GIPR antibody, fusion protein of GIPR antibody and FGF21, and pharmaceutical composition and application of GIPR antibody and FGF21

Abstract

Provided are GIPR antibodies and fusion proteins thereof with FGF21, and pharmaceutical compositions thereof. Also provided are methods for treating, preventing or ameliorating one or more symptoms of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, obesity or type 2 diabetes mellitus by using a GIPR antibody and a fusion protein thereof with FGF21.

Description

GIPR antibody and its fusion protein with FGF21, as well as its pharmaceutical composition and application Technical Field

[0001] Provided herein are GIPR antibodies and fusion proteins thereof with FGF21, as well as pharmaceutical compositions thereof. Also provided herein are methods for using GIPR antibodies and fusion proteins thereof with FGF21 to treat, prevent, or ameliorate one or more symptoms of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, obesity, or type 2 diabetes.

[0002] Background of the Invention

[0003] Gastric inhibitory polypeptide (GIP) is a 42-amino acid peptide secreted by intestinal K cells after a meal. GIP stimulates insulin secretion by activating the gastric inhibitory polypeptide receptor (GIPR) on the surface of pancreatic β cells (Tseng et al., 1996, J. Clin. Invest. 98:2440-2445; Ravn et al., 2013, J. Biol. Chem. 288:19760-72). GIPR is widely distributed beyond the pancreas, including in bone, heart, stomach, intestine, and adipose tissue (Peter et al., 2013, J. Biol. Chem. 288:19760-72). This diverse distribution suggests that the GIP / GIPR pathway has numerous biological functions beyond blood glucose regulation. Experimental evidence indicates that the GIP / GIPR signaling pathway is closely related to fat metabolism in at least these tissues (Yip et al., 2000, Life Sci. 66:91–103). Experimental data also show that GIP concentrations in the blood circulation of patients with obesity or diabetes are increased (Creutzfeldt et al., 1978, Diabetologia 14:15–24; Flatt et al., 1984, J. Endocrinol. 101:249–256; Salera et al., 1982, J. Clin. Endocrinol. Metab. 55:329–336; et al., 2003, J. Clin. Endocrinol. Metab. 88:2706–2713).

[0004] Fibroblast Growth Factor (FGF21) is an endogenous protein synthesized in the liver and a member of the FGF family. It plays an important role in regulating sugar and lipid metabolism (Schlein et al., 2016, Cell Metabolism. 23:441-453; Habegger et al., 2013, Diabetes. 62:1453-1463). FGF21 can act on the liver, adipocytes, pancreatic β cells, muscle tissue, and the hypothalamus of the brain, exerting its physiological functions by activating FGF21-specific receptors (Kharitonenkov et al., 2005, J. Clin. Invest. 115:1627-1635; Dunshe et al., 2016, J. Biol. Chem. 291:5986-5996). FGF21 activates a receptor complex on the cell membrane that includes β-Klotho and one of the FGF receptors, FGFR1c, FGFR2c, or FGFR3c. The expression of these receptors is tissue-specific. For example, FGFR1c is primarily expressed in adipose tissue, while FGFR2c and FGFR3c are primarily expressed in the liver (Yie et al., 2012, Chem. Biol. Drug Des. 79:398-410). Endogenous FGF21 concentrations in adipose tissue are approximately 100 pg / mL in healthy individuals, but can increase tenfold in patients with obesity and non-alcoholic fatty liver disease, suggesting FGF21 resistance in these patients. Dietary changes also affect circulating FGF21 levels. Excessive consumption of fructose or alcohol can dramatically increase plasma FGF21 levels. Prolonged protein restriction or excessive carbohydrate intake can also elevate FGF21 levels.

[0005] This article combines the lipid-reducing effects of GIPR antibodies with the effects of FGF21 in reducing liver inflammation, fibrosis, and improving lipoproteins, thereby treating non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, obesity, and type 2 diabetes through multiple actions. This article provides a fusion protein drug for the treatment of patients suffering from one or more of these conditions: non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, obesity, and type 2 diabetes.

[0006] Summary of the Invention

[0007] Provided herein are antibodies that specifically bind to GIPR and are antagonists of GIPR.

[0008] The present invention also provides an antibody that can specifically bind to GIPR, wherein the antibody comprises one, two, three, four, five, or six amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0009] a. Light chain CDR1 amino acid sequences: SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11;

[0010] b. Light chain CDR2 amino acid sequence: YAS, SEQ ID NO: 2;

[0011] c. Light chain CDR3 amino acid sequences: SEQ ID NO: 3 and SEQ ID NO: 4;

[0012] d. Heavy chain CDR1 amino acid sequence: SEQ ID NO: 12;

[0013] e. Heavy chain CDR2 amino acid sequence: SEQ ID NO: 13; and

[0014] f. Heavy chain CDR3 amino acid sequence: SEQ ID NO: 14.

[0015] The present invention further provides an FGF21 fusion protein comprising an antibody that can specifically bind to GIPR and one, two, three, four, five, six, seven, or eight FGF21 fragments; the fusion protein connects the amino terminus of an FGF21 fragment to the carboxyl terminus of the light chain or heavy chain of the GIPR antibody via a peptide linker sequence.

[0016] This article provides an FGF21 fusion protein, which comprises an antibody that can specifically bind to GIPR and one, two, three, or four FGF21 fragments; the fusion protein connects the amino terminus of an FGF21 fragment to the carboxyl terminus of the light chain or heavy chain of the GIPR antibody through a peptide linker sequence.

[0017] This article provides an FGF21 fusion protein, which comprises an antibody that can specifically bind to GIPR and one or two FGF21 fragments; the fusion protein connects the amino terminus of an FGF21 fragment to the carboxyl terminus of the light chain or heavy chain of the GIPR antibody through a peptide linker sequence.

[0018] This article provides an FGF21 fusion protein, which comprises a GIPR antibody and one or two FGF21 fragments; the fusion protein connects the amino terminus of the FGF21 fragment to the carboxyl terminus of the GIPR antibody light chain through a peptide linker sequence (Linker): N'-R-Linker-FGF21-C'; or connects the amino terminus of an FGF21 fragment to the carboxyl terminus of the GIPR antibody heavy chain: N'-R-Linker-FGF21-C'; wherein: N' represents the amino terminus of the fusion protein polypeptide chain, C' represents the carboxyl terminus of the fusion protein polypeptide chain, FGF21 represents the FGF21 fragment, R is the amino acid sequence of the light chain or heavy chain of the GIPR antibody, and Linker represents the peptide linker sequence.

[0019] Provided herein is a polynucleotide encoding a GIPR antibody described herein.

[0020] Provided herein is a polynucleotide encoding a fusion protein of a GIPR antibody described herein and FGF21.

[0021] Provided herein is a vector comprising a polynucleotide encoding a GIPR antibody described herein.

[0022] Provided herein is a vector comprising a polynucleotide encoding a fusion protein of a GIPR antibody described herein and FGF21.

[0023] Provided herein is a host cell comprising a vector described herein.

[0024] Provided herein is a pharmaceutical composition comprising a GIPR antibody described herein and a pharmaceutically acceptable carrier.

[0025] Provided herein is a pharmaceutical composition comprising a fusion protein of a GIPR antibody and FGF21 as described herein and a pharmaceutically acceptable carrier.

[0026] Provided herein is a use of a GIPR antibody described herein in the preparation of a medicament for treating, preventing or ameliorating non-alcoholic steatohepatitis-related diseases.

[0027] Provided herein is a use of a fusion protein of a GIPR antibody and FGF21 described herein in the preparation of a medicament for treating, preventing or ameliorating non-alcoholic fatty liver disease.

[0028] Provided herein is a use of a GIPR antibody described herein in the preparation of a medicament for treating, preventing or ameliorating type 2 diabetes.

[0029] Provided herein is a use of a fusion protein of a GIPR antibody and FGF21 described herein in the preparation of a medicament for treating, preventing or ameliorating type 2 diabetes.

[0030] Provided herein is a use of a GIPR antibody described herein in the preparation of a medicament for weight loss or the treatment, prevention or improvement of obesity and obesity-related disorders.

[0031] Provided herein is a use of a fusion protein of a GIPR antibody and FGF21 as described herein in the preparation of a medicament for weight loss or the treatment, prevention or improvement of obesity and obesity-related disorders.

[0032] Provided herein is the use of a GIPR antibody described herein in the preparation of a medicament for simultaneously treating, preventing, or ameliorating two or more conditions of non-alcoholic steatohepatitis, obesity, or type 2 diabetes.

[0033] Provided herein is the use of a fusion protein of a GIPR antibody and FGF21 described herein in the preparation of a medicament for simultaneously treating, preventing or ameliorating two or more conditions of non-alcoholic steatohepatitis, obesity or type 2 diabetes.

[0034] Provided herein are methods for treating, preventing, or ameliorating one or more symptoms of nonalcoholic steatohepatitis-like diseases, comprising administering to a subject a therapeutically effective amount of a GIPR antibody described herein.

[0035] Provided herein are methods for treating, preventing, or ameliorating one or more symptoms of non-alcoholic steatohepatitis-related diseases, comprising administering to a subject a therapeutically effective amount of a fusion protein of a GIPR antibody and FGF21 as described herein.

[0036] Provided herein are methods of treating, preventing, or ameliorating one or more symptoms of obesity comprising administering to a subject a therapeutically effective amount of a GIPR antibody described herein.

[0037] Provided herein are methods for treating, preventing, or ameliorating one or more symptoms of obesity, comprising administering to a subject a therapeutically effective amount of a fusion protein of a GIPR antibody and FGF21 as described herein.

[0038] Provided herein are methods of treating, preventing, or ameliorating one or more symptoms of type 2 diabetes comprising administering to a subject a therapeutically effective amount of a GIPR antibody described herein.

[0039] Provided herein are methods for treating, preventing, or ameliorating one or more symptoms of type 2 diabetes, comprising administering to a subject a therapeutically effective amount of a fusion protein of a GIPR antibody and FGF21 as described herein.

[0040] BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 shows the activation curve of the hFGFR1c / hKLB signaling pathway activated by the reporter gene assay using the Fc-FGF21 fusion protein Fc-linker-FGF21 (containing SEQ ID NO: 79, SEQ ID NO: 82, SEQ ID NO: 85 and SEQ ID NO: 86). 50 Fc-FGF21 (RGE): 18.61 nM, Fc-FGF21 (SEQ ID NO: 79): 5.57 nM, Fc-FGF21 (SEQ ID NO: 82): 41.40 nM; Figure 1 (b) EC 50 They are Fc-FGF21 (RGE): 8.13 nM, Fc-FGF21 (SEQ ID NO: 85): 156.50 nM, and Fc-FGF21 (SEQ ID NO: 86): 31.44 nM respectively.

[0042] Figure 2 shows the concentration inhibition curves of the reporter gene assay for GIPR antibody / FGF21 fusion proteins V1W1-Linker-FGF21 (containing SEQ ID NO: 89) and V1W2-Linker-FGF21 (containing SEQ ID NO: 89) in antagonizing GIP-activated hGIPR signaling pathway, IC 50 They are LH: 6.50 nM, V1W1-FGF21 (SEQ: 89): 91.14 nM, and V1W2-FGF21 (SEQ: 89): 15.30 nM respectively.

[0043] Figure 3 shows the activation curves of the hFGFR1C / hKLB signaling pathway activated by GIPR antibody / FGF21 fusion proteins V1W1-Linker-FGF21 (containing SEQ ID NO: 89) and V1W2-Linker-FGF21 (containing SEQ ID NO: 89) detected by reporter gene experiments. 50 They are Fc-FGF21 (RGE): 11.65 nM, V1W1-FGF21 (SEQ ID NO: 89): 16.44 nM, and V1W2-FGF21 (SEQ ID NO: 89): 37.80 nM, respectively.

[0044] Figure 4 shows the time curves of mouse body weight (a, c) and body weight change rate (b, d) at high, medium and low doses of hGIPR antibody / FGF21 fusion protein V1W1-Linker-FGF21 (which contains SEQ ID NO: 89) and V1W2-Linker-FGF21 (which contains SEQ ID NO: 89) during the efficacy experiment period of C57BL / 6 obese mice induced by high-fat diet.

[0045] Figure 5: Shows the OGTT time curves (a, c) and blood glucose area under the curve values ​​(b, d) of mice at high, medium and low doses of hGIPR antibody / FGF21 fusion protein V1W1-Linker-FGF21 (which contains SEQ ID NO: 89) during the efficacy experiment period of high-fat diet-induced obese mice.

[0046] Figure 6 shows the effects of high, medium, and low doses of hGIPR antibody / FGF21 fusion protein V1W1-Linker-FGF21 (comprising SEQ ID NO: 89) on serum liver function indicators (a) and lipid metabolism indicators (b) in mice induced by high-fat diet during the efficacy experiment period.

[0047] Figure 7 shows the effects of high, medium, and low doses of hGIPR antibody / FGF21 fusion protein V1W2-Linker-FGF21 (comprising SEQ ID NO: 89) on serum indicators of liver function (a) and serum indicators of lipid metabolism (b) during the efficacy experiment period in high-fat diet-induced obese mice.

[0048] Figure 8 shows the activation curves of the reporter gene experiment detecting the activation of the mFGFR1C / hKLB signaling pathway by GIPR antibody / FGF21 fusion proteins V1W2-Linker-FGF21 (which contains SEQ:89), V1W2-Linker-FGF21 (which contains SEQ ID NO:115), V1W2-Linker-FGF21 (which contains SEQ ID NO:116), V1W2-Linker-FGF21 (which contains SEQ ID NO:117), and V1W2-Linker-FGF21 (which contains SEQ ID NO:118), EC 50They are V1W2-FGF21 (SEQ: 89): 0.80 nM, V1W2-FGF21 (SEQ: 115): 1.33 nM, V1W2-FGF21 (SEQ: 116): 1.35 nM, V1W2-FGF21 (SEQ: 117): 1.29 nM, V1W2-FGF21 (SEQ: 118): 1.71 nM.

[0049] Figure 9 shows the effects of high, medium and low doses of hGIPR antibody / FGF21 fusion protein V1W2-Linker-FGF21 (containing SEQ ID NO: 118) on serum indicators of liver function (a), serum indicators of lipid metabolism (b), NAS scores and fibrosis scores (c) during the efficacy experiment period in mice with non-alcoholic steatohepatitis induced by a high-fat diet.

[0050] Figure 10 shows the sequences of variants of wild-type FGF21 shown in SEQ ID NO:77 to SEQ ID NO:89, SEQ ID NO:115 to SEQ ID NO:118 and SEQ ID NO:122.

[0051] Figure 11: shows the amino acid or nucleotide sequences shown in SEQ ID NO:29 to SEQ ID NO:51, SEQ ID NO:61 to SEQ ID NO:68, SEQ ID NO:103 to SEQ ID NO:106, SEQ ID NO:111 to SEQ ID NO:114, SEQ ID NO:119 to SEQ ID NO:121 and SEQ ID NO:123 to SEQ ID NO:125. DETAILED DESCRIPTION

[0052] definition

[0053] Unless otherwise defined herein, scientific and technical terms used herein shall have the meanings understood by those of ordinary skill in the art. Generally, the nomenclature and techniques associated with the pharmacology, biology, biochemistry, cell and tissue culture, biology, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are well known and frequently used in the art.

[0054] Standard single-letter and three-letter abbreviations are used herein to identify polynucleotide and polypeptide sequences. Polypeptide sequences are written with the first amino acid residue (N') bearing an amino group on the left and the last amino acid residue (C') bearing a carboxyl group on the right, such as the FGF21 fragment sequences referred to herein: SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, 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:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, and SEQ ID NO:122. Single-stranded and double-stranded nucleic acid sequences are written with the 5' end of the top strand on the left and the 3' end on the right. Specific portions of a polypeptide may be referred to by amino acid residue number, such as amino acids 80 to 130, or by the actual residue at that position, such as Lys 80 to Lys 130. Specific polypeptide or polynucleotide sequences may also be described by explaining how they differ from a reference sequence.

[0055] The terms "peptide," "polypeptide," and "protein" all refer to molecules comprising two or more amino acids interconnected by peptide bonds. These terms encompass, for example, polypeptide analogs (e.g., muteins, variants, and fusion proteins) of natural and artificial proteins and protein sequences, as well as proteins that have been post-transcriptionally or otherwise covalently or non-covalently modified. A peptide, polypeptide, or protein can be monomeric or multimeric.

