (which comprises SEQ ID NO: 70 and SEQ ID NO: 79) to hGIPR by flow cytometry (FACS), wherein the gray peak and the dotted peak are negative controls, the gray peak is the background peak of blank cells CHO-DHFR-, the dotted peak represents the negative binding peak of L10H8 to blank cells CHO-DHFR-, and the solid peak represents the specific binding peak of L10H8 to CHO-DHFR-hGIPR.

[0038] Figure 2: Shows the concentration inhibition curve (IC 50 = 7.6 nM, R 2 = 0.99) of the hGIPR antibody L7H6 (which contains SEQ ID NO: 67 and SEQ ID NO: 77) antagonizing GIP activation of the hGIPR signaling pathway in a direct cAMP experiment.

[0039] Figure 3 : Shows the inhibitory curve (IC 50 = 14.9 nM, R 2 = 0.99) of the GIPR antibody / GLP-1 fusion protein GLP-1-Linker-L7H6 (which contains SEQ ID NO: 67, SEQ ID NO: 77, SEQ ID NO: 106, SEQ ID NO: 111) antagonizing GIP activation of the hGIPR signaling pathway in a direct cAMP experiment.

[0040] Figure 4 : Shows the activation curve (EC 50 = 0.04 nM, R 2 = 0.99) of the GIPR antibody / GLP-1 fusion protein GLP-1-Linker-L7H6 activating the hGLP-1R signaling pathway in a reporter gene experiment.

[0041] Figure 5 : Shows the time curve of the body weight change rate of each group of mice during the pharmacodynamic experiment period of high-fat diet-induced C57BL / 6 obese mice.

[0042] Figure 6 : Shows the activation curve (EC50 = 17.40 pM, R 2 = 0.99) of the GIPR antibody / GLP-1 fusion protein GLP-1-Linker-V1W5 (which contains SEQ ID NO: 106, SEQ ID NO: 111, SEQ ID NO: 125 and SEQ ID NO: 131) activating the human GLP-1 receptor (hGLP-1R) signaling pathway in a reporter gene experiment.

[0043] Figure 7 : Shows the concentration inhibition curve (IC50 = 7.03 nM, R 2 = 0.99) of the hGIPR antibody GLP-1-Linker-V1W5 antagonizing GIP activation of the hGIPR signaling pathway in a direct cAMP experiment.

[0044] Figure 8: Shows the inhibitory curve of the hGIPR antibody / GLP-1 fusion protein GLP-1-Linker-V1W5 antagonizing the GIP-activated signal pathway of the macaque GIP receptor (maGIPR) in a direct cAMP experiment (IC50 = 4.30 nM, R 2 = 0.99).

[0045] Figure 9 : Shows the pharmacokinetic (PK) time curve of the antibody part of the hGIPR antibody / GLP-1 fusion protein during the pharmacokinetic experiment in rhesus monkeys.

[0046] Figure 10 : Shows the pharmacokinetic (PK) time curve of the GLP-1 part of the hGIPR antibody / GLP-1 fusion protein during the pharmacokinetic experiment in rhesus monkeys.

[0047] Figure 11 : Shows the time curve of the effect of the hGIPR antibody / GLP-1 fusion protein on the change in food intake during the pharmacodynamic experiment in obese cynomolgus monkeys induced by a high-fat diet.

[0048] Figure 12 : Shows the time curve of the effect of the hGIPR antibody / GLP-1 fusion protein on the change in body weight during the pharmacodynamic experiment in obese cynomolgus monkeys induced by a high-fat diet.

[0049] Figure 13 : Shows the time curve of the effect of the hGIPR antibody / GLP-1 fusion protein on the body weight change rate during the pharmacodynamic experiment in obese cynomolgus monkeys induced by a high-fat diet. Day 28: Compared with the formulation control group, the statistical P values of GLP-1-Linker-V1W5 and the positive control group were 0.000 and 0.003, respectively; Day 56: Compared with the GLP-1-Linker-V1W5 group, the statistical P values of the formulation control and the positive control groups were 0.003 and 0.028, respectively.

[0050] Figure 14 : Shows the time curve of the effect of the hGIPR antibody / GLP-1 fusion protein on the change in trunk fat during the pharmacodynamic experiment in obese cynomolgus monkeys induced by a high-fat diet.

[0051] Figure 15 : Shows the time curve of the effect of the hGIPR antibody / GLP-1 fusion protein on the change in total fat during the pharmacodynamic experiment in obese cynomolgus monkeys induced by a high-fat diet.

[0052] Figure 16 : Shows the time curve of the effect of the hGIPR antibody / GLP-1 fusion protein on the total fat change rate during the pharmacodynamic experiment in obese cynomolgus monkeys induced by a high-fat diet.

[0053] Figure 17 : It shows the time curve of the effect of hGIPR antibody / GLP-1 fusion protein on the change in total fat mass per 1 kg body weight during the pharmacodynamic experiment period of high-fat diet-induced obese cynomolgus monkeys.

[0054] Figure 18 : It shows the time curve of the effect of hGIPR antibody / GLP-1 fusion protein on the change in total lean tissue mass per 1 kg body weight during the pharmacodynamic experiment period of high-fat diet-induced obese cynomolgus monkeys. Detailed implementation mode

[0055] Definition

[0056] Unless otherwise defined herein, scientific and technical terms related to this article shall have the meanings understood by those of ordinary skill in the art. Generally, the nomenclature and techniques related to pharmacology, biology, biochemistry, cell and tissue culture, biology, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, as well as hybridization described herein, are well-known and commonly used in the art.

[0057] In this article, standard single-letter or three-letter abbreviations are used to indicate polynucleotide and polypeptide sequences. When writing polypeptide sequences, the first amino acid residue with an amino group (N′) is on the far left and the last amino acid residue with a carboxyl group (C′) is on the far right. For example, the GLP-1 fragment sequences involved in this article: SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109. The reverse polypeptide sequence refers to the sequence formed by arranging the amino acid occurrence order of the polypeptide sequence in reverse. For example, the reverse GLP-1 fragment sequences formed by the above GLP-1 fragment sequences: SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123. The 5′ end of the single-stranded nucleic acid sequence and the upstream strand of the double-stranded nucleic acid sequence is on the left and their 3′ end is on the right. Specific parts of polypeptides can be represented by amino acid residue numbers, such as amino acids 80 to 130, or by the actual residues at that site, such as Lys80 to Lys130. Specific polypeptide or polynucleotide sequences can also be described by explaining their differences from a reference sequence.

[0058] The terms "peptide", "polypeptide", and "protein" all refer to molecules containing two or more amino acids connected to each other by peptide bonds. These terms cover, for example, natural and artificial proteins and polypeptide analogs of protein sequences (such as mutant proteins, variants, and fusion proteins) as well as post-transcriptionally or otherwise covalently or non-covalently modified proteins. Peptides, polypeptides, or proteins can be monomers or polymers.

[0059] The term "polypeptide fragment" refers to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion as compared to the corresponding full-length protein. The fragment length can be, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 50, 70, 80, 90, 100, 150, or 200 amino acids. The fragment length can be, for example, at most 1000, 750, 500, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 14, 13, 12, 11, or 10 amino acids. The fragment can further comprise one or more additional amino acids at one or both of its ends, for example, amino acid sequences from different native proteins (e.g., Fc or leucine zipper domains) or artificial amino acid sequences (e.g., artificial linker sequences).

[0060] The polypeptides herein include polypeptides modified for any reason and by any method, e.g., to: (1) reduce proteolytic susceptibility, (2) reduce oxidative susceptibility, (3) alter the affinity for forming protein complexes, (4) alter binding affinity, and (5) confer or modify other physicochemical or functional properties. Analogs include mutant proteins of the polypeptide. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in the native sequence (e.g., in polypeptide portions outside of domains forming intramolecular contacts). A "conservative amino acid substitution" is one that does not significantly alter the structural properties of the parent sequence (e.g., the substituted amino acid should not disrupt a helix present in the parent sequence or interfere with other secondary structure types that confer properties to the parent sequence or are essential for its function).

[0061] A "variant" of a polypeptide comprises an amino acid sequence having an insertion, deletion, and / or substitution of one or more amino acid residues in the amino acid sequence as compared to another polypeptide sequence. Variants herein include fusion proteins.

[0062] A "derivative" of a polypeptide is a chemically modified polypeptide, e.g., by conjugation with other chemical moieties such as polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.

[0063] Unless otherwise indicated, the term "antibody" includes antibodies of two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and mutant proteins thereof, examples of which are provided below.

[0064] The term "antibody" refers to a protein that contains an antigen-binding portion and optionally a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that facilitates binding of the antibody to the antigen. Examples of antibodies include intact antibodies, antibody fragments (e.g., the antigen-binding portion of an antibody), antibody derivatives, and antibody mimetics. The antibody may comprise, for example, a selected protein scaffold or an artificial scaffold with grafted CDRs or CDR derivatives. The scaffold includes, but is not limited to, antibody-derived scaffolds that contain, for example, introduced elements to stabilize the three-dimensional structure of the antibody and fully synthetic scaffolds that contain, for example, biocompatible polymers. See, e.g., Korndorfer et al., 2003, Proteins 53:121-129; Roque et al., 2004, Biotechnol. Prog. 20:639-654. In addition, the antibody may be a peptide antibody mimetic ("PAMs") or a scaffold that contains an antibody mimetic that utilizes fibronectin as a scaffold.

[0065] An antibody can have, for example, the structure of a native immunoglobulin. An "immunoglobulin" is a tetrameric molecule. In a native immunoglobulin, each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal end of each chain includes a variable domain of about 100 to 110 amino acids that is primarily involved in antigen recognition. The carboxyl-terminal portion of each chain defines a constant region that is primarily involved in effector functions. Human antibody light chains are divided into κ and λ light chains. Heavy chains are divided into μ, δ, α, or ε and define the isotype of the antibody, e.g., IgM, IgD, IgG, IgA, and IgE, respectively. In both the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids. See, Fundamental Immunology Ch. 7 (Paul ed., 2nd ed., Raven Press, 1989). The variable regions of each light / heavy chain pair form the antibody binding site, such that a complete immunoglobulin has two binding sites.

[0066] Native immunoglobulin chains exhibit the same basic structure of relatively conserved framework regions (FRs) connected by three highly variable regions, also known as complementarity-determining regions or CDRs. From the N-terminus to the C-terminus, both light and heavy chains contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain is consistent with the definition in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, U.S. Dept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991.

[0067] Unless otherwise specified, "antibody" refers to a complete immunoglobulin or an antigen-binding portion thereof that can compete with the complete antibody for specific binding. The antigen-binding portion can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the complete antibody. Antigen-binding portions include, in particular, Fab, Fab’, F(ab’)2, Fv, domain antibodies (dAbs), fragments comprising complementarity-determining regions (CDRs), single-chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides comprising at least a portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide.

[0068] The Fab fragment is a monovalent fragment having the V L , V H , C L , and C H1 domains; the F(ab’)2 fragment is a divalent fragment having two Fab fragments linked by a disulfide bond in the hinge region; the Fv fragment has V H and V L domains; the dAb fragment has V H domains, V L domains, or an antigen-binding fragment of V H or V L domains (U.S. Patent Nos. US 6,846,634 and US 6,696,245; U.S. Patent Application Publication Nos. US 2005 / 0202512, US 2004 / 0202995, US 2004 / 0038291, US 2004 / 0009507, and US 2003 / 0039958; Ward et al., 1989, Nature 341:544-546).

[0069] A single-chain antibody (scFv) is a fusion protein in which V L and VH Antibodies in which the regions are joined by a linker (e.g., a synthetic amino acid residue sequence) to form a continuous protein, where the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen-binding site (see, e.g., Bird et al., 1988, Science 242:423-26; and Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5879-83).

[0070] A diabody is a bivalent antibody that comprises two polypeptide chains, where each polypeptide chain comprises a V H and a V L domain joined by a linker that is short enough so as not to allow pairing of the two domains on the same chain, thus allowing each domain to pair with a complementary domain on the other polypeptide chain (see, e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:6444-48; and Poljak et al., 1994, Structure 2:1121-23). If the two polypeptide chains of a diabody are identical, then the diabody resulting from their pairing will have the same antigen-binding site. Polypeptide chains with different sequences can be used to prepare diabodies with different antigen-binding sites. Similarly, a triabody and a tetrabody are antibodies that comprise three and four polypeptide chains, respectively, and form three and four antigen-binding sites, which can be the same or different.

[0071] The methods described by Kabat et al. in Sequences of Proteins of Immunological Interest, 5th ed., U.S. Dept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991 are used herein to identify the complementarity-determining regions (CDRs) and framework regions (FRs) of a given antibody. One or more CDRs can be incorporated into the molecule covalently or non-covalently to make it an antibody. An antibody can incorporate the CDR(s) into a larger polypeptide chain. The CDR(s) can be covalently linked to another polypeptide chain, or the CDR(s) can be incorporated non-covalently. The CDRs allow the antibody to specifically bind to a particular related antigen.

[0072] An antibody can have one or more binding sites. If there is more than one binding site, the binding sites can be the same or different from one another. For example, a native human immunoglobulin typically has two identical binding sites, while a "bispecific" or "bifunctional" antibody has two different binding sites.

[0073] The term "murine antibody" includes antibodies having one or more variable and constant regions derived from murine immunoglobulin sequences.

[0074] The term "humanized antibody" refers to an antibody produced by grafting the complementarity-determining region sequences of a murine antibody molecule into the framework of a human antibody variable region.

[0075] The terms "antigen-binding domain", "antigen-binding region" or "antigen-binding site" refer to the part of an antibody that contains the amino acid residues that interact with an antigen and contribute to the specificity and affinity of the antibody for the antigen. For an antibody that binds specifically to its antigen, this will include at least part of at least one of its CDR domains.

[0076] The term "epitope" refers to the molecular moiety that binds to an antibody (e.g., via the antibody). An epitope can contain non-contiguous portions of a molecule (e.g., in a polypeptide, amino acid residues that are not contiguous in the primary sequence of the polypeptide are close enough to each other in the tertiary and quaternary structures of the polypeptide to be bound by an antibody).

[0077] The "percent identity" of two polynucleotide or two polypeptide sequences is determined by comparing the sequences using the GAP computer program (part of the GCG Wisconsin Package; version 10.3 (Accelrys, San Diego, CA)) with its default parameters.

[0078] The terms "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably throughout and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA or RNA analogs generated using nucleotide analogs (e.g., peptide nucleic acids and non-natural nucleotide analogs) and their hybrids. Nucleic acid molecules can be single-stranded or double-stranded. In one embodiment, the nucleic acid molecules herein contain a continuous open reading frame encoding an antibody or a fragment, derivative, mutant protein or variant provided herein.

[0079] Two single-stranded polynucleotides are "complementary" to each other if their sequences can be aligned in an antiparallel fashion such that each nucleotide in one polynucleotide is opposite the complementary nucleotide in the other polynucleotide, no gaps are introduced and there are no unpaired nucleotides at the 5' or 3' ends of each sequence. A polynucleotide is "complementary" to another polynucleotide if the two polynucleotides can hybridize to each other under moderately stringent conditions. Thus, a polynucleotide can be complementary to another polynucleotide, but not to its complementary sequence.

[0080] The term "vector" refers to a nucleic acid that can be used to introduce another nucleic acid linked thereto into a cell. One type of vector is a "plasmid", which refers to a linear or circular double-stranded DNA molecule to which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), into which additional DNA segments can be introduced into the viral genome. Some vectors can replicate autonomously in the host cells into which they are introduced (e.g., bacterial vectors containing a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) integrate into the genome of the host cell upon introduction into the host cell and are thus replicated along with the host genome. An "expression vector" is a type of vector that can direct the expression of a selected polynucleotide.

[0081] A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g., expression level, timing, or location) of the nucleotide sequence. A "regulatory sequence" is a nucleic acid that can affect the expression (e.g., expression level, timing, or location) of a nucleic acid to which it is operably linked. Regulatory genes act directly on the nucleic acid being regulated or through the action of one or more other molecules (e.g., polynucleotides that bind to the regulatory sequence and / or the nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Further examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology, Volume 185, Academic Press, San Diego, CA; and Baron et al., 1995, Nucleic Acids Res. 23: 3605-06.

[0082] The term "host cell" refers to a cell used for expressing nucleic acids such as the nucleic acids provided herein. The host cell can be a prokaryote, such as Escherichia coli, or it can be a eukaryote, such as a unicellular eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cells), an animal cell (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, or insect cells), or a hybridoma. Generally, the host cell is a cultured cell that can be transformed or transfected with a nucleic acid encoding a polypeptide, which can then be expressed in the host cell. The phrase "recombinant host cell" can be used to refer to a host cell that has been transformed or transfected with a nucleic acid to be expressed. The host cell can also be a cell that contains the nucleic acid but does not express it at the desired level, unless regulatory sequences are introduced into the host cell such that they are operably linked to the nucleic acid. It should be understood that the term host cell refers not only to a specific subject cell but also to the progeny or potential progeny of that cell. Due to, for example, mutations or environmental influences, subsequent generations may have certain modifications, and the progeny may in fact be different from the parental cell but still fall within the scope of the term as used herein.

[0083] Gastric inhibitory polypeptide receptor

[0084] The gastric inhibitory polypeptide receptor (GIPR) belongs to class B of the seven-transmembrane G protein-coupled receptor family and is coupled to one or more intracellular signaling pathways through heterotrimeric guanine nucleotide-binding proteins (G proteins) (Drucker et al., 2006, Cell Metab. 3:153-65). To date, studies have found that GIPR is mainly expressed on the surface of pancreatic islet β cells and adipocytes (Ravn et al., 2013, J. Biol. Chem. 288:19760-72), and is involved in glucose and lipid metabolism processes in the human body, and is thus also closely associated with diabetes, obesity, and related disorders (Skaw et al., 2016, Diabetes Obes. Metab. 18:847-854). As used herein, both "human GIPR" and "hGIPR" refer to the human gastric inhibitory polypeptide receptor and can be used interchangeably. As used herein, both "mouse GIPR" and "mGIPR" refer to the mouse gastric inhibitory polypeptide receptor and can also be used interchangeably.

