GIPR antibody and fusion protein between same and GLP-1, and pharmaceutical composition and application thereof

Abstract

To provide an antibody, a fusion protein, a pharmaceutical composition, and use thereof for treating, preventing, or improving one or more symptoms among non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, type 2 diabetes, or obesity.SOLUTION: An antibody that specifically binds to human GIPR is provided, wherein each amino acid sequence is independently selected from the amino acid sequences listed below: specific light chain CDR1, CDR2, and CDR3 amino acid sequences and specific heavy chain CDR1, CDR2, and CDR3 amino acid sequences. Further provided is a GLP-1 fusion protein comprising: an antibody that specifically binds to GIPR; and 1, 2, 3, 4, 5, 6, 7 or 8 GLP-1 fragments.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Described herein are antibodies that specifically bind to GIPR and their fusion proteins with GLP-1. The present invention provides a method for treating non-alcoholic fatty liver disease (NFAD), a method for treating non-alcoholic fatty liver disease (NFAD), and a pharmaceutical composition thereof. For treating, preventing, or ameliorating one or more symptoms of fatty liver disease, type 2 diabetes, or obesity, Methods for using GIPR antibodies and their fusion proteins with GLP-1 are also described herein. It is provided at. [Background technology]

[0002] Gastrointestinal inhibitory polypeptide (GIP) is a polypeptide secreted by intestinal K cells after feeding. It is a peptide hormone and contains two isoforms: a 42- and a 30-amino acid peptide. GIP activates the gastric inhibitory polypeptide receptor (GIPR) on the surface of pancreatic β cells. It is involved in the physiological process of insulin secretion by activating the ,1996,J.Clin.Invest.98:2440-2445;Ravn et. al., 2013, J. Biol. Chem. 288:19760-72). G.I.P. Because their classical biological functions are similar to those of GLP-1, these peptide hormones GIPRs are collectively known as incretins. They are found in the pancreas, bone, heart, stomach, intestine, and adipose tissue. It is widely distributed in many tissues (Peter et al., 2013, J. Biol. Chem. 288:19760-72), this diverse distribution is due to the GIP / GIPR pathway. Experimental evidence suggests that GIP / GIP has biological functions beyond blood glucose regulation. The IPR signaling pathway is at least closely related to lipid metabolism in these tissues. (Yip and Wolfe, 2000, Life Sci. 66:9 1-103) Experimental data also support increased circulating GIP concentrations in obese or diabetic patients. It has also been shown that there is an increase in ologia 14:15-24;Flatt et al., 1984, J. Endo crinol.101:249-256;Salera et al.,1982,J. Clin.Endocrinol.Metab.55:329-336;Vilsboe ll et al.,2003,J.Clin.Endocrinol.Metab.8 8:2706-2713). After blocking GIPR signaling with a GIPR inhibitor, significant Significant weight loss, decreased insulin resistance, and even improved type 2 diabetes were observed with a high-fat diet. observed in induced obese mice (Ravn et al., 2013, JB iol.Chem.288:19760-72).

[0003] Long-acting glucagon-like peptide-1 analogs (GLP-1 analogs) are a new generation and It is one of the most effective drugs for type 2 diabetes (Tomlinson et al., 2015, Ex pert Opin.Investig.Drugs 25:1744-7658;Ga llwitz, 2015, Eur. Endocr. 11:21-25). long-acting G LP-1 drugs are also in clinical trials for the treatment of nonalcoholic fatty liver disease (NAFLD). Studies have shown that long-acting GLP-1 drugs may be effective in patients with NAFLD. Improved liver tissue morphology, alanine aminotransferase / glutathione aminotransferase activity The results show that the α-glucanase inhibitor has a significant effect on reducing the fatty acid transferase ratio and liver fat content (Sa mson et al.,2013,J.Diabetes Complication s 27:401-6;Portillo-Sanchez and Cusi,201 6, Clin. Diabetes Endocrinol. 2:9). Summary of the Invention [Problem to be solved by the invention]

[0004] If GLP-1 drugs and GIPR inhibitors can be used together, and combinations or fusions thereof, which improve insulin resistance while simultaneously The effect of reducing excess fat accumulation (obesity) can be achieved. In this regard, blood glucose It decreases and increases lipid metabolism, but here the GLP-1 part improves glucose metabolism and promotes food intake. It reduces appetite and weight, and the GIPR antibody moiety reduces further accumulation of fat, improving liver function. GIPR antibody's fat-reducing effect and GLP-1's weight-reducing effect are used to improve the function of non- The present disclosure can synergistically treat alcoholic fatty liver disease / non-alcoholic steatohepatitis. One of the following: non-alcoholic fatty liver disease / non-alcoholic steatohepatitis, type 2 diabetes, and obesity The present invention provides fusion protein drugs that provide benefit to patients suffering from one or more diseases. [Means for solving the problem]

[0005] An antibody that specifically binds to GIPR and is an antagonist of GIPR, Provided herein.

[0006] An antibody that specifically binds to GIPR, comprising a sequence of 1, 2, 3, 4, 5, or 6 amino acids. wherein each amino acid sequence is independently an amino acid sequence listed below: 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; 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; 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; d. Heavy chain CDR1 amino acid sequences: SEQ ID NO:18, SEQ ID NO:23, and SEQ ID NO:26; 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; 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 Also provided herein is an antibody selected from:

[0007] An antibody that specifically binds to GIPR and 1, 2, 3, 4, 5, 6, 7, or 8 GLPs a GLP-1 fusion protein comprising a GLP-1 fragment; The carboxy terminus of the -1 fragment is linked to the amino terminus of the light or heavy chain of the GIPR antibody. or by attaching the amino terminus of the GLP-1 fragment to the light chain or A GLP-1 fusion protein, which binds the carboxy terminus of the heavy chain, is further defined herein. will be provided to.

[0008] It is a GLP-1 fusion protein containing a GIPR antibody and two GLP-1 fragments. The fusion protein connects the carboxy terminus of the GLP-1 fragment to the carboxy terminus of the GIPR antibody. linked to the amino terminus of the light chain: N'-GLP-1-linker-R-C'; or GLP-1 The carboxy terminus of the fragment is attached to the amino terminus of the heavy chain of the GIPR antibody: N'-GL P-1-Linker-R-C'; where N' is an amino acid of the polypeptide chain of the fusion protein. C' represents the carboxy terminus of the polypeptide chain of the fusion protein; GL P-1 represents a GLP-1 fragment, and R represents an amino acid sequence of the light or heavy chain of the GIPR antibody. and the linker represents a peptide linker. Provided in the specification.

[0009] Polynucleotides encoding the GIPR antibodies described herein are referred to herein as It is provided.

[0010] A polynucleotide encoding a fusion protein of a GIPR antibody and GLP-1 as described herein. Nucleotides are provided herein.

[0011] A vector comprising a polynucleotide encoding a GIPR antibody described herein, Provided herein.

[0012] A polynucleotide encoding a fusion protein of a GIPR antibody and GLP-1 as described herein. Vectors comprising the nucleotides are provided herein.

[0013] Provided herein are host cells comprising the vectors described herein.

[0014] A pharmaceutical composition comprising a GIPR antibody described herein and a pharmaceutically acceptable carrier. , provided herein.

[0015] The fusion proteins of GIPR antibodies and GLP-1 described herein and pharmaceutically acceptable salts thereof and a carrier capable of dissolving the non-alcoholic fatty acid. a GI agonist as described herein in the preparation of a medicament for the treatment, prevention or amelioration of hepatitis disease; Further provided herein are uses of PR antibodies.

[0016] In the preparation of a medicament for treating, preventing or ameliorating non-alcoholic steatohepatitis disease, The use of the fusion proteins of GIPR antibodies and GLP-1 described herein is It is provided at.

[0017] In the preparation of a medicament for treating, preventing or ameliorating type 2 diabetes, Uses of GIPR antibodies are provided herein.

[0018] In the preparation of a medicament for treating, preventing or ameliorating type 2 diabetes, Use of a fusion protein of a GIPR antibody and GLP-1 is provided herein. .

[0019] For reducing weight or treating, preventing or ameliorating obesity and obesity-related disorders The use of a GIPR antibody as described herein in the preparation of a medicament is provided herein. It is served.

[0020] For reducing weight or treating, preventing or ameliorating obesity and obesity-related disorders A fusion protein of a GIPR antibody and GLP-1 as described herein in the preparation of a medicament. The use of quality is provided herein.

[0021] Simultaneous presence of two or more of the following diseases: nonalcoholic steatohepatitis, obesity, or type 2 diabetes a GIP as described herein in the preparation of a medicament for treating, preventing, or ameliorating Uses of R antibodies are provided herein.

[0022] Simultaneous presence of two or more of the following diseases: nonalcoholic steatohepatitis, obesity, or type 2 diabetes a GIP as described herein in the preparation of a medicament for treating, preventing, or ameliorating The use of a fusion protein of R antibody and GLP-1 is provided herein.

[0023] Methods for treating, preventing, or ameliorating one or more symptoms of non-alcoholic steatohepatitis The method comprises administering to a subject a therapeutically effective dose of a GIPR antibody described herein. A method is provided herein that includes:

[0024] Methods for treating, preventing, or ameliorating one or more symptoms of non-alcoholic steatohepatitis The method comprises administering to a subject a therapeutically effective dose of a GIPR antibody described herein and GLP-1. Provided herein are methods that include administering the fusion protein to a subject.

[0025] A method for treating, preventing, or ameliorating one or more symptoms of type 2 diabetes, comprising administering to a subject a therapeutically effective amount of a compound selected from the group consisting of acetaminophen, benzodiazepine ... and administering to a subject a therapeutically effective dose of a GIPR antibody described herein. Methods are provided herein.

[0026] A method for treating, preventing, or ameliorating one or more symptoms of type 2 diabetes, comprising administering to a subject a therapeutically effective amount of a compound selected from the group consisting of acetaminophen, benzodiazepine ... a therapeutically effective dose of a fusion protein of a GIPR antibody and GLP-1 described herein; Provided herein are methods comprising administering to a subject

[0027] A method for treating, preventing, or ameliorating one or more symptoms of obesity, comprising therapeutically A method comprising administering to a subject an effective dose of a GIPR antibody described herein, Provided herein.

[0028] A method for treating, preventing, or ameliorating one or more symptoms of obesity, comprising therapeutically The subjects were tested for an effective dose of a fusion protein of a GIPR antibody and GLP-1 described herein. Provided herein are methods comprising administering to the body. [Brief explanation of the drawings]

[0029] [Figure 1] 1 shows the results of a FACS test for the specific binding of recombinantly expressed hGIPR antibody L10H8 (comprising SEQ ID NO: 70 and SEQ ID NO: 79) to hGIPR. The gray and dotted peaks are negative controls, the gray peak represents 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. [Figure 2] 1 shows the concentration inhibition curve of hGIPR antibody L7H6 (comprising SEQ ID NO: 67 and SEQ ID NO: 77) antagonizing GIP activation of the hGIPR signaling pathway as determined by a direct cAMP assay (IC50=7.6 nM, R2=0.99). [Figure 3] 1 shows the inhibition curve of the GIPR antibody / GLP-1 fusion protein GLP-1-linker-L7H6 (comprising SEQ ID NO: 67, SEQ ID NO: 77, SEQ ID NO: 106, SEQ ID NO: 111) antagonizing GIP activation of the hGIPR signaling pathway as determined by a direct cAMP assay (IC50=14.9 nM, R2=0.99). [Figure 4]1 shows the activation curve of a reporter gene experiment to test the GIPR antibody / GLP-1 fusion protein GLP-1-linker-L7H6, which activates the hGLP-1R signaling pathway as determined by reporter gene assay (EC50=0.04 nM, R2=0.99). [Figure 5] 1 shows the time curves of the percentage of weight change in different groups of C57BL / 6 obese mice induced by a high-fat diet during the efficacy study. [Figure 6] 1 shows the activation curve of a reporter gene experiment testing the activation of the human GLP-1 receptor (hGLP-1R) signaling pathway by the GIPR antibody / GLP-1 fusion protein GLP-1-linker-V1W5 (comprising SEQ ID NO: 106, SEQ ID NO: 111, SEQ ID NO: 125 and SEQ ID NO: 131) as determined by reporter gene assay (EC50=17.40 pM, R2=0.99). [Figure 7] 1 shows the inhibition curve of hGIPR antibody GLP-1-linker-V1W5 antagonizing GIP activation of the hGIPR signaling pathway as determined by a direct cAMP assay (IC50=7.03 nM, R2=0.99). [Figure 8] 1 shows the inhibition curve of the human GIP receptor (hGIPR) antibody / GLP-1 fusion protein GLP-1-linker-V1W5 antagonizing GIP activation of the monkey GIPR receptor (maGIPR) signaling pathway as determined by a direct cAMP assay (IC50=4.30 nM, R2=0.99). [Figure 9] 1 shows the pharmacokinetic (PK) time curves of the antibody portion of the hGIPR antibody / GLP-1 fusion protein in rhesus monkeys during a pharmacokinetic study. [Figure 10] 1 shows the pharmacokinetic (PK) time curves of the GLP-1 portion of the hGIPR antibody / GLP-1 fusion protein in rhesus monkeys during a pharmacokinetic study. [Figure 11] 1 shows the time curve of the effect of hGIPR antibody / GLP-1 fusion protein on change in food intake during an efficacy study in high-fat diet-induced obese cynomolgus macaques. [Figure 12] 1 shows the time curve of the effect of hGIPR antibody / GLP-1 fusion protein on change in body weight during an efficacy study in high-fat diet-induced obese cynomolgus macaques. [Figure 13] Figure 1 shows the time curve of the effect of hGIPR antibody / GLP-1 fusion protein on percent weight change during an efficacy study in high-fat diet-induced obese cynomolgus macaques. Day 28: When compared to the formulation control group, the GLP-1-linker-V1W5 and positive control groups showed statistical P values ​​of 0.000 and 0.003, respectively; Day 56: When compared to the GLP-1-linker-V1W5 group, the formulation and positive control groups showed statistical P values ​​of 0.003 and 0.028, respectively. [Figure 14] 1 shows the time curve of the effect of hGIPR antibody / GLP-1 fusion protein on change in trunk fat mass during an efficacy study in high-fat diet-induced obese cynomolgus macaques. [Figure 15] 1 shows the time curve of the effect of hGIPR antibody / GLP-1 fusion protein on change in total fat mass during an efficacy study in high-fat diet-induced obese cynomolgus macaques. [Figure 16] 1 shows the time curve of the effect of hGIPR antibody / GLP-1 fusion protein on percent change in total fat mass during an efficacy study in high-fat diet-induced obese cynomolgus macaques. [Figure 17] 1 shows the time curve of the effect of hGIPR antibody / GLP-1 fusion protein on change in total body fat mass per kilogram of body weight during an efficacy study in high-fat diet-induced obese cynomolgus macaques. [Figure 18] 1 shows the time curve of the effect of hGIPR antibody / GLP-1 fusion protein on change in total lean tissue mass per kilogram of body weight during an efficacy study in high-fat diet-induced obese cynomolgus macaques. DETAILED DESCRIPTION OF THE INVENTION

[0030] definition Unless otherwise defined herein, scientific and technical terms are understood by those of ordinary skill in the art. Generally, the term "pharmacology," "biology," "biochemistry," "cell and tissue culture" is used in the fields of , Biology, Molecular Biology, Immunology, Microbiology, Genetics and Protein, Nucleic Acid Chemistry and Hybridization The nomenclature and techniques associated with redox synthesis are well known in the art and are generally It is intended to be used for practical purposes.

[0031] The present invention uses standard single-letter or triple-letter methods to designate polynucleotide and polypeptide sequences. When a polypeptide sequence is written, the first amino acid sequence bearing the amino group is The amino acid residue (N') is at the left end and is the last amino acid residue (C') that has a carboxyl group. is at the right end, for example, a GLP-1 fragment sequence according to the present invention: SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109. A peptide sequence is a polypeptide sequence in which the amino acids are arranged in reverse order relative to the original. , for example, a reverse GLP-1 fragment converted from the above GLP-1 fragment sequence Sequences: SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: Refers to No. 123. The upstream chain of a single-stranded or double-stranded nucleic acid sequence The 5' ends are on the left and their 3' ends are on the right. It is represented by the number of amino acid residues, such as amino acids 80 to 130, or Lys80 to Ly The specific polypeptide or polypeptide may be represented by the actual residue at that site, such as s130. A nucleotide sequence may also be represented by showing its differences from a reference sequence.

[0032] The terms "peptide," "polypeptide," and "protein" refer to peptide bonds. Thus, these terms refer to molecules containing two or more amino acids linked together. , natural and artificial proteins and peptide analogues of protein sequences (mutated proteins, variants and fusion proteins) and post-translationally, or covalently or non-covalently. A peptide, polypeptide, or protein is a compound that is synthesized from a monomer or a modified protein. It may be a polymer.

[0033] The term "polypeptide fragment" refers to an amino acid sequence from the corresponding full-length protein. refers to a polypeptide having a terminal and / or carboxyl terminal deletion. Length must be at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 The fragment may be 50, 70, 80, 90, 100, 150, or 200 amino acids. The maximum length is, for example, 1000, 750, 500, 250, 200, 175, 15 0, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 14, The fragment may be 13, 12, 11, or 10 amino acids. and one or more additional amino acids, e.g., amino acids from different naturally occurring proteins. amino acid sequences (e.g., Fc or leucine zipper domains) or artificial amino acid sequences (e.g., The nucleic acid sequence may further comprise an artificial binding sequence.

[0034] The peptides of the present invention can be used for any reason and by any means, for example, (1) to synthesize proteins (2) Reduced susceptibility to oxidation; (3) Reduced susceptibility to protein degradation. (4) modifying the binding affinity to form a protein complex; and (5) modifying the binding affinity to form a protein complex. Includes peptides modified by imparting or modulating physicochemical or functional properties. Analogs include muteins of polypeptides, e.g., single or multiple amino acids. Substitutions (e.g., conservative amino acid substitutions) can be made within the native sequence (e.g., within the molecular (outside the domain of the polypeptide that forms the contact). A "conservative amino acid substitution" is one that does not substantially alter the structural characteristics (e.g., amino acid substitutions are not required to It should not disrupt helices that are essential for conferring its properties or functions to the parent sequence. (It should not interfere with other secondary structural types that are considered to be

[0035] A "mutant" of a polypeptide is one in which the amino acid sequence is different from that of another polypeptide sequence. The mutations of the present invention include insertions, deletions, and / or substitutions of one or more residues in the amino acid sequence. The bodies contained the fusion protein.

[0036] "Derivatives" of polypeptides include polyethylene glycol, albumin (human serum albumin), by conjugation to other chemical moieties, such as amino acids, phosphorylation, and glycosylation. It is a chemically modified polypeptide.

[0037] Unless otherwise specified, the term "antibody" refers to an antibody that contains two full-length heavy chains and two full-length light chains. and antibodies, and derivatives, variants, fragments, and muteins thereof, comprising Examples of which are given below.

[0038] The term "antibody" refers to an antigen-binding portion and, optionally, a molecule that promotes binding of the antibody to the antigen. The antigen-binding moiety may comprise a scaffold or framework portion that enables the antigen-binding moiety to adopt a conformation that conforms to the antigen-binding moiety. Examples of antibodies include complete antibodies and antibody fragments (antigen-binding portions of antibodies). Antibodies include antibody derivatives and antibody analogs. These may include alternative protein scaffolds or artificial scaffolds that contain derivatives of the CDRs. Although not specified, antibody-derived scaffolds introduced to stabilize the three-dimensional structure of antibodies, and These include fully synthetic scaffolds containing biocompatible polymers and other materials. r et al.,2003,Proteins 53:121-129; See et al., 2004, Biotechnol. Prog. 20:639-654. Additionally, antibodies may be used as mock peptide antibodies ("PAMs") or as scaffold-like fibrils. The scaffold may be either a mock antibody-containing scaffold using linconnexin.

[0039] An antibody may have a structure similar to that of a native immunoglobulin. In native immunoglobulins, each tetramer consists of two identical pairs of polypeptide chains. Each pair has a "light" chain (approximately 25 kDa) and a "heavy" chain (approximately 50-70 kDa). The amino terminus of each chain contains approximately 100-110 possible amino acids that are primarily involved in antigen recognition. The carboxyl terminus of each chain is primarily responsible for the effector activity. The constant region is defined by the α, δ, and κ light chains. ε, and the same type of IgM, IgD, IgG, IgA, and IgE. Within the light and heavy chains, the variable and constant regions are "J" regions of about 12 or more amino acids. The heavy chains are joined by a "D" region, and the heavy chains also contain a "D" region of about 10 more amino acids. mental Immunology ch.7(edited by Paul,2n (D edition, Raven Press, 1989). The variable regions of the chain pairs form the antibody binding site, and thus a complete immunoglobulin consists of two It has two binding sites.