[0056] The term "polypeptide fragment" refers to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion compared to the corresponding full-length protein. The fragment length can be, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 50, 70, 80, 90, 100, 150, or 200 amino acids. The fragment length can be, for example, at most 1000, 750, 500, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 14, 13, 12, 11, or 10 amino acids. The fragment can further comprise one or more additional amino acids at one or both ends, for example, an amino acid sequence from a different natural protein (e.g., an Fc or leucine zipper domain) or an artificial amino acid sequence (e.g., an artificial linker sequence).

[0057] The polypeptides herein include polypeptides that have been modified for any reason and by any method, for example, to: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter affinity for forming protein complexes, (4) alter binding affinity, and (5) confer or modify other physicochemical or functional properties. Analogs include muteins of the polypeptides. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in the native sequence (e.g., in portions of the polypeptide outside of the domains that form intramolecular contacts). "Conservative amino acid substitutions" are those that do not significantly alter the structural properties of the parent sequence (e.g., the substituted amino acid should not disrupt the helices present in the parent sequence or interfere with other secondary structure types that confer properties to the parent sequence or are necessary for its function).

[0058] A "variant" of a polypeptide comprises an amino acid sequence in which one or more amino acid residues are inserted, deleted, and / or substituted relative to another polypeptide sequence. Variants herein include protein variants, antibody variants, and antibody fusion protein variants. In various embodiments herein, the antibody fusion proteins used comprise a variety of different variants of the FGF21 portion. For example, SEQ ID NO:77 to SEQ ID NO:89, SEQ ID NO:115 to SEQ ID NO:118, and SEQ ID NO:122 describe variant sequences of wild-type FGF21 that retain comparable biological activity and protein structure to wild-type FGF21, while having insertions, deletions, and / or substitutions of one or more amino acid residues relative to wild-type FGF21. When these FGF21 variants are fused with the antibodies described later in this article, "variants" of the wild-type FGF21 fusion protein are formed. For example, SEQ ID NO:104 to SEQ ID NO:106, SEQ ID NO:119 to SEQ ID NO:121, and SEQ ID NO:123 to SEQ ID NO:125 describe the heavy chain sequences of such variants of the FGF21 fusion protein.

[0059] "Derivatives" of polypeptides are polypeptides that have been chemically modified, for example by conjugation to other chemical moieties such as polyethylene glycol, albumin (eg, human serum albumin), phosphorylation, and glycosylation.

[0060] Unless otherwise indicated, the term "antibody" includes antibodies with two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and muteins thereof, examples of which are described below.

[0061] The term "antibody" is a protein comprising an antigen-binding portion and optionally a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that promotes the binding of the antibody to the antigen. Examples of antibodies include whole antibodies, antibody fragments (e.g., the antigen-binding portion of an antibody), antibody derivatives, antibody analogs, and antibody mutants, and fusion proteins thereof. The antibody may comprise, for example, an optional protein scaffold or an artificial scaffold having transplanted CDRs or CDR derivatives. The scaffold includes, but is not limited to, antibody-derived scaffolds introduced, for example, to stabilize the three-dimensional structure of the antibody and fully synthetic scaffolds comprising, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins 53:121-129; Roque et al., 2004, Biotechnol. Prog. 20:639-654. In addition, the antibody may be a peptidomimetic antibody ("PAM") or a scaffold comprising an antibody-mimicking antibody that utilizes fibrinogen as a scaffold.

[0062] Antibodies can have the structure of, for example, natural immunoglobulins. "Immunoglobulins" are tetrameric molecules. In natural immunoglobulins, each tetramer is composed of two identical pairs of polypeptide chains, each pair having a "light" chain (about 25 kDa) and a "heavy" chain (about 50-70 kDa). The amino terminus of each chain includes a variable domain of about 100 to 110 amino acids, which is primarily involved in antigen recognition. The carboxyl terminus of each chain defines a constant region, which is primarily involved in effector functions. Human antibody light chains are divided into kappa and lambda light chains. Heavy chains are divided into μ, δ, α, or ε, and determine the isotype of the antigen, for example, IgM, IgD, IgG, IgA, and IgE, respectively. In the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids. See, Fundamental Immunology Ch. 7 (Paul ed., 2nd ed., Raven Press, 1989). The variable regions of each light / heavy chain pair form the antibody binding site, so that an intact immunoglobulin has two binding sites.

[0063] Natural immunoglobulin chains show the same basic structure of relatively conserved framework regions (FRs) connected by three highly variable regions, also known as complementarity determining regions or CDRs. From N-terminus to C-terminus, both light and heavy chains contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The distribution of amino acids in each domain is consistent with the definition of Kabat et al. in Sequences of Proteins of Immunological Interest, 5th edition, US Patent No. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991.

[0064] Unless otherwise indicated, an "antibody" refers to an intact immunoglobulin or an antigen-binding portion thereof that can compete with an intact antibody for specific binding. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions include, among others, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), fragments including complementary determining regions (CDRs), single-chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides comprising at least a portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide.

[0065] Fab fragment has V L 、V H 、C L , and C H1 The F(ab')2 fragment is a bivalent fragment with two Fab fragments connected by a disulfide bond in the hinge region; the Fv fragment has a V H and V L Domain; dAb fragment has V H domain, V L domain, or V H or V L Antigen-binding fragments of the leukemia / antigen binding domain (U.S. Patent Nos. 6,846,634 and 6,696,245; U.S. Patent Application Publication Nos. 2005 / 0202512, 2004 / 0202995, 2004 / 0038291, 2004 / 0009507, and 2003 / 0039958; Ward et al., 1989, Nature 341:544-546).

[0066] Single-chain antibody (scFv) is a fusion protein in which V L and VH The regions are connected by a linker (e.g., a synthetic sequence of amino acid residues) to form an antibody contiguous protein, wherein the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen-binding site (see, e.g., Bird et al., 1988, Science 242:423-26; and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83).

[0067] Diabodies are bivalent antibodies comprising two polypeptide chains, each comprising a V H and V L In the invention, the linker is a domain that is so short as to not allow the pairing of the two domains on the same chain, thereby allowing each domain to pair with a complementary domain on another polypeptide chain (see, e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48; and Poljak et al., 1994, Structure 2:1121-23). ​​If the two polypeptide chains of a double-chain antibody are identical, the double-chain antibody obtained by pairing them will have the same antigen-binding site. Polypeptide chains with different sequences can be used to prepare double-chain antibodies with different antigen-binding sites. Similarly, three-chain antibodies and four-chain antibodies are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen-binding sites, respectively, which may be the same or different.

[0068] The complementary determining regions (CDRs) and framework regions (FRs) of a given antibody are identified herein using the method described by Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Edition, U.S. Patent No. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991. One or more CDRs can be covalently or non-covalently incorporated into a molecule to make it an antibody. An antibody can incorporate CDR(s) into a larger polypeptide chain. CDR(s) can be covalently linked to another polypeptide chain or non-covalently incorporated into the CDR(s). CDRs allow an antibody to specifically bind to a specific antigen of interest.

[0069] Antibodies can have one or more binding sites. If there is more than one binding site, the binding site can be the same as or different from the other. For example, natural human immunoglobulins typically have two identical binding sites, while "bispecific" or "bifunctional" antibodies have two different binding sites.

[0070] The term "murine antibody" includes antibodies having one or more variable and constant regions derived from mouse immunoglobulin sequences.

[0071] The term "humanized antibody" refers to an antibody produced by transplanting the complementarity determining region sequences of a mouse antibody molecule into the framework of a human antibody variable region.

[0072] The terms "antigen binding domain," "antigen binding region," or "antigen binding site" are the portion of an antibody comprising the amino acid residues that interact with an antigen and contribute to the antibody's specificity and affinity for the antigen. For an antibody that specifically binds to its antigen, this will include at least a portion of at least one of its CDR domains.

[0073] The term "epitope" is the portion of a molecule that is bound by an antibody (e.g., by an antibody). An epitope may comprise a non-contiguous portion of a molecule (e.g., in a polypeptide, amino acid residues that are not contiguous in the primary sequence of the polypeptide are sufficiently close to each other in the tertiary and quaternary structures of the polypeptide to be bound by an antibody).

[0074] The "percent identity" of two polynucleotide or two polypeptide sequences is determined by comparing the sequences using the GAP computer program, part of the GCG Wisconsin Package; version 10.3 (Accelrys, San Diego, CA), using its default parameters.

[0075] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably throughout the text and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA or RNA analogs generated using nucleotide analogs (e.g., peptide nucleic acids and non-natural nucleotide analogs), and hybrids thereof. Nucleic acid molecules can be single-stranded or double-stranded. In one embodiment, the nucleic acid molecules herein comprise a continuous open reading frame encoding an antibody provided herein, or a fragment, derivative, mutein, or variant thereof.

[0076] Two single-stranded polynucleotides are "complementary" to each other if their sequences are aligned antiparallel, such that each nucleotide in one polynucleotide is opposite the complementary nucleotide in the other polynucleotide, no gaps are introduced, and there are no unpaired nucleotides at the 5' or 3' ends of each sequence. A polynucleotide is "complementary" to another if the two polynucleotides hybridize to each other under moderately stringent conditions. Thus, a polynucleotide can be complementary to another polynucleotide without being its complementary sequence.

[0077] The term "vector" is a nucleic acid that can be used to introduce another nucleic acid connected thereto into a cell. One type of vector is a "plasmid," which refers to a linear or circular double-stranded DNA molecule to which additional nucleic acid segments can be attached. Another type of vector is a viral vector (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), in which additional DNA segments can be introduced into the viral genome. Certain vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors comprising bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell when introduced into the host cell and therefore replicate together with the host genome. An "expression vector" is a type of vector that can direct the expression of a selected polynucleotide.

[0078] A nucleotide sequence is "operably linked to a regulatory sequence" if the regulatory sequence affects the expression (e.g., level, time, or site) of the nucleotide sequence. A "regulatory sequence" is a nucleic acid that can affect the expression (e.g., level, time, or site) of a nucleic acid to which it is operably linked. A regulatory gene, for example, acts directly on the regulated nucleic acid or through the action of one or more other molecules (e.g., a polynucleotide that binds to the regulatory sequence and / or nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Further examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology, Volume 185, Academic Press, San Diego, CA; and Baron et al., 1995, Nucleic Acids Res. 23:3605-06.

[0079] The term "host cell" is a cell for expressing nucleic acids, such as nucleic acids provided herein. The host cell can be a prokaryotic organism, such as Escherichia coli, or it can be a eukaryotic organism, such as a unicellular eukaryotic organism (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell) or a hybridoma. Typically, a host cell is a cultured cell that can be transformed or transfected with a polypeptide-encoding nucleic acid, which can then be expressed in the host cell. The phrase "recombinant host cell" can be used to describe a host cell transformed or transfected with a nucleic acid to be expressed. A host cell can also be a cell that contains the nucleic acid but does not express it at a desired level, unless a regulatory sequence is introduced into the host cell so that it is operably connected to the nucleic acid. It should be understood that the term host cell refers not only to a specific subject cell but also to the progeny or possible progeny of the cell. Due to, for example, mutations or environmental influences, certain modifications may occur in subsequent generations, and the progeny may in fact be different from the parent cell but still fall within the scope of the term used herein.

[0080] gastric inhibitory peptide receptor

[0081] The gastric inhibitory peptide receptor (GIPR) belongs to the B-type family of seven-transmembrane G protein-coupled receptors (GPCRs). It couples to one or more intracellular signaling pathways via heterotrimeric guanine nucleotide-binding proteins (G proteins) (Drucker et al., 2006, Cell Metab. 3:153-65). Studies to date have found that GIPR is primarily expressed on the surface of pancreatic β cells and adipocytes (Ravn et al., 2013, J. Biol. Chem. 288:19760-72). It is involved in both glucose and lipid metabolism in humans and is therefore closely associated with diabetes, obesity, and related conditions (Skaw et al., 2016, Diabetes Obes. Metab. 18:847–854). As used herein, "human GIPR" and "hGIPR" refer to the human GIPR and are used interchangeably. As used herein, "mouse GIPR" and "mGIPR" refer to the mouse GIPR and are used interchangeably.

[0082] In one embodiment, the antibodies provided herein are antibodies that specifically bind to human GIPR. In another embodiment, the antibodies provided herein are antibodies that specifically bind to GIPR on the cell membrane, and the antibodies can inhibit or block the conduction of GIP signals in these cells. In another embodiment, the antibodies provided herein are antibodies that specifically bind to human GIPR, and the antibodies can bind to GIPR of other species (e.g., monkey or mouse) and block the signal transduction of GIP in these species. In a further embodiment, the antibodies provided herein are mouse antibodies that bind to human GIPR, and the antibodies can bind to GIPR of other species (e.g., monkey).

[0083] In one embodiment, the amino acid and polynucleotide sequences of GIPR are listed below, and the sequence data are derived from the GeneBank database of the National Center for Biotechnology Information and the Uniprot database of the European Bioinformatics Institute:

[0084] Human (Homo sapiens) polynucleotide, accession number: S79852;

[0085] Human (Homo sapiens) amino acids, accession number: AAB35419.2.

[0086] Gastric inhibitory peptide receptor antibodies (GIPR antibodies)

[0087] In one embodiment, provided herein are GIPR antibodies. In another embodiment, provided herein are GIPR antibodies that are intact GIPR antibodies. In another embodiment, provided herein are GIPR antibodies that are fragments of GIPR antibodies. In another embodiment, provided herein are GIPR antibodies that are derivatives of GIPR antibodies. In another embodiment, provided herein are GIPR antibodies that are mutant proteins of GIPR antibodies. In a further embodiment, provided herein are GIPR antibodies that are variants of GIPR antibodies.

[0088] In one embodiment, the GIPR antibodies provided herein comprise one, two, three, four, five, or six amino acid sequences, wherein each amino acid sequence is independently selected from the group consisting of:

[0089] a. Light chain CDR1 amino acid sequence: SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11;

[0090] b. Light chain CDR2 amino acid sequence: YAS, SEQ ID NO: 2;

[0091] c. Light chain CDR3 amino acid sequences: SEQ ID NO: 3, SEQ ID NO: 4;

[0092] d. Heavy chain CDR1 amino acid sequence: SEQ ID NO: 12;

[0093] e. Heavy chain CDR2 amino acid sequence: SEQ ID NO: 13; and

[0094] f. Heavy chain CDR3 amino acid sequence: SEQ ID NO: 14.

[0095] Table 1 lists the amino acid sequences of the light chain CDRs of the GIPR antibodies provided herein, and their corresponding polynucleotide encoding sequences. Table 2 lists the amino acid sequences of the heavy chain CDRs of the GIPR antibodies provided herein, and their corresponding polynucleotide encoding sequences.

[0096] Table 1: Amino acid sequences and polynucleotide encoding sequences of light chain CDRs

[0097] Table 2: Amino acid sequences and polynucleotide encoding sequences of heavy chain CDRs

[0098] In one embodiment, the antibodies provided herein comprise a sequence that differs from one of the CDR amino acid sequences listed in Tables 1 and 2 by 5, 4, 3, 2, or 1 single amino acid additions, substitutions, and / or deletions. In another embodiment, the antibodies provided herein comprise a sequence that differs from one of the CDR amino acid sequences listed in Tables 1 and 2 by 4, 3, 2, or 1 single amino acid additions, substitutions, and / or deletions.

[0099] In another embodiment, an antibody provided herein comprises a sequence that differs from one of the CDR amino acid sequences listed in Tables 1 and 2 by 3, 2, or 1 single amino acid additions, substitutions, and / or deletions.

[0100] In another embodiment, an antibody provided herein comprises a sequence that differs from one of the CDR amino acid sequences listed in Tables 1 and 2 by 2 or 1 single amino acid additions, substitutions and / or deletions.

[0101] In further embodiments, the antibodies provided herein comprise a sequence that differs from one of the CDR amino acid sequences listed in Tables 1 and 2 by a single amino acid addition, substitution, and / or deletion.

[0102] In one embodiment, the GIPR antibodies provided herein comprise one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0103] a. Light chain CDR1 amino acid sequence: SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; and

[0104] b. Heavy chain CDR1 amino acid sequence: SEQ ID NO: 12.

[0105] In another embodiment, the GIPR antibodies provided herein comprise one or two amino acid sequences, wherein each amino acid sequence is independently selected from the group consisting of:

[0106] a. Light chain CDR2 amino acid sequence: YAS, SEQ ID NO: 2 and

[0107] b. Heavy chain CDR2 amino acid sequence: SEQ ID NO: 13.

[0108] In another embodiment, the GIPR antibodies provided herein comprise one or two amino acid sequences, wherein each amino acid sequence is independently selected from the group consisting of:

[0109] a. Light chain CDR3 amino acid sequence: SEQ ID NO: 3, SEQ ID NO: 4; and

[0110] b. Heavy chain CDR3 amino acid sequence: SEQ ID NO: 14.