[0085] In one embodiment, the antibodies provided herein are antibodies that specifically bind to human GIPR. In another embodiment, the antibodies provided herein are antibodies that specifically bind to GIPR on the cell membrane, and the antibody can inhibit or block the conduction of GIP signals within these cells. In another embodiment, the antibodies provided herein are antibodies that specifically bind to human GIPR, and the antibody can bind to GIPR of other species (such as monkeys or mice) and block the signal transduction of GIP in these species. In a further embodiment, the antibodies provided herein are murine antibodies that bind to human GIPR, and the antibody can bind to GIPR of other species (such as monkeys).

[0086] In one embodiment, the amino acid and polynucleotide sequences of GIPR are listed below, and the sequence data is from the GeneBank database of the National Center for Biotechnology Information in the United States and the Uniprot database of the European Bioinformatics Institute:

[0087] Human (Homo sapiens) polynucleotide (SEQ ID NO: 114); accession number: S79852; Human (Homo sapiens) amino acid (SEQ ID NO: 113); accession number: AAB35419.2; Rhesus macaque polynucleotide (SEQ ID NO: 116); accession number: XM_015124289.1;

[0088] Rhesus macaque amino acid (SEQ ID NO: 115); accession number: XP_014979775; Mouse (Mus musculus) polynucleotide (SEQ ID NO: 118); accession number: CCDS39795; and Mouse (Mus musculus) amino acid (SEQ ID NO: 117); accession number: Q0P543.

[0089] Gastric inhibitory polypeptide receptor antibody (GIPR antibody)

[0090] In one embodiment, the GIPR antibodies provided herein are provided. In another embodiment, the GIPR antibodies provided herein are intact GIPR antibodies. In another embodiment, the GIPR antibodies provided herein are GIPR antibody fragments. In another embodiment, the GIPR antibodies provided herein are GIPR antibody derivatives. In another embodiment, the GIPR antibodies provided herein are GIPR antibody mutant proteins. In a further embodiment, the GIPR antibodies provided herein are GIPR antibody variants.

[0091] In one embodiment, the GIPR antibodies provided herein comprise one, two, three, four, five, or six amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0092] a. Light chain CDR1 amino acid sequences: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 15;

[0093] b. Light chain CDR2 amino acid sequences: SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, and SEQ ID NO: 16;

[0094] c. Light chain CDR3 amino acid sequences: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 17;

[0095] d. Heavy chain CDR1 amino acid sequences: SEQ ID NO: 18, SEQ ID NO: 23, and SEQ ID NO: 26;

[0096] e. Heavy chain CDR2 amino acid sequences: SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, and SEQ ID NO: 29; and

[0097] f. Heavy chain CDR3 amino acid sequences: SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, and SEQ ID NO: 30.

[0098] Table 1 lists the amino acid sequences of the light chain CDRs of the GIPR antibodies provided herein, as well as their corresponding polynucleotide coding sequences. Table 2 lists the amino acid sequences of the heavy chain CDRs of the GIPR antibodies provided herein, as well as their corresponding polynucleotide coding sequences.

[0099] Table 1: Amino acid sequences of light chain CDRs and their polynucleotide coding sequences

[0100]

[0101]

[0102] Table 2: Amino acid sequences of heavy chain CDRs and their polynucleotide coding sequences

[0103]

[0104]

[0105] In one embodiment, the antibodies provided herein comprise a sequence that differs from one of the CDR amino acid sequences listed in Tables 1 and 2 by 5, 4, 3, 2, or 1 single amino acid addition, substitution, and / or deletion. In another embodiment, the antibodies provided herein comprise a sequence that differs from one of the CDR amino acid sequences listed in Tables 1 and 2 by 4, 3, 2, or 1 single amino acid addition, substitution, and / or deletion.

[0106] In another embodiment, the antibodies provided herein comprise a sequence that differs from one of the CDR amino acid sequences listed in Tables 1 and 2 by 3, 2, or 1 single amino acid addition, substitution, and / or deletion.

[0107] In another embodiment, the antibodies provided herein comprise a sequence that differs from one of the CDR amino acid sequences listed in Tables 1 and 2 by 2 or 1 single amino acid addition, substitution, and / or deletion.

[0108] In a further embodiment, the antibodies provided herein comprise a sequence that differs from one of the CDR amino acid sequences listed in Tables 1 and 2 by 1 single amino acid addition, substitution, and / or deletion.

[0109] In one embodiment, the GIPR antibodies provided herein comprise one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0110] a. Light chain CDR1 amino acid sequences: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 15; and

[0111] b. Heavy chain CDR1 amino acid sequences: SEQ ID NO: 18, SEQ ID NO: 23, and SEQ ID NO: 26.

[0112] In another embodiment, the GIPR antibodies provided herein comprise one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0113] a. Light chain CDR2 amino acid sequences: SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, and SEQ ID NO: 16; and

[0114] b. Amino acid sequences of heavy chain CDR2: SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, and SEQ ID NO: 29.

[0115] In another embodiment, the GIPR antibodies provided herein comprise one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0116] a. Amino acid sequences of light chain CDR3: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 17; and

[0117] b. Amino acid sequences of heavy chain CDR3: SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, and SEQ ID NO: 30.

[0118] In another embodiment, the GIPR antibodies provided herein comprise one, two, three, or four amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0119] a. Amino acid sequences of light chain CDR1: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 15;

[0120] b. Amino acid sequences of heavy chain CDR1: SEQ ID NO: 18, SEQ ID NO: 23, and SEQ ID NO: 26;

[0121] c. Amino acid sequences of light chain CDR2: SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, and SEQ ID NO: 16; and

[0122] d. Amino acid sequences of heavy chain CDR2: SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, and SEQ ID NO: 29.

[0123] In another embodiment, the GIPR antibodies provided herein comprise one, two, three, or four amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0124] a. Amino acid sequences of light chain CDR1: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 15;

[0125] b. Amino acid sequences of heavy chain CDR1: SEQ ID NO: 18, SEQ ID NO: 23, and SEQ ID NO: 26;

[0126] c. Amino acid sequences of light chain CDR3: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 17; and

[0127] d. Amino acid sequences of heavy chain CDR3: SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, and SEQ ID NO: 30.

[0128] In a further embodiment, the GIPR antibodies provided herein comprise one, two, three, or four amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0129] a. Amino acid sequences of light chain CDR2: SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, and SEQ ID NO: 16;

[0130] b. Amino acid sequences of heavy chain CDR2: SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, and SEQ ID NO: 29;

[0131] c. Amino acid sequences of light chain CDR3: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 17; and

[0132] d. Amino acid sequences of heavy chain CDR3: SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, and SEQ ID NO: 30.

[0133] In one embodiment, the GIPR antibodies provided herein comprise one, two, or three amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17.

[0134] In another embodiment, the GIPR antibodies provided herein comprise one, two, or three amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30.

[0135] In one embodiment, the GIPR antibodies provided herein comprise a combination of light and heavy chain CDR1 amino acid sequences independently selected from the following: SEQ ID NO: 1 and SEQ ID NO: 18, SEQ ID NO: 4 and SEQ ID NO: 18, SEQ ID NO: 7 and SEQ ID NO: 23, SEQ ID NO: 10 and SEQ ID NO: 26, SEQ ID NO: 13 and SEQ ID NO: 26, and SEQ ID NO: 15 and SEQ ID NO: 26.

[0136] In another embodiment, the GIPR antibodies provided herein comprise a combination of light and heavy chain CDR2 amino acid sequences independently selected from the following: SEQ ID NO: 2 and SEQ ID NO: 19, SEQ ID NO: 5 and SEQ ID NO: 21, SEQ ID NO: 8 and SEQ ID NO: 24, SEQ ID NO: 11 and SEQ ID NO: 27, and SEQ ID NO: 16 and SEQ ID NO: 29.

[0137] In a further embodiment, the GIPR antibodies provided herein comprise a combination of light and heavy chain CDR3 amino acid sequences independently selected from the following: SEQ ID NO: 3 and SEQ ID NO: 20, SEQ ID NO: 6 and SEQ ID NO: 22, SEQ ID NO: 9 and SEQ ID NO: 25, SEQ ID NO: 12 and SEQ ID NO: 28, SEQ ID NO: 14 and SEQ ID NO: 28, and SEQ ID NO: 17 and SEQ ID NO: 30.

[0138] In one embodiment, the GIPR antibodies provided herein comprise:

[0139] a. A combination of light and heavy chain CDR1 amino acid sequences independently selected from the following: SEQ ID NO: 1 and SEQ ID NO: 18, SEQ ID NO: 4 and SEQ ID NO: 18, SEQ ID NO: 7 and SEQ ID NO: 23, SEQ ID NO: 10 and SEQ ID NO: 26, SEQ ID NO: 13 and SEQ ID NO: 26, and SEQ ID NO: 15 and SEQ ID NO: 26; and

[0140] b. A combination of light and heavy chain CDR2 amino acid sequences independently selected from the following: SEQ ID NO: 2 and SEQ ID NO: 19, SEQ ID NO: 5 and SEQ ID NO: 21, SEQ ID NO: 8 and SEQ ID NO: 24, SEQ ID NO: 11 and SEQ ID NO: 27, and SEQ ID NO: 16 and SEQ ID NO: 29.

[0141] In another embodiment, the GIPR antibodies provided herein comprise:

[0142] a. A combination of light and heavy chain CDR1 amino acid sequences independently selected from the following: SEQ ID NO: 1 and SEQ ID NO: 18, SEQ ID NO: 4 and SEQ ID NO: 18, SEQ ID NO: 7 and SEQ ID NO: 23, SEQ ID NO: 10 and SEQ ID NO: 26, SEQ ID NO: 13 and SEQ ID NO: 26, and SEQ ID NO: 15 and SEQ ID NO: 26; and

[0143] b. A combination of light and heavy chain CDR3 amino acid sequences independently selected from the following: SEQ ID NO: 3 and SEQ ID NO: 20, SEQ ID NO: 6 and SEQ ID NO: 22, SEQ ID NO: 9 and SEQ ID NO: 25, SEQ ID NO: 12 and SEQ ID NO: 28, SEQ ID NO: 14 and SEQ ID NO: 28, and SEQ ID NO: 17 and SEQ ID NO: 30.

[0144] In another embodiment, the GIPR antibodies provided herein comprise:

[0145] a. A combination of light and heavy chain CDR2 amino acid sequences independently selected from the following: SEQ ID NO: 2 and SEQ ID NO: 19, SEQ ID NO: 5 and SEQ ID NO: 21, SEQ ID NO: 8 and SEQ ID NO: 24, SEQ ID NO: 11 and SEQ ID NO: 27, and SEQ ID NO: 16 and SEQ ID NO: 29; and

[0146] b. A combination of light and heavy chain CDR3 amino acid sequences independently selected from the following: SEQ ID NO: 3 and SEQ ID NO: 20, SEQ ID NO: 6 and SEQ ID NO: 22, SEQ ID NO: 9 and SEQ ID NO: 25, SEQ ID NO: 12 and SEQ ID NO: 28, SEQ ID NO: 14 and SEQ ID NO: 28, and SEQ ID NO: 17 and SEQ ID NO: 30.

[0147] In a further embodiment, the GIPR antibodies provided herein comprise:

[0148] a. A combination of light and heavy chain CDR1 amino acid sequences independently selected from the following: SEQ ID NO: 1 and SEQ ID NO: 18, SEQ ID NO: 4 and SEQ ID NO: 18, SEQ ID NO: 7 and SEQ ID NO: 23, SEQ ID NO: 10 and SEQ ID NO: 26, SEQ ID NO: 13 and SEQ ID NO: 26, and SEQ ID NO: 15 and SEQ ID NO: 26;

[0149] b. A combination of light and heavy chain CDR2 amino acid sequences independently selected from the following: SEQ ID NO: 2 and SEQ ID NO: 19, SEQ ID NO: 5 and SEQ ID NO: 21, SEQ ID NO: 8 and SEQ ID NO: 24, SEQ ID NO: 11 and SEQ ID NO: 27, and SEQ ID NO: 16 and SEQ ID NO: 29; and

[0150] c. A combination of light and heavy chain CDR3 amino acid sequences independently selected from the following: SEQ ID NO: 3 and SEQ ID NO: 20, SEQ ID NO: 6 and SEQ ID NO: 22, SEQ ID NO: 9 and SEQ ID NO: 25, SEQ ID NO: 12 and SEQ ID NO: 28, SEQ ID NO: 14 and SEQ ID NO: 28, and SEQ ID NO: 17 and SEQ ID NO: 30.

[0151] In one embodiment, the GIPR antibodies provided herein comprise:

[0152] a. A combination of light and heavy chain CDR1, CDR2, and CDR3 amino acid sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20;

[0153] b. A combination of light and heavy chain CDR1, CDR2, and CDR3 amino acid sequences: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 18, SEQ ID NO: 21, and SEQ ID NO: 22;

[0154] c. A combination of light and heavy chain CDR1, CDR2, and CDR3 amino acid sequences: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25;

[0155] d. A combination of light and heavy chain CDR1, CDR2, and CDR3 amino acid sequences: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28;

[0156] e. Combinations of light and heavy chain CDR1, CDR2, and CDR3 amino acid sequences: SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28; or

[0157] f. Combinations of light and heavy chain CDR1, CDR2, and CDR3 amino acid sequences: SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 29, and SEQ ID NO: 30.

[0158] In one embodiment, the GIPR antibodies provided herein comprise one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0159] a. Light chain variable domain amino acid sequences: SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71; and amino acid sequences having at least 80%, at least 85%, at least 90%, or at least 95% identity to any one of these sequences; and

[0160] b. Heavy chain variable domain amino acid sequences: SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80; and amino acid sequences having at least 80%, at least 85%, at least 90%, or at least 95% identity to any one of these sequences.

[0161] In another embodiment, the polynucleotide coding sequences of the GIPR antibodies provided herein comprise one or two polynucleotide coding sequences, wherein each polynucleotide coding sequence is independently selected from the polynucleotide sequences listed below:

[0162] a. Polynucleotide coding sequences for light chain variable domains: SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, and SEQ ID NO: 91; and polynucleotide coding sequences having at least 80%, at least 85%, at least 90%, or at least 95% identity to any one of these sequences; and

[0163] b. Polynucleotide coding sequences for heavy chain variable domains: SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, and SEQ ID NO: 100; and polynucleotide coding sequences having at least 80%, at least 85%, at least 90%, or at least 95% identity to any one of these sequences.

[0164] In one embodiment, the GIPR antibodies provided herein comprise an amino acid sequence independently selected from the following: SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71.

[0165] In another embodiment, the GIPR antibodies provided herein comprise an amino acid sequence independently selected from the following: SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80.

[0166] In one embodiment, the GIPR antibodies provided herein comprise a combination of light and heavy chain variable domain amino acid sequences independently selected from the following: SEQ ID NO: 61 and SEQ ID NO: 72, SEQ ID NO: 62 and SEQ ID NO: 73, SEQ ID NO: 63 and SEQ ID NO: 74, SEQ ID NO: 64 and SEQ ID NO: 74, SEQ ID NO: 65 and SEQ ID NO: 75, SEQ ID NO: 66 and SEQ ID NO: 76, SEQ ID NO: 67 and SEQ ID NO: 77, SEQ ID NO: 68 and SEQ ID NO: 77, SEQ ID NO: 69 and SEQ ID NO: 78, SEQ ID NO: 70 and SEQ ID NO: 79, and SEQ ID NO: 71 and SEQ ID NO: 80.

[0167] In one embodiment, the GIPR antibodies provided herein comprise an amino acid sequence independently selected from the following: SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 76, and SEQ ID NO: 77.

[0168] In another embodiment, the GIPR antibodies provided herein comprise a combination of light and heavy chain variable domain amino acid sequences independently selected from the following: SEQ ID NO: 61 and SEQ ID NO: 72 (L1H1), SEQ ID NO: 62 and SEQ ID NO: 73 (L2H2), SEQ ID NO: 63 and SEQ ID NO: 74 (L3H3), SEQ ID NO: 64 and SEQ ID NO: 74 (L4H3), SEQ ID NO: 65 and SEQ ID NO: 75 (L5H4), SEQ ID NO: 66 and SEQ ID NO: 76 (L6H5), SEQ ID NO: 67 and SEQ ID NO: 77 (L7H6), SEQ ID NO: 68 and SEQ ID NO: 77 (L8H6), SEQ ID NO: 69 and SEQ ID NO: 78 (L9H7), SEQ ID NO: 70 and SEQ ID NO: 79 (L10H8), and SEQ ID NO: 71 and SEQ ID NO: 80 (L11H9).

[0169] This text can also use the "LxHy" notation to refer to the provided GIPR antibodies, where "x" corresponds to the light chain variable region sequence identifier; "y" corresponds to the heavy chain variable region sequence identifier. For example, L2H2 refers to a full antibody having a light chain variable region comprising the amino acid sequence of SEQ ID NO: 62 (L2) and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 73 (H2).

[0170] In one embodiment, the GIPR antibodies provided herein comprise one or two amino acid sequences, where each amino acid sequence is independently selected from the amino acid sequences listed below:

[0171] a. Light chain constant amino acid sequences: SEQ ID NO: 101 and SEQ ID NO: 102; and

[0172] b. Heavy chain constant amino acid sequences: SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 124.

[0173] In one embodiment, the GIPR antibodies provided herein comprise one or two amino acid sequences, where each amino acid sequence is independently selected from the combinations of light and heavy chain constant amino acid sequences listed below: SEQ ID NO: 101 and SEQ ID NO: 103, SEQ ID NO: 101 and SEQ ID NO: 104, SEQ ID NO: 102 and SEQ ID NO: 103, and SEQ ID NO: 102 and SEQ ID NO: 104. In another embodiment, the GIPR antibodies provided herein comprise one or two amino acid sequences, where each amino acid sequence is independently selected from the combinations of light and heavy chain constant amino acid sequences listed below: SEQ ID NO: 101 and SEQ ID NO: 124 and SEQ ID NO: 102 and SEQ ID NO: 124.