[0040] Native immunoglobulin chains consist of three superunits, also known as complementarity-determining regions or CDRs. The same basic structure of relatively conserved framework regions (FR) bounded by variable regions From the N-terminus to the C-terminus, the light and heavy chains are composed of the structural domains FR1, CDR1, F Amino acids in all structural domains, including R2, CDR2, FR3, CDR3, and FR4. The distribution of acids is shown 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 was.

[0041] Unless otherwise specified, an "antibody" refers to an intact immunoglobulin or a It means any antigen-binding portion of an intact antibody that can compete specifically with the intact antibody. Binding moieties can be generated by recombinant DNA techniques, enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions can be produced by, among others, Fab, Fab', F(ab)2, Fv and structural domain antibodies (dAbs), which are fragments containing complementarity-determining regions (CDRs). single-chain antibody (scFv), chimeric antibody, double-chain antibody (bispecific antibody), triple-chain antibody (triabodies), quadruplex antibodies (tetrabodies), and polypeptides that bind to specific antigens The antibody comprises a polypeptide comprising at least a portion of an immunoglobulin.

[0042] Fab fragments are V L , V H , C L , and C H1 Monovalent fragments with domains F(ab')2 fragments are fragments of the same nucleotide sequence as the F(ab')2 fragment, which is a fragment of the same nucleotide sequence as the F(ab')2 fragment. a bivalent fragment having two Fab fragments linked by an Fv flag; Mention is V H and V L domain; the dAb fragment has a V H Domain, V L Do Main or V H or V L The antigen-binding fragment of the domain (U.S. Patent No. 6,449,299) ,846,634 and U.S. Pat. No. 6,696,245; U.S. Pat. Publication No. 2005 / 0202512, U.S. Patent Application Publication No. 2004 / 0202995 No. 2004 / 0038291, U.S. Patent Application Publication No. No. 2004 / 0009507 and U.S. Patent Application Publication No. 2003 / 0039958 No. specification; Ward et al., 1989, Nature 341:544-546 ).

[0043] Single-chain antibodies (scFv) are V L and V H If the region is a linker (e.g., a combination of amino acid residues), a fusion protein in which the two proteins are linked via a sequence (sequence) to form a continuous protein antibody, Here, the linker acts as a bridge between the protein chains, allowing them to fold back on themselves to form a monovalent antigen-binding site. long enough to allow for the ence 242:423-26; and Huston et al., 1988, Pro. c. Natl. Acad. Sci. USA 85:5879-83).

[0044] A double-chain antibody is a bivalent antibody containing two polypeptide chains, each of which is and connected by a linker that is too short to allow pairing of the two domains on the same chain. V H and V L Each domain therefore contains a complementary domain on another polypeptide chain. This allows pairing with the target domain (e.g., Holliger et al., 1 993,Proc.Natl.Acad.Sci.USA90:6444-48; Poljak et al., 1994, Structure 2:11 21-23 If the two polypeptide chains of a double-chain antibody are identical, the resulting antibody chains are Double-chain antibodies have identical antigen-binding sites. By using the same antibody, it is possible to create double-chain antibodies with different antigen-binding sites. Quadruplex antibodies contain three and four polypeptide chains, the three and four chains of which may be the same or different. and an antibody that forms four antigen-binding sites.

[0045] As used herein, Sequences of Proteins of Immunol ogical Interest,5th edition,USDept.Of Health and Human Services,PHS,NIH,NIH Pu In Publication No. 91-3242, 1991, Kabat et al. The complementarity determining regions (CDRs) and framework regions (FDRs) of a given antibody can be identified using the methods described in One or more CDRs are covalently or non-covalently incorporated into the molecule. Antibodies can be made by incorporating larger polypeptide chains into the CDRs. The CDRs can be covalently linked to another polypeptide chain, or can be non-covalently linked to the CDRs. The CDRs are the components that allow an antibody to bind specifically to a particular antigen of interest. Allows for bonding.

[0046] An antibody may have one or more binding sites. If there is more than one binding site, the binding sites The positions may be identical to or different from each other. For example, in naturally occurring human immunoglobulins typically have two identical binding sites, while "bispecific" or "bifunctional" antibodies , which has two different binding sites.

[0047] The term "murine antibody" refers to one or more possible antibodies derived from murine immunoglobulin sequences. The term "antibody" includes antibodies having both variable and constant regions.

[0048] The term "humanized antibody" refers to a method for modifying the sequences of the complementarity determining regions of a murine antibody molecule to make them compatible with a human antibody. These are antibodies created by grafting variable regions onto a framework.

[0049] The terms "antigen-binding domain," "antigen-binding region," or "antigen-binding site" refer to the binding site of an antigen. and containing amino acid residues that interact with and contribute to the specificity and affinity of the antibody for the antigen. For antibodies that specifically bind their antigen, this is the portion of their CDR domain. It includes at least a portion of at least one of the main

[0050] The term "epitope" refers to the portion of a molecule that can be bound to (e.g., by) an antibody. An epitope is a non-contiguous portion of a molecule (e.g., in a polypeptide, are not adjacent in the primary sequence of the antibody, but in the tertiary and quaternary structure of the polypeptide The amino acid residues may be sufficiently close to each other to be joined by

[0051] The "percent identity" of two polynucleotide or two polypeptide sequences is determined by the GA P computer program (GCG Wisconsin Package; versi Default on 10.3 (part of Accelrys, San Diego, CA) The parameters are determined using a comparison sequence.

[0052] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used throughout the text. and are used interchangeably, and refer to DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), nucleotide analogs (e.g., peptide nucleic acids and non-natural nucleosides DNA or RNA analogs and their hybrids produced using The nucleic acid molecule may be single-stranded or double-stranded. The nucleic acid molecule contained therein is an antibody or a fragment, derivative, mutein, or The variant encodes a contiguous open reading frame.

[0053] Two single-stranded nucleotides are "complementary" to each other if their sequences can be antiparallel. ", where each nucleotide in one polynucleotide is a nucleotide in the other polynucleotide. The complementary nucleotides are opposite, no gaps are introduced, and the 5' or 3' end of each sequence Unpaired nucleotides are not visible. Two polynucleotides If one polynucleotide is capable of hybridizing to the other under moderately stringent conditions, the other polynucleotide is A polynucleotide is "complementary" to another polynucleotide. A polynucleotide may be complementary to another polynucleotide, but not its complementary sequence.

[0054] The term "carrier" refers to a nucleic acid that is linked to another nucleic acid and that is introduced into a cell. One type of carrier is a "plasmid," and It refers to a linear or circular double-stranded DNA molecule that can bind to a nucleic acid segment. Types include viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adenosine adeno-associated viruses), in which additional DNA segments are present within the viral genome Some carriers are capable of self-replicating in the host cells into which they are introduced. (e.g., bacterial carriers containing bacterial origins of replication and free-type mammalian carriers) Other carriers (e.g., non-free mammalian carriers) may be introduced into host cells to induce cell proliferation. It integrates into the genome and thereby replicates along with the host genome. , a type of carrier capable of directing the expression of a selected polynucleotide.

[0055] Regulatory sequences affect the expression (e.g., level, time, or location of expression) of a nucleotide sequence. A nucleotide sequence is "operably linked" to a regulatory sequence if it exerts an effect on the transcription of the target gene. " refers to the expression (e.g., level, time, or location of expression) of a nucleic acid to which it is operably linked. A regulatory gene is a nucleic acid that affects, for example, the regulated nucleic acid directly or The polynucleotide is linked to one or more other molecules (e.g., regulatory sequences and / or nucleic acids). Examples of regulatory sequences include promoters, enhancers, and other expression regulators. Further examples of regulatory sequences include transcription control elements (e.g., polyadenylation signals). , Goeddel, 1990, Gene Expression Technology :Methods in Enzymology,Volume 185,Academ ic Press, San Diego, CA; and Baron et al., 199 5, Nucleic Acids Res. 23:3605-06, etc.

[0056] The term "host cell" refers to a cell in which a nucleic acid, such as those provided herein, can be expressed. The host cell is a prokaryotic organism, such as E. coli. or it may be a eukaryote, such as a unicellular eukaryote (e.g., yeast or other fungi). ), plant cells (e.g., tobacco or tomato plant cells), animal cells (e.g., human cells, human cells, hamster cells, rat cells, mouse cells or insect cells) or hybridomas Typically, the host cell is pre-transfected with a peptide-encoding nucleic acid that can then be expressed in the host cell. A cultured cell that can be transformed or transfected. The term "recombinant host cell" The phrase is used to refer to a host cell transformed or transfected with a nucleic acid for which expression is anticipated. A host cell can also be used that contains a nucleic acid, but in which a control sequence is operably linked to the nucleic acid. A cell that does not express the nucleic acid at a desired level unless introduced into the host cell in such a way that The term "host cell" includes not only the cell of a particular subject, but also the progeny or It should be understood that the term also refers to potential descendants. Such progeny may, in fact, differ from the parent cell, due, for example, to mutations or environmental influences. , still within the scope of this term as used in the present invention.

[0057] Gastrointestinal inhibitory peptide receptor Gut inhibitory peptide receptors are a family of receptors coupled to seven-transmembrane G proteins The receptor belongs to type B of the ATP-dependent ATPase inhibitors. The receptor is a heterotrimeric guanine nucleotide-binding protein ( G proteins) are coupled to one or more intracellular signaling pathways (Druc ker et al., 2006, Cell Metab. 3: 153-65). In this study, GIPR was found to be expressed primarily on the surface of pancreatic beta cells and adipocytes (Ravne et al., 2013). t al., 2013, J. Biol. Chem. 288: 19760-72), in humans It is involved in both glucose and lipid metabolism in diabetes, obesity and related diseases. are closely related (Skaw et al., 2016, Diabetes Obes Metab. 18:847-854). "GIPR" and "hGIPR" both refer to human intestinal inhibitory peptide receptor and are used interchangeably. As used herein, "mouse GIPR" and "mGIPR" refer to both Both terms refer to mouse gastric inhibitory peptide receptor and may be used interchangeably.

[0058] In one embodiment, the antibodies provided herein specifically bind to human GIPR. In another embodiment, the antibody provided herein is an antibody that binds to a cell membrane. and an antibody that specifically binds to GIPR in these cells. In another embodiment, the transduction of IP signals can be inhibited or blocked. The provided antibodies are antibodies that specifically bind to human GIPR, and antibodies may also bind to other species (e.g., It binds to the GIPR of the rhesus monkey or mouse and blocks GIP signaling in these species. In a further embodiment, the antibodies provided herein target human GIPR. It is a murine antibody that binds to GIPRs of other species (e.g., monkeys) and can bind to GIPRs of other species (e.g., monkeys).

[0059] In one embodiment, the amino acid and polynucleotide sequences of GIPR are listed below. The sequence data are available from the US National Center for Biotechnology Information. Gene -Bank database and the European Bioinformatics Institute institute for biological information) Obtained from the iprot database: Human (Homo sapiens) polynucleotide (SEQ ID NO:1 14);Accession number: S79852; Human (Homo sapiens) amino acid (SEQ ID NO: 113); Accession number: AAB35419.2; Monkey (Rhesus macaque) polynucleotide (SEQ ID NO: 11 6);Accession number: XM_015124289.1; Monkey (Rhesus macaque) amino acid (SEQ ID NO: 115); Consignment number:XP_014979775; Mouse (Mus musculus) polynucleotide (SEQ ID NO: 11 8); Accession number: CCDS39795; and mouse (Mus musculus lus) amino acid (SEQ ID NO: 117); Accession number: Q0P543.

[0060] Gut inhibitory peptide receptor (GIPR) antibodies In one embodiment, a GIPR antibody is provided herein. In another embodiment, the GIPR antibodies provided herein are full GIPR antibodies. In embodiments, the GIPR antibodies provided herein comprise a GIPR antibody fragment. In another embodiment, the GIPR antibody provided herein is a GIP In another embodiment, the GIPR antibodies provided herein are derivatives of the GIPR antibodies. In a further embodiment, the antibody is a GIPR antibody mutein. The GIPR antibodies provided herein are variants of GIPR antibodies.

[0061] In one embodiment, the GIPR antibodies provided herein are 1, 2, 3, 4, 5, or 6 amino acid sequences, each of which independently corresponds to one of the amino acids listed below. Amino acid sequence: 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; 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; 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; d. Heavy chain CDR1 amino acid sequences: SEQ ID NO:18, SEQ ID NO:23, and SEQ ID NO:26; 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 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 is selected from.

[0062] Table 1 shows the amino acid sequences of the light chain CDRs of the GIPR antibodies provided herein, as well as Table 2 lists the polynucleotide coding sequences corresponding to the sequences provided herein. The amino acid sequence of the heavy chain CDR of the GIPR antibody to be used, as well as the corresponding polynucleotide code The code array is listed.

[0063] [Table 1] TIFF2025165930000002.tif144170

[0064] [Table 2] TIFF2025165930000004.tif213170

[0065] In one embodiment, the antibodies provided herein comprise 5, 4, 3, 2, or 1 Amino acid additions, substitutions, and / or deletions are those of the CDR amino acid sequences listed in Tables 1 and 2. In another embodiment, the antibody provided herein comprises a sequence different from one of The 4, 3, 2, or 1 amino acid additions, substitutions, and / or deletions are listed in Tables 1 and 2. It contains a sequence that differs from one of the CDR amino acid sequences.

[0066] In another embodiment, the antibodies provided herein have 3, 2, or 1 amino acid residues. The amino acid additions, substitutions, and / or deletions are identical to one of the CDR amino acid sequences listed in Tables 1 and 2. Contains different sequences.

[0067] In another embodiment, the antibodies provided herein comprise two or one amino acid residue. additions, substitutions, and / or deletions that differ from one of the CDR amino acid sequences listed in Tables 1 and 2. Contains an array of

[0068] In a further embodiment, the antibodies provided herein comprise one amino acid addition , substitutions, and / or deletions differ from one of the CDR amino acid sequences listed in Tables 1 and 2. Contains arrays.

[0069] In one embodiment, the GIPR antibodies provided herein contain one or two amino acids. and a sequence of amino acids, wherein each amino acid sequence is independently selected from the amino acid sequences listed below. : 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 b. Heavy chain CDR1 amino acid sequences: SEQ ID NO:18, SEQ ID NO:23, and SEQ ID NO:26 is selected from.

[0070] In another embodiment, the GIPR antibodies provided herein contain one or two antigens. amino acid sequences, wherein each amino acid sequence is independently an amino acid sequence listed below: Column: 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 b. 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 is selected from.

[0071] In another embodiment, the GIPR antibodies provided herein are 1, 2, 3, or comprises four amino acid sequences, wherein each amino acid sequence is independently: Amino acid sequence: a. 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; and b. 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 is selected from.

[0072] In another embodiment, the GIPR antibodies provided herein are 1, 2, 3, or comprises four amino acid sequences, wherein each amino acid sequence is independently: Amino acid sequence: 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; b. Heavy chain CDR1 amino acid sequences: SEQ ID NO:18, SEQ ID NO:23, and SEQ ID NO:26; c. 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 d. 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 is selected from.

[0073] In another embodiment, the GIPR antibodies provided herein are 1, 2, 3, or comprises four amino acid sequences, wherein each amino acid sequence is independently: Amino acid sequence: 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; b. Heavy chain CDR1 amino acid sequences: SEQ ID NO:18, SEQ ID NO:23, and SEQ ID NO:26; 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; and d. 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 is selected from.

[0074] In further embodiments, the GIPR antibodies provided herein are or four amino acid sequences, wherein each amino acid sequence is independently selected from the group consisting of: The amino acid sequence: 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; b. 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; 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; and d. 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 is selected from.

[0075] In one embodiment, the GIPR antibodies provided herein comprise one, two, or three wherein each amino acid sequence is independently selected from the amino acids listed below: Acid sequences: 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 can be.

[0076] In another embodiment, the GIPR antibodies provided herein are 1, 2, or 3 and wherein each amino acid sequence independently comprises one of the amino acids listed below. Acid sequences: 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: 2 8, SEQ ID NO:29, and SEQ ID NO:30.

[0077] In one embodiment, the GIPR antibodies provided herein are selected from the following list: Sequence number 1 and sequence number 18, sequence number 4 and sequence number 18, sequence number 7 and sequence number 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 and a combination of light and heavy chain CDR1 amino acid sequences independently selected from SEQ ID NO: 26. nothing.

[0078] In another embodiment, the GIPR antibody provided herein is selected from the following list: 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: 2 4, SEQ ID NO: 11 and SEQ ID NO: 27, and SEQ ID NO: 16 and SEQ ID NO: 29, independently selected from The present invention includes a combination of light chain and heavy chain CDR2 amino acid sequences selected from the above.

[0079] In further embodiments, the GIPR antibodies provided herein are G: 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: No. 25, SEQ ID NO: 12 and SEQ ID NO: 28, SEQ ID NO: 14 and SEQ ID NO: 28, and SEQ ID NO: A combination of light and heavy chain CDR3 amino acid sequences independently selected from SEQ ID NO: 17 and SEQ ID NO: 30. This includes.

[0080] In one embodiment, the GIPR antibody provided herein is a. The following list: SEQ ID NO:1 and SEQ ID NO:18, SEQ ID NO:4 and SEQ ID NO:18, SEQ ID NO: 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 light and heavy chain CDR1 amino acids independently selected from SEQ ID NO: 15 and SEQ ID NO: 26. a combination of amino acid sequences; and b. The following list: SEQ ID NO:2 and SEQ ID NO:19, SEQ ID NO:5 and SEQ ID NO:21, SEQ ID NO: 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. A combination of light chain and heavy chain CDR2 amino acid sequences independently selected from Includes:

[0081] In another embodiment, the GIPR antibody provided herein is a. The following list: SEQ ID NO:1 and SEQ ID NO:18, SEQ ID NO:4 and SEQ ID NO:18, SEQ ID NO: 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 light and heavy chain CDR1 amino acids independently selected from SEQ ID NO: 15 and SEQ ID NO: 26. a combination of amino acid sequences; and b. The following list: SEQ ID NO:3 and SEQ ID NO:20, SEQ ID NO:6 and SEQ ID NO:22, SEQ ID NO: 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 light and heavy chain CDR3 amino acids independently selected from SEQ ID NO: 17 and SEQ ID NO: 30. Combination of amino acid sequences Includes:

[0082] In another embodiment, the GIPR antibody provided herein is a. The following list: SEQ ID NO:2 and SEQ ID NO:19, SEQ ID NO:5 and SEQ ID NO:21, SEQ ID NO: 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: a combination of light chain and heavy chain CDR2 amino acid sequences independently selected from 29; and b. The following list: SEQ ID NO:3 and SEQ ID NO:20, SEQ ID NO:6 and SEQ ID NO:22, SEQ ID NO: 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 light and heavy chain CDR3 amino acids independently selected from SEQ ID NO: 17 and SEQ ID NO: 30. Combination of amino acid sequences Includes:

[0083] In a further embodiment, the GIPR antibody provided herein: a. The following list: SEQ ID NO:1 and SEQ ID NO:18, SEQ ID NO:4 and SEQ ID NO:18, SEQ ID NO: 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 light and heavy chain CDR1 amino acids independently selected from SEQ ID NO: 15 and SEQ ID NO: 26. Combination of amino acid sequences; b. The following list: SEQ ID NO:2 and SEQ ID NO:19, SEQ ID NO:5 and SEQ ID NO:21, SEQ ID NO: 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: a combination of light chain and heavy chain CDR2 amino acid sequences independently selected from 29; and c. The following list: SEQ ID NO:3 and SEQ ID NO:20, SEQ ID NO:6 and SEQ ID NO:22, SEQ ID NO: 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 light and heavy chain CDR3 amino acids independently selected from SEQ ID NO: 17 and SEQ ID NO: 30. Combination of amino acid sequences Includes:

[0084] In one embodiment, the GIPR antibody provided herein is a. Light and heavy chain CDR1, CDR2, and CDR3 amino acid sequences: SEQ ID NO: 1, SEQ ID NO: SEQ ID NO:2, a combination of SEQ ID NO:3, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; b. Light and heavy chain CDR1, CDR2, and CDR3 amino acid sequences: SEQ ID NO: 4, SEQ ID NO: No. 5, a combination of SEQ ID NO: 6, SEQ ID NO: 18, SEQ ID NO: 21, and SEQ ID NO: 22; c. Light and heavy chain CDR1, CDR2, and CDR3 amino acid sequences: SEQ ID NO: 7, SEQ ID NO: SEQ ID NO: 8, a combination of SEQ ID NO: 9, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25; d. Light and heavy chain CDR1, CDR2, and CDR3 amino acid sequences: SEQ ID NO: 10, SEQ ID NO: A combination of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28; e. Light and heavy chain CDR1, CDR2, and CDR3 amino acid sequences: SEQ ID NO: 13, SEQ ID NO: a combination of SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28; or teeth f. Light and heavy chain CDR1, CDR2, and CDR3 amino acid sequences: SEQ ID NO: 15, SEQ ID NO: Combination of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 29, and SEQ ID NO: 30 Includes:

[0085] In one embodiment, the GIPR antibodies provided herein contain one or two amino acids. and a sequence of amino acids, wherein each amino acid sequence is independently selected from the amino acid sequences listed below. : a. Light chain variable domain amino acid sequences: SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO: 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 sequences that are at least 80%, at least an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical; and b. Heavy chain variable domain amino acid sequences: SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO: No. 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 8 0; and any of the above sequences and at least 80%, at least 85%, at least 90% %, or at least 95% identical amino acid sequence is selected from.