[0111] In another embodiment, the GIPR antibodies provided herein comprise one, two, three, or four amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0112] a. Light chain CDR1 amino acid sequence: SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11;

[0113] b. Heavy chain CDR1 amino acid sequence: SEQ ID NO: 12;

[0114] c. Light chain CDR2 amino acid sequence: YAS, SEQ ID NO: 2; and

[0115] d. Heavy chain CDR2 amino acid sequence: SEQ ID NO: 13.

[0116] In another embodiment, the GIPR antibodies provided herein comprise one, two, three, or four amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0117] a. Light chain CDR1 amino acid sequence: SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11;

[0118] b. Heavy chain CDR1 amino acid sequence: SEQ ID NO: 12;

[0119] c. Light chain CDR3 amino acid sequence: SEQ ID NO: 3, SEQ ID NO: 4; and

[0120] d. Heavy chain CDR3 amino acid sequence: SEQ ID NO: 14.

[0121] In further embodiments, the GIPR antibodies provided herein comprise one, two, three, or four amino acid sequences, wherein each amino acid sequence is independently selected from the group consisting of:

[0122] a. Light chain CDR2 amino acid sequence: YAS, SEQ ID NO: 2;

[0123] b. Heavy chain CDR2 amino acid sequence: SEQ ID NO: 13;

[0124] c. Light chain CDR3 amino acid sequence: SEQ ID NO: 3, SEQ ID NO: 4; and

[0125] d. Heavy chain CDR3 amino acid sequence: SEQ ID NO: 14.

[0126] In one embodiment, the GIPR antibodies provided herein comprise one, two, or three amino acid sequences, wherein each amino acid sequence is independently selected from the group consisting of SEQ ID NO: 1, YAS, 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, and SEQ ID NO: 11.

[0127] In another embodiment, the GIPR antibodies provided herein comprise one, two, or three amino acid sequences, wherein each amino acid sequence is independently selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

[0128] In one embodiment, the GIPR antibodies provided herein comprise a combination of light and heavy chain CDR1 amino acid sequences independently selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 12, SEQ ID NO: 5 and SEQ ID NO: 12, SEQ ID NO: 6 and SEQ ID NO: 12, SEQ ID NO: 7 and SEQ ID NO: 12, SEQ ID NO: 8 and SEQ ID NO: 12, SEQ ID NO: 9 and SEQ ID NO: 12, SEQ ID NO: 10 and SEQ ID NO: 12, and SEQ ID NO: 11 and SEQ ID NO: 12.

[0129] In another embodiment, the GIPR antibodies provided herein comprise a combination of light and heavy chain CDR2 amino acid sequences independently selected from the group consisting of YAS and SEQ ID NO:13, and SEQ ID NO:2 and SEQ ID NO:13.

[0130] In further embodiments, the GIPR antibodies provided herein comprise a combination of light chain and heavy chain CDR3 amino acid sequences independently selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:14, and SEQ ID NO:4 and SEQ ID NO:14.

[0131] In one embodiment, the GIPR antibody provided herein comprises:

[0132] a. a combination of light chain and heavy chain CDR1 amino acid sequences independently selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 12, SEQ ID NO: 5 and SEQ ID NO: 12, SEQ ID NO: 6 and SEQ ID NO: 12, SEQ ID NO: 7 and SEQ ID NO: 12, SEQ ID NO: 8 and SEQ ID NO: 12, SEQ ID NO: 9 and SEQ ID NO: 12, SEQ ID NO: 10 and SEQ ID NO: 12, and SEQ ID NO: 11 and SEQ ID NO: 12; and

[0133] b. A combination of light chain and heavy chain CDR2 amino acid sequences independently selected from the following: YAS and SEQ ID NO: 13, and SEQ ID NO: 2 and SEQ ID NO: 13.

[0134] In another embodiment, the GIPR antibody provided herein comprises:

[0135] a. a combination of light chain and heavy chain CDR1 amino acid sequences independently selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 12, SEQ ID NO: 5 and SEQ ID NO: 12, SEQ ID NO: 6 and SEQ ID NO: 12, SEQ ID NO: 7 and SEQ ID NO: 12, SEQ ID NO: 8 and SEQ ID NO: 12, SEQ ID NO: 9 and SEQ ID NO: 12, SEQ ID NO: 10 and SEQ ID NO: 12, and SEQ ID NO: 11 and SEQ ID NO: 12; and

[0136] b. A combination of light chain and heavy chain CDR3 amino acid sequences independently selected from the following: SEQ ID NO: 3 and SEQ ID NO: 14, SEQ ID NO: 4 and SEQ ID NO: 14.

[0137] In another embodiment, the GIPR antibody provided herein comprises:

[0138] a. a combination of light chain and heavy chain CDR2 amino acid sequences independently selected from the following: YAS and SEQ ID NO: 13, and SEQ ID NO: 2 and SEQ ID NO: 13; and

[0139] b. A combination of light chain and heavy chain CDR3 amino acid sequences independently selected from the following: SEQ ID NO: 3 and SEQ ID NO: 14, SEQ ID NO: 4 and SEQ ID NO: 14.

[0140] In further embodiments, the GIPR antibodies provided herein comprise:

[0141] a. a combination of light chain and heavy chain CDR1 amino acid sequences independently selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 12, SEQ ID NO: 5 and SEQ ID NO: 12, SEQ ID NO: 6 and SEQ ID NO: 12, SEQ ID NO: 7 and SEQ ID NO: 12, SEQ ID NO: 8 and SEQ ID NO: 12, SEQ ID NO: 9 and SEQ ID NO: 12, SEQ ID NO: 10 and SEQ ID NO: 12, and SEQ ID NO: 11 and SEQ ID NO: 12;

[0142] b. a combination of light chain and heavy chain CDR2 amino acid sequences independently selected from the group consisting of: YAS and SEQ ID NO: 13, and SEQ ID NO: 2 and SEQ ID NO: 13; and

[0143] c. A combination of light chain and heavy chain CDR3 amino acid sequences independently selected from the following: SEQ ID NO: 3 and SEQ ID NO: 14, SEQ ID NO: 4 and SEQ ID NO: 14.

[0144] In one embodiment, the GIPR antibody provided herein comprises:

[0145] a. A combination of light chain and heavy chain CDR1, CDR2, and CDR3 amino acid sequences: SEQ ID NO: 1, YAS or SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14;

[0146] b. A combination of light and heavy chain CDR1, CDR2, and CDR3 amino acid sequences: SEQ ID NO: 5, YAS or SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14;

[0147] c. A combination of light and heavy chain CDR1, CDR2, and CDR3 amino acid sequences: SEQ ID NO: 6, YAS or SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14;

[0148] d. A combination of light chain and heavy chain CDR1, CDR2, and CDR3 amino acid sequences: SEQ ID NO: 7, YAS or SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14;

[0149] e. A combination of light and heavy chain CDR1, CDR2, and CDR3 amino acid sequences: SEQ ID NO: 8, YAS or SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14;

[0150] f. A combination of light and heavy chain CDR1, CDR2, and CDR3 amino acid sequences: SEQ ID NO: 9, YAS or SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14;

[0151] g. A combination of light and heavy chain CDR1, CDR2, and CDR3 amino acid sequences: SEQ ID NO: 10, YAS or SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14; or

[0152] h. Combination of light chain and heavy chain CDR1, CDR2, and CDR3 amino acid sequences: SEQ ID NO: 11, YAS or SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14.

[0153] i. Combination of light and heavy chain CDR1, CDR2, and CDR3 amino acid sequences: SEQ ID NO: 1, YAS or SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14.

[0154] In one embodiment, the GIPR antibodies provided herein comprise one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0155] a. light chain variable domain amino acid sequence: SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37; and an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to any of the sequences thereof; and

[0156] b. Heavy chain variable domain amino acid sequence: SEQ ID NO: 38; and amino acid sequences that are at least 80%, at least 85%, at least 90%, or at least 95% identical thereto.

[0157] In another embodiment, the polynucleotide coding sequence for a GIPR antibody provided herein comprises one or two polynucleotide coding sequences, wherein each polynucleotide coding sequence is independently selected from the polynucleotide sequences listed below:

[0158] a. a light chain variable domain polynucleotide encoding sequence: SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47; and a polynucleotide encoding sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to any of the sequences thereof; and

[0159] b. Heavy chain variable domain polynucleotide encoding sequence: SEQ ID NO: 48; and polynucleotide encoding sequences that are at least 80%, at least 85%, at least 90%, or at least 95% identical thereto.

[0160] In one embodiment, the GIPR antibody provided herein comprises an amino acid sequence independently selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37.

[0161] In another embodiment, the GIPR antibody provided herein comprises an amino acid sequence independently selected from the group consisting of SEQ ID NO:38.

[0162] In one embodiment, the GIPR antibodies provided herein comprise a combination of light and heavy chain variable domain amino acid sequences independently selected from the group consisting of SEQ ID NO: 29 and SEQ ID NO: 38, SEQ ID NO: 30 and SEQ ID NO: 38, SEQ ID NO: 31 and SEQ ID NO: 38, SEQ ID NO: 32 and SEQ ID NO: 38, SEQ ID NO: 33 and SEQ ID NO: 38, SEQ ID NO: 34 and SEQ ID NO: 38, SEQ ID NO: 35 and SEQ ID NO: 38, SEQ ID NO: 36 and SEQ ID NO: 38, and SEQ ID NO: 37 and SEQ ID NO: 38.

[0163] In one embodiment, the GIPR antibody provided herein comprises an amino acid sequence independently selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37.

[0164] In another embodiment, the GIPR antibodies provided herein comprise a combination of light and heavy chain variable domain amino acid sequences independently selected from the group consisting of SEQ ID NO: 29 and SEQ ID NO: 38 (L1H1), SEQ ID NO: 30 and SEQ ID NO: 38 (L2H1), SEQ ID NO: 31 and SEQ ID NO: 38 (L3H1), SEQ ID NO: 32 and SEQ ID NO: 38 (L4H1), SEQ ID NO: 33 and SEQ ID NO: 38 (L5H1), SEQ ID NO: 34 and SEQ ID NO: 38 (L6H1), SEQ ID NO: 35 and SEQ ID NO: 38 (L7H1), SEQ ID NO: 36 and SEQ ID NO: 38 (L8H1), and SEQ ID NO: 37 and SEQ ID NO: 38 (L9H1).

[0165] The GIPR antibodies provided herein may also be referred to herein by the notation "LxHy," where "x" corresponds to the light chain variable region sequence designation; and "y" corresponds to the heavy chain variable region sequence designation. For example, L2H2 refers to an intact antibody having a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30 (L2) and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38 (H1).

[0166] In one embodiment, the GIPR antibodies provided herein comprise one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0167] a. Light chain constant region amino acid sequence: SEQ ID NO: 49; and

[0168] b. Heavy chain constant region amino acid sequence: SEQ ID NO: 50, SEQ ID NO: 51.

[0169] In one embodiment, the GIPR antibodies provided herein comprise one or two amino acid sequences, wherein each amino acid sequence is independently selected from the combination of the light chain and heavy chain constant region amino acid sequences listed below: SEQ ID NO: 49 and SEQ ID NO: 50, SEQ ID NO: 49 and SEQ ID NO: 51.

[0170] In one embodiment, the GIPR antibodies provided herein comprise the amino acid sequences of the light and heavy chain CDRs and FRs (framework) listed herein. The amino acid sequences of the FRs are contained within the amino acid sequences of the light or heavy chain variable domains and are not listed separately. In one embodiment, the antibody comprises a light chain CDR1 sequence listed herein. In another embodiment, the antibody comprises a light chain CDR2 sequence listed herein. In another embodiment, the antibody comprises a light chain CDR3 sequence listed herein. In another embodiment, the antibody comprises a heavy chain CDR1 sequence listed herein. In another embodiment, the antibody comprises a heavy chain CDR2 sequence listed herein. In another embodiment, the antibody comprises a heavy chain CDR3 sequence listed herein. In another embodiment, the antibody comprises a light chain FR1 sequence listed herein. In another embodiment, the antibody comprises a light chain FR2 sequence listed herein. In another embodiment, the antibody comprises a light chain FR3 sequence listed herein. In another embodiment, the antibody comprises a light chain FR4 sequence listed herein. In another embodiment, the antibody comprises a heavy chain FR1 sequence described herein. In another embodiment, the antibody comprises a heavy chain FR2 sequence described herein. In another embodiment, the antibody comprises a heavy chain FR3 sequence described herein. In a further embodiment, the antibody comprises a heavy chain FR4 sequence described herein.

[0171] In one embodiment, the light chain CDR3 sequence of the antibody does not differ from one of the light chain CDR3 amino acid sequences listed herein as SEQ ID NO: 3 or SEQ ID NO: 4 by more than 6, 5, 4, 3, 2, or 1 single amino acid additions, substitutions, and / or deletions. In another embodiment, the antibody further comprises 1, 2, 3, 4, 5, or 6 combinations of the light and heavy chain CDR sequences listed herein.

[0172] In one embodiment, the GIPR antibodies provided herein comprise a light chain variable domain amino acid sequence selected from the group consisting of L1 (SEQ ID NO: 29), L2 (SEQ ID NO: 30), L6 (SEQ ID NO: 34), L7 (SEQ ID NO: 35), L8 (SEQ ID NO: 36), and L9 (SEQ ID NO: 37) light chain variable domain sequences listed herein. In one embodiment, the light chain variable domain amino acid sequence of the GIPR antibody differs from the light chain variable domain amino acid sequence of one of L1 (SEQ ID NO: 29), L2 (SEQ ID NO: 30), L6 (SEQ ID NO: 34), L7 (SEQ ID NO: 35), L8 (SEQ ID NO: 36), and L9 (SEQ ID NO: 37) by 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues, wherein each difference is independently a deletion, insertion, or substitution of a single amino acid residue. In another embodiment, the light chain variable domain amino acid sequence of the GIPR antibody is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the light chain variable domain amino acid sequence of one of L1 (SEQ ID NO:29), L2 (SEQ ID NO:30), L6 (SEQ ID NO:34), L7 (SEQ ID NO:35), L8 (SEQ ID NO:36), and L9 (SEQ ID NO:37). In another embodiment, the light chain variable domain polynucleotide encoding sequence of the GIPR antibody is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the polynucleotide encoding sequence of one of L1 (SEQ ID NO: 29), L2 (SEQ ID NO: 30), L6 (SEQ ID NO: 34), L7 (SEQ ID NO: 35), L8 (SEQ ID NO: 36), and L9 (SEQ ID NO: 37). In another embodiment, the light chain variable domain polynucleotide encoding sequence of the GIPR antibody comprises a polynucleotide sequence that hybridizes under moderate conditions to the complement of the polynucleotide encoding sequence of the light chain variable domain of one of L1 (SEQ ID NO: 29), L2 (SEQ ID NO: 30), L6 (SEQ ID NO: 34), L7 (SEQ ID NO: 35), L8 (SEQ ID NO: 36), and L9 (SEQ ID NO: 37).In a further embodiment, the polynucleotide encoding sequence of the light chain variable domain of the GIPR antibody comprises a polynucleotide sequence that hybridizes under stringent conditions to a complementary sequence of a polynucleotide encoding sequence of the light chain variable domain of one of L1 (SEQ ID NO:29), L2 (SEQ ID NO:30), L6 (SEQ ID NO:34), L7 (SEQ ID NO:35), L8 (SEQ ID NO:36), and L9 (SEQ ID NO:37).

[0173] In one embodiment, the GIPR antibodies provided herein comprise a heavy chain variable domain amino acid sequence selected from the H1 (SEQ ID NO: 38) heavy chain variable domain sequence set forth herein.

[0174] In one embodiment, the antibody provided herein is an antibody comprising a combination of SEQ ID NO: 29 and SEQ ID NO: 38 (L1H1), SEQ ID NO: 30 and SEQ ID NO: 38 (L2H1), SEQ ID NO: 31 and SEQ ID NO: 38 (L3H1), SEQ ID NO: 32 and SEQ ID NO: 38 (L4H1), SEQ ID NO: 33 and SEQ ID NO: 38 (L5H1), SEQ ID NO: 34 and SEQ ID NO: 38 (L6H1), SEQ ID NO: 35 and SEQ ID NO: 38 (L7H1), SEQ ID NO: 36 and SEQ ID NO: 38 (L8H1), SEQ ID NO: 37 and SEQ ID NO: 38 (L9H1), or a desired phenotype thereof (e.g., IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, IgE, or IgD), or a Fab or F(ab')2 fragment thereof.