[0174] In one embodiment, the GIPR antibodies provided herein comprise the light and heavy chain CDRs listed herein, as well as the amino acid sequences of the FRs (frameworks). The amino acid sequences of the FRs are included in the amino acid sequences of the light or heavy chain variable domains and are not listed separately. In one embodiment, the antibody comprises a light chain CDR1 sequence listed herein. In another embodiment, the antibody comprises a light chain CDR2 sequence listed herein. In another embodiment, the antibody comprises a light chain CDR3 sequence listed herein. In another embodiment, the antibody comprises a heavy chain CDR1 sequence listed herein. In another embodiment, the antibody comprises a heavy chain CDR2 sequence listed herein. In another embodiment, the antibody comprises a heavy chain CDR3 sequence listed herein. In another embodiment, the antibody comprises a light chain FR1 sequence listed herein. In another embodiment, the antibody comprises a light chain FR2 sequence listed herein. In another embodiment, the antibody comprises a light chain FR3 sequence listed herein. In another embodiment, the antibody comprises a light chain FR4 sequence listed herein. In another embodiment, the antibody comprises a heavy chain FR1 sequence listed herein. In another embodiment, the antibody comprises a heavy chain FR2 sequence listed herein. In another embodiment, the antibody comprises a heavy chain FR3 sequence listed herein. In a further embodiment, the antibody comprises a heavy chain FR4 sequence listed herein.

[0175] In one embodiment, the light chain CDR3 sequence of the antibody differs from one of the light chain CDR3 amino acid sequences SEQ ID NO: 6, SEQ ID NO: 12, and SEQ ID NO: 14 listed herein by no more than 6, 5, 4, 3, 2, or 1 single amino acid addition, substitution, and / or deletion. In another embodiment, the heavy chain CDR3 sequence of the antibody differs from the heavy chain CDR3 amino acid sequence SEQ ID NO: 22 or SEQ ID NO: 28 listed herein by no more than 6, 5, 4, 3, 2, or 1 single amino acid addition, substitution, and / or deletion. In a further embodiment, the light chain CDR3 sequence of the antibody differs from one of the light chain CDR3 amino acid sequences SEQ ID NO: 6, SEQ ID NO: 12, and SEQ ID NO: 14 listed herein by no more than 6, 5, 4, 3, 2, or 1 single amino acid addition, substitution, and / or deletion, and the heavy chain CDR3 sequence of the antibody differs from the heavy chain CDR3 amino acid sequence SEQ ID NO: 22 or SEQ ID NO: 28 listed herein by no more than 6, 5, 4, 3, 2, or 1 single amino acid addition, substitution, and / or deletion. In another embodiment, the antibody further comprises 1, 2, 3, 4, 5, or 6 of the light and heavy chain CDR light and heavy chain sequence combinations listed herein.

[0176] In one embodiment, the GIPR antibodies provided herein comprise a light chain variable domain amino acid sequence selected from the light chain variable domain sequences of L2 (SEQ ID NO: 62), L3 (SEQ ID NO: 63), L4 (SEQ ID NO: 64), L6 (SEQ ID NO: 66), L7 (SEQ ID NO: 67), and L8 (SEQ ID NO: 68) listed herein. In one embodiment, the light chain variable domain amino acid sequence of the GIPR antibody differs from the light chain variable domain amino acid sequence of one of L2 (SEQ ID NO: 62), L3 (SEQ ID NO: 63), L4 (SEQ ID NO: 64), L6 (SEQ ID NO: 66), L7 (SEQ ID NO: 67), and L8 (SEQ ID NO: 68) by 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, wherein the difference in each sequence is independently a deletion, insertion, or substitution of one amino acid residue. In another embodiment, the light chain variable domain amino acid sequence of the GIPR antibody is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the light chain variable domain amino acid sequence of one of L2 (SEQ ID NO: 62), L3 (SEQ ID NO: 63), L4 (SEQ ID NO: 64), L6 (SEQ ID NO: 66), L7 (SEQ ID NO: 67), and L8 (SEQ ID NO: 68). In another embodiment, the polynucleotide coding sequence of the light chain variable domain of the GIPR antibody is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the polynucleotide coding sequence of one of L2 (SEQ ID NO: 62), L3 (SEQ ID NO: 63), L4 (SEQ ID NO: 64), L6 (SEQ ID NO: 66), L7 (SEQ ID NO: 67), and L8 (SEQ ID NO: 68). In another embodiment, the polynucleotide coding sequence of the light chain variable domain of the GIPR antibody comprises a polynucleotide sequence that hybridizes under moderate conditions to the complementary sequence of the polynucleotide coding sequence of the light chain variable domain of one of L2 (SEQ ID NO: 62), L3 (SEQ ID NO: 63), L4 (SEQ ID NO: 64), L6 (SEQ ID NO: 66), L7 (SEQ ID NO: 67), and L8 (SEQ ID NO: 68).In a further embodiment, the polynucleotide coding sequence of the light chain variable domain of the GIPR antibody comprises a polynucleotide sequence that hybridizes under stringent conditions to a complementary sequence of the polynucleotide coding sequence of the light chain variable domain of one of L2 (SEQ ID NO: 62), L3 (SEQ ID NO: 63), L4 (SEQ ID NO: 64), L6 (SEQ ID NO: 66), L7 (SEQ ID NO: 67), and L8 (SEQ ID NO: 68).

[0177] In one embodiment, the GIPR antibodies provided herein comprise a heavy chain variable domain amino acid sequence selected from the heavy chain variable domain sequences of H2 (SEQ ID NO: 73), H3 (SEQ ID NO: 74), H5 (SEQ ID NO: 76), and H6 (SEQ ID NO: 77) listed herein. In another embodiment, the heavy chain variable domain amino acid sequence of the GIPR antibody has 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid differences from the heavy chain variable domain sequence of one of H2 (SEQ ID NO: 73), H3 (SEQ ID NO: 74), H5 (SEQ ID NO: 76), and H6 (SEQ ID NO: 77), wherein the differences in each sequence are independently a deletion, insertion, or substitution of one amino acid residue. In another embodiment, the heavy chain variable domain amino acid sequence of the GIPR antibody is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the heavy chain variable domain sequence of one of H2 (SEQ ID NO: 73), H3 (SEQ ID NO: 74), H5 (SEQ ID NO: 76), and H6 (SEQ ID NO: 77). In another embodiment, the heavy chain variable domain polynucleotide coding sequence of the GIPR antibody is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the heavy chain variable domain polynucleotide coding sequence of one of H2 (SEQ ID NO: 73), H3 (SEQ ID NO: 74), H5 (SEQ ID NO: 76), and H6 (SEQ ID NO: 77). In another embodiment, the heavy chain variable domain polynucleotide coding sequence of the GIPR antibody comprises a polynucleotide sequence that hybridizes under moderately stringent conditions to a complementary sequence of the polynucleotide coding sequence of the heavy chain variable domain of one of H2 (SEQ ID NO: 73), H3 (SEQ ID NO: 74), H5 (SEQ ID NO: 76), and H6 (SEQ ID NO: 77). In one embodiment, the heavy chain variable domain polynucleotide coding sequence of the GIPR antibody comprises a polynucleotide sequence that hybridizes under stringent conditions to a complementary sequence of the polynucleotide coding sequence of the heavy chain variable domain of one of H2 (SEQ ID NO: 73), H3 (SEQ ID NO: 74), H5 (SEQ ID NO: 76), and H6 (SEQ ID NO: 77).

[0178] In one embodiment, the antibodies provided herein are antibodies comprising a combination of L1H1 (SEQ ID NO: 61 and SEQ ID NO: 72), L2H2 (SEQ ID NO: 62 and SEQ ID NO: 73), L3H3 (SEQ ID NO: 63 and SEQ ID NO: 74), L4H3 (SEQ ID NO: 64 and SEQ ID NO: 74), L5H4 (SEQ ID NO: 65 and SEQ ID NO: 75), L6H5 (SEQ ID NO: 66 and SEQ ID NO: 76), L7H6 (SEQ ID NO: 67 and SEQ ID NO: 77), L8H6 (SEQ ID NO: 68 and SEQ ID NO: 77), L9H7 (SEQ ID NO: 69 and SEQ ID NO: 78), L10H8 (SEQ ID NO: 70 and SEQ ID NO: 79), or L11H9 (SEQ ID NO: 71 and SEQ ID NO: 80), or a desired phenotype thereof (e.g., IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, IgE, or IgD), or a Fab or F(ab′)2 fragment thereof.

[0179] In one embodiment, the antibodies provided herein are antibodies comprising a combination of L2H2 (SEQ ID NO: 62 and SEQ ID NO: 73), L3H3 (SEQ ID NO: 63 and SEQ ID NO: 74), L4H3 (SEQ ID NO: 64 and SEQ ID NO: 74), L6H5 (SEQ ID NO: 66 and SEQ ID NO: 76), L7H6 (SEQ ID NO: 67 and SEQ ID NO: 77) or L8H6 (SEQ ID NO: 68 and SEQ ID NO: 77), or a class - switched antibody thereof (e.g., IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, IgE, and IgD), or a Fab or F(ab′)2 fragment thereof.

[0180] The antibodies provided herein may comprise any one of the constant regions known in the art. The light - chain constant region may be, for example, a κ or λ type light - chain constant region, such as a murine κ or λ type light - chain constant region. The heavy - chain constant region may be, for example, an α, δ, ε, γ, or μ type heavy - chain constant region, such as a murine α, δ, ε, γ, or μ type heavy - chain constant region. In one embodiment, the light - chain or heavy - chain constant region is a fragment, derivative, variant, or mutant protein of the native constant region.

[0181] In one embodiment, the antibodies provided herein further comprise a human constant light chain κ or λ domain or a fragment thereof. The amino acid sequences of the light chain constant regions are as follows:

[0182] Amino acid sequence of the human constant light chain κ domain: (SEQ ID NO: 101); and

[0183] Amino acid sequence of the human constant light chain λ domain: (SEQ ID NO: 102).

[0184] In one embodiment, the antibodies provided herein further comprise a human heavy chain constant domain or a fragment thereof.

[0185] The amino acid sequences of the heavy chain constant regions are as follows:

[0186] Amino acid sequence of the human heavy chain constant region (hIgG2): (SEQ ID NO: 103);

[0187] Amino acid sequence of the human heavy chain constant region (hIgG4): (SEQ ID NO: 104); and

[0188] Amino acid sequence of the human heavy chain constant region (hIgG4): (SEQ ID NO: 124).

[0189] In one embodiment, the GIPR antibodies provided herein comprise a light chain domain amino acid sequence selected from V1 (SEQ ID NO: 125) and V2 (SEQ ID NO: 126) listed herein. In one embodiment, the light chain domain amino acid sequence of the GIPR antibody differs from the light chain domain amino acid sequence of one of V1 and V2 by 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, where the difference in each sequence is independently a deletion, insertion, or substitution of an amino acid residue. In another embodiment, the light chain domain amino acid sequence of the GIPR antibody is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the light chain domain amino acid sequence of one of V1 and V2. In another embodiment, the light chain domain polynucleotide coding sequence of the GIPR antibody is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the polynucleotide coding sequence of one of V1 and V2. In another embodiment, the light chain domain polynucleotide coding sequence of the GIPR antibody comprises a polynucleotide sequence that hybridizes under moderate conditions to a complementary sequence of the polynucleotide coding sequence of the light chain domain of one of V1 and V2. In a further embodiment, the light chain domain polynucleotide coding sequence of the GIPR antibody comprises a polynucleotide sequence that hybridizes under stringent conditions to a complementary sequence of the polynucleotide coding sequence of the light chain domain of one of V1 and V2.

[0190] In one embodiment, the GIPR antibody provided herein comprises a heavy chain domain amino acid sequence selected from W1 (SEQ ID NO: 127), W2 (SEQ ID NO: 128), W3 (SEQ ID NO: 129), W4 (SEQ ID NO: 130), W5 (SEQ ID NO: 131), W6 (SEQ ID NO: 132), W7 (SEQ ID NO: 133), W8 (SEQ ID NO: 134), and W9 (SEQ ID NO: 135) listed herein. In another embodiment, the heavy chain domain amino acid sequence of the GIPR antibody differs from the heavy chain domain sequence of one of W1, W2, W3, W4, W5, W6, W7, W8, and W9 by 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, wherein the difference in each sequence is independently a deletion, insertion, or substitution of an amino acid residue. In another embodiment, the heavy chain domain amino acid sequence of the GIPR antibody is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the heavy chain domain sequence of one of W1, W2, W3, W4, W5, W6, W7, W8, and W9. In another embodiment, the polynucleotide coding sequence of the heavy chain domain of the GIPR antibody is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the polynucleotide coding sequence of one of W1, W2, W3, W4, W5, W6, W7, W8, and W9. In another embodiment, the polynucleotide coding sequence of the heavy chain domain of the GIPR antibody comprises a polynucleotide sequence that hybridizes under moderate stringency conditions to a complementary sequence of the polynucleotide coding sequence of the heavy chain domain of one of W1, W2, W3, W4, W5, W6, W7, W8, and W9. In one embodiment, the polynucleotide coding sequence of the heavy chain domain of the GIPR antibody comprises a polynucleotide sequence that hybridizes under stringent conditions to a complementary sequence of the polynucleotide coding sequence of the heavy chain domain of one of W1, W2, W3, W4, W5, W6, W7, W8, and W9.

[0191] In another embodiment, the antibodies provided herein are antibodies comprising a combination of V1W1 (SEQ ID NO: 125 and SEQ ID NO: 127), V1W2 (SEQ ID NO: 125 and SEQ ID NO: 128), V1W3 (SEQ ID NO: 125 and SEQ ID NO: 129), V1W4 (SEQ ID NO: 125 and SEQ ID NO: 130), V1W5 (SEQ ID NO: 125 and SEQ ID NO: 131), V1W6 (SEQ ID NO: 125 and SEQ ID NO: 132), V1W7 (SEQ ID NO: 125 and SEQ ID NO: 133), V1W8 (SEQ ID NO: 125 and SEQ ID NO: 134), V1W9 (SEQ ID NO: 125 and SEQ ID NO: 135), V2W1 (SEQ ID NO: 126 and SEQ ID NO: 127), V2W2 (SEQ ID NO: 126 and SEQ ID NO: 128), V2W3 (SEQ ID NO: 126 and SEQ ID NO: 129), V2W4 (SEQ ID NO: 126 and SEQ ID NO: 130), V2W5 (SEQ ID NO: 126 and SEQ ID NO: 131), V2W6 (SEQ ID NO: 126 and SEQ ID NO: 132), V2W7 (SEQ ID NO: 126 and SEQ ID NO: 133), V2W8 (SEQ ID NO: 126 and SEQ ID NO: 134), or V2W9 (SEQ ID NO: 12 and SEQ ID NO: 135).

[0192] In one embodiment, the antibodies provided herein are antibodies comprising the V1W1 combination (SEQ ID NO: 125 and SEQ ID NO: 127). In one embodiment, the antibodies provided herein are antibodies comprising the V1W2 combination (SEQ ID NO: 125 and SEQ ID NO: 128). In one embodiment, the antibodies provided herein are antibodies comprising the V1W3 combination (SEQ ID NO: 125 and SEQ ID NO: 129). In one embodiment, the antibodies provided herein are antibodies comprising the V1W4 combination (SEQ ID NO: 125 and SEQ ID NO: 130). In one embodiment, the antibodies provided herein are antibodies comprising the V1W5 combination (SEQ ID NO: 125 and SEQ ID NO: 131). In one embodiment, the antibodies provided herein are antibodies comprising the V1W6 combination (SEQ ID NO: 125 and SEQ ID NO: 132). In one embodiment, the antibodies provided herein are antibodies comprising the V1W7 combination (SEQ ID NO: 125 and SEQ ID NO: 133). In one embodiment, the antibodies provided herein are antibodies comprising the V1W8 combination (SEQ ID NO: 125 and SEQ ID NO: 134). In one embodiment, the antibodies provided herein are antibodies comprising the V1W9 combination (SEQ ID NO: 125 and SEQ ID NO: 135). In one embodiment, the antibodies provided herein are antibodies comprising the V2W1 combination (SEQ ID NO: 126 and SEQ ID NO: 127). In one embodiment, the antibodies provided herein are antibodies comprising the V2W2 combination (SEQ ID NO: 126 and SEQ ID NO: 128). In one embodiment, the antibodies provided herein are antibodies comprising the V2W3 combination (SEQ ID NO: 126 and SEQ ID NO: 129). In one embodiment, the antibodies provided herein are antibodies comprising the V2W4 combination (SEQ ID NO: 126 and SEQ ID NO: 130). In one embodiment, the antibodies provided herein are antibodies comprising the V2W5 combination (SEQ ID NO: 126 and SEQ ID NO: 131). In one embodiment, the antibodies provided herein are antibodies comprising the V2W6 combination (SEQ ID NO: 126 and SEQ ID NO: 132). In one embodiment, the antibodies provided herein are antibodies comprising the V2W7 combination (SEQ ID NO: 126 and SEQ ID NO: 133). In one embodiment, the antibodies provided herein are antibodies comprising the V2W8 combination (SEQ ID NO: 126 and SEQ ID NO: 134).In one embodiment, the antibodies provided herein are antibodies comprising the V2W9 (SEQ ID NO: 126 and SEQ ID NO: 135) combination.

[0193] In one embodiment, the GIPR antibodies provided herein are selected from murine antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, antigen-binding antibody fragments, single-chain antibodies, double-chain antibodies, triple-chain antibodies, quadruple-chain antibodies, Fab fragments, F(ab’)x fragments, domain antibodies, IgD antibodies, IgE antibodies, IgM antibodies, IgGl antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies.

[0194] In one embodiment, the GIPR antibody provided herein is a GIPR monoclonal antibody.

[0195] In another embodiment, the GIPR antibody provided herein is a monoclonal antibody comprising a combination of amino acid sequences selected from the following: SEQ ID NO: 61 and SEQ ID NO: 72, SEQ ID NO: 62 and SEQ ID NO: 73, SEQ ID NO: 63 and SEQ ID NO: 74, SEQ ID NO: 64 and SEQ ID NO: 74, SEQ ID NO: 65 and SEQ ID NO: 75, SEQ ID NO: 66 and SEQ ID NO: 76, SEQ ID NO: 67 and SEQ ID NO: 77, SEQ ID NO: 68 and SEQ ID NO: 77, SEQ ID NO: 69 and SEQ ID NO: 78, SEQ ID NO: 70 and SEQ ID NO: 79, and SEQ ID NO: 71 and SEQ ID NO: 80.