[0086] In another embodiment, the polynucleotides of the GIPR antibodies provided herein The coding sequence may comprise one or two polynucleotide coding sequences, where each polynucleotide The nucleotide coding sequences are independently selected from the polynucleotide sequences listed below: a. Light chain variable domain polynucleotide coding sequences: SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO: 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 any of the above sequences and at least Polypeptides that are at least 80%, at least 85%, at least 90%, or at least 95% identical to the nucleotide sequence; and b. Heavy chain variable domain polynucleotide coding sequences: SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO: 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 at least 80%, at least 85% of any of the above sequences , at least 90%, or at least 95% identical polynucleotide sequences is selected from.

[0087] In one embodiment, the GIPR antibodies provided herein are selected from the following list: Sequence number 61, sequence number 62, sequence number 63, sequence number 64, sequence number 65, sequence number 66 , SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71 The amino acid sequence may be selected arbitrarily.

[0088] In another embodiment, the GIPR antibody provided herein is selected from the following list: SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 7 7, an amino acid sequence independently selected from SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80 Contains columns.

[0089] In one embodiment, the GIPR antibodies provided herein are those listed below. Light and heavy chain variable domain amino acid sequences: 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, Sequence number 65 and sequence number 75, sequence number 66 and sequence number 76, sequence number 67 and sequence number 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 an amino acid independently selected from SEQ ID NO: 71 and SEQ ID NO: 80 It includes a combination of sequences.

[0090] In one embodiment, the GIPR antibodies provided herein are selected from the following list: Sequence number 62, sequence number 63, sequence number 64, sequence number 66, sequence number 67, sequence number 68 , SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 76, and SEQ ID NO: 77. It contains an amino acid sequence.

[0091] In another embodiment, the GIPR antibody provided herein is selected from the group consisting of those listed below. The light and heavy chain variable domain amino acid sequences are 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 (L3H 3), SEQ ID NO: 64 and SEQ ID NO: 74 (L4H3), SEQ ID NO: 65 and SEQ ID NO: 75 (L 5H4), 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 and SEQ ID NO: 80 (L11H9).

[0092] The designation "LxHy" is used to refer to the GIPR antibodies provided herein. where "x" corresponds to the light chain variable region sequence code and "y" corresponds to the heavy chain variable region sequence code. For example, L2H2 contains the amino acid sequence of SEQ ID NO: 62 (L2). A complete antibody having a light chain variable region comprising the amino acid sequence of SEQ ID NO: 73 (H2) and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 74 (H3). It is an antibody.

[0093] In one embodiment, the GIPR antibodies provided herein contain one or two amino acids. and a sequence of amino acids, wherein each amino acid sequence is independently selected from the amino acid sequences listed below. : a. light chain constant region amino acid sequences: SEQ ID NO:101 and SEQ ID NO:102; and b. Heavy chain constant region amino acid sequences: SEQ ID NO: 103 and SEQ ID NO: 104, and SEQ ID NO: 12 4 is selected from.

[0094] In one embodiment, the GIPR antibodies provided herein contain one or two amino acids. and wherein each amino acid sequence independently corresponds to a light chain and a heavy chain as listed below. Combination of constant region amino acid sequences: SEQ ID NO: 101 and SEQ ID NO: 103, SEQ ID NO: 101 and and SEQ ID NO: 104, SEQ ID NO: 102 and SEQ ID NO: 103, and SEQ ID NO: 102 and SEQ ID NO: No. 104. In another embodiment, the GIPR provided herein is selected from The antibody comprises one or two amino acid sequences, wherein each amino acid sequence is independently: A combination of light and heavy chain constant region amino acid sequences listed in SEQ ID NO: 101 and SEQ ID NO: 124, and SEQ ID NO: 102 and SEQ ID NO: 124.

[0095] In one embodiment, the GIPR antibodies provided herein are those listed herein. The amino acid sequences of the light and heavy chain CDRs and FR (framework) regions are included. The amino acid sequences are included in the light or heavy chain variable domain sequences and are not shown separately. In another 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 as recited herein. In another embodiment, the antibody comprises a heavy chain CDR2 sequence as recited herein. In another embodiment, the antibody comprises a heavy chain CDR3 sequence as set forth herein. In another embodiment, the antibody comprises a light chain FR1 sequence as defined herein. In another embodiment, the antibody comprises a light chain FR3 sequence herein. In another embodiment, the antibody comprises a light chain FR4 sequence herein. In another embodiment, the antibody comprises a heavy chain FR1 sequence as described herein. In another embodiment, the antibody comprises a heavy chain FR2 sequence as described herein. In a further embodiment, the antibody comprises a heavy chain FR3 sequence as described herein. It contains the heavy chain FR4 sequence.

[0096] In one embodiment, the light chain CDR3 sequences of the antibody have no more than 6, 5, 4, 3, 2 or 1 The amino acid additions, substitutions, and / or deletions are the light chain CDR3 sequences SEQ ID NO: 6, SEQ ID NO: 12 and SEQ ID NO: 14. In another embodiment, the heavy chain CDR3 of the antibody The sequence may have no more than 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, and / or deletions. The heavy chain CDR3 sequences shown in SEQ ID NO: 22 and SEQ ID NO: 28 are different from those shown in SEQ ID NO: 23 and SEQ ID NO: 24. In some embodiments, the antibody light chain CDR3 sequence contains no more than 6, 5, 4, 3, 2, or 1 amino acid residues. The additions, substitutions, and / or deletions are the same as those of the light chain CDR3 sequences shown above, SEQ ID NO: 6, SEQ ID NO: 1 2, and the heavy chain CDR3 sequence of the antibody is different from SEQ ID NO: 14 and is 6, 5, 4, 3, 2 or 1. Not more than two amino acid additions, substitutions, and / or deletions are present in the heavy chain CDR3 sequences shown above. In another embodiment, the antibody has a light chain sequence different from SEQ ID NO: 22 and SEQ ID NO: 28. and a combination of one, two, three, four, five or six of the heavy chain CDR sequences.

[0097] In one embodiment, the GIPR antibodies provided herein are those listed herein. L2 (SEQ ID NO: 62), L3 (SEQ ID NO: 63), L4 (SEQ ID NO: 64), L6 (SEQ ID NO: 65) L7 (SEQ ID NO: 66), L7 (SEQ ID NO: 67), and L8 (SEQ ID NO: 68) light chain variable domain sequences In one embodiment, the GIPR antibody comprises a light chain variable domain amino acid sequence selected from the group consisting of: The amino acid sequences of the light chain variable domains of 7, 6, 5, 4, 3, 2 or 1 amino acid difference between L2 (SEQ ID NO: 62), L3 (SEQ ID NO: 63), L4 (sequence number 64), L6 (sequence number 66), L7 (sequence number 67), and L8 (SEQ ID NO: 68), wherein: Each sequence difference is independently a deletion, insertion, or substitution of an amino acid residue. In this case, the light chain variable domain of the GIPR antibody is L2 (SEQ ID NO: 62), L3 (SEQ ID NO: 6 3), L4 (SEQ ID NO: 64), L6 (SEQ ID NO: 66), L7 (SEQ ID NO: 67), and L8 at least 70%, at least 10% identical to the amino acid sequence of the light chain variable domain of one of SEQ ID NO: 68 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 amino acid sequence of the target gene. In one embodiment, the polynucleotide coding sequence for the light chain variable domain of the GIPR antibody is 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) 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 In another embodiment, the light chain variable domain of the GIPR antibody comprises a nucleotide coding sequence The polynucleotide coding sequences of L1 (SEQ ID NO: 62), L2 (SEQ ID NO: 63), L3 (SEQ ID NO: 64), L4 (SEQ ID NO: 64), L6 (SEQ ID NO: 66), L7 (SEQ ID NO: 67), and L8 (SEQ ID NO: 68) Hybridizes under moderate conditions with the complementary polynucleotide coding sequence of one of In a further embodiment, the light chain of the GIPR antibody comprises a polynucleotide sequence encoding the light chain of the GIPR antibody. The polynucleotide coding sequences of the variant domains are L2 (SEQ ID NO: 62), L3 (SEQ ID NO: 6 3), L4 (SEQ ID NO: 64), L6 (SEQ ID NO: 66), L7 (SEQ ID NO: 67), and L8 The complementary polynucleotide coding sequence of one light chain variable domain of (SEQ ID NO: 68) and It comprises a polynucleotide sequence that hybridizes under divergent conditions.

[0098] In one embodiment, the GIPR antibodies provided herein are those listed herein. H2 (SEQ ID NO: 73), H3 (SEQ ID NO: 74), H5 (SEQ ID NO: 76), and H6 ( SEQ ID NO: 77) a heavy chain variable domain amino acid sequence selected from the heavy chain variable domain sequence In another embodiment, the heavy chain variable domain amino acid sequence of the GIPR antibody is 15,1 4, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid H2 (SEQ ID NO: 73), H3 (SEQ ID NO: 74), H5 (SEQ ID NO: 76), and H6 (SEQ ID NO: 77) 77), wherein each sequence difference is independently In another embodiment, the GIPR antibody is a deletion, insertion or substitution of one amino acid residue. The heavy chain variable domains of the antibody are H2 (SEQ ID NO: 73), H3 (SEQ ID NO: 74), H5 (SEQ ID NO: No. 76), and H6 (SEQ ID NO: 77), and at least 70% , at least 75%, at least 80%, at least 85%, at least 90%, less The amino acid sequence may be 95%, at least 97%, or at least 99% identical to the amino acid sequence of the target gene. In another embodiment, the heavy chain variable domain of the GIPR antibody is H2 (SEQ ID NO: 73), H H3 (SEQ ID NO: 74), H5 (SEQ ID NO: 76), and H6 (SEQ ID NO: 77) The variant domain polynucleotide coding sequence is at least 70%, at least 75%, or 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 the present invention. In the present specification, the polynucleotide coding sequence for the GIPR antibody heavy chain variable domain is H2 (sequence No. 73), H3 (SEQ ID NO: 74), H5 (SEQ ID NO: 76), and H6 (SEQ ID NO: 77) and a complementary polynucleotide coding sequence for one heavy chain variable domain of In one embodiment, the polynucleotide sequence comprises a polynucleotide sequence that hybridizes under optimal conditions. The polynucleotide coding sequence for the GIPR antibody heavy chain variable domain is H2 (SEQ ID NO: 73) , H3 (SEQ ID NO: 74), H5 (SEQ ID NO: 76), and one overlap of H6 (SEQ ID NO: 77) The complementary polynucleotide coding sequence of the chain variable domain is hybridized under stringent conditions. The polynucleotide sequence includes a polynucleotide sequence that is hybridized with the polynucleotide sequence.

[0099] In one embodiment, the antibody provided herein is L1H1 (SEQ ID NO: 61 and and SEQ ID NO: 72), L2H2 (SEQ ID NO: 62 and SEQ ID NO: 73), L3H3 (SEQ ID NO: 6 3 and SEQ ID NO: 74), L4H3 (SEQ ID NO: 64 and SEQ ID NO: 74), L5H4 (SEQ ID NO: SEQ ID NO: 65 and SEQ ID NO: 75), L6H5 (SEQ ID NO: 66 and SEQ ID NO: 76), L7H6 (SEQ ID NO: L8H6 (sequence number 67 and sequence number 77), L9H7 (sequence number 68 and sequence number 77), (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 its desired phenotype (e.g., Ig A, IgG1, IgG2a, IgG2b, IgG3, IgM, IgE, or IgD) or a combination of Fab or F(ab')2 fragments thereof.

[0100] In one embodiment, the antibodies provided herein are L2H2 (SEQ ID NO: 62 and and SEQ ID NO: 73), L3H3 (SEQ ID NO: 63 and SEQ ID NO: 74), L4H3 (SEQ ID NO: 6 4 and SEQ ID NO: 74), L6H5 (SEQ ID NO: 66 and SEQ ID NO: 76), L7H6 (SEQ ID NO: SEQ ID NO: 67 and SEQ ID NO: 77), or L8H6 (SEQ ID NO: 68 and SEQ ID NO: 77), or its of the desired phenotype (e.g., IgA, IgG1, IgG2a, IgG2b, IgG3, IgM , IgE, or IgD), or a combination of Fab or F(ab')2 fragments thereof It is an antibody containing the combination.

[0101] The antibodies provided herein may comprise any of the constant regions known in the art. The light chain constant region can comprise, for example, a kappa or lambda light chain constant region, e.g., a mouse kappa or lambda light chain constant region. The heavy chain constant region can be, for example, an α, δ, ε, γ, or μ heavy chain constant region. The heavy chain constant region may be, for example, a mouse α, δ, ε, γ, or μ heavy chain constant region. Thus, the light or heavy chain constant region may be a fragment, derivative, variant, or mutant of a native constant region. It is a mutant.

[0102] In one embodiment, the antibodies provided herein comprise a human light chain kappa or lambda constant domain. The amino acid sequence of the light chain constant region is as follows: Is: Human light chain kappa constant domain amino acid sequence: (SEQ ID NO: 101); and Human light chain lambda constant domain amino acid sequence: (SEQ ID NO: 102).

[0103] In one embodiment, the antibodies provided herein comprise a human heavy chain constant domain or It further includes fragments thereof.

[0104] The amino acid sequence of the heavy chain constant region is as follows: Human heavy chain constant region amino acid sequence (hIgG2): (SEQ ID NO: 103); Human heavy chain constant region amino acid sequence (hIgG4): (SEQ ID NO: 104); and Human heavy chain constant region amino acid sequence (hIgG4): (SEQ ID NO: 124).

[0105] In one embodiment, the GIPR antibody provided herein comprises one light chain domain and an amino acid sequence similar to that of V1 (SEQ ID NO:1) provided herein. 25) and V2 (SEQ ID NO: 126). In one embodiment, the GIPR antibody The amino acid sequence of the light chain domain of V1 is 15 times the amino acid sequence of one of the light chain domains of V1 and V2. , 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid where each sequence difference is independently a deletion, insertion, or substitution of one amino acid residue. In another embodiment, the light chain domain amino acid sequence of the GIPR antibody is V1 and and V2 light chain domain amino acid sequence, at least 80%, at least 85%, at least 90%, at least 95%, at least 9 In another embodiment, the GI comprises an amino acid sequence that is at least 7% and at least 99% identical. The light chain domain polynucleotide coding sequence of the PR antibody is one of the light chain domains V1 and V2. At least 70%, at least 75%, at least 8% of the polynucleotide coding sequence 0%, at least 85%, at least 90%, at least 95%, at least 97% and In another embodiment, the GI comprises a polynucleotide sequence that is at least 99% identical to the GI The light chain domain polynucleotide coding sequence of the PR antibody is one of the light chain domains V1 and V2. The complement of the polynucleotide coding sequence is hybridized under moderately stringent conditions. In another embodiment, the light chain of the GIPR antibody comprises a polynucleotide sequence that encodes The domain polynucleotide coding sequence is a light chain domain polynucleotide coding sequence for one of V1 and V2. A polynucleotide that hybridizes under stringent conditions with the complement of the nucleotide coding sequence Contains an octide sequence.

[0106] In one embodiment, the GIPR antibody provided herein comprises one heavy chain domain The amino acid sequence is W1 (SEQ ID NO: 127) as provided herein. , 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). The heavy chain domain amino acid sequences of the antibody are W1, W2, W3, W4, W5, W6, W7, W8 and W9, and the heavy chain domain amino acid sequence of 15, 14, 13, 12, 11, 1 differ by 0, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, where The differences are independently the deletion, insertion, or substitution of one amino acid residue. The heavy chain domain amino acid sequence of the GIPR antibody is W1, W2, W3, W4, W5, W6 a heavy chain domain amino acid sequence of at least 70%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, and at least 99% identical amino acid sequences. In embodiments, the heavy chain domain polynucleotide coding sequence of the GIPR antibody is W2, W3, W4, W5, W6, W7, W8 and W9 heavy chain domain polynucleotides At least 70%, at least 75%, at least 80%, or at least 85%, at least 90%, at least 95%, at least 97% and at least 9 In another embodiment, the polynucleotide sequences of the GIPR antibody are 9% identical to or greater than 10% of the total polynucleotide sequences of the GIPR antibody. The strand domain polynucleotide coding sequences are W1, W2, W3, W4, W5, W6, W7 the complement and middle of the heavy chain domain polynucleotide coding sequence of one of W8 and W9 It includes polynucleotide sequences that hybridize under the most stringent conditions. In embodiments, the heavy chain domain polynucleotide coding sequence of the GIPR antibody is W1, one of the heavy chain domain polynucleotides W2, W3, W4, W5, W6, W7, W8, and W9; A polynucleotide that hybridizes under stringent conditions with the complement of the nucleotide coding sequence. Contains the nucleotide sequence.

[0107] In another embodiment, the antibody provided herein is V1W1 (SEQ ID NO: 12 5 and SEQ ID NO: 127), V1W2 (SEQ ID NO: 125 and SEQ ID NO: 128), V1W3 ( V1W4 (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: 131) No. 132), V1W7 (SEQ ID NO: 125 and SEQ ID NO: 133), V1W8 (SEQ ID NO: 1 25 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: 129) Sequence number 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), V2W 8 (SEQ ID NO: 126 and SEQ ID NO: 134), or V2W9 (SEQ ID NO: 126 and SEQ ID NO: 135).

[0108] In one embodiment, the antibody provided herein is V1W1 (SEQ ID NO: 125 and SEQ ID NO: 127). The antibody provided herein is an antibody comprising a combination of V1W2 (SEQ ID NO: 125 and SEQ ID NO: 128). In one embodiment, the antibody provided herein is V1W3 (SEQ ID NO: In one embodiment, the antibody comprises a combination of SEQ ID NO: 125 and SEQ ID NO: 129. The antibody provided herein is a combination of V1W4 (SEQ ID NO: 125 and SEQ ID NO: 130). In one embodiment, the antibody provided herein is an antibody comprising V1W5 (SEQ ID NO: 125 and SEQ ID NO: 131). The antibodies provided herein are V1W6 (SEQ ID NO: 125 and SEQ ID NO: 132) In one embodiment, the antibody provided herein is an antibody comprising a combination of: In one embodiment, the antibody comprises a combination of V1W7 (SEQ ID NO: 125 and SEQ ID NO: 133). In the present specification, the antibody is V1W8 (SEQ ID NO: 125 and SEQ ID NO: 134). In one embodiment, the antibody provided herein comprises a combination of The antibody is an antibody comprising a combination of V1W9 (SEQ ID NO: 125 and SEQ ID NO: 135). In embodiments, the antibodies provided herein are directed to V2W1 (SEQ ID NO: 126 and In one embodiment, the antibody comprises a combination of the sequences provided herein (SEQ ID NO: 127). The provided antibody is an antibody comprising a combination of V2W2 (SEQ ID NO: 126 and SEQ ID NO: 128). In one embodiment, the antibody provided herein is V2W3 (SEQ ID NO: 1 26 and SEQ ID NO: 129). The antibodies provided herein include a combination of V2W4 (SEQ ID NO: 126 and SEQ ID NO: 130). In one embodiment, the antibody provided herein is an antibody containing V2W5 (sequence In one embodiment, the antibody comprises a combination of SEQ ID NO: 126 and SEQ ID NO: 131. The antibodies provided herein are a combination of V2W6 (SEQ ID NO: 126 and SEQ ID NO: 132). In one embodiment, the antibody provided herein is an antibody comprising a V2 In one embodiment, the antibody comprises a combination of W7 (SEQ ID NO: 126 and SEQ ID NO: 133). The antibodies provided herein are V2W8 (SEQ ID NO: 126 and SEQ ID NO: 13) 4). In one embodiment, the antibody provided herein is an antibody comprising a combination of is an antibody comprising a combination of V2W9 (SEQ ID NO: 126 and SEQ ID NO: 135).