[0175] In one embodiment, the antibody provided herein is an antibody comprising a combination of L1H1 (SEQ ID NO:29 and SEQ ID NO:38), L2H1 (SEQ ID NO:30 and SEQ ID NO:38), L6H1 (SEQ ID NO:34 and SEQ ID NO:38), L7H1 (SEQ ID NO:35 and SEQ ID NO:38), L8H1 (SEQ ID NO:36 and SEQ ID NO:38), or L9H1 (SEQ ID NO:37 and SEQ ID NO:38), or a class-switched antibody thereof (e.g., IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, IgE, and IgD), or a Fab or F(ab')2 fragment thereof.

[0176] The antibodies provided herein may comprise any of the constant regions known in the art. The light chain constant region may be, for example, a kappa or lambda type light chain constant region, such as a mouse kappa or lambda type light chain constant region. The heavy chain constant region may be, for example, an α, δ, ε, γ, or μ type heavy chain constant region, such as a mouse α, δ, ε, γ, or μ type heavy chain constant region. In one embodiment, the light chain or heavy chain constant region is a fragment, derivative, variant, or mutant protein of a native constant region.

[0177] In one embodiment, the antibody provided herein further comprises a human constant light chain kappa domain or a fragment thereof. The amino acid sequence of the light chain constant region is as follows:

[0178] Human constant light chain kappa domain amino acid sequence: (SEQ ID NO: 49).

[0179] In one embodiment, an antibody provided herein further comprises a human heavy chain constant domain or a fragment thereof.

[0180] The amino acid sequence of the heavy chain constant region is as follows:

[0181] Human heavy chain constant region amino acid sequence (hIgG2): (SEQ ID NO: 50); and

[0182] Human heavy chain constant region amino acid sequence (hIgG4): (SEQ ID NO: 51).

[0183] In one embodiment, the GIPR antibodies provided herein comprise a light chain domain amino acid sequence selected from V1 (SEQ ID NO: 103) as set forth herein. In one embodiment, the light chain domain amino acid sequence of the GIPR antibody differs from the light chain domain amino acid sequence of V1 by 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues, wherein each difference is independently a deletion, insertion, or substitution of a single amino acid residue. In another embodiment, the light chain domain amino acid sequence of the GIPR antibody is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the light chain domain amino acid sequence of V1. In another embodiment, the polynucleotide encoding sequence of the light chain domain of the GIPR antibody is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the polynucleotide encoding sequence of one of V1 and V2. In another embodiment, the polynucleotide encoding sequence of the light chain domain of the GIPR antibody comprises a polynucleotide sequence that hybridizes under moderate conditions to the complement of the polynucleotide encoding sequence of the light chain domain of V1. In a further embodiment, the polynucleotide encoding sequence of the light chain domain of the GIPR antibody comprises a polynucleotide sequence that hybridizes under stringent conditions to the complement of the polynucleotide encoding sequence of the light chain domain of V1.

[0184] In one embodiment, the GIPR antibodies provided herein comprise a heavy chain domain amino acid sequence selected from W1 (SEQ ID NO: 111) and W2 (SEQ ID NO: 112) listed herein. In another embodiment, the heavy chain domain amino acid sequence of the GIPR antibody differs from the heavy chain domain sequence of one of W1 and W2 by 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues, wherein each difference is independently a deletion, insertion, or substitution of a single amino acid residue. In another embodiment, the heavy chain domain amino acid sequence of the GIPR antibody is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the heavy chain domain sequence of one of W1 and W2. In another embodiment, the heavy chain domain polynucleotide coding sequence of the GIPR antibody is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the polynucleotide coding sequence of one of W1 and W2. In another embodiment, the heavy chain domain polynucleotide coding sequence of the GIPR antibody comprises a polynucleotide sequence that hybridizes under moderate stringency conditions to the complement of the polynucleotide coding sequence of the heavy chain domain of one of W1 and W2. In one embodiment, the heavy chain domain polynucleotide coding sequence of the GIPR antibody comprises a polynucleotide sequence that hybridizes under stringent conditions to the complement of the polynucleotide coding sequence of the heavy chain domain of one of W1 and W2.

[0185] In another embodiment, the antibody provided herein is an antibody comprising a combination of V1W1 (SEQ ID NO: 103 and SEQ ID NO: 111) and V1W2 (SEQ ID NO: 103 and SEQ ID NO: 112).

[0186] In one embodiment, the GIPR antibodies provided herein are selected from a murine 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 diabody, a triabody, a tetrabody, a Fab fragment, a F(ab')x fragment, a domain antibody, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

[0187] In one embodiment, the GIPR antibodies provided herein are GIPR monoclonal antibodies.

[0188] In another embodiment, the GIPR antibody provided herein is a monoclonal antibody comprising a combination of amino acid sequences selected from the group consisting of SEQ ID NO:29 and SEQ ID NO:38, SEQ ID NO:30 and SEQ ID NO:38, SEQ ID NO:31 and SEQ ID NO:38, SEQ ID NO:32 and SEQ ID NO:38, SEQ ID NO:33 and SEQ ID NO:38, SEQ ID NO:34 and SEQ ID NO:38, SEQ ID NO:35 and SEQ ID NO:38, SEQ ID NO:36 and SEQ ID NO:38, and SEQ ID NO:37 and SEQ ID NO:38.

[0189] In one embodiment, the GIPR antibodies provided herein are murine GIPR antibodies. In another embodiment, the GIPR antibodies provided herein are humanized GIPR antibodies.

[0190] In one embodiment, the GIPR antibodies provided herein reduce the IC of human GIP signaling. 50 The value is about 1 nM to about 200 nM or about 1 nM to about 100 nM.

[0191] Antibodies and antibody fragments

[0192] In one embodiment, the antibodies provided herein are complete antibodies (including polyclonal, monoclonal, chimeric, humanized, or human antibodies with full-length heavy and / or light chains). In another embodiment, the antibodies provided herein are antibody fragments, such as F(ab')2, Fab, Fab', Fv, Fc, or Fd fragments, single domain antibodies, single chain antibodies, maximum antibodies (maxibodies), micro antibodies (minibodies), internal antibodies (intrabodies), two-chain antibodies, three-chain antibodies, four-chain antibodies, v-NAR, or bis-scFv (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology 23: 1126-1136). In another embodiment, the antibodies provided herein include antibody polypeptides as disclosed in U.S. Patent No. 6703199, including fibronectin polypeptide monoclonal antibodies. In a further embodiment, the antibodies provided herein include single chain polypeptides as disclosed in U.S. Patent Publication 2005 / 0238646.

[0193] In one embodiment, the variable region of the monoclonal antibody gene is amplified using nucleotide primers to express in the hybridoma. These primers can be synthesized by one of ordinary skill in the art or purchased from commercial sources. Mouse and human variable region primers include V Ha 、V Hb 、V Hc 、V Hd 、C H1 、V L , and C L Primers for the heavy and light chain variable regions can be purchased from commercial sources. These primers can be used to amplify the heavy or light chain variable regions, which are then inserted into vectors such as IMMUNOZAP. TM H or IMMUNOZAP TM These vectors are then introduced into E. coli, yeast, or mammalian-based expression systems. These methods can be used to produce large quantities of V H and V L Domain-fused single-chain proteins (see Bird et al., 1988, Science 242:423-426).

[0194] It will be appreciated by those skilled in the art that some proteins, such as antibodies, may be subjected to a variety of post-transcriptional modifications. The type and extent of these modifications depend on the host cell line and culture conditions used to express the protein. Such modifications include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation. The carboxyl terminal basic residues (e.g., lysine or arginine) of antibodies may be lost due to frequent modification by carboxypeptidases (see, Harris, 1995, Journal of Chromatography 705:129-134).

[0195] Mouse monoclonal antibodies can be produced using conventional hybridoma cell methods. The monoclonal antibody can be separated and purified by a variety of established techniques. Such separation techniques include affinity chromatography, size exclusion chromatography, and ion exchange chromatography (see, for example, Coligan pp. 2.7.1-2.7.12 and pp. 2.9.1-2.9.3; Baines et al., "Purification of Immunoglobulin G (IgG), " Methods in Molecular Biology, Vol. 10, pp. 79-104 (The Humana Press, Inc., 1992)). The monoclonal antibody can be purified by affinity chromatography using a suitable ligand screened based on the specific properties of the antibody (e.g., heavy chain or light chain isotype, binding specificity, etc.). Examples of suitable ligands for affinity chromatography include protein A, protein G, anti-constant region (light chain or heavy chain) antibodies, anti-idiotypic antibodies, and FGF21-associated proteins or fragments thereof or variants.

[0196] The complementary determining regions (CDRs) in the center of the antibody binding site can be used to perform affinity maturation to generate antibodies with increased affinity, such as antibodies with increased affinity for c-erbB-2 (Schier et al., 1996, J. Mol. Biol. 263:551-567). Therefore, this type of technology can be used to prepare antibodies against human GIPRs.

[0197] For example, antibodies against human GIPR can be used in in vitro or in vivo assays to detect the presence or absence of human GIPR.

[0198] Antibodies can also be prepared by any conventional technique. For example, they can be purified from cells that naturally express these antibodies (e.g., they can be purified from hybridomas that produce the antibodies) or produced in a recombinant expression system using any technique known in the art. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, edited by Kennet et al., Plenum Press (1980); and Antibodies: A Laboratory Manual, edited by Harlow and Land, Cold Spring Harbor Laboratory Press (1988). This is discussed in the nucleic acid section below.

[0199] Antibodies can be prepared and screened for desired properties by any known technique. Some techniques involve isolating a nucleic acid encoding a polypeptide chain (or portion thereof) of a relevant antibody (e.g., an anti-GIPR antibody) and manipulating the nucleic acid by recombinant DNA techniques. The nucleic acid can be fused to another related nucleic acid or modified (e.g., by mutagenesis or other conventional techniques) to add, delete, or replace one or more amino acid residues.

[0200] When it is desired to improve the affinity of an antibody according to the present invention comprising one or more of the above-described CDRs, affinity maturation can be achieved by various approaches including maintaining the CDRs (Yang et al., 1995, J. Mol. Biol. 254:392-403), chain replacement (Marks et al., 1992, Bio / Technology 10:779-783), using mutant strains of E. coli (Low et al., 1996, J. Mol. Biol. 250:350-368), DNA shuffling (Patten et al., 1997, Curr. Opin. Biotechnol. 8:724-733), phage display (Thompson et al., 1996, J. Mol. Biol. 256:7-88), and other PCR techniques (Crameri et al., 1998, Nature 391:288-291). All of these affinity maturation methods are discussed in Vaughan et al., 1998, Nature Biotechnology 16:535-539.

[0201] In one embodiment, the antibodies provided herein are anti-GIPR fragments. The fragments may consist entirely of antibody-derived sequences or may contain additional sequences. Examples of antigen-binding fragments include Fab, F(ab')2, single-chain antibodies, diabodies, triabodies, tetrabodies, and domain antibodies. Other examples are provided in Lunde et al., 2002, Biochem. Soc. Trans. 30:500-06.

[0202] Single-chain antibodies can be formed by connecting the heavy and light chain variable domains (Fv regions) via an amino acid bridge (short peptide linker), thereby obtaining a single polypeptide chain. L and V H ) DNAs. The resulting polypeptides can fold back on themselves to form antigen-binding monomers, or they can form multimers (e.g., dimers, trimers, or tetramers), depending on the length of the flexible linker between the two variable domains (Kortt et al., 1997, Prot. Eng. 10:423; Kortt et al. 2001, Biomol. Eng. 18:95-108). By combining different V domains containing polypeptides L and VH , can form multimeric scFvs with different binding phenotypes (Kriangkum et al., 2001, Biomol. Eng. 18: 31-40). Technologies developed for the production of single-chain antibodies include those described in U.S. Patent No. 4,946,778; Bird, 1988, Science 242: 423; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; Ward et al., 1989, Nature 334: 544-546; de Graaf et al., 2002, Methods Mol Biol. 178: 379-87. Single-chain antibodies derived from the antibodies provided herein include, but are not limited to, scFvs comprising the variable domain combination L1H1, all of which are encompassed herein.

[0203] Antigen-binding fragments derived from antibodies can also be obtained by proteolytic hydrolysis of the antibody, for example, by digestion of intact antibodies with pepsin or papain according to conventional methods. For example, antibodies can be enzymatically cleaved with pepsin to provide an SS fragment known as F(ab')2 to produce antibody fragments. This fragment can be further cleaved using a sulfhydryl reducing agent to produce a 3.5S Fab' monovalent fragment. Alternatively, this cleavage reaction can be performed using a sulfhydryl protecting group to cleave disulfide bonds; alternatively, enzymatic cleavage using papain can be used to directly produce two monovalent Fab fragments and an Fc fragment. These methods are described, for example, in Goldenberg, U.S. Patent No. 4,331,647, Nisonoff et al., 1960, Arch. Biochem. Biophys. 89:230; Porter, 1959, Biochem. J. 73:119; Edelman et al., Methods in Enzymology 1:422 (Academic Press, 1967); and Andrews and Titus, JA Current Protocols in Immunology (Coligan et al., eds., John Wiley & Sons, 2003), pp. 2.8, 1-2.8.10 and pp. 2.10A.1-2.10A.5. Other methods of cleaving antibodies, such as preparing heavy chains to form monovalent heavy and light chain fragments (Fd), further cleaving the fragments or using other enzymatic, chemical or genetic techniques, can also be used, as long as the fragments bind to an antigen that can be recognized by the intact antibody.

[0204] Another form of antibody fragment is a peptide comprising one or more antibody complementary determining regions (CDRs). CDRs can be obtained by constructing polypeptides encoding the relevant CDRs. For example, such polypeptides can be prepared by using polymerase chain reaction to synthesize variable regions using mRNA from antibody-producing cells as a template, see, for example, Larrick et al., 1991, Methods: A Companion to Methods in Enzymology 2: 106; Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al., ed., 166 pages (Cambridge University Press, 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," Monoclonal Antibodies: Principles and Applications, Birch et al., ed., 137 pages (Wiley-Liss, Inc., 1995). The antibody fragment may further comprise at least one variable domain of an antibody described herein. Thus, for example, a V region domain may be monomeric and be a V H or V L domain, which may be at least equal to 1 x 10 -7 M or higher affinity independently binds to GIPR.

[0205] The variable region domain may be any naturally occurring variable domain or a genetically engineered version thereof. A genetically engineered version refers to a variable region domain produced using recombinant DNA engineering techniques. Such genetically engineered versions include, for example, those generated from a specific antibody variable region by insertion, deletion, or alteration of the specific antibody amino acid sequence. Specific examples include variable region domains comprising only one CDR, optionally one or more framework amino acids from one antibody, and the remainder of a variable region domain from another antibody, assembled by genetic engineering.

[0206] The variable region domain may be covalently linked to at least one other antibody domain or fragment thereof at the C-terminal amino acid. Thus, for example, the V H Immunoglobulin C H1 Similarly, V L The domain can be combined with C KIn this manner, for example, the antibody may be a Fab fragment wherein the antigen binding domains comprise their C-termini connected to the C H1 and C K Domain covalently linked joint V H and V L Domain. Other amino acids can be used to extend C H1 domains, for example to provide a hinge region or a portion of a hinge domain as in a Fab' fragment or to provide other domains, such as an antibody C H2 and C H3 domain.

[0207] Antibody derivatives and variants

[0208] For example, the nucleotide sequences encoding the amino acid sequences L1 and H can be altered by random mutagenesis or by site-directed mutagenesis (e.g., oligonucleotide-induced site-directed mutagenesis) to produce altered polynucleotides comprising one or more specific nucleotide substitutions, deletions, or insertions compared to the non-mutated polynucleotide. Examples of techniques for producing such alterations are described in Walder et al., 1986, Gene 42:133; Bauer et al., 1985, Gene 37:73; Craik, 1985, BioTechniques, 3:12-19; ​​Smith et al., 1981, Genetic Engineering: Principles and Methods, Plenum Press; and U.S. Patent Nos. 4,518,584 and 4,737,462. These and other methods can be used to produce derivatives of anti-GIPR antibodies that have desired properties, such as increased affinity, avidity, or specificity for GIPR, increased in vivo or in vitro activity or stability, or reduced in vivo side effects compared to the underivatized antibody.