[0196] In one embodiment, the GIPR antibody provided herein is a murine GIPR antibody. In another embodiment, the GIPR antibody provided herein is a humanized GIPR antibody.

[0197] In one embodiment, the GIPR antibody provided herein reduces the IC 50 value of human GIP signaling to about 1 nM to about 200 nM or about 1 nM to about 100 nM.

[0198] Antibodies and antibody fragments

[0199] In one embodiment, the antibodies provided herein are intact antibodies (including polyclonal, monoclonal, chimeric, humanized, or human antibodies having full-length heavy and / or light chains). In another embodiment, the antibodies provided herein are antibody fragments, such as F(ab’)2, Fab, Fab’, Fv, Fc, or Fd fragments, single domain antibodies, single-chain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetra-bodies, v-NAR, or bis-scFv (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology 23:1126-1136). In another embodiment, the antibodies provided herein include antibody polypeptides as disclosed in U.S. Patent No. 6,703,199, including fibronectin polypeptide single-chain antibodies. In a further embodiment, the antibodies provided herein include single-chain polypeptides as disclosed in U.S. Patent Publication 2005 / 0238646.

[0200] In one embodiment, the variable regions of the genes of monoclonal antibodies are amplified using nucleotide primers expressed in hybridomas. These primers can be synthesized by those of ordinary skill in the art or purchased from commercial sources. Murine and human variable region primers include primers for the V Ha 、V Hb 、V Hc 、V Hd 、C H1 、V L 、and C L regions that can be purchased from commercial sources. These primers can be used to amplify the heavy or light chain variable regions, which are then inserted separately into vectors such as IMMUNOZAP TM H or IMMUNOZAP TM L (Stratagene). These vectors are then introduced into E. coli, yeast, or mammalian-based expression systems. These methods can be used to produce large amounts of single-chain proteins containing fusions of the V H and V L domains (see Bird et al., 1988, Science 242:423-426).

[0201] Those skilled in the art will understand that some proteins, such as antibodies, may undergo a variety of post-translational modifications. The type and extent of these modifications depend on the host cell line used for expressing the protein and the culture conditions. Such modifications include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation. The carboxyl-terminal basic residues of an antibody (e.g., lysine or arginine) may be lost due to the frequent modification by carboxypeptidase (see, Harris, 1995, Journal of Chromatography 705: 129-134).

[0202] Murine monoclonal antibodies can be produced using conventional hybridoma cell methods. The monoclonal antibodies can be isolated and purified by a variety of established techniques. Such isolation techniques include affinity chromatography on protein A-agarose, size exclusion chromatography, and ion exchange chromatography (see, e.g., Coligan pp. 2.7.1-2.7.12 and 2.9.1-2.9.3; Baines et al., “Purification of Immunoglobulin G (IgG),” Methods in Molecular Biology, Vol. 10, pp. 79-104 (The Humana Press, Inc., 1992)). The monoclonal antibodies can be purified by affinity chromatography using an appropriate ligand selected based on the specific properties of the antibody (e.g., heavy chain or light chain isotype, binding specificity, etc.). Examples of appropriate ligands for affinity chromatography include protein A, protein G, anti-constant region (light chain or heavy chain) antibodies, anti-idiotype antibodies, and TGF-binding protein or its fragments or variants.

[0203] The molecule can be affinity matured by engineering the complementarity determining regions (CDRs) in the center of the antibody binding site to obtain an antibody with increased affinity, such as an antibody with increased affinity for c-erbB-2 (Schier et al., 1996, J. Mol. Biol. 263: 551-567). Thus, such techniques can be used to prepare antibodies against human GIPR.

[0204] For example, antibodies against human GIPR can be used in in vitro or in vivo assays for detecting the presence of human GIPR.

[0205] Antibodies can also be prepared by any conventional technique. For example, they can be purified from cells that naturally express these antibodies (e.g., purified from hybridomas that produce the antibodies) or produced in a recombinant expression system using any known technique in the art. See, e.g., Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, edited by Kennet et al., Plenum Press (1980); and Antibodies: A Laboratory Manual, edited by Harlow and Land, Cold Spring Harbor Laboratory Press (1988). This is discussed in the nucleic acid section below.

[0206] Antibodies can be prepared and screened for desired properties by any known technique. Some techniques involve isolating the nucleic acid encoding the polypeptide chain (or a portion thereof) of the relevant antibody (e.g., an anti-GIPR antibody) and manipulating the nucleic acid by recombinant DNA techniques. The nucleic acid can be fused to another relevant nucleic acid or modified (e.g., by mutagenesis or other conventional techniques) to add, delete, or substitute one or more amino acid residues.

[0207] When it is desired to improve the affinity of an antibody that includes one or more of the above-described CDRs herein, various affinity maturation protocols can be employed including CDR maintenance (Yang et al., 1995, J. Mol. Biol. 254:392-403), chain shuffling (Marks et al., 1992, Bio / Technology 10:779-783), use of mutant strains of Escherichia coli (Low et al., 1996, J. Mol. Biol. 250:350-368), DNA shuffling (Patten et al., 1997, Curr. Opin. Biotechnol. 8:724-733), phage display (Thompson et al., 1996, J. Mol. Biol. 256:7-88), and other PCR techniques (Crameri et al., 1998, Nature 391:288-291). All of these affinity maturation methods are discussed in Vaughan et al., 1998, Nature Biotechnology 16:535-539.

[0208] In one embodiment, the antibodies provided herein are anti-GIPR fragments. The fragment can consist entirely of antibody-derived sequences or can contain additional sequences. Examples of antigen-binding fragments include Fab, F(ab’)2, single-chain antibodies, diabodies, triabodies, tetra-bodies, and domain antibodies, with other examples provided in Lunde et al., 2002, Biochem. Soc. Trans. 30: 500-06.

[0209] The heavy and light chain variable domains (Fv regions) can be joined via an amino acid bridge (short peptide linker) to form single-chain antibodies, resulting in a single polypeptide chain. Single-chain Fvs (scFvs) have been prepared by fusing DNA encoding the peptide linker between DNAs encoding the two variable domain polypeptides (V L and V H ). The resulting polypeptides can fold back on themselves to form antigen-binding monomers, or they can form multimers (e.g., dimers, trimers, or tetramers), depending on the length of the flexible linker between the two variable domains (Kortt et al., 1997, Prot. Eng. 10: 423; Kortt et al. 2001, Biomol. Eng. 18: 95-108). By combining different V L and V H containing polypeptides, multimeric scFvs that bind to different epitopes can be formed (Kriangkum et al., 2001, Biomol. Eng. 18: 31-40). Techniques developed for producing single-chain antibodies include those described in U.S. Patent No. 4,946,778; Bird, 1988, Science 242: 423; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; Ward et al., 1989, Nature 334: 544-546; de Graaf et al., 2002, Methods Mol Biol. 178: 379-87. Single-chain antibodies derived from the antibodies provided herein include, but are not limited to, scFvs containing the variable domain combination L1H1, all of which are encompassed herein.

[0210] Antigen-binding fragments derived from antibodies can also be obtained by proteolytic cleavage of the antibody, e.g., by digesting the intact antibody with pepsin or papain according to conventional methods. For example, an antibody can be enzymatically cleaved with pepsin to provide an SS fragment called F(ab’)2 to produce antibody fragments. This fragment can be further cleaved using a sulfhydryl reducing agent to generate 3.5S Fab’ monovalent fragments. Alternatively, the cleavage reaction can be carried out using a sulfhydryl protecting group to effect cleavage of the disulfide bonds; additionally, enzymatic cleavage with papain can be used to directly produce two monovalent Fab fragments and one Fc fragment. These methods are described, e.g., in Goldenberg, U.S. Patent No. 4,331,647, Nisohoff et al., 1960, Arch. Biochem. Biophys. 89:230; Porter, 1959, Biochem. J. 73:119; Edelman et al., Methods in Enzymology 1:422 (Academic Press, 1967); and Andrews and Titus, J.A.Current Protocols in Immunology (edited by Coligan et al., John Wiley & Sons, 2003), pages 2.8, 1-2.8.10 and pages 2.10A.1-2.10A.5. Other methods of cleaving antibodies, e.g., preparing heavy chains to form monovalent heavy and light chain fragments (Fd), further cleaving the fragments or also using other enzymatic, chemical or genetic techniques can be used as long as the fragments bind to the antigen that can be recognized by the intact antibody.

[0211] Another form of antibody fragment is a peptide comprising one or more antibody complementarity determining regions (CDRs). The CDRs can be obtained by constructing polypeptides encoding the relevant CDRs. For example, such polypeptides can be prepared by using polymerase chain reaction with mRNA from antibody-producing cells as a template to synthesize variable regions. See, for example, Larrick et al., 1991, Methods: A Companion to Methods in Enzymology 2:106; Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. eds., page 166 (Cambridge University Press, 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," Monoclonal Antibodies: Principles and Applications, Birch et al. eds., page 137 (Wiley-Liss, Inc., 1995). The antibody fragment can further comprise at least one variable domain of the antibodies described herein. Thus, for example, the V domain can be monomeric and be a V H or V L domain, which can bind to the GIPR independently with an affinity of at least equal to 1 x 10 -7 M or higher as described below.

[0212] The variable domain can be any natural variable domain or its genetically engineered form. The genetically engineered form refers to a variable domain produced using recombinant DNA engineering techniques. The genetically engineered form includes, for example, those generated from a specific antibody variable region by insertion, deletion or alteration of the amino acid sequence of the specific antibody. Specific examples include variable domains assembled by genetic engineering that contain only one CDR and optionally one or more framework amino acids from one antibody and the remaining part of the variable domain from another antibody.

[0213] The variable domain can be covalently linked at the C-terminal amino acid to at least one other antibody domain or a fragment thereof. Thus, for example, the V H domain present in the variable domain can be linked to the immunoglobulin C H1 domain or a fragment thereof. Similarly, the V L domain can be linked to the C Kassociated with a domain or a fragment thereof. In this way, for example, the antibody can be a Fab fragment, wherein the antigen-binding domain contains their C-termini covalently linked to the combined V H1 and C K domains covalently linked to the V H and V L domains. The C H1 domain can be extended with other amino acids, for example to provide a hinge region or a partial hinge domain as in a Fab' fragment or to provide other domains, such as the antibody C H2 and C H3 domains.

[0214] Derivatives and variants of antibodies

[0215] For example, the nucleotide sequences encoding the amino acid sequences L1 and H1 can be altered by random mutagenesis or by site-directed mutagenesis (e.g., oligonucleotide-directed site-directed mutagenesis) to produce an altered polynucleotide that contains one or more specific nucleotide substitutions, deletions, or insertions compared to the unmutated polynucleotide. Examples of techniques for generating such alterations are described in Walder et al., 1986, Gene 42:133; Bauer et al., 1985, Gene 37:73; Craik, 1985, BioTechniques, 3:12-19; Smith et al., 1981, Genetic Engineering: Principles and Methods, Plenum Press; and U.S. Patent Nos. 4,518,584 and 4,737,462. These and other methods can be used to generate derivatives of anti-GIPR antibodies that have desired properties such as enhanced affinity, avidity, or specificity for GIPR, enhanced in vivo or in vitro activity or stability, or reduced in vivo side effects compared to the underivatized antibody.

[0216] Other anti-GIPR antibody derivatives in this art include covalent or aggregated conjugates of anti-GIPR antibodies or fragments thereof with other proteins or polypeptides, such as by expressing a recombinant fusion protein comprising a heterologous polypeptide fused to the N-terminus or C-terminus of an anti-GIPR antibody polypeptide. For example, the binding peptide can be a heterologous signal (or leader) polypeptide, such as the yeast α-factor leader peptide or a peptide such as an epitope tag. Antibodies comprising the fusion protein can include peptides (such as polyhistidine) added to assist in the purification or identification of the antibody. The antibody can also be linked to a FLAG peptide, as described by Hopp et al., 1988, Bio / Technology 6:1204 and U.S. Patent 5,011,912. The FLAG peptide has high antigenicity and provides an epitope that is reversibly bound by a specific monoclonal antibody (mAb), allowing for the rapid detection and convenient purification of the expressed recombinant protein. Reagents for preparing fusion proteins in which the FLAG peptide is fused to a given polypeptide are commercially available (Sigma-Aldrich, St. Louis, MO). In another embodiment, oligomers comprising one or more antibodies can be used as GIPR antagonists or higher-order oligomers. The oligomers can be in the form of covalently linked or non-covalently linked dimers, trimers, or higher oligomers. Oligomers comprising two or more antibodies can be used, one example being a homodimer. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, etc.

[0217] One embodiment is directed to oligomers comprising multiple antibodies that are linked by covalent or non-covalent interactions between peptide moieties fused to the antibodies. Such peptides can be peptide spacers or peptides having properties that promote oligomerization. Leucine zippers and certain polypeptides derived from antibodies are peptides that can promote antibody oligomerization, as described in detail below.

[0218] In a specific embodiment, the oligomer comprises two to four antibodies. The antibodies of the oligomer can be in any form, such as any of the forms described above, such as variants or fragments. Preferably, the oligomer comprises antibodies having GIPR binding activity.

[0219] In one embodiment, oligomers are prepared using polypeptides derived from immunoglobulins. The preparation of heterologous polypeptides fused to various portions of antibody-derived polypeptides, including the Fc domain, has been described, for example, in Ashkenazi et al., 1991, PNAS USA 88:10535; Byrn et al., 1990, Nature 344:677; and Hollenbaugh et al., Construction of Immunoglobulin Fusion Proteins, Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11. One embodiment herein is directed to a dimer comprising two fusion proteins generated by fusing the GIP-binding fragments of an anti-GIPR antibody to the Fc region of the antibody. The dimer can be prepared, for example, by inserting a gene fusion encoding the fusion protein into a suitable expression vector, expressing the fusion gene in a host cell transformed with the recombinant expression vector, and allowing the expressed fusion proteins to assemble like antibody molecules, wherein the interchain disulfide bonds between the Fc portions form the dimer.

[0220] As used herein, the term "Fc polypeptide" includes polypeptides in the native and mutant protein forms derived from the Fc region of an antibody. Also included are truncated forms of such polypeptides that contain a hinge region that promotes dimerization. Fusion proteins containing an Fc portion (and oligomers formed therefrom) offer the advantage of convenient purification by affinity chromatography on protein A or protein G columns.

[0221] One suitable Fc polypeptide described in PCT application W0 93 / 10151 (incorporated herein by reference) is a single-chain polypeptide extending from the N-terminal hinge region to the native C-terminus of the Fc region of a human IgG1 antibody. Another available Fc polypeptide is the Fc mutant protein described in U.S. Patent 5,457,035 and Baum et al., 1994, EMBO J. 13:3992-4001. The amino acid sequence of this mutant protein is the same as the amino acid sequence of the native Fc sequence shown in W0 93 / 10151, except that amino acid 19 is changed from leucine to alanine, amino acid 20 is changed from leucine to glutamine, and amino acid 22 is changed from glycine to alanine. This mutant protein exhibits a reduced affinity for Fc receptors. In other embodiments, the heavy and / or light chains of the anti-GIPR antibody can be replaced with the variable portions of the antibody heavy and / or light chains.

[0222] Alternatively, the oligomer is a fusion protein comprising multiple antibodies, with or without spacer peptides. Suitable spacer peptides are described in U.S. Patents 4,751,180 and 4,935,233.

[0223] Another method for preparing oligomeric antibodies involves the use of leucine zippers. Leucine zipper domains are peptides that promote the oligomerization of the proteins in which they are present. Leucine zippers were initially discovered in several DNA-binding proteins (Landschulz et al., 1988, Science 240:1759) and have since been found in a variety of different proteins. Among the known leucine zippers are native peptides or their derivatives that can dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT application WO 94 / 10308, and a leucine zipper derived from pulmonary surfactant protein D (SPD) is described in Hoppe et al., 1994, FEBS Letters 344:191, which is incorporated herein by reference. The use of modified leucine zippers that allow the stable trimerization of heterologous proteins fused thereto is described in Fanslow et al., 1994, Semin. Immunol. 6:267-78. In one method, a recombinant fusion protein comprising an anti-GIPR antibody fragment or derivative fused to a leucine zipper peptide is expressed in a suitable host cell, and the soluble oligomeric anti-GIPR antibody fragment or its derivative is collected from the culture supernatant.

[0224] In another embodiment, the antibody derivative may comprise at least one of the CDRs disclosed herein. For example, one or more CDRs can be integrated into a known antibody framework region (IgG1, IgG2, etc.) or conjugated to a suitable carrier to enhance its half-life. Suitable carriers include, but are not limited to, Fc, albumin, transferrin, and the like. These and other suitable carriers are known in the art. The conjugated CDR peptides can be monomers, dimers, tetramers, or other forms. In one embodiment, one or more water-soluble polymers bind to one or more specific sites of the binder, such as at the amino terminus. In one example, the antibody derivative comprises one or more water-soluble polymer attachments including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. See, for example, U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, and 4,179,337. In some embodiments, the derivative comprises one or more methoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate-based polymers, poly(N-vinylpyrrolidone)-polyethylene glycol, polyoxyethylene polyols (e.g., glycerol), and polyvinyl alcohol, as well as mixtures of such polymers. In some embodiments, one or more water-soluble polymers bind randomly to one or more side chains. In some embodiments, PEG can enhance the therapeutic effect of a binder such as an antibody. Some such methods are described, for example, in U.S. Patent No. 6,133,426, which is incorporated herein by reference for any purpose.

[0225] It should be understood that the antibodies provided herein may have at least one amino acid substitution, provided that the antibody retains binding specificity. Accordingly, modifications to the antibody structure are within the scope of this disclosure. These may include amino acid substitutions that do not disrupt the ability of the antibody to bind GIPR, which may be conservative or non-conservative. Conservative amino acid substitutions may include non-natural amino acid residues, which are typically incorporated by chemical peptide synthesis rather than biological systems. These include peptidomimetics and other reverse or inverted forms of amino acid moieties. Conservative amino acid substitutions may also involve replacing a natural amino acid residue with a non-natural residue that has little or no effect on the polarity or charge of the amino acid residue at that site. Non-conservative substitutions may involve the exchange of a member of one class of amino acids or amino acid analogs with a member of another class having different physical properties (e.g., volume, polarity, hydrophobicity, charge).