[0109] In one embodiment, the GIPR antibody provided herein is a murine-derived antibody, Humanized antibodies, chimeric antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, anti Antibody fragments, single chain antibodies, double chain antibodies, triple chain antibodies, quadruplex antibodies, Fab fragments fragments, F(ab')x fragments, structural domain antibodies, IgD antibodies, IgE antibodies , IgM antibody, IgG1 antibody, IgG2 antibody, IgG3 antibody, or IgG4 antibody will be done.

[0110] In one embodiment, the GIPR antibody provided herein is a GIPR monoclonal antibody. It is a clonal antibody.

[0111] In another embodiment, the GIPR antibody provided herein is selected from the following list: 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: No. 74, SEQ ID NO: 64 and SEQ ID NO: 74, SEQ ID NO: 65 and SEQ ID NO: 75, SEQ ID NO: 6 6 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 A monoclonal antibody comprising a combination of amino acid sequences selected from SEQ ID NO:80.

[0112] In one embodiment, the GIPR antibody provided herein is a murine GIPR antibody. In another embodiment, the GIPR antibody provided herein is a humanized It is a GIPR antibody.

[0113] In one embodiment, the GIPR antibodies provided herein have a titer of about 1 nM to 200 nM or approximately 1 nM to 100 nM and reduce human GIP signaling .

[0114] Antibodies and antibody fragments In one embodiment, the antibodies provided herein are full-length antibodies (full-length heavy chains and and / or light chains of polyclonal, monoclonal, chimeric, humanized or human antibodies. In another embodiment, the antibody provided herein is an antibody flag. fragments, e.g., F(ab')2, Fab, Fab', Fv, Fc, or Fd fragments antibodies, single domain antibodies, single chain antibodies, maxibodies, minibodies minibodies, intrabodies, double-chain antibodies , triplex antibodies, quadruplex antibodies, v-NAR or bis-scFv (e.g., Hollis et al., J. Immunol. 2012, 11:111-112, 2012). Nger and Hudson,2005,Nature Biotechnolog y, 23:1126-1136). In another embodiment, The antibodies used are fibronectin polypeptide monobodies. Antibody polypeptides such as those disclosed in U.S. Pat. No. 6,703,199, including In another embodiment, the antibodies provided herein comprise a single polypeptide chain. Other antibody polypeptides disclosed in US Patent Application Publication No. 2005 / 0238646 are Also contains lipids.

[0115] In one embodiment, the Ig expressing the monoclonal antibody of interest in a hybridoma The variable region of the G gene is amplified using nucleotide primers. V can be synthesized by one skilled in the art or purchased commercially. Ha , V Hb , V Hc , V Hd , C H1 , V L and C L Primers for mouse and human variable regions, including the These primers may be purchased commercially. )H or IMMUNOZAP™ L (Stratagene) or other vectors. These vectors can then be used to amplify heavy or light chain variable regions that can be inserted. The vector may be introduced into E. coli, yeast, or mammalian-based systems for expression. Get. V H and V L Large numbers of single-chain proteins containing fusions of domains have been synthesized using these methods. (Bird et al., 1988, Science 242:423- (See 426).

[0116] It is well known in the art that certain proteins, such as antibodies, can undergo a variety of post-translational modifications. The type and extent of these modifications should be understood by those skilled in the art. Such modifications often depend on the host cell line and culture conditions used to produce the desired product. glycosylation, methionine oxidation, diketopiperidine These changes may include α-aspartate formation, aspartate isomerization, and asparagine deamidation. The frequency modification is due to the action of carboxypeptidase on the carboxyl-terminal basic residue (lysine). The loss of amino acids (such as glutamate or arginine) is due to the as described in Chromatography 705:129-134).

[0117] A common method for producing mouse monoclonal antibodies is by hybridoma cells. Monoclonal antibodies can be isolated and purified by a variety of well-established techniques. A suitable isolation technique is affinity chromatography using protein-A sepharose. Examples of suitable chromatography include filtration, size exclusion chromatography, and ion exchange chromatography. (e.g., Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3;Baines et al.,“Purific ation of Immunoglobulin G(IgG),”in Metho ds in Molecular Biology,Vol.10,pages 79- 104 (The Humana Press, Inc. 1992). A cloned antibody may be a clone that has specific characteristics of the antibody (e.g., heavy or light chain isotype, binding specificity, etc.). by affinity chromatography using appropriate ligands selected based on Examples of suitable ligands immobilized on a solid support include Protein A, Protein G, anti-constant region (light or heavy chain) antibodies, anti-idiotypic antibodies, and TGF- β-binding protein, or a fragment or variant thereof.

[0118] Schier et al.,1996,J.Mol.Biol.263:551-5 The complementarity determining regions (CDRs) at the center of the antibody binding site, as described by 67 Molecular evolution of antibodies with increased affinity, e.g., increased affinity for c-erbB-2, has also been reported. These techniques have been used to reshape antibodies with different affinities. , can be used to prepare antibodies against GIPR.

[0119] Antibodies against human GIPR can inhibit, for example, GIP either in vitro or in vivo. It can be used in assays to detect the presence of R.

[0120] The antibodies may also be prepared by any conventional technique. For example, the antibodies may be prepared by Can be purified from naturally expressing cells (e.g., antibodies can be isolated from the hybridoma that produces them) purified from mice) or recombinantly expressed using any technique known in the art For example, Monoclonal Antibodies, Hy bridomas:A New Dimension in Biological A nalyses, Kennet et al. (eds.), Plenum Press , New York (1980); and Antibodies: A Laboratory y Manual,Harlow and Land(eds.),Cold Spri ng Harbor Laboratory Press,Cold Spring H Arbor, NY (1988). This is explained in the section on nucleic acids below. .

[0121] Antibodies can be prepared and screened for desired properties by any known technique. Several techniques can be used to identify polypeptide chains of relevant antibodies (e.g., anti-GIPR antibodies). (or portions thereof) and the manipulation of nucleic acids by recombinant DNA techniques. The nucleic acid may be fused to another related nucleic acid or may have one or more amino acid residues It can be modified (e.g., by induced mutagenesis or other conventional techniques) to add, delete, or substitute. do.

[0122] Improving the affinity of antibodies of the present invention containing one or more of the above-described CDRs When required, such antibodies may be prepared by maintaining the CDRs (Yang et al., 1999). 995, J. Mol. Biol., 254:392-403), chain shuffling (Ma rks et al.,1992,Bio / Technology,10:779-78 3), the use of mutant strains of Escherichia coli (E. coli) (Low et al., 1996, J. Mol. Biol., 250:350-368), DNA shuffling (Patt en et al.,1997,Curr.Opin.Biotechnol.,8:7 24-733), phage display (Thompson et al., 1996, J. Mol. Biol., 256:7-88) and further PCR techniques (Crameri et al., 1998, Nature, 391:288-291). All of these methods or affinity maturation are ughan et al.,1998,Nature Biotechnology,1 6:535-539.

[0123] In one embodiment, fragments of GIPR antibodies are provided herein. Such fragments may contain sequences entirely derived from the antibody or additional sequences. Examples of antigen-binding fragments include Fab, F(ab'), single-chain antibodies, bispecific antibodies, and the like. Other examples include monospecific antibodies, trispecific antibodies, tetraspecific antibodies, and domain antibodies. Lunde et al.,2002,Biochem.Soc.Trans.30:5 It is available in 00-06.

[0124] Single-chain antibodies are made by cleaving the polypeptide chains together using an amino acid bridge (a short peptide linker) to produce a single polypeptide chain. By linking heavy and light chain variable domain (Fv region) fragments via Such single-chain Fvs (scFvs) can be formed by combining two variable domain polypeptides. The fusion DNA encodes a peptide linker between the DNAs encoding the VL and VH domains. The resulting polypeptide folds back on itself to form an antigen-binding moiety. They can form monomers or they can be separated by a flexible linker between the two variable domains. Depending on the length, it can form multimers (e.g., dimers, trimers, or tetramers) (Kort t et al.,1997,Prot.Eng.10:423;Kortt et a (I., 2001, Biomol. Eng. 18:95-108). By combining various polypeptides, multimeric scFs that bind to various epitopes can be produced. v can be formed (Kriangkum et al., 2001, Biomol. En g.18:31-40). Techniques developed for the production of single-chain antibodies include U.S. Pat. Specification No. 4946778;Bird,1988,Science 242:423;H uston et al.,1988,Proc.Natl.Acad.Sci.USA 85:5879;Ward et al.,1989,Nature 334:544 ;de Graaf et al.,2002,Methods Mol.Biol.1 78:379-87. Single chains derived from the antibodies provided herein, including scFvs containing the combined L1H1 Antibodies are encompassed by the present invention.

[0125] Multiple antigen-binding fragments derived from a single antibody can also be prepared by conventional methods, such as For example, proteolytic hydrolysis of the antibody, e.g., pepsin or papain digestion of the whole antibody. For example, antibody fragments can be obtained by the S-S fragment, designated F(ab')2. This fragment can be produced by enzymatic cleavage of the antibody with pepsin to produce the fragment. The fragment is further cleaved using a thiol reducing agent to give the 3.5S Fab' monovalent Flag. Optionally, the cleavage reaction may result in a cleavage of a disulfide bond. Alternatively, papain can be used to block the hydroxyl groups. Enzymatic cleavage directly produces two monovalent Fab fragments and an Fc fragment These methods are described, for example, in Goldenberg, U.S. Pat. No. 4,331,647. ,Nisonoffet et al.,1960,Arch.Biochem.Bio phys.89:230;Porter,1959,Biochem.J.73:119 ;Edelman et al.,Methods in Enzymology 1: 422 (Academic Press 1967); and Andrews, S.M. nd Titus, JAin Current Protocols in Imm unology(Coligan JE, et al., eds), John Wi ley & Sons, New York (2003), pages 2.8.1-2. 8.10 and 2.10A.1-2.10A.5. Other methods for cleaving antibodies, such as separating the heavy and light chains to form monovalent light-heavy chain fragments (Fd), are also available. The method, other cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used. Any fragment may be used as long as it binds to the antigen recognized by the intact antibody.

[0126] Another form of antibody fragment contains one or more complementarity-determining regions (CDRs) of an antibody. CDRs are peptides containing CDRs. Such polynucleotides can be obtained, for example, by using mRNA or antibody production as a template. It is prepared by using polymerase chain reaction to synthesize the variable region using living cells. (e.g., Larrick et al., 1991, Methods: A Comp Anion to Methods in Enzymology 2:106;Cou rtenay-Luck,“(Genetic Manipulation of Mo noclonal Antibodies,”in Monoclonal Antib odies:Production,Engineering and Clinica l Application, Ritter et al. (eds.), page 1 66 (Cambridge University Press 1995); and Wa rd et al., “Genetic Manipulation and Expr. ession or Antibodies,”in Monoclonal Anti bodies:Principles and Applications,Birch et al.,(eds.),page 137(Wiley-Liss,Inc.1 995). Antibody fragments contain at least one possible antibody fragment described herein. Thus, for example, a V region domain may comprise a monomer and BiV H or V L domain, which can be 1x10 as described below. -7 Affinity below M It can bind to GIPR with high affinity.

[0127] The variable region domain may be any naturally occurring variable domain or an engineered form thereof. By engineered form is meant a variable region domain produced using recombinant DNA techniques. Such engineered forms may include, for example, modifications in or to the amino acid sequence of a particular antibody. including those generated from a particular antibody variable region by insertions, deletions, or changes thereto. A specific example is a fragment of a first antibody that contains one CDR and optionally one or more fragments. an engineered variable region domain containing framework amino acids and a variable region domain from a second antibody; Contains the remainder of the region domain.

[0128] The variable region domains are those that, at the C-terminal amino acid, have at least one other antibody domain or Thus, for example, a fragment of a nucleotide sequence present in a variable region domain may be covalently linked to the fragment. Existing V H The domain is immunoglobulin C H1 linked to the domain or its fragment Similarly, V L The domain is C K It can be linked to a domain or a fragment thereof. For example, the antibody can be a Fab fragment, in which the antigen-binding domain is , and at their C-termini, respectivelyH1 and covalently linked to the Cκ domain Related V H and V L Contains domains. C H1 The domain is extended with additional amino acids For example, the hinge region or hinge region domain as found in the Fab' fragment antibody C H2 and C H3 domains, and more domains. You can.

[0129] Antibody Derivatives and Variants The nucleotide sequences of L1 and H1 may be determined, for example, by random mutagenesis or by by directed mutagenesis (e.g., oligonucleotide-directed site-directed mutagenesis) The polynucleotide is modified to have one or more specific nucleotides as compared to the non-mutated polynucleotide. Modified polynucleotides containing nucleotide substitutions, deletions, or insertions can be generated. Examples of techniques for doing this are described in Walder et al., 1986, Gene 42:1 33;Bauer et al.,1985,Gene 37:73;Craik,19 85,BioTechniques,3:12-19;Smith et al.,19 81,Genetic Engineering:Principles and Me Methods, Plenum Press; and U.S. Pat. Nos. 4,518,584 and These and other methods are described in U.S. Patent No. 4,737,462. Compared to the antibody, the desired properties, such as affinity for GIPR, avidity (a improved vidity, or specificity, or in vivo or in vitro stability, or It can be used to generate derivatives of, for example, GIPR antibodies that have reduced in vivo side effects. do.

[0130] Other derivatives of GIPR antibodies within the scope of the present invention include derivatives of the anti-GIPR antibody polypeptide, such as or expressed or recombinant fusion proteins containing a heterologous polypeptide fused to the C-terminus. Covalent or non-covalent binding of anti-GIPR antibodies with other proteins or polypeptides, etc. The conjugated peptide may comprise a heterologous aggregating conjugate, or a fragment thereof. A signal (or leader) polypeptide, such as the yeast α-factor leader or epitope The antibody containing the fusion protein may be an antigen-binding protein, such as a peptide tag. It may contain peptides added to facilitate purification or identification (e.g., poly-His). Antibodies are also described by Hopp et al., 1988, Bio / Technology 6:1204 and U.S. Pat. No. 5,011,912, The FLAG peptide is highly antigenic and can be linked to a specific monoclonal antibody. The recombinantly expressed mAb provides an epitope that is reversibly bound by a mAb. This allows for rapid assay and easy purification of proteins. Reagents useful for preparing fusion proteins fused to a polypeptide are commercially available (S In another embodiment, one or more antibodies Oligomers containing the covalently linked GIPR polypeptide can be used as GIPR antagonists. Covalently or non-covalently linked dimers, trimers, or higher order oligomers. The use of oligomers comprising two or more antibodies is contemplated, an example of which is It is a homodimer. Other oligomers include heterodimers, homotrimers, and heterotrimers. , homotetramers, heterotetramers, etc.

[0131] One embodiment involves covalent or non-covalent interactions between peptide moieties fused to an antibody. The present invention relates to an oligomer comprising multiple antibodies linked via a peptide bond. peptide linkers (spacers), or peptides with properties that promote oligomerization. Certain polypeptides derived from leucine zippers and antibodies are described in more detail below. As described, it is a peptide that can promote oligomerization of antibodies bound to it. .

[0132] In certain embodiments, the oligomer comprises two to four antibodies. , in any of the forms described above, e.g., mutants or fragments, Preferably, the oligomer comprises an antibody that exhibits GIPR binding activity.

[0133] In one embodiment, the oligomer is prepared using polypeptides derived from immunoglobulins. The antibodies are prepared by fusion with various portions of antibody-derived polypeptides (including the Fc domain). The preparation of fusion proteins containing certain heterologous polypeptides is possible, for example, by the use of Ashkenazi e t al.,1991,PNAS USA 88:10535;Byrn et al. , 1990, Nature 344:677; and Hollenbaugh et al. .,1992“Construction of Immunoglobulin Fu sion Proteins”,in Current Protocols in I mmunology,Suppl.4,pages 10.19.1-10.19.11 One embodiment provided herein is an anti-GIPR antibody Two GIPR-binding fragments were generated by fusing them to the Fc region of an antibody. The dimer may be, for example, a dimer encoding a fusion protein. The gene fusion is inserted into an appropriate expression vector and transformed with the recombinant expression vector. The gene fusion is expressed in the selected host cell, and an antibody that mimics the expressed fusion protein is produced. The molecules can be assembled together, immediately after which the interchain disulfide bonds are formed. Formed between the Fc portions results in a dimer.

[0134] As used herein, the term "Fc polypeptide" refers to the Fc region of an antibody. This includes native and mutein forms of polypeptides derived from Also included are truncated forms of such polypeptides containing the Fc portion (and its associated fragments). The fusion protein containing the oligomer formed from Protein A or Protein G It offers the advantage of easy purification by affinity chromatography on ram.

[0135] PCT Application WO 93 / 10151 (incorporated herein by reference) One suitable Fc polypeptide described in (see below) is a human IgG1 antibody N-terminal hinge fragment. Another useful Fc polypeptide is a single-chain polypeptide stretching from the Fc region to the native C-terminus of the Fc region. The peptides are described in U.S. Pat. No. 5,457,035 and Baum et al., 1999 4, EMBO J. 13:3992-4001. The amino acid sequence of this mutant protein is a change from Leu to Ala at amino acid 19. Amino acid 20 has been changed from Leu to Glu, and amino acid 22 has been changed to Gly WO 93 / 10151 except that The mutein is identical to that of the native Fc sequence shown in Figure 1. The mutein has a specific binding affinity to the Fc receptor. In other embodiments, the heavy and / or light chains of the anti-GIPR antibody exhibit reduced affinity. The variable portions of the antibody may be substituted for the variable portions of the antibody heavy and / or light chains.

[0136] Alternatively, the oligomer may be a nucleotide sequence with or without a peptide linker (spacer peptide). A fusion protein containing multiple antibodies. Suitable peptide linkers include those described in U.S. Pat. Nos. 4,751,180 and 4,935,233. .

[0137] Another method for preparing oligomeric antibodies involves the use of leucine zippers. Zipper domains are peptides that promote oligomerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins. (Landschulz et al., 1988, Science 240:175 9), and has since been found in a variety of different proteins. Among the pers are natural peptides and their derivatives that dimerize or trimerize. Examples of leucine zipper domains suitable for producing mer proteins are described in PCT application WO 02 / 04999. Pulmonary surfactant protein D (SPD) is described in Publication No. 94 / 10308. ) is a leucine zipper derived from Hoppe et al., 1994, FEBS L Etc. 344:191, which is incorporated herein by reference. Use of a modified leucine zipper that allows stable trimerization of heterologous proteins fused to it However, Fanslow et al., 1994, Semin. Immunol. 6:26 In one method, an anti-GI antibody fused to a leucine zipper peptide is used. The recombinant fusion protein containing the PR antibody fragment or derivative is then grown in a suitable host cell. soluble oligomeric anti-GIPR antibody fragments or derivatives expressed and formed using It is recovered from the culture supernatant.

[0138] In another embodiment, the antibody derivative comprises at least one of the CDRs disclosed herein. For example, one or more CDRs may comprise a known antibody framework region (e.g., IgG1, IgG2, etc.) or a suitable vehicle to increase its half-life. Suitable vehicles include, but are not limited to, Fc, albumin, and thiamin. These and other suitable vehicles are known in the art. Such conjugated CDR peptides may be in the form of a monomer, dimer, tetramer, or In one embodiment, the one or more water-soluble polymers may be one or The binding agent is attached at multiple specific positions, for example, at the amino terminus of the binding agent. Conductors include, but are not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. For example, U.S. Pat. No. 4,640,835, U.S. Pat. No. 4,496,689, U.S. Pat. No. 4,301,144, U.S. Pat. No. 4,670,417, U.S. Pat. No. 4,7911 See U.S. Pat. No. 4,179,337 and U.S. Pat. No. 4,179,337. In the present invention, the derivatives are monomethoxy-polyethylene glycol, dextran, cellulose , or other carbohydrate-based polymers, poly-(N-vinylpyrrolidone)-polyethylene Glycol, Propylene Glycol Homopolymer, Polypropylene Oxide / Ethylene Oxide oxide copolymers, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohols The polymers include one or more of the following: vinyl alcohols, as well as mixtures of such polymers. In this embodiment, the one or more water-soluble polymers have one or more side chains randomly linked thereto. In certain embodiments, PEG enhances the therapeutic potential of a binding agent, such as an antibody. Several such methods may be used for any purpose, e.g. No. 6,133,426, which is incorporated herein by reference. do.