[0209] Other anti-GIPR antibody derivatives in the field include covalent or aggregated conjugates of anti-GIPR antibodies or fragments thereof with other proteins or polypeptides, for example, by expressing a recombinant fusion protein comprising a heterologous polypeptide fused to the N-terminus or C-terminus of an anti-GIPR antibody polypeptide. For example, the binding peptide can be a heterologous signal (or guide) polypeptide, such as a yeast alpha factor leader peptide or a peptide such as an epitope tag. Antibodies comprising fusion proteins can contain peptides (e.g., polyhistidine) added to aid in the purification or identification of the antibody. Antibodies can also be linked to FLAG peptides, as described in Hopp et al., 1988, Bio / Technology 6:1204 and U.S. Patent No. 5,011,912. FLAG peptides have high antigenicity and provide an epitope that is reversibly bound by a specific monoclonal antibody (mAb), allowing rapid detection and convenient purification of expressed recombinant proteins. Reagents for preparing fusion proteins in which the FLAG peptide is fused to a given polypeptide are commercially available (Sigma-Aldrich, St. Louis, MO). In another embodiment, oligomers comprising one or more antibodies can be used as GIPR antagonists or higher order oligomers can be used. The oligomers can be in the form of covalently or non-covalently linked dimers, trimers, or higher oligomers. Oligomers comprising two or more antibodies can be used, one example of which is a homodimer. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, and the like.

[0210] One embodiment is directed to oligomers comprising multiple antibodies linked by covalent or non-covalent interactions between peptide moieties fused to the antibodies. Such peptides can be peptide spacers or peptides having properties that promote oligomerization. Leucine zippers and certain antibody-derived polypeptides are peptides that can promote antibody oligomerization, as described in detail below.

[0211] In a specific embodiment, the oligomer comprises two to four antibodies. The antibodies in the oligomer can be in any form, such as any form described above, such as a variant or fragment. Preferably, the oligomer comprises an antibody with GIPR binding activity.

[0212] In one embodiment, oligomers are prepared using polypeptides derived from immunoglobulins. Preparations comprising heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) have been described, for example, in Ashkenazi et al., 1991, PNAS USA 88:10535; Byrn et al., 1990, Nature 344:677; and Hollenbaugh et al., Construction of Immunoglobulin Fusion Proteins, Current Protocols in Immunology, Suppl. 4, pp. 10.19.1-10.19.11. One embodiment herein is directed to a dimer comprising two fusion proteins produced by fusion of a GIP-binding fragment of an anti-GIPR antibody with the Fc region of an antibody. Dimers can be prepared, for example, by inserting a gene encoding the fusion protein into an appropriate expression vector, expressing the fusion gene in a host cell transformed with the recombinant expression vector, and allowing the expressed fusion protein to assemble like an antibody molecule, wherein interchain disulfide bonds between the Fc portions form a dimer.

[0213] As used herein, the term "Fc polypeptide" includes polypeptides derived from the Fc region of an antibody in both native and mutein forms. Truncated forms of such polypeptides that include a hinge region that promotes dimerization are also included. Fusion proteins containing the Fc portion (and oligomers formed therefrom) offer the advantage of convenient purification by affinity chromatography on Protein A or Protein G columns.

[0214] One suitable Fc polypeptide described in PCT application WO 93 / 10151 (incorporated herein by reference) is a single-chain polypeptide extending from the N-terminal hinge region to the native C-terminus of the Fc region of a human IgG1 antibody. Another useful Fc polypeptide is the Fc mutein described in U.S. Patent No. 5,457,035 and Baum et al., 1994, EMBO J. 13:3992-4001. The amino acid sequence of this mutein is identical to the amino acid sequence of the native Fc sequence set forth in WO 93 / 10151, except that amino acid 19 is changed from leucine to alanine, amino acid 20 is changed from leucine to glutamine, and amino acid 22 is changed from glycine to alanine. This mutein exhibits reduced affinity for Fc receptors. In other embodiments, the heavy and / or light chains of an anti-GIPR antibody may be substituted with the variable portion of the heavy and / or light chain of an antibody.

[0215] Alternatively, the oligomer is a fusion protein comprising multiple antibodies, with or without spacer peptides. Suitable spacer peptides are described in US Pat. Nos. 4,751,180 and 4,935,233.

[0216] Another method for preparing oligomeric antibodies involves the use of leucine zippers. Leucine zipper domains are peptides that promote the oligomerization of proteins in which they are present. Leucine zippers were initially discovered in several DNA-binding proteins (Landschulz et al., 1988, Science 240:1759) and have since been found in a variety of different proteins. Among the known leucine zippers are natural peptides or derivatives thereof that can dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT application WO 94 / 10308, and a leucine zipper derived from surfactant protein D (SPD) is described in Hoppe et al., 1994, FEBS Letters 344:191, incorporated herein by reference. The use of modified leucine zippers that allow stable trimerization of heterologous proteins fused thereto is described in Fanslow et al., 1994, Semin. Immunol. 6:267-78. In one method, a recombinant fusion protein comprising an anti-GIPR antibody fragment or derivative fused to a leucine zipper peptide is expressed in a suitable host cell, and soluble oligomeric anti-GIPR antibody fragments or derivatives are collected from the culture supernatant.

[0217] In another embodiment, the antibody derivative may comprise at least one of the CDRs disclosed herein. For example, one or more CDRs may be integrated into known antibody framework regions (IgG1, IgG2, etc.) or combined with an appropriate carrier to enhance its half-life. Suitable carriers include, but are not limited to, Fc, albumin, transferrin and similar substances. These and other suitable carriers are known in the art. The CDR-binding peptide may be a monomer, dimer, tetramer or other form. In one embodiment, one or more water-soluble polymers are combined at one or more specific sites of the binding agent, for example, at the amino terminus. In one example, the antibody derivative comprises one or more water-soluble polymer attachments including, but not limited to, polyethylene glycol, polyoxyethylene glycol or polypropylene glycol. See, for example, U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, and 4,179,337. In some embodiments, the derivatives comprise one or more monomethoxy polyethylene glycols, dextran, cellulose, or other carbohydrate-based polymers, poly(N-vinylpyrrolidone), polyethylene glycol, polyoxyethylene polyols (e.g., glycerol), and polyvinyl alcohol, as well as mixtures of such polymers. In some embodiments, one or more water-soluble polymers are randomly attached to one or more side chains. In some embodiments, PEG can enhance the therapeutic effect of binding agents, such as antibodies. Some such methods are described, for example, in U.S. Patent No. 6,133,426, which is incorporated herein by reference for any purpose.

[0218] It should be understood that the antibodies provided herein may have at least one amino acid replacement, as long as the antibody retains binding specificity. Therefore, modifications of antibody structure are included in the scope of this article. These may include amino acid replacements that do not destroy the antibody GIPR binding ability, which may be conservative or non-conservative. Conservative amino acid replacements may include non-natural amino acid residues, which are typically integrated through chemical peptide synthesis rather than biological system synthesis. These include peptidomimetics and other reverse or inverted forms of amino acid moieties. Conservative amino acid replacements may also involve replacing natural amino acid residues with non-natural residues, which have little or no effect on the polarity or charge of the amino acid residues at the site. Non-conservative replacements may involve exchanging a member of a class of amino acids or amino acid analogs with a member of another class of amino acids having different physical properties (e.g., volume, polarity, hydrophobicity, charge).

[0219] Furthermore, those skilled in the art can generate variants to be tested that include amino acid substitutions at each desired amino acid residue. Such variants can be screened using activity assays known to those skilled in the art. Such variants can be used to collect information about appropriate variants. For example, if a certain amino acid residue is found to cause activity destruction, undesirable reduction, or inappropriate activity, variants with such changes can be avoided. In other words, based on the information collected from these routine tests, those skilled in the art can easily determine which amino acids should be avoided from further substitution (alone or in combination with other mutations).

[0220] A skilled artisan can use known techniques to determine suitable variants of polypeptides such as those listed herein. In some embodiments, one skilled in the art can identify suitable regions of the molecule that, when altered, will not destroy the activity by targeting regions that are not important for activity. In some embodiments, residues or portions of the molecule that are conserved in similar polypeptides can be identified. In some embodiments, even regions important for biological activity or structure can be conservatively substituted without destroying the biological activity or having adverse effects on the polypeptide structure. In addition, one skilled in the art can examine structure-function studies to identify residues in similar polypeptides that are important for activity or structure. Given this comparison, the importance of amino acid residues in the protein corresponding to amino acid residues important for activity or structure in similar proteins can be predicted. One skilled in the art can select chemically similar amino acid replacements for these predicted important amino acid residues.

[0221] Those skilled in the art can also analyze the three-dimensional structure and amino acid sequence related to the structure of similar polypeptides. Given this information, those skilled in the art can predict the amino acid residue alignment of the antibody with respect to the three-dimensional structure. In some embodiments, those skilled in the art may choose not to make significant changes to amino acid residues predicted to be on the surface of the protein because such residues may be involved in important interactions with other molecules. Many scientific publications are devoted to the prediction of secondary structure. See, Moult, 1996, Curr. Op. Biotech. 7: 422-427, Chou et al., 1974, Biochemistry 13: 222-245; Chou et al., 1974, Biochemistry 113: 211-222; Chou et al., 1978, Adv. Enzymol. Relat. Areas Mol. Biol. 47: 45-148; Chou et al., 1979, Ann. Rev. Biochem. 47: 251-276 and Chou et al., Biophys. J. 26: 367-384. In addition, computer programs are currently available to assist in the prediction of secondary structure. For example, two polypeptides or proteins with a sequence identity greater than 30% or a similarity greater than 40% generally have similar higher-order structures. The recent growth of the Protein Structure Database (PDB) has enhanced the predictability of secondary structure, including the potential number of folds in a polypeptide or protein structure. See, Holm et al., 1999, Nucl. Acid. Res. 27: 244-247. It has been shown (Brenner et al., 1997, Curr. Op. Struct. Biol. 7: 369-376) that there are a finite number of folds in a given polypeptide or protein and once a critical number of structures are determined, structure prediction will become significantly more accurate.

[0222] Other methods for predicting secondary structure include "threading" (Jones, 1997, Curr. Opin. Struct. Biol., 7:377-87; Sippl et al., 1996, Structure 4:15-19; "profile analysis" (Bowie et al., 1991, Science 253:164-170; Gribskov et al., 1990, Meth. Enzym. 183:146-159; Gribskov et al., 1987, Proc. Nat. Acad. Sci. USA 84:4355-4358) and "evolutionary linkage" (see Holm, supra (1999), and Brenner, supra (1997). In some embodiments, antibody variants include glycosylation variants in which the number and / or type of glycosylation sites are altered compared to the amino acid sequence of the parent polypeptide. In some embodiments, the variant has a greater or lesser number of N-linked glycosylation sites compared to the native protein. Alternatively, substitutions that remove the sequence can remove existing N-linked carbohydrate chains. Also provided are rearrangements of N-linked carbohydrate chains in which one or more N-linked carbohydrate sites (typically those occurring in nature) are removed and one or more new N-linked sites are created. Other preferred antibody variants include cysteine ​​variants in which one or more cysteine ​​residues are deleted or replaced by another amino acid (e.g., serine) compared to the parent amino acid sequence. Cysteine ​​variants can be used when the antibody must fold into a biologically active conformation (e.g., after isolating soluble inclusion bodies). Cysteine ​​variants typically have fewer cysteine ​​residues than the native protein and typically have an even number of cysteines to minimize interactions caused by unpaired cysteines.

[0223] One skilled in the art can determine the desired amino acid substitution (conservative or non-conservative) when such substitution is desired. In some embodiments, amino acid substitutions can be used to identify important residues of human GIPR antibodies or to increase or decrease the affinity of the human GIPR antibodies described herein.

[0224] According to some embodiments, preferred amino acid substitutions are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity and / or (5) confer or modify other physicochemical or functional properties on such polypeptides. According to some embodiments, single or multiple amino acid substitutions (in some embodiments, conservative amino acid substitutions) may be made in the naturally occurring sequence (in some embodiments, in portions of the polypeptide outside of the domains forming intermolecular contacts). In some embodiments, conservative amino acid substitutions generally do not substantially alter the structural properties of the parent sequence (e.g., the replacement amino acid should not disrupt helices present in the parent sequence or interfere with other types of secondary structure that characterize the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structure are described in Proteins, Structures and Molecular Principles, Creighton, ed., WH Freeman and Company (1984); Introduction to Protein Structure, Branden and Tooze, eds., Garland Publishing (1991); and Thornton et al., 1991, Nature 354:105, which are incorporated herein by reference.

[0225] In some embodiments, the antibodies provided herein may be chemically bonded to polymers, lipids, or other moieties.

[0226] Antigen binding agents may comprise at least one CDRs described herein incorporated into a biocompatible framework structure. In one example, the biocompatible framework structure comprises a polypeptide or portion thereof sufficient to form a conformationally stable structural support or framework or scaffold that can display one or more amino acid sequences (e.g., CDRs, variable regions, etc.) that can bind to an antigen in a confined surface area. Such structures may be naturally occurring polypeptides or polypeptide "folds" (structural motifs), or may have one or more modifications relative to a natural polypeptide or fold, such as amino acid additions, deletions, or substitutions. These scaffolds may be derived from polypeptides of any species (or more than one species), for example, humans, other mammals, other vertebrates, invertebrates, bacteria, or viruses.

[0227] Biosoluble backbone structures are typically based on protein scaffolds or backbones rather than immunoglobulin domains. For example, those based on fibronectin, ankyrin, lipocalin, neotumor suppressor protein, cytochrome b, CP1 zinc finger protein, PST1, coiled-coil, LACI-D1, Z domains, and amylase inhibitory peptide domains can be used (see, e.g., Nygren and Uhlen, 1997, Current Opinion in Structural Biology 7:463-469).

[0228] In addition, those skilled in the art will recognize that suitable binding agents include portions of these antibodies, such as one or more heavy chain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3, as specifically disclosed herein. At least one heavy chain CDR1, CDR2, CDR3, or CDR1, CDR2, and CDR3 region has at least one amino acid substitution, as long as the antibody retains the binding specificity of the non-substituted CDR. The non-CDR portion of the antibody can be a non-protein molecule, wherein the binding agent cross-blocks the binding of the antibody disclosed herein to human GIPR and / or inhibits GIP signaling through the receptor. The non-CDR portion of the antibody can be a non-protein molecule, wherein the antibody exhibits a similar binding pattern to human GIP peptide as exhibited by at least one of antibodies L1H1 / L8H1 in a competitive binding assay, and / or neutralizes GIP activity. The non-CDR portion of the antibody can be composed of amino acids, wherein the antibody is a recombinant binding protein or a synthetic peptide, and wherein the recombinant binding protein cross-blocks the binding of the antibody disclosed herein to human GIPR and / or neutralizes GIP activity in vivo or in vitro. The non-CDR portion of the antibody may be composed of amino acids, wherein the antibody is a recombinant antibody and the recombinant antibody exhibits a similar type of binding to a human GIPR peptide as exhibited by at least one of antibodies L1H1 / L8H1 in a competition binding assay, and / or neutralizes GIP signaling.

[0229] Fusion protein of GIPR antibody and FGF21

[0230] In one embodiment, the present invention provides a fusion protein of a GIPR antibody and FGF21, which comprises an antibody that can specifically bind to GIPR, and one, two, three, four, five, six, seven or eight FGF21 fragments; the fusion protein connects the amino terminus of an FGF21 fragment to the carboxyl terminus of the light chain or heavy chain of the GIPR antibody through a peptide linker sequence.

[0231] In one embodiment, the present invention provides a fusion protein of a GIPR antibody and FGF21, which comprises an antibody that can specifically bind to GIPR, and one, two, three or four FGF21 fragments; the fusion protein connects the amino terminus of an FGF21 fragment to the carboxyl terminus of the light chain or heavy chain of the GIPR antibody through a peptide linker sequence.

[0232] In one embodiment, the present invention provides a fusion protein of a GIPR antibody and FGF21, which comprises an antibody that can specifically bind to GIPR and one or two FGF21 fragments; the fusion protein connects the amino terminus of an FGF21 fragment to the carboxyl terminus of the light chain or heavy chain of the GIPR antibody through a peptide linker sequence.

[0233] In another embodiment, the present invention provides an FGF21 fusion protein, which comprises a GIPR antibody and one or two FGF21 fragments; the fusion protein connects the amino terminus of the FGF21 fragment to the carboxyl terminus of the GIPR antibody light chain through a peptide linker sequence (Linker): N'-R-Linker-FGF21-C'; or connects the amino terminus of the FGF21 fragment to the carboxyl terminus of the GIPR antibody heavy chain: N'-R-Linker-FGF21-C'; wherein: N' represents the amino terminus of the fusion protein polypeptide chain, C' represents the carboxyl terminus of the fusion protein polypeptide chain, FGF21 represents the FGF21 fragment, R is the amino acid sequence of the light chain or heavy chain of the GIPR antibody, and Linker represents the peptide linker sequence.

[0234] In one embodiment, in the FGF21 fusion protein provided herein, the FGF21 fragments are each independently selected from the following amino acid sequences: SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, and 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:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118 and SEQ ID NO:122.