[0226] Moreover, one of ordinary skill in the art can generate variant polypeptides containing amino acid substitutions at each desired amino acid residue. Such variants can be screened using activity assays known to those of ordinary skill in the art. Such variants can be used to gather information about suitable variants. For example, if a particular amino acid residue is found to cause loss of activity, an undesired decrease, or inappropriate activity, variants having such changes can be avoided. In other words, based on the information gathered from these routine experiments, one of ordinary skill in the art can readily identify amino acids at which further substitutions (alone or in combination with other mutations) should be avoided.

[0227] One of ordinary skill in the art can use known techniques to identify suitable variants of the polypeptides listed herein. In some embodiments, one of ordinary skill in the art can identify suitable regions of the molecule that can be altered without disrupting activity by targeting regions that are not important for activity. In some embodiments, residues or molecular moieties that are conserved in similar polypeptides can be identified. In some embodiments, even regions that are important for biological activity or structure can be conservatively substituted without disrupting biological activity or having an adverse effect on polypeptide structure. In addition, one of ordinary skill in the art can examine structure-function studies to identify residues in similar polypeptides that are important for activity or structure. In view of this comparison, the importance of amino acid residues in a protein corresponding to amino acid residues that are important for activity or structure in a similar protein can be predicted. One of ordinary skill in the art can select chemically similar amino acid substitutions for these predicted important amino acid residues.

[0228] Those skilled in the art can also analyze the three-dimensional structure and amino acid sequence related to the structure of similar polypeptides. Given such information, those skilled in the art can predict the amino acid residue alignment of the antibody in terms of the three-dimensional structure. In some embodiments, those skilled in the art may choose not to make significant changes to the amino acid residues predicted to be on the protein surface, as such residues may be involved in important interactions with other molecules. Many scientific publications are dedicated to the prediction of secondary structure. See, Moult, 1996, Curr. Op. Biotech. 7:422-427, Chou et al., 1974, Biochemistry 13:222-245; Chou et al., 1974, Biochemistry 113:211-222; Chou et al., 1978, AdV. Enzymol. Relat. Areas Mol. Biol. 47:45-148; Chou et al., 1979, Ann. Rev. Biochem. 47:251-276 and Chou et al., Biophys. J. 26:367-384. In addition, computer programs can currently be used to assist in predicting secondary structure. For example, two polypeptides or proteins with a sequence identity greater than 30% or a similarity greater than 40% generally have similar higher-order structures. The recent growth of the Protein Data Bank (PDB) has enhanced the predictability of secondary structure, including the potential number of folds in a polypeptide or protein structure. See, Holm et al., 1999, Nucl. Acid. Res. 27:244-247. It has been shown (Brenner et al., 1997, Curr. Op. Struct. Biol. 7:369-376) that there is a limited number of folds in a given polypeptide or protein and that once a critical number of structures have been determined, structure prediction becomes significantly more accurate.

[0229] Other methods for predicting secondary structure include "threading" (Jones, 1997, Curr. Opin. Struct. Biol., 7:377-87; Sippl et al., 1996, Structure 4:15-19), "profile analysis" (Bowie et al., 1991, Science 253:164-170; Gribskov et al., 1990, Meth. Enzym. 183:146-159; Gribskov et al., 1987, Proc. Nat. Acad. Sci. USA 84:4355-4358) and "evolutionary linkage" (see Holm, supra (1999), and Brenner, supra (1997)). In some embodiments, antibody variants include glycosylation variants in which the number and / or type of glycosylation sites are altered compared to the amino acid sequence of the parental polypeptide. In some embodiments, the variant has more or fewer N-linked glycosylation sites compared to the native protein. Alternatively, replacement of the sequence can remove existing N-linked glycans. Rearrangement of N-linked glycans is also provided, in which one or more N-linked glycan sites (usually those that are naturally occurring) are removed and one or more new N-linked sites are created. Other preferred antibody variants include cysteine variants in which one or more cysteine residues are deleted or replaced by another amino acid (e.g., serine) compared to the parental amino acid sequence. Cysteine variants can be used when the antibody must fold into a bioactive conformation (e.g., after isolation of soluble inclusion bodies). Cysteine variants typically have fewer cysteine residues than the native protein and typically have an even number of cysteines to minimize interactions caused by unpaired cysteines.

[0230] Those skilled in the art can determine the desired amino acid substitutions (conservative or non-conservative) when such substitutions are needed. In some embodiments, amino acid substitutions can be used to identify important residues of the human GIPR antibody or to increase or decrease the affinity of the human GIPR antibodies described herein.

[0231] According to some embodiments, preferred amino acid substitutions are as follows: (1) reducing proteolytic sensitivity, (2) reducing oxidative sensitivity, (3) altering the binding affinity for forming protein complexes, (4) altering the binding affinity and / or (4) conferring or modifying other physicochemical or functional properties on such polypeptides. According to some embodiments, single or multiple amino acid substitutions (conservative amino acid substitutions in some embodiments) can be made in the naturally occurring sequence (in some embodiments, in the polypeptide portion outside the domain forming intermolecular contacts). In some embodiments, conservative amino acid substitutions generally do not substantially alter the structural properties of the parent sequence (e.g., the substituted amino acid should not break a helix present in the parent sequence or interfere with other types of secondary structure characterizing the parent sequence). Examples of polypeptide secondary and tertiary structures recognized in the art are described in Proteins, Structures and Molecular Principles, edited by Creighton, W.H. Freeman and Company (1984); Introduction to Protein Structure, edited by Branden and Tooze, Garland Publishing (1991); and Thornton et al., 1991, Nature 354:105, which are incorporated herein by reference.

[0232] In some embodiments, the antibodies provided herein can be chemically bonded to polymers, lipids, or other moieties.

[0233] An antigen-binding reagent can comprise at least one of the CDRs described herein incorporated into a biocompatible scaffold structure. In one example, the biocompatible scaffold structure comprises a polypeptide or portion thereof sufficient to form a conformationally stable structure to support or scaffold one or more amino acid sequences (e.g., CDRs, variable regions, etc.) that can bind to an antigen, which can be displayed in a confined surface region. Such structures can be naturally occurring polypeptides or polypeptide "folds" (structural motifs), or can have one or more modifications relative to the natural polypeptides or folds, such as amino acid additions, deletions, or substitutions. These scaffolds can be derived from polypeptides of any species (or more than one species), e.g., human, other mammals, other vertebrates, invertebrates, bacteria, or viruses.

[0234] Biologically soluble scaffold structures are generally based on protein scaffolds or skeletons rather than immunoglobulin domains. For example, those based on fibronectin, ankyrin, lipocalin, neocarcinostatin, cytochrome b, CP1 zinc finger protein, PST1, coiled coil, LACI-D1, Z domain, and amylastatin domain can be used (see, e.g., Nygren and Uhlen, 1997, Current Opinion in Structural Biology 7:463-469).

[0235] In addition, those skilled in the art will recognize that suitable binding agents include portions of these antibodies, such as one or more heavy chain CDR1, CDR2, CDR3, light chain CDR1, CDR2, and CDR3, as specifically disclosed herein. At least one of the heavy chain CDR1, CDR2, CDR3, CDR1, CDR2, and CDR3 regions has at least one amino acid substitution, provided that the antibody retains the binding specificity of the non-substituted CDRs. The non-CDR portion of the antibody can be a non-protein molecule, wherein the binding agent cross-blocks the binding of the antibodies disclosed herein to human GIPR and / or inhibits the signaling of GIP through this receptor. The non-CDR portion of the antibody can be a non-protein molecule, wherein the antibody shows a binding pattern to human GIP peptide similar to that shown by at least one of antibodies L2H2 / L6H5 in a competitive binding assay, and / or neutralizes the activity of GIP. The non-CDR portion of the antibody can be composed of amino acids, wherein the antibody is a recombinant binding protein or a synthetic peptide, and the recombinant binding protein cross-blocks the binding of the antibodies disclosed herein to human GIPR and / or neutralizes GIP activity in vivo or in vitro. The non-CDR portion of the antibody can be composed of amino acids, wherein the antibody is a recombinant antibody, and the recombinant antibody shows a binding pattern to human GIPR peptide similar to that shown by at least one of antibodies L2H2 / L6H5 in a competitive binding assay, and / or neutralizes GIP signaling.

[0236] Fusion protein of GIPR antibody and GLP-1 or reverse GLP-1

[0237] In one embodiment, provided herein is a fusion protein of a GIPR antibody and GLP-1, which comprises an antibody capable of specifically binding to GIPR, and one, two, three, four, five, six, seven, or eight GLP-1 fragments or reverse GLP-1 fragments; the fusion protein connects the carboxyl terminus of a GLP-1 fragment to the amino terminus of a GIPR antibody light chain or heavy chain, or connects the amino terminus of a reverse GLP-1 fragment to the carboxyl terminus of a GIPR antibody light chain or heavy chain through a peptide linker sequence (Linker).

[0238] In another embodiment, provided herein is a fusion protein of a GIPR antibody and GLP-1, which comprises an antibody that can specifically bind to GIPR, and one, two, three, four, five, six, seven, or eight GLP-1 fragments; the fusion protein connects the carboxyl terminus of a GLP-1 fragment to the amino terminus of a GIPR antibody light chain or heavy chain through a peptide linker sequence (Linker).

[0239] In another embodiment, provided herein is a fusion protein of a GIPR antibody and GLP-1, which comprises an antibody that can specifically bind to GIPR, and one, two, three, four, five, six, seven, or eight reverse GLP-1 fragments; the fusion protein connects the amino terminus of a reverse GLP-1 fragment to the carboxyl terminus of a GIPR antibody light chain or heavy chain through a peptide linker sequence (Linker).

[0240] In another embodiment, provided herein is a fusion protein of a GIPR antibody and GLP-1, which comprises an antibody that specifically binds to GIPR, and one, two, three, or four GLP-1 fragments; the fusion protein connects the carboxyl terminus of a GLP-1 fragment to the amino terminus of a GIPR antibody light chain or heavy chain through a peptide linker sequence (Linker).

[0241] In another embodiment, provided herein is a fusion protein of a GIPR antibody and GLP-1, which comprises an antibody that can specifically bind to GIPR, and one, two, three, or four reverse GLP-1 fragments; the fusion protein connects the amino terminus of a reverse GLP-1 fragment to the carboxyl terminus of a GIPR antibody light chain or heavy chain through a peptide linker sequence (Linker).

[0242] In another embodiment, provided herein is a fusion protein of a GIPR antibody and GLP-1, which comprises an antibody that can specifically bind to GIPR, and two GLP-1 fragments; the fusion protein connects the carboxyl terminus of a GLP-1 fragment to the amino terminus of a GIPR antibody light chain or heavy chain through a peptide linker sequence (Linker).

[0243] In another embodiment, provided herein is a fusion protein of a GIPR antibody and GLP-1, which comprises an antibody that can specifically bind to GIPR, and two reverse GLP-1 fragments; the fusion protein connects the amino terminus of a reverse GLP-1 fragment to the carboxyl terminus of a GIPR antibody light chain or heavy chain through a peptide linker sequence (Linker).

[0244] In another embodiment, provided herein is a GLP-1 fusion protein comprising a GIPR antibody and two GLP-1 fragments; the fusion protein connects the carboxyl terminus of a GLP-1 fragment to the amino terminus of the light chain of a GIPR antibody via a peptide linker sequence (Linker): N′-GLP-1-Linker-R-C′; or connects the carboxyl terminus of a GLP-1 fragment to the amino terminus of the heavy chain of a GIPR antibody: N′-GLP-1-Linker-R-C′; wherein: N′ represents the amino terminus of the fusion protein polypeptide chain, C′ represents the carboxyl terminus of the fusion protein polypeptide chain, GLP-1 represents a GLP-1 fragment, R is the amino acid sequence of the light chain or heavy chain of a GIPR antibody, and Linker represents a peptide linker sequence.

[0245] In another embodiment, provided herein is a GLP-1 fusion protein comprising a GIPR antibody and two reverse GLP-1 fragments; the fusion protein connects the amino terminus of a reverse GLP-1 fragment to the carboxyl terminus of the light chain of a GIPR antibody via a peptide linker sequence (Linker): N′-R-Linker-reverse GLP-1-C′; or connects the amino terminus of a reverse GLP-1 fragment to the carboxyl terminus of the heavy chain of a GIPR antibody: N′-R-Linker-reverse GLP-1-C′; wherein: N′ represents the amino terminus of the fusion protein polypeptide chain, C′ represents the carboxyl terminus of the fusion protein polypeptide chain, reverse GLP-1 represents a reverse GLP-1 fragment, R is the amino acid sequence of the light chain or heavy chain of a GIPR antibody, and Linker represents a peptide linker sequence.

[0246] In a further embodiment, provided herein is a GLP-1 fusion protein comprising a GIPR antibody and two GLP-1 fragments; the fusion protein connects the carboxyl terminus of a GLP-1 fragment to the amino terminus of the light chain of a GIPR antibody via a peptide linker sequence (Linker): N′-GLP-1-Linker-R-C′; wherein: N′ represents the amino terminus of the fusion protein polypeptide chain, C′ represents the carboxyl terminus of the fusion protein polypeptide chain, GLP-1 represents a GLP-1 fragment, R is the amino acid sequence of the light chain of a GIPR antibody, and Linker represents a peptide linker sequence.

[0247] In one embodiment, in the GLP-1 fusion proteins provided herein, the GLP-1 fragments are each independently selected from the amino acid sequences of one of the following: SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109. In one embodiment, in the GLP-1 fusion proteins provided herein, the reverse GLP-1 fragments are each independently selected from the amino acid sequences of one of the following: SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123.

[0248] In one embodiment, in the GLP-1 fusion proteins provided herein, the sequence of the peptide linker independently comprises from 1 to 200 amino acid amines, from 2 to 100 amino acid amines, from 5 to 50 amino acid amines, from 6 to 25 amino acid amines, or from 10 to 20 amino acid amines.

[0249] In another embodiment, in the GLP-1 fusion proteins provided herein, the sequences of the peptide linkers are each independently selected from the amino acid sequences of the following: SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112.

[0250] Nucleic acid

[0251] In one aspect, isolated nucleic acid molecules are provided herein. The nucleic acid molecule comprises, for example, a polynucleotide encoding all or part of an antibody, such as one or both chains of an antibody or a GLP-1 fusion protein herein, or a fragment, derivative, mutant, or variant thereof; a polynucleotide sufficient to serve as a hybridization probe; PCR primers or sequencing primers for identifying, analyzing, mutating, or amplifying a polynucleotide encoding a polypeptide; an antisense nucleic acid for inhibiting the expression of a polynucleotide, and its complementary sequence. The nucleic acid can be of any length. For example, their length can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000, or more nucleotides, and / or comprise one or more additional sequences, such as regulatory sequences, and / or be part of a larger nucleic acid, such as a vector. The nucleic acid can be single-stranded or double-stranded and comprise RNA and / or DNA nucleotides, as well as their artificial variants (e.g., peptide nucleic acids).

[0252] Nucleic acids encoding antibody polypeptides (e.g., heavy or light chains, variable domains only, or full length) can be isolated from B cells of mice immunized with GIPR antigen. Nucleic acids of antibodies or GLP-1 fusion proteins can be isolated by conventional methods such as polymerase chain reaction (PCR).

[0253] The nucleic acid sequences encoding the variable regions of the heavy and light chains are shown above. Those skilled in the art will appreciate that due to the degeneracy of the genetic code, each polypeptide sequence disclosed herein can be encoded by a greater number of other nucleic acid sequences. Each degenerate nucleotide sequence encoding the antibodies or GLP-1 fusion proteins provided herein is provided.

[0254] The present invention further provides nucleic acids that hybridize under specific hybridization conditions to other nucleic acids (e.g., nucleic acids comprising nucleotide sequences of any L2H2 / L6H5). Methods of hybridizing nucleic acids are well known in the art. See, e.g., Current Protocols in Molecular Biology, John Wiley & Son (1989), 6.3.1-6.3.6. As defined herein, for example, medium stringency conditions use a prewash solution containing 5x sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), a hybridization buffer containing approximately 50% formamide, 6x SSC and a hybridization temperature of 55°C (or other similar hybridization solutions, e.g., containing approximately 50% formamide, hybridizing at 42°C), and the wash conditions are 60°C, using 0.5x SSC, 0.1% SDS. High stringency hybridization conditions hybridize in 6x SSC at 45°C and then wash one or more times at 68°C in 0.1x SSC, 0.2% SDS. In addition, one of ordinary skill in the art can manipulate the hybridization and / or wash conditions to increase or decrease the hybridization stringency such that nucleic acids comprising nucleotide sequences that are at least 65, 70, 75, 80, 85, 90, 95, 98 or 99% homologous to each other generally can still hybridize to each other. The basic parameters affecting the choice of hybridization conditions and guidance for designing appropriate conditions are set forth, e.g., in Sambrook, Fritsch and Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Chapters 9 and 11; Current Protocols in Molecular Biology, 1995, Ausubel et al. eds., John Wiley & Sons, Inc., Sections 2.10 and 6.3-6.4) and can be readily determined by one of ordinary skill in the art based on, e.g., the length and / or base composition of the DNA. Changes can be introduced into the nucleic acid by mutagenesis, thereby resulting in changes in the amino acid sequence of the polypeptide encoded thereby (e.g., an antigen-binding protein). Any technique known in the art can be used to introduce the mutations. In one embodiment, one or more specific amino acid residues are altered using, e.g., a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are altered using, e.g., a random mutagenesis protocol. Regardless of how it is generated, the mutant polypeptide can be expressed and screened for the desired properties.