[0139] As long as the antibody retains its binding specificity, it is possible for the antibodies provided herein to bind to at least one It will be understood that modifications to the antibody structure may have up to one amino acid substitution. These are included within the scope of the invention. Conservative amino acid substitutions may be either conservative or non-conservative. Conservative amino acid substitutions are used to identify the amino acid sequence of interest in biological systems. Unnatural amino acid residues that are typically incorporated by chemical peptide synthesis rather than by conventional synthesis are also included. This includes peptidomimetics and other reverse or inverted forms of amino acid moieties. Conservative amino acid substitutions may also be made if they affect the polarity or charge of the amino acid residue at that position. It may involve substitution of natural amino acid residues with standard residues so that there is little or no substitution. Conservative substitutions replace members of a class of amino acids or amino acid mimetics with amino acids that have different physical characteristics. exchange with a member from another class that has similar properties (e.g., size, polarity, hydrophobicity, charge) This may include:

[0140] Moreover, one skilled in the art can easily create tested polypeptides containing single amino acid substitutions at each desired amino acid residue. The mutants can then be screened using activity assays known to those skilled in the art. Such variants can be used to gather information about suitable variants. For example, changes to specific amino acid residues may disrupt or otherwise reduce undesirably low If it is found that such changes have resulted in reduced or inappropriate activity, In other words, the information gathered from such routine experiments Based on this, one skilled in the art can determine whether further substitutions are avoided, either alone or in combination with other mutations. The amino acid sequence that should be used can be easily determined.

[0141] Those skilled in the art will be able to identify suitable variants of the polypeptides described herein using well-known techniques. In certain embodiments, one skilled in the art can determine regions that are not important for activity. By targeting specific regions, one can identify suitable areas of the molecule that can be altered without destroying activity. In certain embodiments, residues and conserved molecules among similar polypeptides are obtained. In certain embodiments, moieties can be identified that are useful for biological activity or structure. Potentially important regions may be modified without destroying biological activity or adversely affecting polypeptide structure. Furthermore, those skilled in the art will recognize that substitutions of amino acids that are important for activity or structure may be made conservatively without adversely affecting the overall activity of the polypeptide. Consider structure-function studies that identify residues in similar polypeptides that are essential for In light of such comparisons, it is possible to identify proteins that are important for activity or structure in similar proteins. Predict the importance of amino acid residues in proteins, corresponding to key amino acid residues Those skilled in the art can substitute chemically such predicted important amino acid residues. Similar amino acid substitutions can be selected.

[0142] Those skilled in the art will recognize the three-dimensional structure and amino acid sequence associated with the corresponding structure in similar polypeptides. In view of such information, one skilled in the art can determine its three-dimensional structure. In certain embodiments, the alignment of amino acid residues of the antibody can be predicted with respect to the Vendors should not make radical changes to amino acid residues predicted to be on the surface of the protein. residues may be selected to be low in the nucleotide sequence because such residues are likely to be involved in important interactions with other molecules. Several scientific publications are devoted to the prediction of secondary structure. oult,1996,Curr.Op.Biotech.7:422-427;Chou et al.,1974,Biochemistry 13:222-245;Cho u et al.,1974,Biochemistry 113:211-222;C hou 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., Biop See Phys. J. 26:367-384. Computer programs are now available that assist in the detection of sequences with greater than 30% sequence identity. Two polypeptides or proteins that share a similarity of 40% or more have similar structural topologies. The recent growth of the Protein Structure Database (PDB) has Improved secondary structure, including the potential number of folds within the structure of a polypeptide or protein. Provided predictability. Holm et al., 1999, Nucl. Acid. Re See s.27:244-247. There are a critical number of folds, and once a critical number of structures have been determined, structure prediction becomes remarkably accurate. It has been suggested that this may be the case (Brenner et al., 1997, Curr. O p.Struct.Biol.7:369-376).

[0143] An additional method for predicting secondary structure is "threading." (Jones, 1997, Curr. Opin. Structure. Biol. 7:377 -87;Sippl et al.,1996,Structure 4:15-19) , "Profile Analysis" (Bowie et al., 1991, Science 25 3:164-170;Gribskov et al.,1990,Meth.Enzy m.183:146-159;Gribskov et al.,1987,Proc. Nat. Acad. Sci. USA 84:4355-4358), and "Evolutionary Relationships (evolutionary linkage)" (Holm, op. cit. (1999), and See Brenner, supra (1997). In certain embodiments, Variants of the glycosylation site include glycosylation variants, where the number and / or type of glycosylation site is varied. In certain embodiments, the amino acid sequence of the parent polypeptide is altered compared to the amino acid sequence of the parent polypeptide. In some embodiments, the variant may contain more or fewer N-linked glycosylation sites than the native protein. Alternatively, removal of such sequences by substitution may result in removal of any N-linked carbohydrate chains present. Rearrangements of N-linked carbohydrate chains are also provided, wherein one or more N-linked glycosyl groups The cleavage site (typically a naturally occurring one) is removed and one or more new N-linkages are introduced. Further preferred antibody variants include cysteine ​​variants, wherein: One or more cysteine ​​residues are deleted or altered compared to the parent amino acid sequence. Cysteine ​​variants are substituted with amino acids such as serine, which can be used to make antibodies more soluble, e.g., insoluble. After isolation of inclusion bodies, they must be refolded into a biologically active conformation. Cysteine ​​variants generally have fewer cysteine ​​residues than the native protein. groups, typically an even number to minimize interactions resulting from unpaired cysteines. Has.

[0144] Desired amino acid substitutions (whether conservative or non-conservative) are made in the presence of a nucleotide sequence in which such substitutions are desired. This can be determined by one of skill in the art when desired. In certain embodiments, amino acid substitutions are to identify important residues of antibodies against human GIPR, or to identify the human GIPR-specific antibodies described herein. These may be used to increase or decrease the affinity of an antibody for a target GIPR.

[0145] According to certain embodiments, preferred amino acid substitutions are those that (1) reduce susceptibility to proteolysis. (2) reduce susceptibility to oxidation; and (3) form protein complexes. (4) modifying the binding affinity so that the or modulate other physicochemical or functional properties in the polypeptide. According to certain embodiments, single or multiple amino acid substitutions (in certain embodiments, Conservative amino acid substitutions) may be made in the native sequence (in certain embodiments, molecular indirect substitutions). In certain embodiments, Conservative amino acid substitutions typically can substantially alter the structural characteristics of the parent sequence. (e.g., the replacement amino acid should not disrupt a helix present in the parent sequence, or (It should not disrupt other types of secondary structure that characterize the parent sequence.) Examples of recognized polypeptide secondary and tertiary structures are listed in Proteins, Structures, and Protein Chains. tures and Molecular Principles (Creighton ,Ed.,WH Freeman and Company,New York(19 84));Introduction to Protein Structure(B randen and Tooze,Eds.,Garland Publishing , New York, NY (1991)); and Thornton et al., 1991, Nature 354:105, each of which is incorporated by reference. The present application is incorporated by reference.

[0146] In certain embodiments, the antibodies of the invention are chemically conjugated to polymers, lipids, or other moieties. It can be done.

[0147] The antigen binding agent may be a C12- or C22-based antibody described herein that is incorporated into a biocompatible framework structure. In one embodiment, the biocompatible framework structure may include at least one of: The amino acid sequence is a sequence of amino acids that bind to antigens in localized surface regions (e.g., CDRs, variable regions, etc.). A conformationally stable structural support capable of presenting one or more sequences of amino acids. or a polypeptide or portion thereof sufficient to form a framework or scaffold. Such structures are called natural polypeptides or polypeptide "folds" (structural motifs). or may have amino acid additions, deletions or fold changes compared to the native polypeptide or fold. These scaffolds may have one or more modifications, such as substitutions, that are useful in humans, other mammals, other Any species of vertebrate, invertebrate, plant, bacterium or virus (or two or more species) ) polypeptide.

[0148] Typically, biocompatible framework structures are used to bind proteins other than immunoglobulin domains. These are based on a matrix scaffold or skeleton, e.g., fibronectin, ankyrin, lipocalin, Neocarzinostatin, cytochrome b, CP1 zinc finger, PST1, coiled cofactor Those based on Il, LACI-D1, Z domain and tendamistat domain were used. (e.g., Nygren and Uhlen, 1997, Current Op See inion in Structural Biology 7:463-469 ).

[0149] Furthermore, those skilled in the art will appreciate that suitable binding agents may be selected from the heavy chain CDR1, CDR2, CDR3, CDR4, CDR5, CDR6, CDR7, CDR8, CDR9, CDR10, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19, CDR19, CDR11, CDR12, CDR13, C one or more of: light chain CDR1, CDR2, CDR3, light chain CDR1, CDR2 and CDR3; It will be appreciated that these antibody portions include heavy chain CDR1, CDR2, CDR3 At least one of the light chain CDR1, CDR2 and CDR3 regions is an unsubstituted CDR The antibody may have at least one amino acid substitution as long as it retains the binding specificity of the antibody. The R moiety can be a non-protein molecule, where the binding agent binds to the human GIPR as described herein. and / or inhibit GIP signaling via the receptor. The non-CDR portion of an antibody can be a non-protein molecule, where the antibody The binding of antibodies L2H2 / L6H5 to a human GIPR peptide in a competitive binding assay exhibit a binding pattern similar to that exhibited by at least one of the The non-CDR portions of the antibody may be composed of amino acids, where the antibody is a recombinant binding protein or synthetic peptide, the recombinant binding protein being human GIPR cross-blocking and / or in vitro or in vivo binding of the antibodies disclosed herein to The non-CDR portion of the antibody may be composed of amino acids. wherein the antibody is a recombinant antibody, and the recombinant antibody is capable of binding to human IgG in a competitive binding assay. The antibody L2H2 / L6H5 is directed against the GIPR peptide. and / or neutralize the activity of GIP.

[0150] Fusion proteins of GIPR antibodies and GLP-1 or reverse GLP-1 In one embodiment, an antibody that specifically binds to GIPR, and , 7, or 8 GLP-1 fragments or reverse GLP-1 fragments, A fusion protein of R antibody and GLP-1, wherein the fusion protein comprises a peptide linker The carboxy terminus of the GLP-1 fragment is linked to the GIPR antibody via a linker sequence. or the amino terminus of the reverse GLP-1 fragment. fusion protein, which links the carboxy terminus of the light or heavy chain of a GIPR antibody is provided herein.

[0151] In another embodiment, an antibody that specifically binds to GIPR, and 1, 2, 3, 4, 5, Fusions of GIPR antibodies and GLP-1 containing 6, 7, or 8 GLP-1 fragments a protein; and the fusion protein is formed via a peptide linker sequence (linker): The carboxy terminus of the GLP-1 fragment is linked to the amino terminus of the GIPR antibody light or heavy chain. Fusion proteins that bind to the termini are provided herein.

[0152] In another embodiment, an antibody that specifically binds to GIPR, and 1, 2, 3, 4, 5, Fusion of GIPR antibody and GLP-1 containing 6, 7, or 8 reverse GLP-1 fragments The fusion protein is a fusion protein comprising a peptide linker sequence (linker) The amino terminus of the reverse GLP-1 fragment is linked to the carboxyl group of the light or heavy chain of the GIPR antibody. Fusion proteins are provided herein that are linked to the cis-terminus.

[0153] In another embodiment, an antibody that specifically binds to GIPR and one, two, three, or four It was a fusion protein of a GIPR antibody and GLP-1 containing a GLP-1 fragment of The fusion protein is linked to the GLP-1 flag through a peptide linker sequence (linker). fusion proteins that link the carboxy terminus of the GIPR antibody to the amino terminus of the GIPR antibody light or heavy chain. Synthetic proteins are provided herein.

[0154] In another embodiment, an antibody that specifically binds to GIPR and one, two, three, or four A fusion protein of a GIPR antibody and GLP-1, comprising a reverse GLP-1 fragment of The fusion protein is a reverse GLP-1 fusion protein linked to a peptide linker sequence (linker). The amino terminus of the fragment is linked to the carboxy terminus of the GIPR antibody light or heavy chain. , fusion proteins are provided herein.

[0155] In another embodiment, an antibody that specifically binds to GIPR and two GLP-1 fragments is provided. A fusion protein of a GIPR antibody and GLP-1, comprising a fragment thereof; the fusion protein The molecule is linked to the carboxyl group of the GLP-1 fragment via a peptide linker sequence (linker). a fusion protein linking the carboxy terminus of a GIPR antibody to the amino terminus of a light or heavy chain of the antibody, Provided herein.

[0156] In another embodiment, an antibody that specifically binds to GIPR and two reverse GLP-1 fragments is provided. A fusion protein of a GIPR antibody and GLP-1, comprising a fragment thereof; the fusion protein The protein is linked to the amino acid sequence of the reverse GLP-1 fragment via a peptide linker sequence (linker). fusion proteins linking the carboxy terminus of a GIPR antibody light chain or heavy chain to the carboxy terminus of the GIPR antibody light chain or heavy chain is provided herein.

[0157] In another embodiment, a GIPR antibody and a GLP-1 antibody comprising two GLP-1 fragments are -1 fusion protein; the fusion protein comprises a peptide linker sequence (linker). The carboxy terminus of the GLP-1 fragment is connected to the amino terminus of the GIPR antibody light chain via Linked: N'-GLP-1-linker-R-C'; or GLP-1 fragment carbohydrate The hydroxy terminus of the GIPR antibody heavy chain is linked to the amino terminus of the antibody heavy chain: N'-GLP-1-Linker-R -C'; where N' represents the amino terminus of the polypeptide chain of the fusion protein and C' represents represents the carboxy terminus of the polypeptide chain of the fusion protein, and GLP-1 R represents the amino acid sequence of the light chain or heavy chain of the GIPR antibody, and Provided herein are fusion proteins in which - represents a peptide linker sequence.

[0158] In another embodiment, a GIPR antibody and a GLP-1 antibody comprising two reverse GLP-1 fragments are a GLP-1 fusion protein; wherein the fusion protein is an amino acid sequence of a reverse GLP-1 fragment; The N'-R-linker-reverse GLP- 1-C'; or a reverse GLP-1 fragment via a peptide linker sequence (linker). to the carboxy terminus of the GIPR antibody heavy chain: N'-R-linker-reverse GLP-1-C'; where N' represents the amino terminus of the polypeptide chain of the fusion protein. and C' represents the carboxy terminus of the polypeptide chain of the fusion protein, and reverse GLP-1 is , represents a reverse GLP-1 fragment, and R is the amino acid sequence of the light or heavy chain of the GIPR antibody. and the linker represents a peptide linker sequence. It is provided.

[0159] In a further embodiment, a GLP-1 antibody comprising a GIPR antibody and two GLP-1 fragments. LP-1 fusion protein; wherein the fusion protein comprises a peptide linker sequence (linker ) to connect the carboxy terminus of the GLP-1 fragment to the amino terminus of the GIPR antibody light chain. Attached to the end: N'-GLP-1-linker-R-C'; where: N' is the fusion protein C' represents the amino terminus of the polypeptide chain of the fusion protein, represents the carboxy terminus, GLP-1 represents a GLP-1 fragment, and R represents a GIPR antibody. a fusion protein, wherein the amino acid sequence of the light chain is represented by the linker, and the linker is a peptide linker sequence. is provided herein.

[0160] In one embodiment, in the GLP-1 fusion protein provided herein , the GLP-1 fragments independently have the following amino acid sequences: SEQ ID NO: 105, SEQ ID NO: No. 106, SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109. In one embodiment, the GLP-1 fusion proteins provided herein wherein the reverse GLP-1 fragment independently has the following amino acid sequence: SEQ ID NO: 119 , SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123 is selected from.

[0161] In one embodiment, in the GLP-1 fusion protein provided herein , the peptide linker (linker) sequence is independently 1 to 200 amino acid residues, 2 to 1 00 amino acid residues, 5 to 50 amino acid residues, 6 to 25 amino acid residues, or 10 to 2 Contains 0 amino acid residues.

[0162] In another embodiment, in the GLP-1 fusion proteins provided herein and wherein the peptide linker (linker) sequences are independently selected from the following amino acid sequences: SEQ ID NO:1 10, SEQ ID NO:111, and SEQ ID NO:112.

[0163] nucleic acid In one aspect, the present invention provides an isolated nucleic acid molecule encoding an antibody provided herein. The nucleic acid may be, for example, all or part of an antibody or a GLP-1 fusion protein, e.g. For example, an antibody of the invention, or a fragment, derivative, mutein, or variant thereof Polynucleotides encoding one or both strands of the variant; hybridization probes a polynucleotide sufficient to be used as a primer; a polynucleotide encoding a polypeptide; PCR primers or sequences for identifying, analyzing, mutating, or amplifying peptides an antisense primer; an antisense nucleic acid for inhibiting expression of the polynucleotide; and The nucleic acid may be any length. 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 15 0, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1, 1,000, 1,500, 3,000, 5,000 or more nucleotides in length, and / or may contain one or more additional sequences, e.g., regulatory sequences, and / or may contain larger The nucleic acid may be a large nucleic acid, e.g., part of a vector. The nucleic acid may be single-stranded or double-stranded, Nucleotides containing NA and / or DNA, and artificial variants thereof (e.g., peptide nucleic acids) It can be seen.

[0164] Encoding an antibody polypeptide (e.g., heavy or light chain, variable domain only, or full length) Nucleic acids encoding the antibodies can be isolated from B cells of mice immunized with the GIPR antigen. Nucleic acids of GLP-1 fusion proteins can be isolated using conventional procedures such as polymerase chain reaction (PCR). It can be isolated by

[0165] Nucleic acid sequences encoding the heavy and light chain variable regions are shown above. Those skilled in the art will appreciate that the genetic code Due to degeneracy, each of the polypeptide sequences disclosed herein can be synthesized from a large number of other nucleic acid sequences. It will be understood that the invention is encoded by the Each degenerate nucleotide sequence encoding each antibody or GLP-1 fusion protein is provided.

[0166] The present invention relates to the hybridization of L2H2 and L6 with other nucleic acids (e.g., L2H2 / L6) under specific hybridization conditions. Further provided is a nucleic acid that hybridizes to a nucleic acid comprising any one of the nucleotide sequences of H5. Methods for hybridizing nucleic acids are well known in the art. For example, Current Protocols in Molecular Biology y, John Wiley & Sons, NY (1989), 6.3.1-6.3 See .6. As defined herein, for example, moderately stringent The optimal hybridization conditions were 5x sodium chloride / sodium citrate (S SC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), approximately 50% formaldehyde Hybridization buffer containing 6x SSC, prewashing g) solution, and a hybridization temperature of 55°C (or 42°C). Other similar hybridization media, such as those containing about 50% formamide at the desired temperature, Washing conditions were as follows: 0.5x SSC, 0.1% SDS, and 60°C. Stringent hybridization conditions are 45°C in 6xSSC. The mixture is then resuspended in 0.1xSSC, 0.2% SDS at 68°C and washed one more time. Furthermore, those skilled in the art will appreciate that the ratio of each other is at least 65, 70, 75, 80, 85, 90, 95, 98 Nucleic acids containing nucleotide sequences that are 99% or more identical typically hybridize to each other. Increasing or decreasing the stringency of hybridization so that the hybridization remains Hybridization and / or washing conditions can be manipulated to allow for Developing the fundamental parameters that influence the selection of hybridization conditions and the optimal conditions Guidelines for this purpose are given, for example, in Sambrook, Fritsch, and Maniat is(1989,Molecular Cloning:A Laboratory M anual,Cold Spring Harbor Laboratory Pres. s,Cold Spring Harbor,NY,chapters 9 and 11; and Current Protocols in Molecular Bio John Wiley, 1995, Ausubel et al., Eds. & Sons, Inc., sections 2.10 and 6.3-6.4) and are readily apparent to those skilled in the art based on, for example, the length and / or base composition of the DNA. Changes can be introduced into a nucleic acid by mutation, thereby This results in a change in the amino acid sequence of the polypeptide (e.g., antibody) that it encodes. Mutations can be introduced using any technique known in the art. In the method, one or more specific amino acid residues are mutated, for example, by a site-directed mutagenesis process. In another embodiment, one or more randomly selected The selected residues are then varied, for example, using random mutagenesis protocols. Once generated, the mutant polypeptides are expressed and screened for desired properties. It can be done.