[0235] In one embodiment, in the FGF21 fusion protein provided herein, the sequences of the peptide linkers each independently contain from 1 to 200 amino acid amines, from 2 to 100 amino acid amines, from 5 to 50 amino acid amines, from 6 to 25 amino acid amines, or from 10 to 20 amino acid amines.

[0236] In another embodiment, in the FGF21 fusion protein provided herein, the sequences of the peptide linkers are independently selected from the following amino acid sequences: SEQ ID NO:61, SEQ ID NO:62, and SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67 and SEQ ID NO:68.

[0237] Nucleic Acids

[0238] In one aspect, isolated nucleic acid molecules are provided herein. The nucleic acid molecules include, for example, a polynucleotide encoding all or part of an antibody, such as one or both chains of an antibody or FGF21 fusion protein, or a fragment, derivative, mutein, or variant thereof; a polynucleotide sufficient for use as a hybridization probe; a PCR primer or a sequencing primer for identifying, analyzing, mutating, or amplifying a polynucleotide encoding a polypeptide; an antisense nucleic acid for inhibiting expression of a polynucleotide, and its complementary sequence. The nucleic acids can be of any length. For example, they can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000, or more nucleotides in length, and / or include one or more additional sequences, such as regulatory sequences, and / or be part of a larger nucleic acid, such as a vector. The nucleic acid can be single-stranded or double-stranded and comprise RNA and / or DNA nucleotides, as well as artificial variants thereof (eg, peptide nucleic acids).

[0239] Nucleic acids encoding antibody polypeptides (e.g., heavy or light chain, variable domain only or full length) can be isolated from B cells of mice immunized with GIPR antigens. Nucleic acids encoding antibodies or FGF21 fusion proteins can be isolated by conventional methods such as polymerase chain reaction (PCR).

[0240] The nucleic acid sequences encoding the heavy and light chain variable regions are shown above. A skilled artisan will appreciate that due to the degeneracy of the genetic code, each polypeptide sequence disclosed herein may be encoded by a greater number of other nucleic acid sequences. Provided herein are degenerate nucleotide sequences encoding the antibodies or FGF21 fusion proteins provided herein.

[0241] Further provided herein are nucleic acids that hybridize to other nucleic acids (e.g., nucleic acids comprising any of the nucleotide sequences of L1H1 / L8H1) under specific hybridization conditions. Methods for hybridizing nucleic acids are well known in the art. See, for example, Current Protocols in Molecular Biology, John Wiley & Son (1989), 6.3.1-6.3.6. As defined herein, for example, moderately stringent conditions use a prewash solution comprising 5x sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), a hybridization buffer of approximately 50% formamide, 6x SSC, and a hybridization temperature of 55°C (or other similar hybridization solutions, such as those containing 50% formamide, hybridizing at 42°C), and an elution condition of 60°C using 0.5x SSC, 0.1% SDS. Stringent hybridization conditions involve hybridization in 6x SSC at 45°C, followed by one or more washes in 0.1x SSC, 0.2% SDS at 68°C. In addition, those skilled in the art can manipulate hybridization and / or washing conditions to increase or decrease hybridization stringency so that nucleic acids comprising nucleotide sequences that are at least 65, 70, 75, 80, 85, 90, 95, 98 or 99% homologous to each other can generally still hybridize to each other. The basic parameters affecting the selection of hybridization conditions and guidance for designing appropriate conditions are listed in, for example, Sambrook, Fritsch and Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Chapters 9 and 11; Current Protocols in Molecular Biology, 1995, edited by Ausubel et al., John Wiley & Sons, Inc., Sections 2.10 and 6.3-6.4) and can be easily determined by those with ordinary skill in the art based on, for example, the length and / or base composition of DNA. Changes can be introduced into nucleic acids by mutation, thereby resulting in changes in the amino acid sequence of the polypeptide (e.g., antigen-binding protein) encoded therein. Any technique known in the art can be used to introduce mutations. In one embodiment, one or more specific amino acid residues are changed using, for example, a site-directed mutagenesis scheme. In another embodiment, one or more randomly selected residues are altered using, for example, a random mutagenesis protocol. Regardless of how they are generated, the mutant polypeptides can be expressed and screened for desired properties.

[0242] Mutations can be introduced into nucleic acids without significantly changing the biological activity of their encoded polypeptides. For example, nucleotide substitutions can be performed that result in amino acid replacements at non-essential amino acid residues. In one embodiment, the nucleotide sequences provided herein for L1 to L9 and H1 or FGF21 fusion proteins, or fragments, variants, or derivatives thereof, are mutated such that one or more deletions or replacements of amino acid residues encoding L1 to L9 and H as shown herein are obtained, resulting in two or more residues with different sequences. In another embodiment, mutagenesis inserts an amino acid near one or more amino acid residues of L1 to L9 and H1 or FGF21 fusion proteins as shown herein, resulting in two or more residues with different sequences. Alternatively, one or more mutations can be introduced into nucleic acids to selectively alter the biological activity of their encoded polypeptides (e.g., binding to GIPRs). For example, the mutation can quantitatively or qualitatively alter the biological activity. Examples of quantitative changes include increasing, decreasing, or eliminating the activity. Examples of qualitative changes include altering the antigenic specificity of an antibody or FGF21 fusion protein.

[0243] In another aspect, the present invention provides nucleic acid molecules suitable for use as primers or hybridization probes for detecting nucleic acid sequences herein. The nucleic acid molecules herein may comprise only a portion of a nucleic acid sequence encoding a full-length polypeptide herein, for example, a fragment that can be used as a probe or primer or encodes an active portion of a polypeptide herein (e.g., a GIPR binding portion).

[0244] Probes based on the nucleic acid sequences herein can be used to detect the nucleic acid or similar nucleic acids, such as transcripts encoding the polypeptides herein. The probes can contain labeling groups, such as radioisotopes, fluorescent compounds, enzymes, or enzyme cofactors. Such probes can be used to identify cells expressing the polypeptides.

[0245] In another aspect, provided herein are vectors comprising a nucleic acid encoding a polypeptide herein or a portion thereof. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors.

[0246] The recombinant expression vectors herein may comprise a nucleic acid herein in a form suitable for expression of the nucleic acid in a host cell. The recombinant expression vectors include one or more regulatory sequences, selected based on the host cell to be used for expression, which are operably linked to the pre-expressed nucleic acid sequence. Regulatory sequences include those that direct constitutive expression of the nucleotide sequence in a variety of host cell types (e.g., SV40 early gene enhancer, Rous sarcoma virus promoter, and cytomegalovirus promoter), those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences, see Voss et al., 1986, Trends Biochem. Sci. 11:287, Maniatis et al., 1987, Science 236:1237, the entire contents of which are incorporated herein by reference), and those that direct inducible expression of the nucleotide sequence in response to a specific treatment or condition (e.g., metallothionein promoters in mammalian cells and tetracycline-responsive and / or streptomycin-responsive promoters in both prokaryotic and eukaryotic systems (supra)). Those skilled in the art will appreciate that the design of the expression vector depends on factors such as the choice of host cells to be transformed, the desired protein expression level, etc. The expression vectors herein can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by the nucleic acids described herein.

[0247] On the other hand, the present invention provides a host cell that can be introduced into the expression vector herein. The host cell can be any prokaryotic or eukaryotic cell. Prokaryotic host cells include gram-negative or gram-positive organisms, such as Escherichia coli or bacillus. More advanced eukaryotic cells include insect cells, yeast cells and mammalian-derived cell lines. Examples of suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells or their derivatives such as Veggie CHO and related cell lines grown in serum-free medium (see Rasmussen et al., 1998, Cytotechnology 28:31) or CHO strain DXB-11, which lacks DHFR (see Urlaub et al., 1980, Proc. Natl. Acad, Sci. USA 77:4216-20). Other CHO cell lines include CHO-K1 (ATCC #CCL-61), EM9 (ATCC #CRL-1861), and UV20 (ATCC #CRL-1862), other host cells include the COS-7 line of monkey kidney cells (ATCC #CRL-1651) (see Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL-163), AM-1 / D cells (described in U.S. Patent No. 6,210,924), HeLa cells, BHK (ATCC CRL-10) cell line, CV1 / EBNA cell line derived from African green monkey kidney cell line CV1 (ATCC CCL-70) (see McMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney cells such as 293, 293EBNA or MSR 293, human epithelial A431 cells, human C010205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro cultures of primary tissues, primary transplants, HL-60, U937, HaK, or Jurkat cells. Appropriate cloning and expression vectors for bacterial, fungal, yeast, and mammalian cell hosts are described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, 1985).

[0248] Can be introduced into prokaryotic or eukaryotic cell by traditional transformation or transfection technique.For stable mammalian transfection, depending on the expression vector and transfection technique used, it is known that only a small portion of cells can incorporate foreign DNA into their genome.In order to identify and screen these integrants, usually the gene encoding screening marker (such as antibiotic resistance) is introduced into the host cell together with the gene of interest.Preferred screening markers include those that can confer drug (such as G418, hygromycin and methotrexate) resistance.In other methods, can be identified by drug screening to comprise the stably transfected cell that is introduced into nucleic acid (for example, the cell that has integrated the screening gene can survive, and other cells then die).

[0249] The transformed cells can be cultured under conditions that enhance expression of the polypeptide, and the polypeptide can be recovered by conventional protein purification methods. One such purification method is described in the Examples below. The polypeptides contemplated for use herein include substantially homologous recombinant mammalian anti-GIPR antibodies or FGF21 fusion protein polypeptides that are substantially free of contaminating endogenous materials.

[0250] Activity of GIPR antibodies

[0251] The activity of GIPR antibodies refers to the therapeutic biological effects exhibited by the antibodies provided herein after specifically binding to GIPR and inhibiting or blocking GIP signaling, such as the treatment of non-alcoholic steatohepatitis, obesity, and / or type 2 diabetes. The term "reducing the biological activity of GIP signaling" or "inhibiting or blocking the biological activity of GIP signaling" refers to the binding of GIPR antibodies or their fusion proteins with FGF21 to GIPR in vivo and inhibiting or blocking the cellular stress response downstream of the receptor caused by GIP. Reactions include, but are not limited to, increased insulin secretion, promotion of fat storage, and inhibition of lipolysis. In one embodiment, provided herein are murine or humanized antibodies that can specifically bind to human GIPR. Such antibodies include antagonistic or neutralizing antibodies that can reduce or neutralize GIP signaling.

[0252] In one embodiment, the antibodies provided herein are effective in reducing the IC of human GIP signaling. 50 In another embodiment, the antibodies provided herein have an IC value of about 0.01 nM to about 500 nM, about 0.1 nM to about 200 nM, about 0.5 nM to about 200 nM, about 1 nM to about 200 nM, or about 10 nM to about 100 nM. 50 In another embodiment, the antibodies provided herein have an IC value of about 1 nM to about 200 nM in reducing human GIP signaling. 50In another embodiment, the antibodies provided herein have an IC value of about 10 nM to about 100 nM in reducing human GIP signaling. 50 The value is about 1 nM, about 2 nM, about 5 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, or about 100 nM.

[0253] In one embodiment, the GIPR antibodies provided herein have one or more of the following properties when binding to human GIPR:

[0254] a. When binding to human GIPR with a K substantially similar to that of the reference antibody d ;

[0255] b. When inhibiting GIP activation of human GIPR with an IC substantially similar to that of the reference antibody 50 ;and

[0256] c. Cross-competition with the reference antibody for binding on human GIPR.

[0257] In another embodiment, the GIPR antibody described herein is an antibody having one or more of the following properties:

[0258] a. When inhibiting GIP activation of human GIPR, its IC50 is the same as or better than the reference GIPR antibody; and

[0259] b. The GIPR antibody cross-competes with the reference GIPR antibody for binding to human GIPR.

[0260] In this context, the term "substantially similar" means that the IC 50 or K d IC of comparable or reference antibody 50 or K d In one embodiment, the reference antibody comprises, for example, an antibody having a light chain of SEQ ID NO: 29 and a heavy chain of SEQ ID NO: 38. In another embodiment, the reference antibody comprises GIPR antibodies L1H1, L9H1.

[0261] Biological activity of the fusion protein of GIPR antibody and FGF21

[0262] The biological activity of the fusion protein of GIPR antibody and FGF21 includes two aspects: the biological activity of FGF21 and the activity of GIPR antibody. The activity of GIPR antibody is as described above. "FGF21 biological activity" refers to the biological activity of the fusion protein of GIPR antibody and FGF21 binding to and activating FGF21 receptor in vivo and causing cell stress response, and showing therapeutic effects, such as non-alcoholic steatohepatitis, obesity, or type 2 diabetes. The aforementioned cell stress response includes but is not limited to increasing insulin secretion, inhibiting glucagon secretion, suppressing appetite, weight loss, inducing oversatiety, inhibiting apoptosis, inducing pancreatic cell proliferation, and pancreatic cell differentiation. Combining the biological activities of FGF21 and GIPR antibody, the FGF21 fusion protein described herein can be used to treat a variety of diseases and conditions associated with FGFRs and GIPR. The fusion protein exerts its biological effects by acting on FGF21R ​​and / or GIPR, and thus the FGF21 fusion protein described herein can be used to treat subjects with diseases and conditions that respond favorably to "increasing FGF21R ​​stimulation" or "reducing GIPR stimulation." These subjects are referred to as "subjects in need of FGF21R ​​stimulation treatment" or "subjects in need of reducing GIPR stimulation." These include hepatic steatosis (GIPR, see US 2017 / 0275370A1), nonalcoholic fatty liver disease (FGF21R, see Debra et al., 2016, Hepatobiliary Surg Nutr, 5:515–518; GIPR, see US 2017 / 0275370A1), nonalcoholic steatohepatitis (FGF21, see Armstrong et al., 2013, BMJ Open. 3:e003995; GIPR, see US 2017 / 0275370A1), non-insulin-dependent diabetes mellitus, insulin-dependent diabetes mellitus, stroke (FGF21R, see WO 00 / 16797), myocardial infarction (FGF21R, see WO 98 / 08531), and obesity (FGF21R, see WO 98 / 19698; GIPR, see Furija et al., 2008, PLoS ONE 3:e3163; US2017 / 0275370A1), postoperative catabolic changes (FGF21R ​​see US 6,006,753), functional dyspepsia and irritable bowel syndrome (FGF21R ​​see WO99 / 64060), also including subjects at risk of developing non-insulin-dependent diabetes mellitus (see WO 00 / 07617), subjects with impaired glucose tolerance or impaired fasting glucose, subjects whose weight is about 25% higher than normal weight for the subject's height and weight, and subjects who have undergone partial pancreatectomy.

[0263] In one embodiment, changes in the biological activity of the GIPR antibody or its fusion protein with FGF21 are detected using a reporter gene assay to quantify the ability of the GIPR antibody or FGF21 fusion protein to inhibit GIPR function in vitro.

[0264] Pharmaceutical composition

[0265] In one embodiment, provided herein is a pharmaceutical composition comprising a GIPR antibody provided herein and one or more pharmaceutically acceptable carriers.

[0266] In another embodiment, provided herein is a pharmaceutical composition comprising a fusion protein of a GIPR antibody and FGF21 provided herein, and one or more pharmaceutically acceptable carriers.

[0267] As used herein, the term "carrier" includes vehicles, pharmaceutical excipients, or stabilizers that are not harmful to cells or mammals at the dosages and concentrations employed.

[0268] Treatment

[0269] In one embodiment, provided herein is a method for treating, preventing, or ameliorating non-alcoholic steatohepatitis, comprising administering to a subject a therapeutically effective amount of a GIPR antibody provided herein or a pharmaceutical composition thereof.

[0270] In another embodiment, provided herein is a method for treating, preventing, or ameliorating non-alcoholic steatohepatitis, comprising administering to a subject a therapeutically effective amount of a fusion protein of a GIPR antibody and FGF21 provided herein, or a pharmaceutical composition thereof.

[0271] In another embodiment, provided herein is a method for treating, preventing, or ameliorating obesity, comprising administering to a subject a therapeutically effective amount of a GIPR antibody provided herein, or a pharmaceutical composition thereof.

[0272] In another embodiment, provided herein is a method for treating, preventing, or ameliorating obesity, comprising administering to a subject a therapeutically effective amount of a fusion protein of a GIPR antibody and FGF21 provided herein, or a pharmaceutical composition thereof.

[0273] In another embodiment, provided herein is a method for treating, preventing, or ameliorating type 2 diabetes, comprising administering to a subject a therapeutically effective amount of a GIPR antibody provided herein, or a pharmaceutical composition thereof.