[0255] Mutations can be introduced into nucleic acids without significantly altering the biological activity of the encoded polypeptide. For example, nucleotide substitutions can be made that result in amino acid substitutions at non-essential amino acid residues. In one embodiment, mutations are made in the nucleotide sequences provided herein for L1 to L11 and H1 to H9 or the GLP-1 fusion protein or fragments, variants, or derivatives thereof such that one or more deletions or substitutions of the amino acid residues comprising L1 to L11 and H1 to H9 as shown herein result in two or more residues that differ in sequence. In another embodiment, mutagenesis inserts an amino acid near one or more amino acid residues of L1 to L11 and H1 to H9 or the GLP-1 fusion protein as shown herein to result in two or more residues that differ in sequence. Alternatively, one or more mutations can be introduced into the nucleic acid to selectively alter the biological activity of the encoded polypeptide (e.g., binding to GIPR). For example, the mutation can quantitatively or qualitatively alter the biological activity. Examples of quantitative changes include increasing, decreasing, or eliminating the activity. Examples of qualitative changes include altering the antigenic specificity of an antibody or GLP-1 fusion protein.

[0256] In another aspect, the present invention provides nucleic acid molecules suitable for use as primers or hybridization probes for detecting the nucleic acid sequences herein. The nucleic acid molecules of the present invention can comprise only a portion of the nucleic acid sequence encoding the full-length polypeptide herein, e.g., a fragment that can be used as a probe or primer or that encodes an active portion of the polypeptide herein (e.g., the GIPR-binding portion).

[0257] Probes based on the nucleic acid sequences herein can be used to detect that nucleic acid or similar nucleic acids, e.g., transcripts encoding the polypeptides herein. The probe can comprise a label, such as a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such probes can be used to identify cells that express the polypeptide.

[0258] In another aspect, the present invention provides vectors comprising nucleic acids encoding the polypeptides herein or portions thereof. Examples of vectors include but are not limited to plasmids, viral vectors, episomal mammalian vectors, and expression vectors, such as recombinant expression vectors.

[0259] The recombinant expression vectors of the present invention may comprise the nucleic acids of the present invention in a form suitable for expression in a host cell. The recombinant expression vector comprises one or more regulatory sequences, selected based on the host cell to be used for expression, operably linked to the nucleic acid sequence to be expressed. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in a variety of host cells (e.g., the SV40 early gene enhancer, the Rous sarcoma virus promoter, and the cytomegalovirus promoter), those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences, see Voss et al., 1986, Trends Biochem. Sci. 11:287, Maniatis et al., 1987, Science 236:1237, the entire contents of which are incorporated herein by reference), and those that direct inducible expression of a nucleotide sequence in response to a specific treatment or condition (e.g., the metallothionein promoter in mammalian cells and the tetracycline-responsive promoter and / or streptomycin-responsive promoter in both prokaryotic and eukaryotic systems (ibid.)). One of skill in the art will understand that the design of the expression vector depends on factors such as the choice of host cell to be transformed, the desired level of protein expression, etc. The expression vectors of the present invention can be introduced into host cells, whereby proteins or peptides encoded by the nucleic acids described herein, including fusion proteins or peptides, are produced.

[0260] On the other hand, the present invention provides host cells into which the expression vectors of the present invention can be introduced. The host cells can be any prokaryotic or eukaryotic cells. Prokaryotic host cells include Gram-negative or Gram-positive organisms such as Escherichia coli or Bacillus. More advanced eukaryotic cells include insect cells, yeast cells, and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells or their derivatives such as Veggie CHO and related cell lines grown in serum-free medium (see Rasmussen et al., 1998, Cytotechnology 28:31) or CHO strain DXB-11, which lacks DHFR (see Urlaub et al., 1980, Proc. Natl. Acad, Sci. USA 77:4216-20). Other CHO cell lines include CHO-K1 (ATCC#CCL-61), EM9 (ATCC#CRL-1861), and UV20 (ATCC#CRL-1862). Other host cells include the COS-7 line of monkey kidney cells (ATCC#CRL-1651) (see Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL-163), AM-1 / D cells (described in U.S. Patent Serial No. 6210924), HeLa cells, BHK (ATCC CRL-10) cell line, the CV1 / EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL-70) (see McMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney cells such as 293, 293 EBNA or MSR 293, human epithelial A431 cells, human C010205 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from primary tissue in vitro cultures, primary transplants, HL-60, U937, HaK or Jurkat cells. Suitable cloning and expression vectors for bacterial, fungal, yeast, and mammalian cell hosts are described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, 1985).

[0261] The vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. For stable mammalian transfection, depending on the expression vector and transfection technique used, only a small fraction of cells are known to integrate foreign DNA into their genomes. To identify and select these integrants, a gene encoding a selectable marker (e.g., antibiotic resistance) is typically introduced into the host cell along with the gene of interest. Preferred selectable markers include those that confer resistance to drugs such as G418, hygromycin, and methotrexate. In other methods, stable transfected cells containing the introduced nucleic acid can be identified by drug selection (e.g., cells that have integrated the selectable gene survive while other cells die).

[0262] The transformed cells can be cultured under conditions that enhance polypeptide expression, and the polypeptide can be recovered by conventional protein purification methods. One such purification method is described in the Examples below. Polypeptides pre - used herein include recombinant mammalian anti - GIPR antibodies or GLP - 1 fusion protein polypeptides that are substantially homologous and substantially free of contaminating endogenous materials.

[0263] Activity of the GIPR antibody

[0264] The activity of the GIPR antibody refers to the therapeutic biological effects shown by the antibodies provided herein when they specifically bind to GIPR, inhibit or block GIP signal transduction, such as treating obesity, type 2 diabetes, and / or non - alcoholic fatty liver disease. The term "biological activity of reducing GIP signal transduction" or "biological activity of inhibiting or blocking GIP signal transduction" refers to the ability of a GIPR antibody or its fusion protein with GLP - 1 to bind to GIPR in vivo and inhibit or block the cellular responses downstream of GIP activation of this receptor. The responses include but are not limited to increasing insulin secretion, promoting fat storage, and inhibiting lipolysis. In one embodiment, murine or humanized antibodies that can specifically bind to human GIPR are provided herein. Such antibodies include antagonistic or neutralizing antibodies that can reduce or neutralize GIP signal transduction.

[0265] In one embodiment, the Kd of the antibodies provided herein when binding to human GIPR is from about 0.01 nM to about 1000 nM, about 0.1 nM to about 500 nM, about 0.5 nM to about 200 nM, about 1 nM to about 200 nM, or about 10 nM to about 100 nM. In another embodiment, the Kd of the antibodies provided herein when binding to human GIPR is from about 1 nM to about 200 nM. In another embodiment, the Kd of the antibodies provided herein when binding to human GIPR is from about 1 nM to about 100 nM. In another embodiment, the K of the antibodies provided herein when binding to human GIPR dis about 1 nM, about 2 nM, about 5 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, or about 100 nM.

[0266] In one embodiment, the antibodies provided herein have an IC 50 value for reducing human GIP signaling of from about 0.01 nM to about 500 nM, from about 0.1 nM to about 200 nM, from about 0.5 nM to about 200 nM, from about 1 nM to about 200 nM, or from about 10 nM to about 100 nM. In another embodiment, the antibodies provided herein have an IC 50 value for reducing human GIP signaling of from about 1 nM to about 200 nM. In another embodiment, the antibodies provided herein have an IC 50 value for reducing human GIP signaling of from about 10 nM to about 100 nM. In another embodiment, the antibodies provided herein have an IC 50 value for reducing human GIP signaling of about 1 nM, about 2 nM, about 5 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, or about 100 nM.

[0267] In one embodiment, when the GIPR antibodies provided herein bind to human GIPR, they have one or more of the following properties:

[0268] a. When binding to human GIPR, having a K d ;

[0269] b. When inhibiting GIP activation of human GIPR, having an IC 50 ; and

[0270] c. Cross-competing for binding with the reference antibody on human GIPR.

[0271] In another embodiment, the GIPR antibodies described herein are antibodies having one or more of the following properties:

[0272] a. When binding to human GIPR, having a Kd that is the same as or better than that of a reference GIPR antibody;

[0273] b. When inhibiting GIP activation of human GIPR, having an IC 50 that is the same as or better than that of a reference GIPR antibody; and

[0274] c. The GIPR antibody cross-competes for binding with a reference GIPR antibody on human GIPR.

[0275] In one embodiment, the reference antibody comprises a combination of a light chain variable domain amino acid sequence SEQ ID NO: 66 and a heavy chain variable domain amino acid sequence SEQ ID NO: 76.

[0276] In one embodiment, the reference antibody comprises a combination of a light chain variable domain amino acid sequence SEQ ID NO: 68 and a heavy chain variable domain amino acid sequence SEQ ID NO: 77.

[0277] In another embodiment, the reference antibody is monoclonal antibody L2H2, L6H5, or L10H8.

[0278] As used herein, the term "substantially similar" means having an IC 50 or K d comparable to that of the reference antibody or being about 200%, about 180%, about 160%, about 150%, about 140%, about 120%, about 110%, about 100%, about 99%, about 98%, about 97%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, or about 50% of the IC 50 or K d value of the reference antibody. In one embodiment, the reference antibody includes, for example, an antibody having a combination of light chain SEQ ID NO: 66 and heavy chain SEQ ID NO: 76. In another embodiment, the reference antibody includes GIPR antibodies L2H2, L6H5, or L10H8.

[0279] Biological activity of the fusion protein of GIPR antibody and GLP-1

[0280] The biological activity of the fusion protein of GIPR antibody and GLP-1 includes two aspects: the biological activity of GLP-1 and the GIPR antibody activity. The activity of the GIPR antibody is as described above. "GLP-1 biological activity" refers to the biological activity of the fusion protein of GIPR antibody and GLP-1 that binds to and activates the GLP-1 receptor in vivo, causes a cellular stress response, and exhibits a therapeutic effect, such as obesity, type 2 diabetes, or non-alcoholic steatohepatitis. The aforementioned cellular stress response includes but is not limited to increased insulin secretion, inhibition of glucagon secretion, suppression of appetite, weight loss, induction of satiety, inhibition of apoptosis, induction of pancreatic cell proliferation, and pancreatic cell differentiation. Combining the biological activities of GLP-1 and the GIPR antibody, the GLP-1 fusion protein described herein can be used to treat various diseases and disorders associated with GLP-1R and GIPR. The fusion protein exerts its biological effect by acting on GLP-1R and / or GIPR, and thus can be used to treat subjects with diseases and disorders that respond favorably to "increased GLP-1R stimulation" or to "decreased GIPR stimulation". These subjects are referred to as subjects "in need of GLP-1R stimulation treatment" or "in need of decreased GIPR stimulation". These include non-insulin-dependent diabetes, insulin-dependent diabetes, stroke (GLP-1R see WO 00 / 16797), myocardial infarction (GLP-1R see WO 98 / 08531), obesity (GLP-1R see WO 98 / 19698; GIPR see Furija et al., 2008, PLoS ONE 3: e3163; US 2017 / 0275370 A1), postoperative catabolic changes (GLP-1R see US 6,006,753), functional dyspepsia and irritable bowel syndrome (GLP-1R see WO99 / 64060), hepatic steatosis (GIPR see US 2017 / 0275370A1), non-alcoholic fatty liver disease (GLP-1R see Debra et al., 2016, Hepatobiliary Surg Nutr, 5: 515-518; GIPR see US 2017 / 0275370 A1), non-alcoholic steatohepatitis (GLP-1 see Armstrong et al., 2013, BMJ Open. 3: e003995; GIPR see US 2017 / 0275370 A1), and also includes subjects at risk of developing non-insulin-dependent diabetes (see WO 00 / 07617), subjects with impaired glucose tolerance or impaired fasting glucose, subjects with a body weight that is approximately 25% above normal weight for the subject's height and weight, and subjects with partial pancreatectomy.

[0281] In one embodiment, the biological activity change of the GIPR antibody or its fusion protein with GLP-1 is detected by a direct cAMP assay to quantify the function of the GIPR antibody or GLP-1 fusion protein in inhibiting GIPR in vitro.

[0282] Drug composition

[0283] In one embodiment, provided herein is a drug composition comprising a GIPR antibody provided herein and one or more pharmaceutically acceptable carriers.

[0284] In another embodiment, provided herein is a drug composition comprising a fusion protein of a GIPR antibody provided herein and GLP-1, and one or more pharmaceutically acceptable carriers.

[0285] As used herein, the term "carrier" includes a vehicle, a pharmaceutical excipient, or a stabilizer that is harmless to cells or mammals when exposed thereto at the dosages and concentrations used.

[0286] Therapeutic method

[0287] In one embodiment, provided herein are methods of treating, preventing, or ameliorating type 2 diabetes, which comprise administering to a subject a therapeutically effective amount of a GIPR antibody provided herein or a pharmaceutical composition thereof.

[0288] In another embodiment, provided herein are methods of treating, preventing, or ameliorating non-alcoholic fatty liver disease, which comprise administering to a subject a therapeutically effective amount of a GIPR antibody provided herein, or a pharmaceutical composition thereof.

[0289] In another embodiment, provided herein are methods of treating, preventing, or ameliorating non-alcoholic fatty liver disease, which comprise administering to a subject a therapeutically effective amount of a fusion protein of a GIPR antibody provided herein and GLP-1, or a pharmaceutical composition thereof.

[0290] In another embodiment, provided herein are methods of treating, preventing, or ameliorating non-alcoholic steatohepatitis, which comprise administering to a subject a therapeutically effective amount of a GIPR antibody provided herein, or a pharmaceutical composition thereof.

[0291] In another embodiment, provided herein are methods of treating, preventing, or ameliorating non-alcoholic steatohepatitis, which comprise administering to a subject a therapeutically effective amount of a fusion protein of a GIPR antibody provided herein and GLP-1, or a pharmaceutical composition thereof.

[0292] In another embodiment, provided herein are methods of treating, preventing, or ameliorating type 2 diabetes, which comprise administering to a subject a therapeutically effective amount of a GIPR antibody provided herein, or a pharmaceutical composition thereof.

[0293] In another embodiment, provided herein are methods of treating, preventing, or ameliorating type 2 diabetes, which comprise administering to a subject a therapeutically effective amount of a fusion protein of a GIPR antibody provided herein and GLP-1, or a pharmaceutical composition thereof.

[0294] In another embodiment, provided herein are methods of treating, preventing, or ameliorating obesity, which comprise administering to a subject a therapeutically effective amount of a GIPR antibody provided herein, or a pharmaceutical composition thereof.

[0295] In a further embodiment, provided herein are methods of treating, preventing, or ameliorating obesity, which comprise administering to a subject a therapeutically effective amount of a fusion protein of a GIPR antibody provided herein and GLP-1, or a pharmaceutical composition thereof.

[0296] In any of the uses provided herein, the pharmaceutical composition is for intravenous or subcutaneous injection.

[0297] As used herein, the term "subject" refers to a mammal, including a human, and may be used interchangeably with the term "patient".

[0298] The term "treatment" includes alleviating or preventing at least one symptom or other aspect of a disorder, or alleviating the severity of the disease. A GIPR antibody or a fusion protein of a GIPR antibody and GLP-1 provided herein need not produce a complete cure, or eradicate all symptoms or manifestations of the disease, to constitute an effective therapeutic agent. As is recognized in the relevant art, a drug as a therapeutic agent may reduce the severity of a given disease state, but need not eliminate all manifestations of the disease to be considered an effective therapeutic agent. Similarly, prophylactic administration of a treatment need not be completely effective in preventing the appearance of symptoms to constitute an effective prophylactic agent. It is sufficient to merely reduce the impact of the disease (e.g., by reducing the number or severity of its symptoms, or by enhancing another therapeutic effect, or by producing another beneficial effect), or to reduce the likelihood of the occurrence or exacerbation of the disease in a subject. One embodiment herein relates to a method of administering a GIPR antibody or a fusion protein of a GIPR antibody and GLP-1 to a patient in an amount and for a time sufficient to induce a sustained improvement in an indicator of the severity of the specific disorder above a baseline level.

[0299] The GIPR antibody or the fusion protein drug composition of the GIPR antibody and GLP-1 can be administered by any suitable technique including but not limited to parenteral, topical or inhalation administration. If by injection, the pharmaceutical composition can be administered by, for example, intra-articular, intravenous, intramuscular, intra-lesional, intraperitoneal or subcutaneous routes, either by rapid injection or continuous infusion. Local administration at the site of disease or injury can be considered, such as transdermal administration and sustained release administration by implants. Inhalation administration includes, for example, nasal or oral inhalation, administration using a spray, inhalation of the antibody in the form of an aerosol, etc. Other options include oral formulations including tablets, syrups or lozenges.

[0300] It is advantageous to administer the GIPR antibody or GLP-1 fusion protein provided herein in the form of a composition comprising one or more other components such as a physiologically acceptable carrier, excipient or diluent. The composition may optionally further comprise one or more of the physiologically active agents described below. In several specific embodiments, the composition comprises one, two, three, four, five or six physiologically active agents in addition to one or more of the antibodies (e.g., murine antibodies or humanized antibodies) or GLP-1 fusion proteins provided herein.

[0301] In one embodiment, the pharmaceutical composition comprises a murine antibody or humanized antibody or GLP-1 fusion protein provided herein and one or more of the following: a buffer with a pH suitable for the antibody or GLP-1 fusion protein, an antioxidant such as ascorbic acid, a low molecular weight polypeptide (e.g., a polypeptide containing fewer than 10 amino acids), a protein, an amino acid, a sugar such as dextrin, a complex such as EDTA, glutathione, a stabilizer and an excipient. Preservatives can also be added according to appropriate industrial standards. The composition can be formulated into a lyophilized powder using a suitable excipient solution as a diluent. The appropriate components are non-toxic to the recipient at the doses and concentrations used. Further examples of components that can be used in pharmaceutical formulations can be found in Remington′s Pharmaceutical Sciences, 16th edition (1980) and 20th edition (2000). Mack Publishing Company provides kits for medical practitioners that include one or more of the antibodies or GLP-1 fusion proteins provided herein and labels or other instructions for treating any of the conditions discussed herein. In one embodiment, the kit comprises a sterile preparation of one or more antibodies or GLP-1 fusion proteins in the form of the above-described composition in one or more vials.

[0302] The dosage and frequency of administration can vary depending on the following factors: the route of administration, the specific antibody or GLP-1 fusion protein used, the nature and severity of the disease being treated, whether the symptoms are acute or chronic, and the volume and general condition of the patient. Appropriate dosages can be determined by methods well known in the art, such as in clinical trials including dose escalation studies.