[0167] A mutation may occur in the nucleus without significantly altering the biological activity of the polypeptide it encodes. For example, nucleotide substitutions can be made to introduce amino acids at non-essential amino acid residues. In one embodiment, L1 to L11 and H1 to H9 or the nucleotide sequence provided herein for the GLP-1 fusion protein or a fragment, variant, or derivative thereof, is intended to mean that they are composed of two or more different amino acids. The amino acid sequence may include one or more deletions or substitutions of amino acid residues to produce a sequence having the desired amino acid residues. The amino acid sequences provided herein for L1 to L11 and H1 to H9 are In another embodiment, the mutagenesis is carried out by mutating two or more mutants. L1 to L11 and H1 to H9 or GLP so as to produce a sequence having the amino acid residues adjacent to one or more amino acid residues set forth herein for the -1 fusion protein. Alternatively, one or more mutations can be introduced into the nucleic acid, which However, the biological activity of the polypeptide it encodes (e.g., binding to GIPR) is selected. For example, a mutation may quantitatively or qualitatively alter a biological activity. Examples of quantitative changes include increasing, decreasing, or eliminating activity. Examples include altering the antigen specificity of an antibody or GLP-1 fusion protein.

[0168] In another aspect, the present invention provides a primer or hybrid for the detection of a nucleic acid sequence of the present invention. The present invention provides nucleic acid molecules suitable for use as DNA synthesis probes. The molecule may contain only a portion of a nucleic acid sequence encoding a full-length polypeptide of the invention, e.g., a polypeptide fragment. A fragment that can be used as a probe or primer or a polypeptide of the present invention It may include a fragment encoding a portion (e.g., a GIPR-binding portion).

[0169] Probes based on the sequences of the nucleic acids of the invention can be used to identify nucleic acids or similar nucleic acids, e.g. The probe can be used to detect transcripts encoding polypeptides of the present invention. For example, the probe may include a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. The probe can be used to identify cells that express the polypeptide.

[0170] In another aspect, the vectors provided herein are capable of expressing the polypeptides or Examples of vectors include, but are not limited to, vectors containing nucleic acids encoding portions thereof. vectors, viral vectors, non-episomal mammalian vectors and expression vectors, e.g., recombinant vectors, Examples include recombinant expression vectors.

[0171] The recombinant expression vectors provided herein are used to express nucleic acids in host cells. The recombinant expression vector may contain the nucleic acid of the invention in any suitable form. operably linked, one or more selected based on the host cell to be used for expression Regulatory sequences are used to regulate the organization of nucleotide sequences in many types of host cells. those directing specific expression (e.g., SV40 early gene enhancer, Rous sarcoma virus promoter and cytomegalovirus promoter), which are nucleic acids that are expressed only in specific host cells. Those that direct expression of the nucleotide sequence (e.g., tissue-specific regulatory sequences, Voss et al. .,1986,Trends Biochem.Sci.11:287,Maniati See S. et al., 1987, Science 236:1237. the disclosures of which are incorporated herein by reference in their entireties), and specific processes or conditions. those that direct inducible expression of a nucleotide sequence in response to a given gene (e.g., in mammalian cells) metallothionein promoters, and in both prokaryotic and eukaryotic cell systems. It contains a tet-responsive and / or streptomycin-responsive promoter (see ibid.). The design of the expression vector will depend on the choice of host cell to be transformed, the level of expression of the desired protein, and other factors. It will be understood by those skilled in the art that the expression vectors of the present invention may depend on factors such as the type of gene that can be expressed. The vector is introduced into a host cell, thereby producing a vector encoded by the nucleic acid described herein. Proteins or peptides, including fusion proteins or peptides, can be produced.

[0172] In another aspect, the present invention provides a host cell into which a recombinant expression vector of the present invention has been introduced. The host cell can be any prokaryotic or eukaryotic cell. The cells may be Gram-negative or Gram-positive bacteria, such as E. coli or Bacillus sp. Higher eukaryotic cells include insect cells, yeast cells, and mammalian cells. Examples of suitable mammalian host cell lines include established cell lines derived from Chinese hamster ovary (CHA). CHO cells or their derivatives such as Veggie CHO and grown in serum-free medium related cell lines (Rasmussen et al., 1998, Cytotec 28:31) or the DHFR-deficient CHO strain DXB-11 (U rlaub et al.,1980,Proc.Natl.Acad.Sci.USA 77:4216-20). Additional CHO cell lines include CHO-K1 (AT CC#CCL-61), EM9(ATCC# CRL-1861), and W20(ATC C# CRL-1862). Further host cells include the COS-7 line of monkey kidney cells ( ATCC# CRL-1651)(Gluzman et al.,1981,Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL-16 3), AM-1 / D cells (described in U.S. Pat. No. 6,210,924), HeLa Cells, BHK (ATCC CRL-10) cell line, African green monkey kidney cell line CV1 The CV1 / EBNA cell line (ATCC CCL-70) derived from (McMahan et al. al., 1991, EMBO J. 10:2821), 293, 293 EBN Human embryonic kidney cells such as A or MSR 293, human epidermal A431 cells, human Colo20 5 cells, other transformed primate cell lines, normal diploid cells, primary tissues, primary transplants Cell lines derived from in vitro culture, such as HL-60, U937, HaK, or Jurkat cells Suitable clones for use with bacterial, fungal, yeast, and mammalian cell hosts. Cloning and expression vectors were prepared as described in Pouwels et al. ors:A Laboratory Manual,Elsevier,NY,19 85).

[0173] The vector DNA is introduced into prokaryotes by conventional transformation or transfection techniques. or eukaryotic cells. For stable transfection of mammalian cells , depending on the expression vector and transfection technique used, only a small fraction of cells However, it is known that these components can integrate foreign DNA into their genome. Selectable markers (e.g., for resistance to antibiotics) can be used to identify and select the cells. A gene encoding the target gene is generally introduced into the host cell along with the gene of interest. New selectable markers include drugs such as G418, hygromycin, and methotrexate. The introduced nucleic acid is stably transfected, including those that confer resistance to drugs. Cells can be identified by drug selection (e.g., by using selectable markers), among other methods. Cells that have integrated the marker gene will survive, while other cells will die.

[0174] The transformed cells may be cultured under conditions that promote expression of the polypeptide, The peptide is recovered by conventional protein purification procedures. One such purification procedure is Polypeptides contemplated for use herein are described in the Examples below. The method comprises preparing a substantially homogeneous recombinant mammalian GIPR antibody that is substantially free of contaminating endogenous substances. The present invention also includes a GLP-1 fusion protein polypeptide.

[0175] GIPR antibody activity The activity of GIPR antibodies is to specifically bind to GIPR and inhibit or block GIP signaling. and subsequently exhibiting a therapeutic biological effect, e.g., in the treatment of obesity, T2DM and / or non-alcoholic Efficacy of the antibodies provided herein in treating non-alcoholic steatohepatitis (NASH) "Decreasing the biological activity of GIP signaling" or "Decreasing the biological activity of GIP signaling" The term "inhibits or blocks the biological activity of" refers to binding to GIPR in vivo. and the role of GIPR antibodies or their derivatives in inhibiting or blocking downstream cellular responses to GIP. These responses include, but are not limited to, These include insulin secretion promoting action, promoting fat accumulation, and inhibiting lipolysis. In some embodiments, the murine or humanized antibodies provided herein are directed against human GIPR. Such antibodies specifically bind to or inhibit GIP signaling. Includes neutralizing antibodies.

[0176] In one embodiment, the K of an antibody provided herein that binds to human GIPR d is approximately 0.01nM to 1000nM, 0.1nM to 500nM, 0.5nM to 200nM , 1 nM to 200 nM, or 10 nM to 100 nM. , the K of the antibodies provided herein that bind to GIPR d is approximately 1nM to 200nM In yet another embodiment, the GIPR-binding protein is Antibody K d In yet another embodiment, the concentration of GIPR is about 1 nM to 100 nM. The K of the antibodies provided herein that bind d is approximately 1nM, 2nM, 5nM, 10n M, 20nM, 30nM, 40nM, 50nM, 60nM, 70nM, 80nM, 90n M, or 100 nM.

[0177] In one embodiment, the antibodies provided herein antagonize GIP signaling. IC 50 is approximately 0.01nM to 500nM, 0.1nM to 200nM, 0.5nM to 2 In another embodiment, the concentration is 00 nM, 1 nM to 200 nM, or 10 nM to 100 nM. and the IC of the antibodies provided herein that antagonize GIP signaling. 50 is about 1 In yet another embodiment, the GIP signaling antagonism is IC of the antibodies provided herein 50 is about 10 nM to 100 nM. In another embodiment, the antibodies provided herein antagonize GIP signaling. IC 50 is approximately 1nM, 2nM, 5nM, 10nM, 20nM, 30nM, 40nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM.

[0178] In one embodiment, the antibodies provided herein specifically bind to GIPR. , having one or more of the following characteristics: a. provides a substantially similar Kd as a reference antibody when binding to GIPR; b. Substantially similar I as the reference antibody in antagonizing GIP-activated GIPR providing C50; and c. Cross-compete for binding with a reference antibody against human GIPR.

[0179] In another embodiment, the antibodies provided herein have one or more of the following characteristics: It is an antibody with the properties: a. Provides the same or better Kd as a reference antibody when binding to GIPR ; b. Equivalent or superior to the reference antibody in antagonizing GIP-activated GIPR provide a better IC50; and c. Cross-compete for binding with a reference antibody against human GIPR.

[0180] In one embodiment, the reference antibody has the light chain variable domain amino acid sequence SEQ ID NO: 66 and the heavy chain variable domain amino acid sequence SEQ ID NO: 67. The combination of chain variable domain amino acid sequences SEQ ID NO: 76.

[0181] In one embodiment, the reference antibody comprises the light chain variable domain amino acid sequence SEQ ID NO: 68 and the heavy chain variable domain amino acid sequence SEQ ID NO: 69. The combination of chain variable domain amino acid sequences SEQ ID NO: 77.

[0182] In another embodiment, the reference antibody is monoclonal antibody L2H2, L6H5, or L It is 10H8.

[0183] As used herein, the term "substantially similar" refers to a reference antibody's IC 50 Or K d or approximately 200%, 180%, 160%, 150%, 1 40%, 120%, 110%, 100%, 99%, 98%, 97%, 95%, 90%, 8 This means that the percentage is 5%, 80%, 75%, 70%, 65%, or 50%. In one embodiment, the reference antibody may have, for example, a combination of heavy chain SEQ ID NO: 76 and light chain SEQ ID NO: 66. In another embodiment, the reference antibody is an antibody comprising the GIPR antibody L2H2, L6H5 , or L10H8.

[0184] Biological activity of fusion protein of GIPR antibody and GLP-1 The biological activity of the fusion protein of GIPR antibody and GLP-1 is similar to the biological activity of GLP-1. The activity of the GIPR antibody is as described above. "Biological activity of GLP-1" refers to the ability of a substance to bind to and activate the GLP-1 receptor in vivo. These compounds induce intracellular signaling responses, contributing to obesity, type 2 diabetes, and non-alcoholic lipid Biology of fusion proteins of GIPR antibody and GLP-1, which have therapeutic effects on fatty liver disease Signaling responses include, but are not limited to, increased insulin secretion, increased glucagon secretion, and increased insulin secretion. Suppression of gonorrhea secretion, suppression of appetite, weight loss, induction of satiety, inhibition of apoptosis, pancreatic beta cells The biological activities of GLP-1 and GIPR antibodies were combined, including induction of proliferation and differentiation of pancreatic beta cells. In combination, the GLP-1 fusion proteins provided herein can be used to Various diseases and disorders associated with 1R and GIPR can be treated. The substance exerts its biological effect by acting on GLP-1R and / or GIPR. To address this issue, the GLP-1 fusion protein treatments provided herein can be used to treat diseases or Symptoms may be due to "increased GLP-1R signaling" or "decreased GIPR signaling" Subjects who would benefit can be treated. These subjects are referred to as "GLP-1R stimulating therapy" Subjects who are in need of a reduction in GIPR stimulation are referred to as such. Controls included non-insulin-dependent diabetes mellitus, insulin-dependent diabetes mellitus, and stroke (GLP-1R, See International Publication No. WO 00 / 16797), myocardial infarction (GLP-1R, International Publication 98 / 08531 brochure), obesity (GLP-1R, WO 98 / 08531 brochure) See Brochure No. 19698; GIPR, Furija et al., 2008, PLoS ONE 3:e3163; U.S. Patent Application Publication No. 2017 / 0275370 A 1), catabolic changes after surgery (GLP-1R, see U.S. Patent No. 6,006,7 53), functional dyspepsia and irritable bowel syndrome (GLP-1R, International Publication No. 99 / 64060), fatty liver (GIPR, U.S. Patent Application Publication No. 2017 / 0275370 A1), non-alcoholic fatty liver disease (GL P-1R, Debra et al., 2016, Hepatobiliary Sur See g Nutr 5:515-518; GIPR, U.S. Patent Application Publication No. 2017 / 0 275370 A1), non-alcoholic steatohepatitis (GLP-1, Arm See Strong et al., 2013, BMJ Open 3:e003995 ;GIPR, see U.S. Patent Application Publication No. 2017 / 0275370 A1) and subjects at risk of developing non-insulin dependent diabetes mellitus (WO 00 / 09466). (See Brochure No. 07617), subjects with impaired glucose tolerance or impaired fasting glucose, subjects with a standard body weight Also included are subjects who are approximately 25% over the reference height and weight, and subjects who have had a partial pancreatectomy.

[0185] In one embodiment, the biological activity of a GIPR antibody or its fusion protein with GLP-1 is GIPR antibody or GLP-1 fusion when activity changes inhibit GIPR in vitro Protein function is detected using a direct cAMP assay, which quantifies the activity.

[0186] Pharmaceutical Composition In one embodiment, the pharmaceutical compositions provided herein are The composition comprises a GIPR antibody and one or more pharmaceutically acceptable carriers.

[0187] In another embodiment, the pharmaceutical compositions provided herein are The fusion protein of the provided GIPR antibody and GLP-1 and one or more pharmaceutically acceptable salts thereof and an acceptable carrier.

[0188] The term "carrier" as used herein refers to a cell or mammal that is to be used. a carrier, pharmaceutical excipient, or stabilizer that is harmless to exposure thereto at the dosages and concentrations to be administered; Contains a fixative.

[0189] Treatment method In one embodiment, a method of treating, preventing, or ameliorating type 2 diabetes therapeutically comprises: administering to a subject an effective dose of a GIPR antibody provided herein or a pharmaceutical composition thereof; Provided herein are methods comprising administering

[0190] In another embodiment, the method for treating, preventing, or ameliorating nonalcoholic fatty liver disease comprises a therapeutically effective dose of a GIPR antibody provided herein, or a therapeutic agent thereof Provided herein are methods that include administering the pharmaceutical composition to a subject.

[0191] In another embodiment, the method for treating, preventing, or ameliorating nonalcoholic fatty liver disease comprises a therapeutically effective dose of a GIPR antibody provided herein and a GLP- The present invention relates to a method for administering a fusion protein of any one of the inventions described herein, or a pharmaceutical composition thereof, to a subject. provided in the document.

[0192] In another embodiment, a method for treating, preventing, or ameliorating nonalcoholic steatohepatitis comprises: a therapeutically effective dose of a GIPR antibody provided herein, or a therapeutic agent thereof Provided herein are methods that include administering the pharmaceutical composition to a subject.

[0193] In another embodiment, a method for treating, preventing, or ameliorating nonalcoholic steatohepatitis comprises: a therapeutically effective dose of a GIPR antibody provided herein and a GLP- The present invention relates to a method for administering a fusion protein of any one of the inventions described herein, or a pharmaceutical composition thereof, to a subject. provided in the document.

[0194] In another embodiment, a method of treating, preventing, or ameliorating type 2 diabetes comprises therapeutic and administering to a subject a GIPR antibody provided herein, or a pharmaceutical composition thereof, in an amount effective to treat the disease. Provided herein are methods comprising administering to the body.

[0195] In another embodiment, a method of treating, preventing, or ameliorating type 2 diabetes comprises therapeutic and a fusion protein of a GIPR antibody and GLP-1 provided herein in an amount effective for the treatment of The methods provided herein include administering to a subject a compound, a compound or a pharmaceutical combination thereof. will be done.

[0196] In another embodiment, a method for treating, preventing, or ameliorating obesity, comprising administering to a subject a therapeutically effective amount of and administering to a subject an appropriate dose of a GIPR antibody provided herein, or a pharmaceutical composition thereof. Provided herein are methods comprising administering

[0197] In a further embodiment, there is provided a method of treating, preventing, or ameliorating obesity, comprising therapeutically administering An effective dosage of the fusion protein of a GIPR antibody and GLP-1 provided herein Provided herein are methods comprising administering to a subject a compound, or a pharmaceutical composition thereof. can be.

[0198] In any of the uses provided herein, The pharmaceutical composition is for intravenous or subcutaneous injection.

[0199] As used herein, the term "subject" refers to a mammal, including a human. , are used interchangeably with the term "patient."

[0200] The term "treatment" refers to the alleviation or prevention of at least one symptom or other aspect of a disease. or a reduction in disease severity. Fusion proteins of antibodies and GLP-1 are effective therapeutic agents, providing a cure and As recognized in the relevant art, a therapeutic agent need not eradicate all symptoms or signs of a disease. Although a therapeutic agent may reduce the severity of a given condition, to be effective it is not necessary to treat all symptoms of the disease. Similarly, a prophylactic drug must completely prevent the onset of a disease state in order to be effective. It is not necessary to merely reduce the impact of the disease (e.g., the number or severity of its symptoms). or by reducing the effectiveness of another treatment or by increasing the effectiveness of another treatment. by producing a beneficial effect) or by reducing the likelihood that a disease will occur or worsen in a subject. One embodiment of the present invention is to provide an index that reflects the severity of a particular disease. The antibody is administered to the patient in an amount and for a time sufficient to induce a sustained improvement over baseline in the target. The present invention relates to a method comprising administering

[0201] Pharmaceutical compositions of GIPR antibodies or fusion proteins of GIPR antibodies and GLP-1 are limited It is not administered orally, but may be administered by any suitable technique, such as parenterally, topically, or by inhalation. When injected, the pharmaceutical composition can be administered, for example, intraarticularly, intravenously, intramuscularly, intralesionally, It can be administered via intraperitoneal or subcutaneous routes, by bolus injection or continuous infusion. For example, transdermal administration and sustained release of implants to the site of disease or injury. Delivery by inhalation can be by, for example, nasal or oral inhalation. Other alternatives include inhaling the antibody in aerosol form, using a nebulizer, or inhaling the antibody in aerosol form. Oral formulations include pills, syrups, or lozenges.

[0202] Advantageously, the fusion proteins of GIPR antibodies or GLP-1 provided herein may contain one or more additional ingredients such as a physiologically acceptable carrier, excipient, or diluent. The composition is administered in a composition containing one or more physiologically active agents, as described below. In many specific embodiments, the composition further comprises one or more antibodies (e.g., murine or humanized antibodies) or GLP-1 fusion proteins In addition to the substance, it contains 1, 2, 3, 4, 5, or 6 physiologically active agents.

[0203] In one embodiment, the pharmaceutical composition comprises a suitable buffer for the antibody at a suitable pH, ascorbic acid Antioxidants such as acids, low molecular weight polypeptides (e.g., those with less than 10 amino acids) , proteins, amino acids, carbohydrates such as dextrin, chelating agents such as EDTA, together with one or more substances selected from the group consisting of glutathione, stabilizers, and excipients. , a murine antibody or a humanized antibody or a GLP-1 fusion protein provided herein Preservatives may also be added in accordance with appropriate industry standards. It can be formulated as a lyophilizate using appropriate excipient solutions. The dosages and concentrations are harmless to the recipient. For example, see Remington's Pharmaceutical Sciences, 1 6th Ed. (1980) and 20th Ed. (2000). one or more antibodies or GLP-1 fusion proteins of the invention and a label or as described herein and other instructions for use by a physician in treating any of the conditions described herein. A Pack Publishing Company kit is provided. Thus, the kit includes a sterile preparation of one or more human antibodies or GLP-1 fusion proteins. , which may be in the form of the composition disclosed above and may be in one or more vials. .

[0204] The dosage and frequency of administration will depend on the route of administration and the specific antibody or GLP-1 fusion protein used. the protein, the nature and severity of the disease being treated, whether the condition is acute or chronic, and the size of the subject The appropriate dosage may vary depending on factors such as the size and overall condition of the patient. can be determined in clinical trials, which may include, for example, dose escalation studies.