[0274] In a further embodiment, provided herein is a method for treating, preventing, or ameliorating type 2 diabetes, comprising administering to a subject a therapeutically effective amount of a fusion protein of a GIPR antibody and FGF21 provided herein, or a pharmaceutical composition thereof.

[0275] In any of the uses provided herein, the pharmaceutical composition is for intravenous or subcutaneous injection.

[0276] As used herein, the term "subject" refers to mammals, including humans, and is used interchangeably with the term "patient."

[0277] The term "treat" includes alleviating or preventing at least one symptom or other aspect of a condition, or reducing the severity of the disease. The GIPR antibodies or GIPR antibody and FGF21 fusion proteins provided herein do not need to produce a complete cure, or eradicate all symptoms or manifestations of the disease, to constitute an effective therapeutic agent. As is recognized in the relevant art, drugs as therapeutic agents can reduce the severity of a given disease state, but do not need to eliminate all manifestations of the disease to be considered effective therapeutic agents. Similarly, prophylactic treatment does not need to be completely effective in preventing the occurrence of symptoms to constitute an effective prophylactic agent. It is sufficient to simply reduce the impact of the disease (for example, by reducing the number or severity of its symptoms, or by improving another therapeutic effect, or by producing another effective effect), or to reduce the likelihood of the disease occurring or worsening in the subject. One embodiment herein relates to a method comprising administering a GIPR antibody or a GIPR antibody and FGF21 fusion protein to a patient in an amount and for a time sufficient to induce sustained improvement above baseline levels of an indicator of the severity of the specific condition.

[0278] GIPR antibodies or fusion protein pharmaceutical compositions of GIPR antibodies and FGF21 can be administered using any appropriate technique, including but not limited to parenteral, topical, or inhalation administration. If injected, the pharmaceutical composition can be administered, for example, by intra-articular, intravenous, intramuscular, intralesional, intraperitoneal, or subcutaneous routes, as a rapid injection or continuous infusion. For example, local administration at the site of disease or injury, such as transdermal administration and implant sustained-release administration, can be considered. Inhalation administration includes, for example, nasal or oral inhalation, the use of a spray, inhalation of the antibody in the form of an aerosol, and the like. Other options include oral formulations, including tablets, syrups, or lozenges.

[0279] It is advantageous to administer the GIPR antibodies or FGF21 fusion proteins provided herein in the form of a composition comprising one or more other components, such as a physiologically acceptable carrier, excipient, or diluent. The composition may optionally further comprise one or more physiologically active agents as described below. In various embodiments, the composition comprises one, two, three, four, five, or six physiologically active agents in addition to one or more antibodies (e.g., murine or humanized antibodies) or FGF21 fusion proteins provided herein.

[0280] In one embodiment, a pharmaceutical composition comprises a murine or humanized antibody or FGF21 fusion protein as provided herein and one or more substances selected from the group consisting of a buffer having a pH suitable for the antibody or FGF21 fusion protein, an antioxidant such as ascorbic acid, a low molecular weight polypeptide (e.g., a polypeptide containing less than 10 amino acids), a protein, an amino acid, a sugar such as dextrin, a complex such as EDTA, glutathione, a stabilizer, and an excipient. Preservatives may also be added according to appropriate industry standards. The composition may be formulated as a lyophilized powder using an appropriate excipient solution as a diluent. Appropriate components are non-toxic to the recipient at the dosages and concentrations used. Further examples of components that can be used in pharmaceutical formulations are provided in Remington's Pharmaceutical Sciences, 16th edition (1980) and 20th edition (2000). Mack Publishing Company provides kits for use by medical practitioners that include one or more antibodies or FGF21 fusion proteins as provided herein and labels or other instructions for treating any of the conditions discussed herein. In one embodiment, the kit includes a sterile formulation of one or more antibodies or FGF21 fusion proteins in the form of the above-described compositions, packaged in one or more vials.

[0281] Dosage and frequency of administration may vary depending on factors such as the route of administration, the specific antibody or FGF21 fusion protein used, the nature and severity of the disease being treated, whether the symptoms are acute or chronic, and the patient's size and overall symptoms. Appropriate dosages can be determined by methods well known in the art, such as in clinical trials, including dose escalation studies.

[0282] The antibodies or FGF21 fusion proteins provided herein can be administered, for example, once or multiple times at regular intervals over a period of time. In a specific embodiment, the murine or humanized antibody or FGF21 fusion protein is administered once every at least one month or longer, for example, one, two, or three months, or even indefinitely. For treating chronic symptoms, long-term treatment is generally most effective. However, for treating acute symptoms, short-term administration, for example, from one to six weeks, is sufficient. Typically, the human antibody is administered until the patient demonstrates a medically relevant improvement in the selected sign or indicator above baseline levels.

[0283] An example of a treatment regimen provided herein includes subcutaneous injection of an antibody or FGF21 fusion protein at an appropriate dose once a week or more frequently to treat symptoms caused by nonalcoholic steatohepatitis, obesity, or type 2 diabetes. Administration of the antibody or FGF21 fusion protein can continue weekly or monthly until the desired outcome is achieved, such as resolution of the patient's symptoms. Treatment can be re-administered as needed, or, alternatively, a maintenance dose can be administered.

[0284] Patients may be monitored for liver fat fraction (HFF), liver injury marker alanine aminotransferase (ALT), body weight, and blood glucose levels before, during, and / or after treatment with an antibody or FGF21 fusion protein, such as a human antibody or FGF21 fusion protein, to detect any changes therein. For certain conditions, changes in HFF, ALT, and blood glucose may vary depending on factors such as the course of the disease. Changes in these levels can be measured using known techniques.

[0285] Specific embodiments of the methods and compositions herein involve the use of, for example, an antibody or FGF21 fusion protein and one or more GIP antagonists, two or more antibodies or FGF21 fusion proteins provided herein, or an antibody or FGF21 fusion protein of the invention and one or more other GIP antagonists. In further embodiments, the antibody or FGF21 fusion protein is administered alone or in combination with other agents for treating the symptoms that afflict the patient. Examples of such agents include proteins as well as non-protein drugs. When multiple drugs are administered in combination, their dosages should be adjusted accordingly as is well known in the art. "Combined administration" combination therapy is not limited to simultaneous administration, but also includes treatment regimens in which the antigen and protein are administered at least once during a course of treatment involving administration of at least one other therapeutic agent to the patient.

[0286] In another aspect, provided herein are methods for preparing a medicament for treating nonalcoholic steatohepatitis, obesity, type 2 diabetes, and related conditions, comprising a mixture of the antibody or FGF21 fusion protein provided herein and a pharmaceutically acceptable excipient for treating conditions related to the aforementioned diseases. The medicament preparation method is as described above.

[0287] Further provided herein are compositions, kits, and methods related to antibodies or FGF21 fusion proteins that specifically bind to human GIPR. Also provided are nucleic acid molecules and derivatives and fragments thereof comprising polynucleotides encoding all or part of a polypeptide that binds to GIPR, such as nucleic acids encoding all or part of an anti-GIPR antibody, antibody fragment, antibody derivative, or FGF21 fusion protein. Further provided herein are vectors and plasmids comprising such nucleic acids, and cells and cell lines comprising such nucleic acids and / or vectors and plasmids. The methods provided include, for example, methods for preparing, identifying, or isolating antibodies or FGF21 fusion proteins, such as anti-GIPR antibodies or FGF21 fusion proteins, that bind to human GIPR, methods for determining whether the antibodies or FGF21 fusion proteins bind to GIPR, and methods for administering antibodies or FGF21 fusion proteins that bind to GIPR to animal models.

[0288] The technical solution of this article is further illustrated below through specific examples.

[0289] Unless otherwise specified, all raw materials and equipment used herein are commercially available or commonly used in the art. The methods in the following examples, unless otherwise specified, are all conventional methods in the art.

[0290] 1. Preparation of antigens for immunization

[0291] CHO-DHFR- cells were seeded into 6-well plates. After 24 hours of culture, the pTM15 plasmid containing the hGIPR gene (nucleotide sequence: SEQ ID NO:114, amino acid sequence: SEQ ID NO:113) was transfected into the cells in the 6-well plates. Transfection was performed using the transfection conditions recommended by Invitrogen using Lipofectamine 2000. After 48 hours, the culture medium was replaced with complete medium containing 300 μg / mL hygromycin, and the medium was changed every three days. After approximately two weeks of culture, stably growing colonies emerged. The cell colonies were dissociated by digestion, and the cells were passaged and cultured again until the passaged cells reached 100% confluency. The constructed stable cell lines were analyzed by FACS using an antibody against the V5 tag (Life Technologies). The stressed cell populations were identified based on the FACS results. The selected CHO-DHFR-hGIPR cells showed significant hGIPR expression on their cell membranes. After subcloning and further characterization, three GIPR cell lines were selected as stable, high-expressing cell lines. These high-hGIPR-expressing cell lines can be used as immunogens for antibody production (see Example 2). Furthermore, in certain embodiments, a fusion protein of the hGIPR extracellular domain and hIgG Fc can also be used as an immunogen for antibody production. The preparation method is as follows: The fusion protein sequence of the hGIPR extracellular domain, hIgG2 Fc, and a peptide linker is subcloned into the pTM5 plasmid. Large-scale transient expression is performed in suspended HEK293 cells, and the cell supernatant is obtained. The hGIPR extracellular domain fusion protein is then purified by affinity chromatography.

[0292] 2. Antibody Preparation

[0293] Immunogens including any of the following can be used to generate antibodies against hGIPR. For example, in certain embodiments, whole cells expressing hGIPR are used as immunogens to generate antibodies against hGIPR. Furthermore, in certain embodiments, a fusion protein comprising the amino acid sequence of the N-terminal domain of hGIPR and hFc is used as an immunogen to generate antibodies against hGIPR. The immunogen is mixed with aluminum hydroxide adjuvant and injected subcutaneously into BALB / c mice (6-8 weeks old). Thereafter, the mice are boosted weekly. After a total of six immunizations, blood is collected by tail snip. Serum is separated by centrifugation, and serum titers are determined by FACS. When an appropriate antibody titer is reached, the mice are sacrificed by cervical dislocation, and spleen cells are harvested under sterile conditions. SP2 / 0 cells in the logarithmic growth phase are collected and centrifuged. The pellet is cultured in serum-free water until resuspended, centrifuged again, resuspended, and counted. The spleen cells and SP2 / 0 cells are mixed to ensure that the numbers of SP2 / 0 and spleen cells are similar. After mixing, the cells are washed and centrifuged three times. After the final centrifugation, the cell pellet was flicked off and pre-warmed PEG-1500 was added dropwise. After pipetting up and down, 30 mL of pre-warmed serum-free medium was slowly added to terminate the PEG fusion reaction. After another centrifugation, the cell pellet was flicked off and fusion medium was added. The spleen cells and feeder cells were plated in a 96-well plate, with 100 μL of medium added to each well. The fused hybridoma cells and feeder cells were cultured together in a 96-well plate and HAT (hypoxanthine, methotrexate, and thymidine) selection was performed to remove non-fused cells. After 10 days, the hybridoma supernatant was collected from the culture plate and analyzed by ELISA.

[0294] 3. ELISA screening of antibodies

[0295] hGIPR-overexpressing CHO-DHFR-hGIPR cells and hGIPR-deficient CHO-DHFR- cells were seeded into 96-well plates. After cells reached 90% confluency, the cell culture supernatant was removed, the plates were washed twice with PBS, and fixed with 100% methanol at 4°C. The plates were then added with 100 μL of freshly prepared 0.6% H₂O₂-PBS for 20 minutes at room temperature and washed twice with PBS. After blocking with 1% BSA (dissolved in PBS), hybridoma supernatant was added and incubated at 4°C for 90 minutes. After multiple washes, 100 μL of diluted goat anti-mouse Fc-HRP secondary antibody (Sigma-Aldrich) was added to each well and incubated at 37°C for 30 minutes. After five washes, 100 μL of TMB chromogenic substrate was added to each well and incubated at 37°C for 15 minutes. Color development was terminated with 50 μL of 2M H₂SO₄, and the OD₄50 value was read. Furthermore, in certain embodiments, a fusion protein comprising the amino acid sequence of the hGIPR N-terminal domain and hFc is used as the coating antigen to coat a 96-well plate. After blocking with 1% BSA (dissolved in PBS), hybridoma cell supernatant is added and incubated at 4°C for 90 minutes. Subsequently, the same steps as the above-described ELISA method are used to screen for anti-hGIPR monoclonal antibodies. The positive control is serum from immunized mice; the negative control is cell culture supernatant. Preliminary ELISA testing identified several positive hybridoma cell lines that secrete anti-hGIPR antibodies. These hybridoma lines secreting anti-hGIPR antibodies were selected and cloned to obtain cell lines that stably secrete anti-hGIPR antibodies. Finally, the positive hybridoma cell supernatants were selected for FACS verification (see Example 10).

[0296] 4. Cloning and subcloning of antibody genes

[0297] Antibody-secreting hybridoma cells were harvested, and mRNA was extracted using the QIAGEN mRNA extraction kit. The extracted mRNA was then reverse-transcribed into cDNA using primers specific for the mouse light and heavy chain constant regions: the heavy chain reverse transcription primer (5'-TTTGGRGGGAAGATGAAGAC-3'), and the light chain reverse transcription primers (5'-TTAACACTCTCCCCTGTTGAA-3') and (5'-TTAACACTCATTCCTGTTGAA-3'). RT-PCR reaction conditions were: 25°C for 5 minutes, 50°C for 60 minutes, and 70°C for 15 minutes. The reverse transcribed cDNA was diluted to 500 μL with 0.1 mM TE, added to an ultrafiltration centrifuge tube (Amicon Ultra-0.5), and centrifuged at 2000 g for 10 min; the filtrate was discarded, and 500 μL of 0.1 mM TE was added, and the mixture was centrifuged at 2000 g for 10 min; the filtrate was discarded, and the preparation tube was inverted into a new centrifuge tube, and centrifuged at 2000 g for 10 min to obtain the purified cDNA; 10 μL of the purified cDNA was taken as a template, and 4 μL of 5x tailing buffer (Promega), 4 μL of dATP (1 mM) and 10 U of terminal transferase (Promega) were added and mixed, incubated at 37°C for 5 min and then incubated at 65°C for 5 min; then, the light and heavy chain variable region genes of the antibody were PCR amplified using the cDNA with the PolyA tail as a template. The upstream primers were all OligodT, the heavy chain downstream primers were (5'-TGGACAGGGATCCAGAGTTCC-3') and (5'-TGGACAGGGCTCCATAGTTCC-3'), and the light chain downstream primer was (5'-ACTCGTCCTTGGTCAACGTG-3'). PCR reaction conditions were: 95°C for 5 minutes; 95°C for 30 seconds, 56°C for 30 seconds, 72°C for 1 minute 40 cycles; 72°C for 7 minutes. The PCR product was ligated into the pMD 18-T vector (Takara Bio) and sequenced. PCR primers were designed based on the sequenced antibody DNA sequence to ligate the complete light chain, heavy chain signal peptide and variable domain, and mouse IgG1 constant region into the expression vector pTM5.

[0298] 5. Antibody humanization and optimization

[0299] First, based on the variable region sequences of the mouse antibody light and heavy chains obtained by screening, the NCBI database was used to search for human antibody germline gene sequences (Ig Germline Gene Sequence) that were homologous to the mouse antibody variable region sequences obtained by screening. The human gene sequence with the highest homology, excluding the CDR sequence, was used as a template sequence for CDR grafting to obtain a humanized antibody variable region sequence. The genes of the humanized antibody light and heavy chains were synthesized and spliced ​​with the human IgG2 or IgG4 constant region sequence to obtain a complete recombinant humanized antibody sequence. The recombinant antibody was expressed according to Example 8, and its affinity for GIPR was verified by FACS technology in step 12, and the antibody with the best affinity performance was selected. Finally, the variable region sequence of the humanized antibody was modified by site-directed mutagenesis to further improve its affinity for GIPR.

[0300] 6. Gene cloning and subcloning of humanized hGIPR antibodies

[0301] The optimized humanized antibody heavy and light chain variable region sequences were outsourced for synthesis. During synthesis, the Nhe1 restriction site was incorporated into the heavy chain variable region at its 5' end and the Sal1 restriction site was incorporated into its 3' end. This allowed the complete heavy chain variable region sequence to be ligated to the expression vector pTM5, which already contained the heavy chain constant region. Similarly, the Nhe1 restriction site was incorporated into the light chain variable region at its 5' end and the Bsiw1 restriction site was incorporated into its 3' end. This allowed the complete light chain variable region sequence to be ligated to the expression vector pTM5, which already contained the light chain constant region.