[0303] The antibodies or GLP-1 fusion proteins provided herein can be administered one or more times at regular intervals over a period of time, for example. In a specific embodiment, a murine antibody or a humanized antibody or a GLP-1 fusion protein is administered once every at least one month or longer, such as once a month, twice a month, or three months, or even indefinitely. For treating chronic conditions, long-term treatment is usually most effective. However, for treating acute conditions, short-term administration, such as from one week to six weeks, is sufficient. Generally, a human antibody is administered until the patient shows a medically relevant improvement in the selected signs or indicators above the baseline level.

[0304] An example of the treatment regimen provided herein includes treating symptoms caused by type 2 diabetes, obesity, or non-alcoholic steatohepatitis, etc. by subcutaneous injection of an antibody or a GLP-1 fusion protein once a week or longer at an appropriate dose. The antibody or the GLP-1 fusion protein can be continuously administered weekly or monthly until the desired result, such as the patient's symptoms subside. Retreatment can be carried out as needed, or alternatively, a maintenance dose can be administered.

[0305] The blood glucose concentration and body weight of a patient can be monitored before, during, and / or after treatment with an antibody or a GLP-1 fusion protein, such as a human antibody or a GLP-1 fusion protein, to detect any changes in their stress. For certain conditions, the change in blood glucose can vary with factors such as the disease process. The blood glucose concentration can be determined by known techniques.

[0306] Specific embodiments of the methods and compositions herein involve the use of, for example, an antibody or a GLP-1 fusion protein and one or more GIP antagonists, two or more antibodies or GLP-1 fusion proteins provided herein, or the antibody or GLP-1 fusion protein of the present invention and one or more other GIP antagonists. In a further embodiment, the antibody or the GLP-1 fusion protein is administered alone or in combination with other agents for treating the symptoms that afflict the patient. Examples of such agents include proteins and non-protein drugs. When multiple drugs are administered in combination, their doses should be adjusted accordingly as is well known in the art. "Combined administration" combination therapy is not limited to simultaneous administration and also includes treatment regimens in which the antigen and the protein are administered at least once during a course of treatment involving the administration of at least one other therapeutic agent to the patient.

[0307] On the other hand, the present invention provides a method for preparing a medicament for treating type 2 diabetes, obesity, and non-alcoholic steatohepatitis and related conditions, which comprises a mixture of an antibody or a GLP-1 fusion protein provided herein and a pharmaceutically acceptable excipient for treating the related conditions of the above diseases. The method for preparing the medicament is as described above.

[0308] The present invention further provides compositions, kits and methods related to antibodies or GLP-1 fusion proteins that specifically bind to human GIPR. Nucleic acid molecules, their derivatives and fragments are also provided, which comprise polynucleotides encoding all or part of a polypeptide that binds to GIPR, such as nucleic acids encoding all or part of an anti-GIPR antibody, an antibody fragment, an antibody derivative or a GLP-1 fusion protein. The present invention further provides vectors and plasmids comprising such nucleic acids, and cells and cell lines comprising such nucleic acids and / or vectors and plasmids. The provided methods include, for example, methods for preparing, identifying or isolating an antibody or a GLP-1 fusion protein that binds to human GIPR, such as an anti-GIPR antibody or a GLP-1 fusion protein, methods for determining whether the antibody or GLP-1 fusion protein binds to GIPR, and methods for administering an antibody or GLP-1 fusion protein that binds to GIPR to an animal model.

[0309] The technical solutions of the present invention will be further described below by specific examples.

[0310] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following examples are conventional methods in the art unless otherwise specified.

[0311] 1. Preparation of antigen for immunization

[0312] Inoculate CHO-DHFR- cells into 6-well plates. After culturing for 24 hours (hr), transfect the pTM15 plasmid cloned with the hGIPR gene (nucleotide sequence shown in SEQ ID NO: 114 and amino acid sequence shown in SEQ ID NO: 113) into the cells in the 6-well plates. The transfection is carried out according to the transfection conditions recommended by Invitrogen for Lipofectamine 2000. After 48 hr, change the culture medium to complete medium containing 300 μg / mL hygromycin, and change the medium every 3 days (d). Culture for about two weeks until stably growing clones appear. Digest and disperse the cell colonies, passage the cells, and continue to culture the cells until the passage cells reach 100% confluence. Use an antibody against the V5 tag (Life Technologies) to perform FACS detection on the constructed stable cell lines respectively, and identify the cell population after pressure according to the FACS detection results. A large amount of hGIPR is expressed on the cell membrane of the screened CHO-DHFR-hGIPR. Finally, after subcloning and further identification, 3 GIPR cell lines are selected as highly expressing stable cell lines. These cell lines highly expressing hGIPR can be used as immunogens for antibody preparation (refer to Example 2). In addition, in some embodiments, a fusion protein of the extracellular region of hGIPR and the Fc of hIgG can also be used as an immunogen for antibody preparation, and its preparation method is as follows: Subclone the gene of the fusion protein sequence of the extracellular region of hGIPR, hIgG2 Fc, and a peptide linker (Linker) into the pTM5 plasmid. Perform large-scale transient expression by suspending HEK293 cells to obtain cell supernatant, and then purify the extracellular region fusion protein of hGIPR by affinity chromatography.

[0313] 2. Preparation of Antibodies

[0314] Antibodies against hGIPR can be generated using an immunogen including any one of the following. For example, in certain embodiments, whole cells expressing hGIPR are used as an immunogen to generate antibodies against hGIPR. In addition, in certain embodiments, a fusion protein containing the amino acid sequence of the N-terminal domain of hGIPR and hFc is used as an immunogen to generate antibodies against hGIPR. The immunogen and aluminum hydroxide adjuvant are mixed well and subcutaneously injected into BALB / c mice (6 - 8 weeks old), and then the mice are boosted once a week thereafter. After a total of 6 immunizations, blood is collected by tail snipping. The serum is separated by centrifugation, and the serum titer is detected by FACS. When the appropriate antibody titer is reached, the mice are sacrificed by cervical dislocation, and spleen cells are obtained under sterile conditions. Additionally, SP2 / 0 cells in the logarithmic growth phase are collected, centrifuged, the precipitated cells are resuspended in serum-free medium, centrifuged and resuspended again, and counted. The spleen cells and SP2 / 0 cells are mixed to ensure that the numbers of SP2 / 0 and spleen cells are close. After mixing, they are "washed - centrifuged" 3 times. The cell pellet after the last centrifugation is disrupted, pre-warmed PEG-1500 is added dropwise, and after pipetting up and down, 30 mL of pre-warmed serum-free medium is slowly added to terminate the fusion effect of PEG. After centrifugation again, the cell pellet is disrupted, fusion medium is added, and the spleen cells and feeder cells are plated in a 96-well plate, with 100 μL of medium added to each well. The fused hybridoma cells and feeder cells are cultured together in the 96-well plate and subjected to HAT (hypoxanthine, aminopterin, and thymidine) screening to remove unfused cells. After 10 d, the supernatant of the hybridoma cells in the culture plate is collected for ELISA detection.

[0315] 3. ELISA screening of antibodies

[0316] CHO-DHFR-hGIPR cells overexpressing hGIPR and CHO-DHFR- cells not expressing hGIPR were respectively seeded into 96-well plates. When the cells grew to 90% confluence, the cell culture supernatant was removed, washed twice with PBS, fixed with 100% methanol at 4°C, then 100 μL of freshly prepared 0.6% H2O2-PBS was added and treated at room temperature for 20 min, and then washed twice with PBS. After blocking with 1% BSA (dissolved in PBS), hybridoma cell supernatant was added and incubated at 4°C for 90 min. After multiple washes, 100 μL of diluted goat anti-mouse Fc-HRP secondary antibody (Sigma-Aldrich) was added to each well and incubated at 37°C for 30 min. After washing 5 times, 100 μL of TMB chromogenic substrate was added to each well and reacted at 37°C for 15 min, 50 μL of 2M H2SO4 was added to terminate the chromogenesis, and the OD 450 value was read. In addition, in some embodiments, a fusion protein comprising the amino acid sequence of the N-terminal domain of hGIPR and hFc was used as the coating antigen to coat the 96-well plates. After blocking with 1% BSA (dissolved in PBS), hybridoma cell supernatant was added and incubated at 4°C for 90 min. The subsequent steps were the same as those of the above ELISA method for screening anti-hGIPR monoclonal antibodies. The positive control was the serum of immunized mice; the negative control was the cell culture supernatant. After the preliminary ELISA detection, several positive hybridoma cell lines secreting anti-hGIPR antibodies were screened. These hybridoma strains secreting anti-hGIPR antibodies were selected and cloned to obtain cell lines that could stably secrete anti-hGIPR antibodies. Finally, the positive hybridoma cell supernatant was selected for FACS verification (refer to Example 10).

[0317] 4. Cloning and subcloning of antibody genes

[0318] Hybridoma cells secreting antibodies were collected, and mRNA of the hybridoma cells was extracted according to the operating procedures of the QIAGEN mRNA extraction kit. Then the extracted mRNA was reverse-transcribed into cDNA, and the reverse transcription primers were specific primers for the constant regions of mouse light and heavy chains. The heavy chain reverse transcription primer was (5’-TTTGGRGGGAAGATGAAGAC-3’), and the light chain reverse transcription primers were (5’-TTAACACTCTCCCCTGTTGAA-3’) and (5’-TTAACACTCATTCCTGTTGAA-3’). The reaction conditions for RT-PCR were: 25°C for 5 min; 50°C for 60 min; 70°C for 15 min. The reverse-transcribed cDNA was diluted to 500 μL with 0.1 mM TE and added to an ultrafiltration centrifugal tube (Amicon Ultra-0.5), and centrifuged at 2000 g for 10 min; the filtrate was discarded, and another 500 μL of 0.1 mM TE was added and centrifuged at 2000 g for 10 min; the filtrate was discarded, the preparation tube was inverted into a new centrifuge tube, and centrifuged at 2000 g for 10 min to obtain purified cDNA; 10 μL of the purified cDNA was taken as a template, 4 μL of 5x tailing buffer (Promega), 4 μL of dATP (1 mM) and 10 U of terminal transferase (Promega) were added and mixed evenly, incubated at 37°C for 5 min and then at 65°C for 5 min; then, using the cDNA with a PolyA tail as a template, the variable region genes of the light and heavy chains of the antibody were amplified by PCR. The upstream primers were all OligodT, and the heavy chain downstream primers were (5’-TGGACAGGGATCCAGAGTTCC-3’) and (5’-TGGACAGGGCTCCATAGTTCC-3’), and the light chain downstream primer was (5’-ACTCGTCCTTGGTCAACGTG-3’). The PCR reaction conditions were: 95°C for 5 min; 95°C for 30 s, 56°C for 30 s, 72°C for 1 min for 40 cycles; 72°C for 7 min; the PCR product was ligated to the PMD 18-T vector (Takara Bio) and then sequenced. PCR primers were designed based on the DNA sequence of the antibody obtained by sequencing, so as to connect the complete light chain, heavy chain signal peptide and variable domain, and the mouse IgG1 constant region to the expression vector pTM5.

[0319] 5. Humanization and optimization of the antibody

[0320] First, according to the retrieved murine antibody light and heavy chain variable region sequences, use the NCBI database to search for human germline gene sequences (Ig Germline Gene Sequence) homologous to the retrieved murine antibody variable region sequences. Except for the CDR sequences, use the human gene sequence with the highest homology as the template sequence for CDR grafting to obtain humanized antibody variable region sequences. Synthesize the genes of humanized antibody light and heavy chains, and splice them with the human IgG2 or IgG4 constant region sequence to obtain the complete recombinant humanized antibody sequence. The recombinant antibody is expressed according to Example 8, and its affinity for GIPR is verified by the FACS technique in Step 10 to select the antibody with the best affinity performance. Finally, through site-directed mutagenesis, the variable region sequence of the humanized antibody is modified to further improve its affinity for GIPR.

[0321] 6. Gene Cloning and Subcloning of Humanized hGIPR Antibody

[0322] The optimized humanized antibody heavy and light chain variable region sequences are outsourced for synthesis. When synthesizing, the Nhe1 restriction enzyme site is introduced at the 5' end of the heavy chain variable region, and the Sal1 restriction enzyme site is introduced at the 3' end, so as to connect the complete heavy chain variable region sequence with the expression vector pTM5 loaded with the heavy chain constant region. Similarly, the Nhe1 restriction enzyme site is introduced at the 5' end of the light chain variable region, and the Bsiw1 restriction enzyme site is introduced at the 3' end when synthesizing, so as to connect the complete light chain variable region sequence with the expression vector pTM5 loaded with the light chain constant region.

[0323] 7. Construction of Humanized hGIPR Antibody and GLP-1 Fusion Protein

[0324] The optimized humanized antibody is fused with GLP-1 and its derivative sequences at the N-terminus or C-terminus of the light chain to form a GLP-1 fusion protein. The sequences of both are connected by a peptide linker sequence (Linker) as a bridge. The nucleotide sequence of signal peptide-GLP-1-Linker is synthesized by GenScript Biotech Corporation. Using the synthesized gene as a template, the sequence of the "signal peptide-GLP-1-Linker" part is amplified by PCR. Another template is the nucleotide sequence of the humanized antibody to amplify the antibody part of the fusion protein sequence. Then, the "signal peptide-GLP-1-Linker" part of the fusion protein nucleic acid sequence is connected to the antibody part by overlapping PCR, and Nhe1 and Not1 restriction enzyme sites are added at both ends of the primer, so as to connect the complete fusion protein sequence with the expression vector pTM5.

[0325] 8. Transient Expression of hGIPR Antibody and GLP-1 Fusion Protein

[0326] Inoculate 5×10 5Suspend HEK293 or CHO expression cell lines at [X] cells / mL into spinner flasks. After rotating culture at 37°C and 5% CO₂ for 24 hours, when the density reaches 1×10 6 / mL, it is used for transfection. During the transfection process, polyethylenimine (PEI) is used as the transfection medium and mixed with DNA. The mixture is incubated statically for 15 minutes and then added to the cell culture. After the cells receive the PEI and DNA mixture, they continue to be cultured by rotation at 37°C and 5% CO₂ for 24 hours. Then, tryptone is added to the cell culture medium as an additive required for expression. Finally, after the expression is completed (more than 96 hours), the cell supernatant is collected for the purification and separation of antibodies.

[0327] 9. Purification and separation of hGIPR antibody and GLP-1 fusion protein

[0328] The collected cell supernatant in Example 8 is centrifuged at high speed (8000 rpm) to remove cells and cell debris, and then filtered through a 0.22 µm filter membrane to clarify. The clarified supernatant is used for purification. The purification process is completed by a chromatograph. The supernatant first flows through a Protein A / G affinity chromatography column. The antibodies contained in the supernatant bind to the ligand of the Protein A / G affinity chromatography column during this period and are retained in the column. Then, the chromatography column is washed with an elution buffer with a low pH value (less than or equal to 3.0) to dissociate the antibodies bound to the column. The collected antibody eluate is quickly neutralized with 1 M Tris-HCl. The obtained antibody eluate is dialyzed and replaced with PBS or other buffer systems.

[0329] 10. Verification of the binding activity of functional hGIPR antibody by flow cytometry

[0330] Digest and collect 10 5 CHO-DHFR-hGIPR cells with PBS containing 10 mM EDTA, and add them to 1.5 mL EP tubes respectively. After centrifugation, discard the supernatant. The negative control samples are resuspended with flow cytometry loading buffer (PBS, 2% FBS). For the positive treatment group, 200 μL of hGIPR antibody at a specific concentration is added to each tube and incubated at room temperature; after incubation, centrifuge at 1500 rpm, discard the supernatant, wash the cell pellet once with flow cytometry loading buffer, centrifuge again, and resuspend the cells; add 1:50 diluted FITC-labeled goat anti-mouse fluorescent secondary antibody or PE-labeled goat anti-human fluorescent secondary antibody to the cell suspension, 200 μL / well, and incubate at room temperature in the dark for 30 minutes; centrifuge, discard the supernatant, wash once again with flow cytometry loading buffer, centrifuge, and finally resuspend the cell pellet with flow cytometry loading buffer and detect on the machine. The functional antibody against hGIPR specifically binds to CHO-DHFR-GIPR cells expressing GIPR. At Figure 1Among the experimental results shown, the grey peak and the dotted line peak are negative controls, while the solid line peak corresponding to 1 μM of L10H8 has an obvious right shift, demonstrating the specific binding of L10H8 and CHO-DHFR-GIP.

[0331] 11. cAMP assay to detect the biological activity of hGIPR antibody or hGIPR antibody / GLP-1 fusion protein in antagonizing GIPR in vitro

[0332] Seed 30,000 CHO-DHFR cells expressing hGIPR per well into a 96-well cell culture plate and place it in an incubator at 37 °C and 5% CO2 overnight. The next day, remove the cell supernatant and add 45 μL / well of hybridoma cell culture supernatant or serially diluted antibody. Incubate at room temperature for 30 min, then add 45 μL / well of GIP polypeptide (Phoenix Pharmaceuticals, 50 pM). Then place the 96-well cell culture plate in an incubator at 37 °C and 5% CO2 and continue to incubate for 30 min. After that, add 10 μL / well of 10% Triton X-100, lyse at room temperature, and mix well with a multi-channel pipette. Use a cAMP assay kit (CisBio) to detect the cAMP generated in the experiment. Take 10 μL / well of the above cell lysate into a white 384-well plate, add 5 μL / well of cAMP-d2 diluted 1:20, and finally add 5 μL / well of Anti-cAMP-Eu3+ cryptate diluted 1:20. Incubate at room temperature for 1 hr. Read the time-resolved fluorescence 665 nm / 620 nm signal ratio on an Envision 2103 microplate reader, and then calculate the IC 50 value. Figure 2 Show that L7H6 antagonizes GIPR with an IC 50 = 7.6 nM. Figure 3 Show that GLP-1-Linker-L7H6 antagonizes GIPR with an IC 50 = 14.9 nM.