[0205] The antibodies or GLP-1 fusion proteins provided herein can be used, for example, to The dose may be administered at regular intervals over a period of time, for example, once or more than once. In this study, the murine antibody or humanized antibody or GLP-1 fusion protein was administered for at least 1 month. or once for a longer period, for example, for one, two, or three months, or indefinitely For treating chronic conditions, long-term treatment is generally most effective. However, to treat acute illnesses, administration over a shorter period, e.g., 1 to 6 weeks, Generally, humanized antibodies are based on one or more selected indicators. It is administered until the patient experiences a medically relevant degree of improvement in the lines.

[0206] An example of a treatment regimen provided herein is for type 2 diabetes, obesity, or non-alcoholic For the treatment of symptoms caused by chronic steatohepatitis, the drug is administered at an appropriate dose for one week or Each dose includes one subcutaneous injection of antibody or GLP-1 fusion protein. The GLP-1 fusion protein may be administered until the desired result is achieved, for example, until the patient's symptoms subside. Treatment may be resumed as needed, or may be administered once a week or once a month until the desired effect is achieved. Alternatively, a maintenance dose may be administered.

[0207] The patient's blood glucose concentration and weight are measured using human antibodies or GLP to detect changes in the patient's blood pressure. before and during treatment with an antibody or GLP-1 fusion protein, such as a GLP-1 fusion protein, and / or thereafter. For some diseases, changes in blood glucose may be a sign of disease progression. Blood glucose concentrations can be determined using known techniques.

[0208] Certain embodiments of the methods and compositions herein include, for example, antibodies or GLP-1 fusion proteins. The present invention relates to a fusion protein and one or more GIP antagonists, one or more antibodies or GLP-1 fusion proteins, or an antibody or It involves the use of a GLP-1 fusion protein and one or more other GIP antagonists. In a further embodiment, the antibody or GLP-1 fusion protein is administered alone or in combination with a GLP-1 fusion protein administered to a patient. It is administered in combination with other drugs used to treat the condition from which it is afflicted. Examples of drugs include proteinaceous and non-proteinaceous drugs. If so, dosages should be adjusted accordingly, as is well known in the art. "Combined administration" Combination therapy is not limited to simultaneous administration, but includes administration of at least one other treatment. The antigen and protein are administered at least once during the course of administration, which comprises administering the therapeutic agent to the patient. It also includes the treatment plan to be administered.

[0209] On the other hand, for treating type 2 diabetes, obesity and non-alcoholic steatohepatitis and related diseases A method for preparing a medicament, the medicament comprising administering to the patient a compound of the present invention for the treatment of a disease related to the above-mentioned disease. The antibody or GLP-1 fusion protein provided herein and a pharmaceutically acceptable excipient Methods for preparing the drug are provided herein, including the admixture of excipients. This is true.

[0210] Compositions related to antibodies or GLP-1 fusion proteins capable of specifically binding to human GIPR Further provided herein are products, kits, and methods. Nucleic acid molecules containing polynucleotides encoding all or part of peptides and derivatives thereof and fragments, such as GIPR antibodies, antibody fragments, antibody derivatives, or GLP Nucleic acids encoding all or part of the fusion protein are also provided. vectors and plasmids containing such nucleic acids and / or vectors and plasmids Further provided herein are cells and cell lines comprising the The method includes, for example, preparing an antibody or a GLP-1 fusion protein that binds to human GIPR, Methods for identifying or isolating, antibodies or GLP-1 fusion proteins that bind to GIPR and a method for determining whether an antibody or GLP-1 fusion protein that binds to GIPR is The present invention also includes a method of administering a protein to an animal model.

[0211] The technical solutions described herein will be further understood by the following examples. [Example]

[0212] Unless specified, the starting materials and equipment described herein are either commercially available or The methods in the following examples are all those commonly used in the art. and, unless otherwise specified, are conventional methods in the art.

[0213] 1: Preparation of antigen for immunization CHO-DHFR-cells were seeded in 6-well plates. After 24 hours, the cells were incubated with GIPR (human GIPR) gene (SEQ ID NO: 114 for the nucleotide sequence, and The pTM15 plasmid containing the nucleotide sequence (see SEQ ID NO: 113) was transformed into Lipofectamine was injected according to the manufacturer's recommended protocol. Transfection was performed using 2000 (Invitrogen). 48 hours after infection, the medium was changed to 100 μg / mL of hygromycin containing The medium was replaced with complete medium and maintained every 3 days for approximately 2 weeks of culture until stable clones appeared. The dispersed cell colonies were removed from the plate and incubated until the cells were 100% confluent. The established stable cell lines were subcultured until they reached the end of the phenotype. To confirm positive clones, a monoclonal antibody against the V5 tag (Life Technologies) was used. The abundance of cell surface hGIP was analyzed by FACS using a fluorochrome-based immunoassay (FACS). R expression was detected in selected CHO-DHFR-hGIPR cells. Three high hGIPR-expressing stable cell lines were identified through subcloning and further validation. These cell lines were used to produce immunogens for antibody preparation (see Experimental (See Example 2.) Furthermore, in one embodiment, cells of hGIPR and hIgG Fc Fusion proteins of the exodomains can also be used as immunogens for antibody preparation. The components are: hGIPR extracellular domain, hIgG Fc and peptide phosphorylation. Subcloning of the linker fusion protein gene into the pTM5 plasmid The cell supernatant was used for mass transient expression in suspended HEK293 cells. Transient expression of hGIPR was then The main fusion protein was obtained by affinity chromatography purification.

[0214] 2: Preparation of antibodies Antibodies to hGIPR can be produced using immunogens including any of the following: For example, in certain embodiments, whole cells expressing hGIPR are immunized with antibodies to hGIPR. Furthermore, in certain embodiments, hGIP is used as an immunogen to produce A fusion protein comprising the N-terminal extracellular domain of hFc and an antibody against hGIPR is Immunogen and aluminum hydroxide adjuvant The mixture was subcutaneously injected into BALB / c mice (6-8 weeks old) and boosted once a week. After a total of six immunizations, blood samples were collected from the tail vein and serum was separated by centrifugation. Serum titers were then analyzed by FACS. After the highest titer was obtained, mice were sacrificed. SP2 / 0 cells in the logarithmic growth phase were collected and centrifuged. The cells were centrifuged and the cell pellet was resuspended in serum-free medium, then centrifuged and resuspended a second time. The spleen cells and SP2 / 0 cells were suspended and counted. SP2 / 0 cells: spleen cells ≥ After mixing at a 1:1 ratio, three wash-centrifugation steps were performed. After removing the pellet, 1 mL of pre-warmed PEG-1500 was added dropwise and allowed to stand for 1 minute. Mix by pipetting and slowly add 30 mL of pre-warmed serum-free medium to The cell pellet was resuspended in fusion medium. Spleen cells in 100 μL The fused hamster and feeder layer cells were plated in each well of a 96-well plate. Hybridoma cells and feeder layer cells were treated with HAT (sarcin, amethopterin, and thymidylidene nitrate). Non-fused cells were removed by co-culture in 96-well plates using chromatin selection. Afterwards, the supernatant of the hybridoma cells in the culture plate was collected for ELISA analysis.

[0215] 3: ELISA screening of antibodies CHO-DHFR-hGIPR cells overexpressing hGIPR and CHO-DHFR- Blank cells were transferred separately into 96-well plates and allowed to reach 90% confluence. The culture medium supernatant was removed, and the attached cells were washed twice with PBS and then with 100% methanol. The cells were fixed at 4°C by adding 100 μL of freshly prepared 0.6% H After adding 2O2-PBS and incubating at room temperature for 20 minutes, the cells were resuspended in PBS. After blocking with 1% BSA solution (dissolved in PBS), the hybridization was performed. The supernatant was added to the cells and incubated at 4°C for 90 minutes. After several washes, 100 μL of diluted Diluted goat anti-mouse Fc-HRP secondary antibody (Sigma-Aldrich) was added to each well. After washing five times, 100 μL of TMB was added to the plate and incubated at 37°C for 30 minutes. Add chromogenic substrate and incubate at 37°C for 15 minutes, then add 50 μL of 2M H2S The reaction was stopped by adding O4 and then read at 450 nm. In the present study, a fusion protein of the N-terminal extracellular domain of hGIPR and hFc was After blocking with 1% BSA (dissolved in PBS), Hybridoma cell supernatant was added and incubated for 90 minutes at 4°C. Same as above ELISA method to screen hGIPR monoclonal antibodies The positive control was serum from immunized mice; the negative control was cell culture medium. After preliminary screening by ISA, several positive hives secreting hGIPR antibodies were identified. These hybridoma cell lines secreting hGIPR antibodies were obtained. The positive hybridoma cells were selected and subcloned by limiting dilution. The supernatant was confirmed by FACS analysis (see Example 10).

[0216] 4: Cloning and subcloning of antibody genes Antibody-secreting hybridoma cells were collected. Hybridoma mRNA was analyzed by QIAG The mRNA was extracted according to the manufacturer's protocol for the EN mRNA extraction kit. The isolated mRNA was reverse transcribed into cDNA. The reverse transcription primers were the mouse light and heavy chain constants. The primer specific to the normal region, in particular, the heavy chain reverse transcription primer (5'-T TTGGRGGGAAGATGAAGAC-3'), and the light chain reverse transcription primer was ( 5'-TTAACACTCTCCCCTGTTGAA-3') and (5'-TTAACA The RT-PCR reaction conditions were as follows: The incubation times were specified as follows: 25°C for 5 minutes, 50°C for 60 minutes, and 70°C for 15 minutes. The cDNA was diluted to 500 μL with 0.1 mM TE and placed in an ultrafiltration centrifuge tube. (Amicon Ultra-0.5) and centrifuge at 2,000 g for 10 minutes. The filtrate was removed, and 500 μL of 0.1 mM TE was added and the mixture was incubated for 10 minutes. The filtrate was removed and the preparation tube was inverted into a new centrifuge tube. The purified cDNA was obtained by centrifugation at 2,000 g for 10 minutes. The cDNA (10 μL) was taken as a template, and then 4 μL of 5x tailing buffer ( Promega), 4 μL of dATP (1 mM) and 10 U of terminal transferase (P romega) was added, mixed evenly, and incubated at 37°C for 5 minutes, then at 65°C for 5 minutes. The poly(A)-tailed cDNA was used as a template to clone the light and heavy chain variable regions of the antibody. PCR was performed to amplify the gene. All upstream primers were oligo dT. The heavy chain downstream primer was (5'-TGGACAGGGATCCAGAGTTCC-3') and (5'-TGGACAGGGCTCCATAGTTCC-3'), and the light chain downstream The primer was (5'-ACTCGTCCTTGGTCAACGTG-3'). The CR reaction conditions were as follows: 95°C for 5 min; 95°C for 30 s; 56°C for 30 s. 40 cycles of 72°C for 1 minute, 72°C for 2 seconds, and 72°C for 7 minutes. PCR products were then sequenced. The PCR primers were: It is designed based on the DNA sequence of the antibody, and therefore contains the complete light chain, heavy chain signal peptide and The variable domains and mouse IgG1 constant region were ligated into the expression vector pTM5.

[0217] 5: Antibody humanization and optimization First, the sequences of the light and heavy chain variable regions of the mouse antibody were obtained from the NCBI online antibody variable region database. Used as input in a search using the regional sequence alignment tool (Ig Blast). The human antibody germline gene sequence (I) is homologous to the mouse antibody variable region sequence for humanization. g Germline Gene sequence) and search for the most homologous sequence excluding the CDR sequence. Human gene sequences having the amino acid sequence of the present invention can be used as templates for CDR grafting to produce humanized antibodies. The variable region sequences were obtained. Humanized antibody light and heavy chain variable region genes were synthesized and used to generate human IgG2 or Combined with the IgG4 constant region sequence, a full-length recombinant humanized antibody sequence was obtained. Antibodies were expressed according to Example 8 and their affinity for GIPR was determined according to Example 1. The antibodies with the best affinity were selected by FACS analysis as described in . The variable region sequences of the humanized antibodies were engineered by site-directed mutagenesis to incorporate GIPRs. Its affinity for ATP was further improved.

[0218] 6: Subcloning of the humanized hGIPR antibody gene The heavy and light chain variable region gene sequences of the optimized humanized antibody were synthesized by outsourcing. During this process, two restriction sites were introduced: NheI at the 5' end and NheI at the 3' end. The SalI site in pTM5 was introduced into the heavy chain variable region sequence. Similarly, NheI was inserted at the 5' end and Bsiw The light chain variable region was expressed in the pTM5 expression vector by introducing I at the 3' end. It was linked to the light chain constant region.

[0219] 7: Construction of a fusion protein of humanized hGIPR antibody and GLP-1 The optimized humanized antibody is linked to a GLP-1 or its derivative sequence via the N-terminus or C-terminus of the light chain. sequence to form a GLP-1 fusion protein, with a peptide bridging the two sequences. The signal peptide was linked via a linker sequence (linker). The nucleotide sequence of The synthetic gene is used as a template to synthesize the signal peptide-GLP The sequence of the "1-linker" portion was amplified using PCR. The antibody sequence of the fusion protein sequence is then amplified using the primer sequence as a template. By Burlap PCR, the nucleic acid sequence of the fusion protein "signal peptide-GLP- The "1-peptide linker" portion is linked to the antibody portion and contains two restriction enzyme sites, Nhe1 and Not1 was introduced at both ends of the primers, thereby determining the complete fusion protein sequence and expression. The present vector pTM5 is ligated together.

[0220] 8: Transient expression of hGIPR antibody and GLP-1 fusion protein HEK293 or CHO suspension cells (5x10 5 / mL) was inoculated into shake flasks After 24 hours of rotation at 37°C, the cell density was 1x10 6 / mL, which is called the trans Polyethylenimine (PEI) was used for the transfection assay. The PEI / DNA mixture is then incubated in a 15-minute incubator. After incubation, the cells were added to the cell culture. The mixture was incubated at 37°C and 5% CO for 24 hours. It was added to the cell culture as a support for expression. Finally, protein expression was completed (9 After >6 hours, cell supernatants were collected for antibody purification.

[0221] 9: Purification and separation of hGIPR antibody and GLP-1 fusion protein Cells and cell debris were removed from the cultures after centrifugation (8000 rpm) and the supernatant was collected at 0.5°C. The supernatant was filtered through a 0.22 μm filter. The clarified supernatant was used for purification. The process was completed by chromatography. The supernatant was first purified by Protein A / G affinity chromatography. The antibody flows through the affinity column, during which it binds to Protein A / G and is deposited on the column. The antibody was then chromatographed using an elution buffer with a low pH (below 3.0). The low pH eluent was immediately neutralized with 1 M Tris-HCl. The purified antibody was then dialyzed against PBS or another buffer system.

[0222] 10: FACS to confirm the binding activity of functional hGIPR antibodies CHO-DHFR-hGIPR cells were incubated with PBS containing 10 mM EDTA. Take out 10 per tube 5 Dispense cells into 1.5 mL EP tubes and centrifuge the supernatant. The negative control sample was prepared by loading the cells in PBS, 2% FBS. For the positive control, 200 μL of GIPR antibody solution at a specific concentration was Add to cells and incubate at room temperature; after incubation, cells are then Centrifuge at 100 rpm, remove the supernatant, wash with FACS loading buffer, and resuspend. The cells were then incubated with FITC-conjugated goat anti-mouse fluorescent antibody at a dilution of 1:50 or Add PE-labeled goat anti-human fluorescent antibody (200 μL / well) and resuspend. The supernatant was removed after centrifugation and the cells were incubated at room temperature for 30 minutes. Wash with FACS loading buffer, centrifuge again, and load for FACS analysis. The recombinant anti-hGIPR functional antibody was used to express GIPR. The experimental results shown in Figure 1 show that the antibody specifically binds to CHO-DHFR-GIPR cells. The gray and dotted peaks are negative controls; they correspond to 1 μM antibody L10H8. The solid peaks corresponding to L10H8 and CHO-DHFR-G show a significant shift to the right. Specific binding of IPR is demonstrated.

[0223] 11: hGIPR antibody or hGIPR antibody for its in vitro antagonism of GIPR / GLP-1 fusion protein cAMP assay test CHO-DHFR cells stably expressing human GIPR were cultured at 30,000 cells per well. Cells were seeded into a 96-well cell culture plate and incubated overnight at 37°C in a 5% CO2 incubator. The next day, the supernatant was removed and 45 μL / well of hybridoma supernatant or serially diluted The cells were allowed to stand at room temperature for 30 minutes, and then the GIP peptide was added. 45 μL / well (Phoenix Pharmaceuticals, 50p Next, place the 96-well plate in an incubator at 37°C and 5% CO2 for 30 minutes. Add 0 μL / well of 10% Triton X-100 to lyse the cells at room temperature. The lysate was mixed uniformly with a pipette. The cAMP produced in the cells was detected. Transfer to a 384-well plate and add 5 μL / well of 1:20 diluted cAMP-d2. Afterwards, 5 μL / well of 1:20 diluted anti-cAMP-Eu3±cryptate was added and plated. The samples were incubated at room temperature for 1 hour. Time-resolved fluorescence 665 nm / 620 nm signal ratio The plate was read on an Envision 2103 microplate reader and then Using m5.0, IC 50 Figure 2 shows that L7H6 has an IC 50 =7.6nM Figure 3 shows that GLP-1-linker-L7H6 antagonizes GIPR at IC 50 =14.9 nM, indicating that it antagonizes GIPR.

[0224] 12: hGIPR antibody / GLP-1 fusion for its in vitro activation of GLP-1R Protein reporter gene assay test CHO-DHFR- cells co-expressing hGLP1R and CRE-luciferase were used. 20,000 cells per well were seeded into a 96-well cell culture plate and cultured overnight at 37°C. The next day, the culture supernatant was removed. The cells were washed twice with serum-free medium, and the remaining liquid was removed in the same manner. Next, 100 μL of serum-free medium containing serially diluted antibodies or GLP-1 was added. The cells were then incubated at 37°C for 4 hours. After stimulation, 100 μL of Bright Glo Chemiluminescent substrate (Promega) was added. Finally, the cell lysates were plated onto a white 96-well plate. The plates were transferred to a plate and the relative light intensity was measured using a SpectraMax L microplate reader (Mo The GLP-1-linker- L7H6 is EC 50 = 0.04 nM, showing that it activates hGLP-1R.

[0225] 13: In vivo efficacy of hGIPR antibody in high-fat diet-induced C57BL / 6 obese mice Sexuality Test A 60% high-fat diet-induced obesity model (DIO mice) was established in C57BL / 6 mice. Mice were purchased and fed a normal diet for one week, after which they were transferred to a normal mouse diet. A certain number of mice were randomly selected as a control group, and the remaining animals were fed a high-fat diet. The animals were fed continuously for 8 weeks, with body weight and food intake assessed weekly. Then, the mice fed a high-fat diet were divided into L10H8 group (10 mg / The mice were randomly divided into two groups: a 100-kg (14.5-lb) group and a model group. The drugs were administered by subcutaneous injection every 2 days for 6 weeks. The normal control group was not administered, and the model group was given the same amount of blank preparation. Data on food intake and behavioral observations were collected during the experiment. After fasting for 1 hour (water was allowed ad libitum), the animals were bled orbitally to isolate serum. The animals were euthanized, and the livers were dissected and weighed, and the liver morphology was observed. T, GLU, TC, and TG, as well as TG in the liver, were examined (see results in Figure 5 and Table 3). ).

[0226] [Table 3]

[0227] After 6 weeks of L10H8 administration, the weight gain of the L10H8 group was significantly greater than that of the model group. Although only slightly lower, the liver weight was significantly lower than that of the model group and normal control. The liver TG of the L10H8 group was significantly lower than that of the model group. These results suggest that L10H8 is a potent anti-inflammatory drug. The results show that the absorption and accumulation of liver fat is significantly slowed down. Table 3 shows the percentage of patients with L10H8 antibody in each group after 6 weeks of treatment. Liver analysis data are summarized.

[0228] 14. hGIPR antibody / GLP-1 fusion protein GLP-1-linker-V1W5 Reporter gene assay study of in vitro GLP-1R activation in 96 CHO-DHFR- cells co-expressing hGLP-1R-CRE-luciferase 20,000 cells were seeded per well in a 100-well cell culture plate and incubated overnight at 37°C. The next day, the culture supernatant was removed. The cells were washed twice with serum-free medium, and the remaining liquid was similarly removed. Next, 100 μL of serum-free medium containing serially diluted antibodies or GLP-1 was added. The cells were incubated at 37°C for 4 hours. After stimulation, 100 μL of Bright Glo chemiluminescence Photosubstrate (Promega) was added. Finally, the cell lysates were transferred to a white 96-well plate. The plates were transferred to a SpectraMax L microplate reader (Molecular De The relative light intensity was recorded at 1000 Hz (vise). Figure 6 shows that GLP-1-linker-V1W5 exhibited an EC5 0=17.40 pM, indicating that it activates hGLP-1R.

[0229] 15. hGIPR antibody or hGIPR antibody / GLP-1 fusion protein GLP-1-phospho cAMP assay study of Car-V1W5 for its in vitro GIPR antagonistic activity CHO-DHFR cells stably expressing human GIPR were cultured in 96-well cell culture plates. Seed 30,000 cells per well and place in an incubator at 37°C, 5% CO2 overnight. The next day, the supernatant was removed and 45 μL / well of hybridoma supernatant or serially diluted antibody was added. The cells were left at room temperature for 30 minutes, and then 45 μL / well of GIP peptide was added. (Phoenix Pharmaceuticals, 50 pM). Place the plates in a 37°C, 5% CO2 incubator for 30 min and add 10 μL / well of 10% T Lyse the cells by adding riton X-100 at room temperature and mix the lysate evenly with a pipette. The cAMP produced during the experiment was detected using a cAMP kit (CisBio). More than 10 μL / well of cell lysate was transferred to a white 384-well plate and 5 μL Add 5 μL / well of 1:20 diluted cAMP-d2 and finally 5 μL / well of 1:20 diluted anti- cAMP-Eu3±cryptate was added and the plate was incubated for 1 hour at room temperature. Time-resolved fluorescence 665nm / 62msec on Envision 2103 microplate reader The signal ratio was read at 0 nm, and then IC was measured using Prism 5.0. 50 The values ​​were calculated. 7 shows that GLP-1-linker-V1W5 is IC50 = 7.03 nM in the human GIP receptor ( Figure 8 shows that GLP-1-linker-V1W5 antagonizes the human GIPR (IC5 0=4.30 nM, indicating that it antagonizes monkey GIP receptor (maGIPR).

[0230] 16: Pharmacokinetic study of GIPR antibody / GLP-1 fusion protein in cynomolgus monkeys A total of six cynomolgus monkeys (three males and three females) were administered GLP- A single subcutaneous injection of 1 / hGIPR antibody fusion protein was given, and a 0.6 mL whole blood sample was collected. The animals were then injected via the vein of the forelimb on the same side of the body as the injection site before (0 min), 2 hours, 4 hours, and 8 hours after injection. 12 hours, 24 hours, 2 days, 4 days, 6 days, 8 days, 10 days, 12 days, 18 days, 28 days The blood samples were collected at each time point and placed in centrifuge tubes on ice. After natural clotting, the blood samples were then centrifuged. Serum was separated by centrifugation and stored at low temperature (-80°C) until use. The GLP-1 portion and the hGIPR antibody portion of the GLP-1 / hGIPR antibody fusion protein Both half-lives in cynomolgus monkeys were quantified separately by ELISA and analyzed by software It was determined by analysis.

[0231] 17: Pharmacokinetic study of GIPR antibody / GLP-1 fusion protein in rhesus monkeys A total of nine healthy male rhesus monkeys were administered the hGIPR antibody / GLP-1 fusion protein (GL P-1-linker-V1W4, GLP-1-linker-V1W5, or GLP-1-linker Three animals in each group received a single subcutaneous injection of 4 mg / kg of Car-V1W6. (0 min) and 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 2 days, 4 days, 6 days after administration 8th, 10th, 12th, 16th, 20th, 24th, 30th, 36th, 42nd, 50th, 6th At day 0, a 0.6 mL whole blood sample was collected from the forelimb vein and administered with 8 μl of DDP-IV inhibitor. After natural clotting, the blood sample was then centrifuged to extract serum. The serum samples were separated and stored at a low temperature (-80°C) until use. The hGIPR antibody and GLP-1 portions of the antibody fusion protein were separately analyzed by ELISA. The half-lives of both drugs in rhesus monkeys were determined by software analysis.

[0232] The PK results showed that the three GLP-1 / hGIPR antibody fusion proteins described above T in the antibody part 1 / 2 were approximately 360, 679, and 614 hours, respectively, and GLP-1 Part T 1 / 2 were approximately 87, 82, and 97 hours, respectively. The PK curves and parameters for monkeys in this group are shown in Figures 9 and 10 and Table 4.

[0233] [Table 4]

[0234] 18: To evaluate the efficacy of hGIPR antibody / GLP-1 fusion protein in primates hGIPR antibody / GLP-1 fusion protein in high-fat diet-induced obese cynomolgus monkeys Quality in vivo efficacy testing An obese cynomolgus monkey model (DIO cynomolgus monkey) was established by inducing a 60% high-fat diet. , and then used it to investigate the in vivo efficacy of GLP-1-linker-L7H6 by subcutaneous administration. High-fat diet-induced monkeys were given GLP-1-linker-L7H according to their body weight. The animals were randomly divided into a 6 (10 mg / kg) group and a model group. The model group was given an equal volume of blank formulation. Nutritional and behavioral observation data were collected during the experiment. After the experiment was completed, the animals were euthanized. The livers were then excised and weighed, and the morphology of the liver was observed. TC and TG, and TC and TG in the liver were examined.

[0235] The GLP-1-linker-V1W5 was determined using the method described above. Full-length cynomolgus monkeys were administered in a formulation control group, a GLP-1-linker-V1W5 group, and a positive control (GL The monkeys were randomly assigned to three groups (using a fusion protein of GLP-1R antibody and GLP-1) The drug was administered twice a week for a total of 8 weeks, with 1 mg / kg in the first week and 1 mg / kg in the second week. The control group received a subcutaneous injection of 2 mg / kg of the blank formulation. During the study, the monkeys' food intake (Fig. 11), body weight and calculated body weight change (Figs. 12 and 13), and The data from the dynamic observation, routine blood tests / blood biochemistry tests and DEXA tests were used to measure the amount of trunk fat. Time curves of change in total fat mass (Figs. 14 and 15), and time curves of change rate of total fat mass (Figure 16) and Figure 17 shows the total body fat mass per kilogram of body weight of monkeys. The time curve of change is shown in Figure 18, while the total lean tissue per kilogram of body weight in monkeys is shown in Figure 19. The time curve of the change in the amount was shown.

[0236] Efficacy studies showed that the efficacy of the compound was compared with the formulation control and the fusion protein of GLP-1R antibody and GLP-1. When compared, GLP-1-linker-V1W5 reduced food intake in animals and significantly reduced body weight in obese cynomolgus monkeys and, more importantly, increased GLP-1 phosphorylation. Car-V1W5 reduced total and trunk fat mass in obese cynomolgus monkeys, whereas On the other hand, behavioral observation, routine blood tests, and No abnormal findings were observed in blood biochemistry tests.

[0237] At the end of the study, the animals' livers were biopsied to detect liver TP, TG, and TC (results are shown in Table 5). The liver biochemistry test results for various groups showed that GLP-1-linker-V1W5 significantly increased the It was shown that the liver TC content was decreased.

[0238] [Table 5]

[0239] The above embodiments should be sufficient to teach one of ordinary skill in the art how to make and use the claimed embodiments. It is intended to disclose and describe the present invention and is not intended to limit the scope of the present disclosure. Modifications obvious to one skilled in the art are intended to be within the scope of the claims herein. All publications, patents, and patent applications cited herein are specifically and individually indicated by reference thereto. No. 60 / 699,999, filed on Dec. 1, 2003, and which is incorporated herein by reference as if incorporated herein by reference.

Claims

1. An antibody that specifically binds to human GIPR, comprising one, two, three, four, five, or six amino acid sequences, wherein each amino acid sequence is independently an amino acid sequence listed below: 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; 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; 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; d. Heavy chain CDR1 amino acid sequences: SEQ ID NO:18, SEQ ID NO:23, and SEQ ID NO:26; 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 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 An antibody selected from:

2. The antibody comprises one or two amino acid sequences, wherein each amino acid sequence is independently an amino acid sequence listed below: 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 b. The antibody of claim 1, wherein the heavy chain CDR1 amino acid sequence is selected from SEQ ID NO: 18, SEQ ID NO: 23, and SEQ ID NO:

26.

3. The antibody comprises or further comprises one or two amino acid sequences, wherein each amino acid sequence is independently an amino acid sequence listed below: 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 b. 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 The antibody of claim 1 or 2, selected from the group consisting of:

4. The antibody comprises or further comprises one or two amino acid sequences, wherein each amino acid sequence is independently an amino acid sequence listed below: a. 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; and b. 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 The antibody according to any one of claims 1 to 3, which is selected from the group consisting of:

5. The antibody of any one of claims 1 to 4, wherein the antibody comprises or further comprises one or two 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.

6. The antibody of any one of claims 1 to 5, wherein the antibody comprises or further comprises one or two 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.

7. The antibody of any one of claims 1 to 6, wherein the antibody comprises or further comprises a combination of light and heavy chain CDR1 amino acid sequences independently selected from the following list: 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.

8. 8. The antibody of any one of claims 1 to 7, wherein the antibody comprises or further comprises a combination of light and heavy chain CDR2 amino acid sequences independently selected from the following list: 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.

9. 9. The antibody of any one of claims 1 to 8, wherein the antibody comprises or further comprises a combination of light and heavy chain CDR3 amino acid sequences independently selected from the following list: 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.

10. The antibody (a) Light chain CDR1 amino acid sequence: SEQ ID NO: 1; Light chain CDR2 amino acid sequence: SEQ ID NO:2; Light chain CDR3 amino acid sequence: SEQ ID NO:3; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 18; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 19; and Heavy chain CDR3 amino acid sequence: SEQ ID NO:20; (b) light chain CDR1 amino acid sequence: SEQ ID NO:4; Light chain CDR2 amino acid sequence: SEQ ID NO:5; Light chain CDR3 amino acid sequence: SEQ ID NO:6; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 18; Heavy chain CDR2 amino acid sequence: SEQ ID NO:21; and Heavy chain CDR3 amino acid sequence: SEQ ID NO:22; (c) light chain CDR1 amino acid sequence: SEQ ID NO:7; Light chain CDR2 amino acid sequence: SEQ ID NO:8; Light chain CDR3 amino acid sequence: SEQ ID NO:9; Heavy chain CDR1 amino acid sequence: SEQ ID NO:23; Heavy chain CDR2 amino acid sequence: SEQ ID NO:24; and Heavy chain CDR3 amino acid sequence: SEQ ID NO:25; (d) 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; (e) 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; (f) light chain CDR1 amino acid sequence: SEQ ID NO: 15; Light chain CDR2 amino acid sequence: SEQ ID NO: 16; Light chain CDR3 amino acid sequence: SEQ ID NO: 17; Heavy chain CDR1 amino acid sequence: SEQ ID NO:26; Heavy chain CDR2 amino acid sequence: SEQ ID NO:29; and Heavy chain CDR3 amino acid sequence: SEQ ID NO: 30 The antibody according to any one of claims 1 to 9, comprising:

11. The antibody Light chain CDR1 amino acid sequence: SEQ ID NO: 15; Light chain CDR2 amino acid sequence: SEQ ID NO: 16; Light chain CDR3 amino acid sequence: SEQ ID NO: 17; Heavy chain CDR1 amino acid sequence: SEQ ID NO:26; Heavy chain CDR2 amino acid sequence: SEQ ID NO:29; and Heavy chain CDR3 amino acid sequence: SEQ ID NO: 30 The antibody of claim 10, comprising:

12. The antibody comprises one or two amino acid sequences, wherein each amino acid sequence is independently an amino acid sequence listed below: a. light chain variable amino acid domain 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 that are at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of the above sequences; and b. Heavy chain variable amino acid domain 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 that are at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of the above sequences. The antibody according to any one of claims 1 to 11, selected from:

13. The antibody-encoding polynucleotide sequence comprises one or two polynucleotide sequences, wherein each polynucleotide sequence is independently a polynucleotide sequence listed below: a. light chain variable domain polynucleotide coding sequences: 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 sequences which are at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of the above sequences; and b. The antibody of any one of claims 1 to 12, wherein the heavy chain variable domain encoding polynucleotide sequence is selected from: 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 a polynucleotide sequence which is at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of the above sequences.

14. 14. The antibody of any one of claims 1 to 13, wherein the antibody comprises or further comprises an amino acid sequence independently selected from the following list: 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.

15. 15. The antibody of any one of claims 1 to 14, wherein the antibody comprises or further comprises an amino acid sequence independently selected from the following list: 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.

16. 16. The antibody of any one of claims 1 to 15, wherein the antibody comprises a combination of light and heavy chain variable domain amino acid sequences independently selected from the following list: 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.

17. 17. The antibody of claim 16, wherein the antibody comprises or further comprises an amino acid sequence independently selected from the following list: 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.

18. 17. The antibody of claim 16, wherein the antibody comprises a combination of light and heavy chain variable domain amino acid sequences independently selected from the following list: 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: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.

19. 17. The antibody of claim 16, wherein the antibody comprises the amino acid sequence SEQ ID NO:67 or SEQ ID NO:

77.

20. The antibody of claim 16, wherein the antibody comprises a combination of amino acid sequences SEQ ID NO:67 and SEQ ID NO:

77.

21. The antibody comprises one or two amino acid sequences, wherein each amino acid sequence is independently an amino acid sequence listed below: a. Light chain constant domain amino acid sequences: SEQ ID NO:101 and SEQ ID NO:102; and b. Heavy chain constant domain amino acid sequences: SEQ ID NO: 103, SEQ ID NO: 104 and SEQ ID NO: 124 The antibody according to any one of claims 1 to 20, selected from:

22. The antibody according to any one of claims 1 to 21, wherein the antibody is a mouse GIPR antibody or a humanized GIPR antibody.

23. The antibody according to any one of claims 1 to 22, wherein the antibody is a GIPR monoclonal antibody.

24. The antibody of any one of claims 1 to 23, wherein the antibody is a monoclonal antibody comprising a combination of amino acid sequences independently selected from the following list: 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.

25. The antibody of any one of claims 1 to 23, wherein the antibody is a monoclonal antibody comprising a combination of amino acid sequences independently selected from the following list: 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.

26. The antibody is selected from the group consisting of the combinations 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), V2W9 (SEQ ID NO: 126 and SEQ ID NO: 135), V2W1 (SEQ ID NO: 126 and SEQ ID NO: 136), V2W2 (SEQ ID NO: 126 and SEQ ID NO: 137 26. The antibody of any one of claims 1 to 25, comprising an antibody of 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: 126 and SEQ ID NO: 135).

27. 26. The antibody of any one of claims 1 to 25, wherein the antibody comprises an antibody of the combination V1W4 (SEQ ID NO: 125 and SEQ ID NO: 130), V1W5 (SEQ ID NO: 125 and SEQ ID NO: 131), or V1W6 (SEQ ID NO: 125 and SEQ ID NO: 132).

28. The antibody of any one of claims 1 to 25, wherein the antibody comprises an antibody of the combination V1W5 (SEQ ID NO: 125 and SEQ ID NO: 131).

29. The antibody has one or more of the following properties: a. provides a Kd that is the same as or better than a reference GIPR antibody when binding to human GIPR; b. IC50 that is the same as or better than a reference GIPR antibody in antagonizing GIP-activated human GIPR ５０ to provide; and c. Cross-compete for binding to human GIPR with a reference GIPR antibody The antibody of any one of claims 1 to 28, comprising:

30. 30. The antibody of claim 29, which cross-competes with the reference GIPR antibody for binding to human GIPR.

31. The antibody of claim 29 or claim 30, wherein the reference GIPR antibody comprises an antibody of any one of claims 1 to 28.

32. The antibody of claim 31 , wherein the reference GIPR antibody comprises a combination of the light chain variable domain amino acid sequence SEQ ID NO: 67 and the heavy chain variable domain amino acid sequence SEQ ID NO:

77.

33. 33. The antibody of any one of claims 1 to 32, characterized in that the antibody is selected from a murine 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 triple-chain antibody, a quadruplex antibody, a Fab fragment, a F(ab')x fragment, a structural domain antibody, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

34. The antibody of any one of claims 1 to 33, wherein the antibody has an IC50 of about 1 nM to 200 nM or 1 nM to 100 nM in reducing human GIP signaling.

35. A GLP-1 fusion protein comprising a GIPR antibody according to any one of claims 1 to 34 and one, two, three, four, five, six, seven or eight GLP-1 fragments or reverse GLP-1 fragments; wherein the fusion protein has the structural feature that the carboxy terminus of the GLP-1 fragment is linked to the amino terminus of the light or heavy chain of the GIPR antibody, or the amino terminus of the reverse GLP-1 fragment is linked to the carboxy terminus of the light or heavy chain of the GIPR antibody, via a peptide linker.

36. The fusion protein of claim 35, wherein the fusion protein comprises a GIPR antibody and one, two, three, or four GLP-1 fragments; and the fusion protein links the carboxy terminus of the GLP-1 fragment to the amino terminus of the light chain or heavy chain of the GIPR antibody via a peptide linker.

37. The fusion protein of claim 35, wherein the fusion protein comprises a GIPR antibody and one, two, three, or four reverse GLP-1 fragments; and the fusion protein links the amino terminus of the reverse GLP-1 fragment to the carboxy terminus of the light chain or heavy chain of the GIPR antibody via a peptide linker.

38. The fusion protein of claim 35, wherein the fusion protein comprises a GIPR antibody and two GLP-1 fragments; and the fusion protein links the carboxy terminus of the GLP-1 fragment to the amino terminus of the light chain or heavy chain of the GIPR antibody via a peptide linker.

39. The fusion protein of claim 35, wherein the fusion protein comprises a GIPR antibody and two reverse GLP-1 fragments; and the fusion protein links the amino terminus of the reverse GLP-1 fragment to the carboxy terminus of the light chain or heavy chain of the GIPR antibody via a peptide linker.

40. The GIPR antibody, GLP-1 fragment and peptide linker are fused to form the fusion protein in one of the following ways: The carboxy terminus of the GLP-1 fragment is fused to the amino terminus of the light chain of the GIPR antibody via a peptide linker: N'-GLP-1-linker-R-C'; The carboxy terminus of the GLP-1 fragment is fused to the amino terminus of the heavy chain of the GIPR antibody via a peptide linker: N'-GLP-1-linker-R-C'; The fusion protein of claim 35, wherein: N' represents the amino terminus of the polypeptide chain of the fusion protein; C' represents the carboxy terminus of the polypeptide chain of the fusion protein; GLP-1 represents a GLP-1 fragment; R represents the amino acid sequence of the light chain or heavy chain of a GIPR antibody described in any one of claims 1 to 34; and linker represents a polypeptide linker.

41. 41. The GLP-1 fusion protein of any one of claims 35 to 40, wherein the peptide linker comprises a full-length, partial, or repeated amino acid sequence independently selected from SEQ ID NO:110, SEQ ID NO:111, and SEQ ID NO:

112.

42. 41. The GLP-1 fusion protein of any one of claims 35 to 40, wherein the GLP-1 fragment comprises an amino acid sequence independently selected from SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, and SEQ ID NO:109; or the reverse GLP-1 fragment comprises an amino acid sequence independently selected from SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, and SEQ ID NO:

123.

43. A polynucleotide encoding the GIPR antibody of any one of claims 1 to 34, or the GLP-1 fusion protein of any one of claims 35 to 42.

44. A vector comprising the polynucleotide of claim 43.

45. 45. A host cell comprising the vector of claim 44.

46. A pharmaceutical composition comprising a GIPR antibody according to any one of claims 1 to 34 or a GLP-1 fusion protein according to any one of claims 35 to 42 in admixture with a pharmaceutically acceptable carrier.

47. Use of a pharmaceutical composition comprising a GIPR antibody according to any one of claims 1 to 34 or a GLP-1 fusion protein according to any one of claims 35 to 42 in the preparation of a medicament for preventing or treating non-alcoholic fatty liver disease.

48. Use of a pharmaceutical composition comprising a GIPR antibody according to any one of claims 1 to 34 or a GLP-1 fusion protein according to any one of claims 35 to 42 in the preparation of a medicament for preventing or treating type 2 diabetes.

49. Use of a pharmaceutical composition comprising a GIPR antibody of any one of claims 1 to 34 or a GLP-1 fusion protein of any one of claims 35 to 42 in the preparation of a medicament for reducing weight or treating obesity and obesity-related disorders.

50. Use of a pharmaceutical composition comprising a GIPR antibody of any one of claims 1 to 34 or a GLP-1 fusion protein of any one of claims 35 to 42 in the preparation of a medicament for simultaneously treating two or more of non-alcoholic fatty liver disease, obesity, or type 2 diabetes.

51. The use according to any one of claims 47 to 50, wherein the pharmaceutical composition is administered intravenously or subcutaneously.

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