[0333] 12. Reporter gene assay to detect the activation of GLP-1R by hGIPR antibody / GLP-1 fusion protein in vitro

[0334] Seed 20,000 CHO-DHFR- cells co-expressing hGLP-1R-CRE-Luciferase per well into a 96-well cell culture plate and culture overnight at 37 °C. The next day, remove the culture medium supernatant, wash the cell surface twice with serum-free medium, aspirate the residual liquid, and then add 100 μL of purified antibody or GLP-1 diluted with serum-free medium and incubate at 37 °C for 4 hours. After the stimulation is completed, add 100 μL of Bright Glo chemiluminescent substrate (Promega), and finally transfer the cell lysate to a white 96-well plate and read the relative fluorescence intensity on a SpectraMax L microplate reader (Molecular Devices). At Figure 4 showed that GLP-1-Linker-L7H6 activated hGLP-1R with an EC 50 = 0.04 nM.

[0335] 13. In vivo pharmacodynamic evaluation of hGIPR antibody in high-fat diet-induced C57BL / 6 obese mice

[0336] Establish an obesity model (DIO mice) in C57BL / 6 mice induced by a 60% high-fat diet. After the mice are purchased and fed a normal diet for one week, randomly select a certain number of mice as the normal control group and feed them with normal mouse feed, and feed the remaining animals with a high-fat diet. Feed continuously for 8 weeks (wk), and weigh the body weight and food intake once a week. Subsequently, randomly divide the mice in the high-fat diet group into the L10H8 group (10 mg / kg) and the model group according to body weight, and inject the drug subcutaneously once every two days for a total of 6 wk. The normal control group is not given the drug, and the model group is given an equal amount of blank preparation. During the experimental period, collect data on the body weight, food intake, and behavioral observation of the mice. Fast the animals for 12 hr (free access to water) before the last day of the experiment, collect blood from the orbital cavity of the animals to separate the serum, and perform euthanasia. Dissect and weigh the liver, observe the liver morphology, detect liver TC and TG, and detect serum ALT, AST, GLU, TC, and TG (the results are shown in Figure 5 and Table 3).

[0337] Table 3: Biochemical test results of each group after drug administration

[0338]

[0339] Note: Mean ± standard error. Compared with the model group, * P < 0.05, ** P < 0.01; liver index = liver weight / animal body weight * 100.

[0340] After administration of the L10H8 antibody for 6 weeks, the weight gain of the L10H8 group was only slightly lower than that of the model group, but its liver weight was significantly lower than that of the model group and was close to that of the normal control group. The liver TG of the L10H8 group was significantly lower than that of the model group, and its serum TG was significantly higher than that of the model group. These test results showed that L10H8 significantly slowed down the absorption and accumulation of lipids in the liver. Figure 5 The weight change rates of mice in each group are shown. Table 3 summarizes the liver indexes of mice in each group after administration of the L10H8 antibody for 6 weeks.

[0341] 14. Detection of the ability of the hGIPR antibody / GLP-1 fusion protein GLP-1-Linker-V1W5 to activate GLP-1R in vitro by reporter gene assay

[0342] Seed 20,000 co-expressing hGLP-1R-CRE-Luciferase CHO-DHFR- cells per well into a 96-well cell culture plate and culture overnight at 37 °C. The next day, remove the culture medium supernatant, wash the cell surface twice with serum-free medium, aspirate the residual liquid, and then add 100 μL of purified antibody or GLP-1 diluted with serum-free medium and incubate at 37 °C for 4 hours. After the stimulation, add 100 μL of Bright Glo chemiluminescent substrate (Promega), and finally transfer the cell lysate to a white 96-well plate and read the relative fluorescence intensity on a SpectraMax L microplate reader (Molecular Devices). Figure 6 Showed that GLP-1-Linker-V1W5 activated hGLP-1R with an EC 50 = 17.40 pM.

[0343] 15. Detection of the ability of the hGIPR antibody or the hGIPR antibody / GLP-1 fusion protein GLP-1-Linker-V1W5 to antagonize the biological activity of GIPR in vitro by cAMP assay

[0344] Seed 30,000 CHO-DHFR cells expressing hGIPR per well into a 96-well cell culture plate and place it in an incubator at 37 °C with 5% CO2 overnight. The next day, remove the cell supernatant and add 45 μL / well of hybridoma cell culture supernatant or serially diluted antibody. Incubate at room temperature for 30 min, then add 45 μL / well of GIP polypeptide (Phoenix Pharmaceuticals, 50 pM). Then place the 96-well cell culture plate in an incubator at 37 °C with 5% CO2 and continue to incubate for 30 min. After that, add 10 μL / well of 10% Triton X-100, lyse at room temperature, and mix well with a multichannel pipette. Detect the cAMP generated in the experiment using a cAMP kit (CisBio). Transfer 10 μL / well of the above cell lysate to a white 384-well plate, add 5 μL / well of cAMP-d2 diluted 1:20, and finally add 5 μL / well of Anti-cAMP-Eu3± cryptate diluted 1:20. Incubate at room temperature for 1 hr. Read the time-resolved fluorescence 665 nm / 620 nm signal ratio on an Envision 2103 microplate reader, and then calculate the IC 50 value using Prism 5.0. Figure 7 It is shown that GLP-1-Linker-V1W5 antagonizes the human GIP receptor (hGIPR) with an IC 50 = 7.03 nM. Figure 8 It is shown that GLP-1-Linker-V1W5 antagonizes the cynomolgus GIP receptor (maGIPR) with an IC 50 = 4.30 nM.

[0345] 16. Pharmacokinetic experiment of hGIPR antibody / GLP-1 fusion protein in cynomolgus monkeys

[0346] Administer a single subcutaneous injection of GLP-1 / hGIPR antibody fusion protein to 6 cynomolgus monkeys (3 males and 3 females) at a dose of 2 mg / kg. Before dosing (0 min) and at 2 hr, 4 hr, 8 hr, 12 hr, 24 hr, 2 d, 4 d, 6 d, 8 d, 10 d, 12 d, 18 d, and 28 d after dosing, collect 0.6 mL of whole blood from the lateral limb vein on the dosing side into a centrifuge tube. After it naturally coagulates on ice, centrifuge to extract serum and store it at ultra-low temperature (-80 °C) until detection. The GLP-1 part and hGIPR antibody part of the GLP-1 / hGIPR antibody fusion protein in the serum samples are quantitatively analyzed separately using ELISA, and the half-lives of both in cynomolgus monkeys are determined through software analysis.

[0347] 17. Pharmacokinetic experiment of hGIPR antibody / GLP-1 fusion protein in rhesus monkeys

[0348] Nine male healthy rhesus monkeys were given a single subcutaneous injection of hGIPR antibody / GLP-1 fusion proteins (GLP-1-Linker-V1W4, GLP-1-Linker-V1W5, or GLP-1-Linker-V1W6), with 3 animals in each group, at a dose of 4 mg / kg. Before drug administration (0 min), and at 2 hr, 4 hr, 8 hr, 12 hr, 24 hr, 2 days, 4 days, 6 days, 8 days, 10 days, 12 days, 16 days, 20 days, 24 days, 30 days, 36 days, 42 days, 50 days, and 60 days after drug administration, 0.6 mL of whole blood samples were collected from the antecubital vein and placed in centrifuge tubes containing 8 L of DDP-IV enzyme inhibitor, and left on ice until natural coagulation occurred, then centrifuged to extract serum, which was stored at ultra-low temperature (-80 °C) until detection. The hGIPR antibody part and the GLP-1 part of the GLP-1 / hGIPR antibody fusion protein in the serum samples were quantitatively analyzed separately by ELISA method, and the half-lives of the two in rhesus monkeys were determined by software analysis.

[0349] The PK results showed that the half-lives T 1 / 2 of the antibody parts of the above three GLP-1 / hGIPR antibody fusion proteins were about 360, 679, and 614 hours respectively, while the half-lives T 1 / 2 of the GLP-1 parts were about 87, 82, and 97 hours respectively. The PK curves and parameters of the monkeys in each group are shown in Figure 9 and 10 and Table 4.

[0350] Table 4: PK parameters of monkeys in each group

[0351]

[0352] 18. Pharmacodynamic experiment evaluation of hGIPR antibody / GLP-1 fusion protein in obese cynomolgus monkeys induced by high-fat diet: Efficacy in primate mammals

[0353] An obese cynomolgus monkey model (DIO cynomolgus monkey) was established by inducing cynomolgus monkeys with 60% high-fat diet to evaluate the in vivo efficacy of subcutaneous injection of GLP-1-Linker-L7H6 in this animal model. The monkeys in the high-fat diet group were randomly divided into a GLP-1-Linker-L7H6 (10 mg / kg) drug administration group and a model group according to body weight. The drug was injected subcutaneously once every two days for a total of 8 wk. The model group was given an equal amount of blank preparation. During the experimental period, data on monkey body weight, food intake, and behavioral observations were collected. After the experimental period ended, the animals were euthanized, the liver was dissected and weighed, the liver morphology was observed, and liver TC and TG were detected; serum ALT, AST, GLU, TC, and TG were detected.

[0354] GLP-1-Linker-V1W5 was measured in the same way as described above. Nine obese cynomolgus monkeys were randomly divided into a formulation control group, a GLP-1-Linker-V1W5 group, and a positive control (a fusion protein control of a GLP-1R antibody and GLP-1) group according to body weight, with 3 monkeys in each group. The drug was injected subcutaneously 2 times a week, 1 mg / kg in the first week and 2 mg / kg starting from the second week, for a total of 8 wk. The formulation control group was given an equal amount of blank formulation. During the experimental period, the food intake of the monkeys was collected (see Figure 11 ), the body weight was collected and the weight change rate was calculated (see Figure 12 and 13 ), behavioral observations, blood routine / biochemical tests, and DEXA test data were collected, including the time curves of trunk fat change and total fat change (see Figure 14 and 15 ), and the time curve of total fat change rate (see Figure 16 ); while Figure 17 shows the time curve of the change in total fat mass per 1 kg of body weight of the monkeys and Figure 18 shows the time curve of the change in total lean tissue mass per 1 kg of body weight of the monkeys.

[0355] The pharmacodynamic study showed that compared with the formulation control and the fusion protein of GLP-1R antibody and GLP-1, GLP-1-Linker-V1W5 reduced the food intake of the animals and more significantly reduced the body weight of obese cynomolgus monkeys. It is worth noting that GLP-1-Linker-V1W5 reduced the total fat and trunk fat of obese cynomolgus monkeys while increasing the lean tissue of the monkeys. At the same time, no abnormalities were found in behavioral observations, blood routine, and biochemical tests.

[0356] After the end of the experimental period, the animals' livers were biopsied, and liver TP, TG, and TC were detected (the results are shown in Table 5). The biochemical test results of the livers of each group after drug administration showed that GLP-1-Linker-V1W5 reduced the TC content in the liver.

[0357] Table 5: Biochemical test results of the livers of each group after drug administration

[0358]

[0359] Note: Mean ± standard error

[0360] The above examples are provided to fully disclose and explain to those of ordinary skill in the art how to make and use the claimed embodiments, and are not meant to limit the scope disclosed herein. Modifications that are obvious to those skilled in the art are within the scope of the claims herein. All publications, patents, and patent applications cited in this specification are hereby incorporated by reference as if each such publication, patent, or patent application was specifically and individually incorporated by reference.

Claims

1. An antibody that can specifically bind to human GIPR, wherein the antibody comprises (a) Light chain CDR1 amino acid sequence: SEQ ID NO: 10; Light chain CDR2 amino acid sequence: SEQ ID NO: 11; Light chain CDR3 amino acid sequence: SEQ ID NO: 12; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 26; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 27; and Heavy chain CDR3 amino acid sequence: SEQ ID NO: 28; or (b) Light chain CDR1 amino acid sequence: SEQ ID NO: 13; Light chain CDR2 amino acid sequence: SEQ ID NO: 11; Light chain CDR3 amino acid sequence: SEQ ID NO: 14; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 26; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 27; and Heavy chain CDR3 amino acid sequence: SEQ ID NO:

28.

2. The antibody of claim 1, wherein the antibody comprises a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the light chain variable region is SEQ ID NO: 66, SEQ ID NO: 67 or SEQ ID NO: 68; and The amino acid sequence of the heavy chain variable region is SEQ ID NO:76 or SEQ ID NO:

77.

3. The antibody of claim 1 or 2, wherein the antibody comprises a combination of light chain and heavy chain variable region amino acid sequences independently selected from the group consisting of SEQ ID NO:66 and SEQ ID NO:76, SEQ ID NO:67 and SEQ ID NO:77, and SEQ ID NO:68 and SEQ ID NO:

77.

4. The antibody of claim 3, wherein the antibody comprises a combination of the amino acid sequences of SEQ ID NO: 67 and SEQ ID NO:

77.

5. The antibody of claim 1 or 2, wherein the antibody further comprises a light chain constant region and a heavy chain constant region, wherein the amino acid sequence of the light chain constant region is SEQ ID NO: 101 or SEQ ID NO: 102; and The amino acid sequence of the heavy chain constant region is SEQ ID NO:103, SEQ ID NO:104 or SEQ ID NO:

124.

6. The antibody of claim 1 or 2, wherein the antibody is a murine GIPR antibody or a humanized GIPR antibody.

7. The antibody of claim 1 or 2, wherein the antibody is a GIPR monoclonal antibody.

8. The antibody of claim 1 or 2, wherein the antibody is a monoclonal antibody comprising a combination of amino acid sequences selected from the group consisting of SEQ ID NO:66 and SEQ ID NO:76, SEQ ID NO:67 and SEQ ID NO:77, and SEQ ID NO:68 and SEQ ID NO:

77.

9. The antibody of claim 1, wherein the antibody comprises SEQ ID NO: 125 and SEQ ID NO: 127, SEQ ID NO: 125 and SEQ ID NO: 128, SEQ ID NO: 125 and SEQ ID NO: 129, SEQ ID NO: 125 and SEQ ID NO: 130, SEQ ID NO: 125 and SEQ ID NO: 131, SEQ ID NO: 125 and SEQ ID NO: 132, SEQ ID NO: 125 and SEQ ID NO: 133, SEQ ID NO: 125 and SEQ ID NO: 134, SEQ ID NO: 125 and SEQ ID NO: 135, SEQ ID NO: 126 and SEQ ID NO: 127, SEQ ID NO: 126 and SEQ ID NO: 128, SEQ ID NO: 126 and SEQ ID NO: 129, SEQ ID NO: 126 and SEQ ID NO: 130, SEQ ID NO: 126 and SEQ ID NO: 131, SEQ ID NO: 126 and SEQ ID NO: 132, NO:132, SEQ ID NO:126 and SEQ ID NO:133, SEQ ID NO:126 and SEQ ID NO:134, or an antibody comprising a combination of SEQ ID NO:126 and SEQ ID NO:

135.

10. The antibody of claim 1, wherein the antibody comprises a combination of SEQ ID NO: 125 and SEQ ID NO: 130, SEQ ID NO: 125 and SEQ ID NO: 131, or SEQ ID NO: 125 and SEQ ID NO:

132.

11. The antibody of claim 1, wherein the antibody comprises a combination of SEQ ID NO: 125 and SEQ ID NO:

131.

12. The antibody according to claim 1 or 2, characterized in that: The antibody is a mouse antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a single-chain antibody, a double-chain antibody, a Fab fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

13. The antibody of claim 1 or 2, wherein the IC of the antibody for reducing human GIP signaling is 50 Values ​​range from 1 nM to 200 nM.

14. The antibody of claim 1 or 2, wherein the antibody has an IC of reducing human GIP signaling. 50 Values ​​range from 1 nM to 100 nM.

15. A GLP-1 fusion protein, characterized in that: the fusion protein comprises the GIPR antibody, a peptide linker and a GLP-1 fragment according to any one of claims 1 to 14; wherein the amino acid sequence of the peptide linker is SEQ ID NO: 111, and the amino acid sequence of the GLP-1 fragment is SEQ ID NO:

106.

16. The fusion protein of claim 15, wherein the fusion protein comprises a GIPR antibody and two GLP-1 fragments; the fusion protein connects the carboxyl terminus of the GLP-1 fragment to the amino terminus of the GIPR antibody light chain via the peptide linker.

17. The fusion protein of claim 15, wherein the GIPR antibody, the GLP-1 fragment, and the peptide linker are fused to form the fusion protein in the following manner: The carboxyl terminus of a GLP-1 fragment and the amino terminus of a GIPR antibody light chain are connected via a peptide linker: N'-GLP-1-Linker-R-C'; Wherein: N' represents the amino terminus of the polypeptide chain, C' represents the carboxyl terminus of the polypeptide chain, GLP-1 represents a GLP-1 fragment, R is the amino acid sequence of the light chain of a GIPR antibody according to claims 1 to 14, and Linker represents a peptide linker.

18. A polynucleotide encoding the GIPR antibody of any one of claims 1 to 14 or the GLP-1 fusion protein of any one of claims 15 to 17. A vector comprising the polynucleotide according to claim 18.

20. A host cell comprising the vector according to claim 19.

21. A pharmaceutical composition comprising the GIPR antibody of any one of claims 1 to 14 or the GLP-1 fusion protein of any one of claims 15 to 17 in admixture with a pharmaceutically acceptable carrier.

22. Use of a pharmaceutical composition comprising the GIPR antibody according to any one of claims 1 to 14 or the GLP-1 fusion protein according to any one of claims 15 to 17 in the preparation of a medicament for preventing or treating non-alcoholic fatty liver disease.

23. Use of a pharmaceutical composition comprising the GIPR antibody according to any one of claims 1 to 14 or the GLP-1 fusion protein according to any one of claims 15 to 17 in the preparation of a medicament for preventing or treating type 2 diabetes.

24. Use of a pharmaceutical composition comprising the GIPR antibody according to any one of claims 1 to 14 or the GLP-1 fusion protein according to any one of claims 15 to 17 in the preparation of a medicament for weight loss or treatment of obesity and obesity-related disorders.

25. Use of a pharmaceutical composition comprising the GIPR antibody according to any one of claims 1 to 14 or the GLP-1 fusion protein according to any one of claims 15 to 17 in the preparation of a medicament for simultaneously treating two or more of non-alcoholic fatty liver disease, obesity or type 2 diabetes.

26. The use according to any one of claims 22 to 25, wherein the pharmaceutical composition is for intravenous or subcutaneous injection.

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