[0302] 7. Construction of fusion protein of humanized hGIPR antibody and FGF21

[0303] The optimized humanized antibody is fused to the FGF21 and its derivative sequences at the C-terminus of the heavy chain to form an FGF21 fusion protein (e.g., SEQ ID NO: 104-106, SEQ ID NO: 119-121, SEQ ID NO: 123 to SEQ ID NO: 125). The sequences of the two are connected by a peptide linker sequence (Linker). The nucleotide sequence of Linker-FGF21 was synthesized by GenScript Biotech Co., Ltd. Using the synthetic gene as a template, the sequence of the "Linker-FGF21" portion was amplified by PCR. Separately, the nucleotide sequence of the humanized antibody was used as a template to amplify the sequence of the signal peptide-antibody portion of the fusion protein. The "Linker-FGF21" portion of the fusion protein nucleic acid sequence was then connected to the antibody portion by overlapping PCR, and Nhe1 and Not1 restriction sites were added to both ends of the primers to connect the complete fusion protein sequence to the expression vector pTM5.

[0304] 8. Transient expression of hGIPR antibody, FGF21 fusion protein, and FC-FGF21(RGE)

[0305] Inoculate 5×10 5 / mL suspended HEK293 or CHO expression cell line into the spinner flask. After 24 hours of rotation culture at 37℃, 5% CO2, the density reached 1×10 6 / mL was used for transfection. Polyethylenimine (PEI) was used as the transfection medium during the transfection process and mixed with the DNA. After a 15-minute static incubation, the mixture was added to the cell culture. After receiving the PEI and DNA mixture, the cells were cultured with rotation at 37°C and 5% CO2 for 24 hours. Tryptone was then added to the cell culture medium as an expression supplement. Finally, after expression was complete (more than 96 hours), the cell supernatant was collected for antibody purification and isolation.

[0306] 9. Purification and Isolation of hGIPR Antibody, FGF21 Fusion Protein, and FC-FGF21(RGE)

[0307] The cell supernatant collected in Example 8 was centrifuged at high speed (8000 rpm) to remove cells and cell debris, and then clarified by filtration with a 0.22 μm filter membrane. The clarified supernatant was used for purification. The purification process was completed by a chromatograph. The supernatant first flowed through a protein A / G affinity chromatography column. The antibodies contained in the supernatant were retained in the column after binding to the ligand of the protein A / G affinity chromatography column during this period. The chromatography column was then washed with an elution buffer with a low pH value (less than or equal to 3.0) to dissociate the antibodies bound to the chromatography column. The collected antibody eluate was rapidly neutralized with 1M Tris-HCl. The obtained antibody eluate was replaced with PBS or other buffer systems after dialysis.

[0308] 10. Construction of a stable cell line expressing the β-Klotho / FGFR1c receptor complex for detection

[0309] CHO-DHFR cells (commercially available) were seeded into 6-well plates and cultured for 24 hours before being transfected with plasmids containing the V5-hKLB gene and the Flag-FGFR1c plasmid. The culture medium was replaced before transfection, and transfection was performed according to the transfection conditions recommended by Invitrogen for Lipofectamine 2000. After 48 hours, the culture medium was replaced with complete medium containing 10 nM methotrexate (MTX). The medium was changed every three days. After approximately two weeks, stable colonies emerged. The cell colonies were then dissected and dispersed. When they reached 50% confluence, pressure selection was performed using gradually increasing MTX concentrations until the concentration reached 10 μM. The constructed stable cell lines were subjected to FACS analysis, and the pressurized cell population was identified using KLB protein N'-terminal V5 tag antibodies (FITC-anti-V5, Invitrogen; Flag-anti-V5, Invitrogen). After 10uM MTX screening, hKLB was abundantly expressed on the CHO-DHFR-V5-hKLB cell membrane. Finally, two stable cell lines with high KLB expression were obtained through subcloning and identification.

[0310] 11. Reporter gene assay to detect the biological activity of hGIPR antibody / FGF21 fusion protein in activating the hFGFR1c / hKLB signaling pathway in vitro

[0311] CHO-DHFR- cells co-expressing hFGFR1c-β-KLB-SRE-Luciferase were seeded at 20,000 cells per well in a 96-well cell culture plate and incubated overnight at 37°C in 5% CO2. The next day, the supernatant was removed, the cell surface was washed twice with serum-free medium, and the residual liquid was aspirated. Then, 100 μL of purified antibody or FGF21 diluted in serum-free medium was added, and the cells were incubated at 37°C for 4 hours. After stimulation, 100 μL of Bright Glo chemiluminescent substrate (Promega) was added, and the cell lysate was transferred to a white 96-well plate. The relative fluorescence intensity was read on a SpectraMax L microplate reader (Molecular Devices) (see Figure 1).

[0312] 12. Reporter gene assay to detect the biological activity of hGIPR antibody / FGF21 fusion proteins V1W1 and V1W2 in antagonizing GIPR in vitro

[0313] CHO-DHFR cells co-expressing hGIPR-CRE-Luciferase were seeded at 30,000 cells per well in a 96-well cell culture plate and incubated overnight at 37°C in a 5% CO2 incubator. The next day, the cell supernatant was removed, and the cell surface was washed twice with serum-free medium. The residual liquid was aspirated, and 50 μL of GIP diluted in serum-free medium and 50 μL of serially diluted hGIPR antibody or hGIPR antibody / FGF21 fusion protein in serum-free medium were added to each well, respectively, and incubated at 37°C for 4 hours. After stimulation, 100 μL of Bright Glo chemiluminescent substrate (Promega) was added, and the cell lysate was transferred to a white 96-well plate. The relative fluorescence intensity was read on a SpectraMax L microplate reader (Molecular Devices) (see Figure 2).

[0314] 13. Reporter gene assay to detect the biological activity of hGIPR antibody / FGF21 fusion proteins V1W1 and V1W2 in activating hβ-Klotho / hFGF1Rc in vitro

[0315] CHO-DHFR- cells co-expressing hFGFR1c-β-KLB-SRE-Luciferase or mFGFR1c-β-KLB-SRE-Luciferase were seeded at 20,000 cells per well in a 96-well cell culture plate and incubated overnight at 37°C. The next day, the supernatant was removed, the cell surface was washed twice with serum-free medium, and the residual liquid was aspirated. Then, 100 μL of purified antibody or FGF21 diluted in serum-free medium was added, and the cells were incubated at 37°C for 4 hours. After stimulation, 100 μL of Bright Glo chemiluminescent substrate (Promega) was added, and the cell lysate was transferred to a white 96-well plate. The relative fluorescence intensity was read on a SpectraMax L microplate reader (Molecular Devices) (see Figures 3 and 8).

[0316] 14. Efficacy study of hGIPR antibody / FGF21 fusion protein in C57BL / 6 obese mice induced by high-fat diet

[0317] Obesity was induced in C57BL / 6 mice (DIO mice) using a 60% high-fat diet. After one week of normal diet, a random number of mice were selected as a control group and fed a standard mouse diet. The remaining animals were fed a high-fat diet for 8 consecutive weeks. Body weight and food intake were measured weekly. Subsequently, the mice in the high-fat diet group were randomly divided into the V1W1-FGF21 (SEQ: 89) group (1 mg / kg), V1W1-FGF21 (SEQ: 89) group (3 mg / kg), V1W1-FGF21 (SEQ: 89) group (10 mg / kg), V1W2-FGF21 (SEQ: 89) group (1 mg / kg), V1W2-FGF21 (SEQ: 89) group (3 mg / kg), and V1W2-FGF21 (SEQ: 89) group (10 mg / kg), a positive drug control group, and a model group. The drug was injected subcutaneously once every three days for a total of 4 weeks. The normal control group was not given the drug, and the model group was given an equal amount of blank preparation. The body weight, food intake, and behavioral observation data of the mice were collected during the experimental period. Fasting blood glucose levels were measured on days 0, 7, 14, 21, and 28 of the experiment. Oral glucose tolerance tests (OGTTs) were performed on days 14 and 28 of the experiment. Before the last day of the experiment, animals were fasted for 12 hours (with free access to water). Orbital blood was collected for serum separation, and the animals were euthanized. Serum ALT, AST, HDL-C, LDL-C, TC, and TG were measured (results are shown in Figures 4, 5, 6, and 7).

[0318] 15. Efficacy study of hGIPR antibody / FGF21 fusion protein in vivo in C57BL / 6 mice with nonalcoholic steatohepatitis fed a choline-deficient, L-amino acid-defined high-fat diet

[0319] A non-alcoholic steatohepatitis model was established in C57BL / 6 mice induced by a choline-deficient, L-amino acid-defined, high-fat diet. After one week of normal diet, a random number of mice were selected as a control group and fed a standard mouse chow diet. The remaining animals were fed a choline-deficient, L-amino acid-defined, high-fat diet. After six weeks of continuous feeding, mice in the choline-deficient, L-amino acid-defined, high-fat diet were randomly divided into a V1W2-FGF21 (SEQ: 118) group (1 mg / kg), a V1W2-FGF21 (SEQ: 118) group (3 mg / kg), a V1W2-FGF21 (SEQ: 118) group (10 mg / kg), a positive control group, and a model group based on serum TC and TG levels. Drugs were administered subcutaneously every three days for a total of 8 weeks. The control group received no drug, while the model group received an equal amount of blank formulation. Body weight, food intake, and behavioral data were collected during the experimental period. At week 8 of the experiment, blood was collected from mice and serum was separated for the detection of ALT, AST, TG, and TC. The livers of mice were collected and weighed, and 95-105 mg of liver tissue was weighed for the detection of TC and TG. The same lobe of liver was fixed in 4% paraformaldehyde and stained with HE and Sirius red (see the results in Figure 9).

[0320] The above examples are provided to fully disclose and illustrate to those skilled in the art how to make and use the claimed embodiments and are not intended to limit the scope of the disclosure herein. Modifications obvious to those skilled in the art are within the scope of the claims herein. All publications, patents, and patent applications cited in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually incorporated herein by reference.

Claims

1. An antibody that specifically binds to a human GIPR, wherein the antibody comprises one, two, three, four, five, or six amino acid sequences, wherein each amino acid sequence is independently selected from the group consisting of: a. Light chain CDR1 amino acid sequences: SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; b. Light chain CDR2 amino acid sequence: YAS, SEQ ID NO: 2; c. Light chain CDR3 amino acid sequences: SEQ ID NO: 3 and SEQ ID NO: 4; d. Heavy chain CDR1 amino acid sequence: SEQ ID NO: 12; e. Heavy chain CDR2 amino acid sequence: SEQ ID NO: 13; and f. Heavy chain CDR3 amino acid sequence: SEQ ID NO:

14.

2. The antibody of claim 1, wherein the antibody comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the group consisting of: a. Light chain CDR1 amino acid sequence: SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; and b. Heavy chain CDR1 amino acid sequence: SEQ ID NO:

12.

3. The antibody of claim 1 or 2, 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: a. Light chain CDR2 amino acid sequence: YAS, SEQ ID NO: 2; and b. Heavy chain CDR2 amino acid sequence: SEQ ID NO:

13.

4. The antibody of any one of claims 1 to 3, 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: a. Light chain CDR3 amino acid sequence: SEQ ID NO: 3 and SEQ ID NO: 4; and b. Heavy chain CDR3 amino acid sequence: SEQ ID NO:

14.

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 group consisting of SEQ ID NO: 1, YAS, 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, and SEQ ID NO:

11.

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 group consisting of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO:

14.

7. The antibody of any one of claims 1 to 6, wherein the antibody comprises or further comprises a combination of light chain and heavy chain CDR1 amino acid sequences independently selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 12, SEQ ID NO: 5 and SEQ ID NO: 12, SEQ ID NO: 6 and SEQ ID NO: 12, SEQ ID NO: 7 and SEQ ID NO: 12, SEQ ID NO: 8 and SEQ ID NO: 12, SEQ ID NO: 9 and SEQ ID NO: 12, SEQ ID NO: 10 and SEQ ID NO: 12, and SEQ ID NO: 11 and SEQ ID NO:

12.

8. The antibody of any one of claims 1 to 7, wherein the antibody comprises or further comprises a combination of light chain and heavy chain CDR2 amino acid sequences independently selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 13 and YAS and SEQ ID NO:

13.

9. The antibody of any one of claims 1 to 8, wherein the antibody comprises or further comprises a combination of light chain and heavy chain CDR3 amino acid sequences independently selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 14 and SEQ ID NO: 4 and SEQ ID NO:

14.

10. The antibody of any one of claims 1 to 9, wherein the antibody comprises (a) Light chain CDR1 amino acid sequence: SEQ ID NO: 1; Light chain CDR2 amino acid sequence: YAS, SEQ ID NO: 2; Light chain CDR3 amino acid sequence: SEQ ID NO: 3; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 12; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 13; and Heavy chain CDR3 amino acid sequence: SEQ ID NO: 14; (b) Light chain CDR1 amino acid sequence: SEQ ID NO: 5; Light chain CDR2 amino acid sequence: YAS, SEQ ID NO: 2; Light chain CDR3 amino acid sequence: SEQ ID NO: 3; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 12; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 13; and Heavy chain CDR3 amino acid sequence: SEQ ID NO: 14; (c) light chain CDR1 amino acid sequence: SEQ ID NO: 6; Light chain CDR2 amino acid sequence: YAS, SEQ ID NO: 2; Light chain CDR3 amino acid sequence: SEQ ID NO: 3; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 12; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 13; and Heavy chain CDR3 amino acid sequence: SEQ ID NO: 14; (d) light chain CDR1 amino acid sequence: SEQ ID NO: 1; Light chain CDR2 amino acid sequence: YAS, SEQ ID NO: 2; Light chain CDR3 amino acid sequence: SEQ ID NO: 4; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 12; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 13; and Heavy chain CDR3 amino acid sequence: SEQ ID NO: 14; (e) light chain CDR1 amino acid sequence: SEQ ID NO: 7; Light chain CDR2 amino acid sequence: YAS, SEQ ID NO: 2; Light chain CDR3 amino acid sequence: SEQ ID NO: 3; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 12; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 13; and Heavy chain CDR3 amino acid sequence: SEQ ID NO: 14; (f) light chain CDR1 amino acid sequence: SEQ ID NO: 8; Light chain CDR2 amino acid sequence: YAS, SEQ ID NO: 2; Light chain CDR3 amino acid sequence: SEQ ID NO: 3; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 12; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 13; and Heavy chain CDR3 amino acid sequence: SEQ ID NO: 14; (g) light chain CDR1 amino acid sequence: SEQ ID NO: 9; Light chain CDR2 amino acid sequence: YAS, SEQ ID NO: 2; Light chain CDR3 amino acid sequence: SEQ ID NO: 3; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 12; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 13; and Heavy chain CDR3 amino acid sequence: SEQ ID NO: 14; (h) light chain CDR1 amino acid sequence: SEQ ID NO: 10; Light chain CDR2 amino acid sequence: YAS, SEQ ID NO: 2; Light chain CDR3 amino acid sequence: SEQ ID NO: 3; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 12; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 13; and Heavy chain CDR3 amino acid sequence: SEQ ID NO: 14; (i) Light chain CDR1 amino acid sequence: SEQ ID NO: 11; Light chain CDR2 amino acid sequence: YAS, SEQ ID NO: 2; Light chain CDR3 amino acid sequence: SEQ ID NO: 3; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 12; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 13; and Heavy chain CDR3 amino acid sequence: SEQ ID NO:

14.

11. The antibody of any one of claims 1 to 10, wherein the antibody comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the group consisting of: a. light chain variable domain amino acid sequence: SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37; and an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to any of the sequences thereof; and b. Heavy chain variable domain amino acid sequence: SEQ ID NO: 38; and amino acid sequences that are at least 80%, at least 85%, at least 90%, or at least 95% identical thereto.

12. The antibody of any one of claims 1 to 11, wherein the polynucleotide encoding sequence of the antibody comprises one or two polynucleotide sequences, wherein each polynucleotide sequence is independently selected from the polynucleotide sequences listed below: a. light chain variable domain polynucleotide encoding sequence: SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47; and a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to any of the sequences thereof; and b. Heavy chain variable domain polynucleotide encoding sequence: SEQ ID NO: 48; and polynucleotide sequences at least 80%, at least 85%, at least 90%, or at least 95% identical thereto.

13. The antibody of any one of claims 1 to 12, wherein the antibody comprises or further comprises an amino acid sequence independently selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO:

37.

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 group consisting of SEQ ID NO:

38.

15. The antibody of any one of claims 1 to 14, wherein the antibody comprises a combination of light and heavy chain variable region amino acid sequences independently selected from the group consisting of SEQ ID NO: 29 and SEQ ID NO: 38, SEQ ID NO: 30 and SEQ ID NO: 38, SEQ ID NO: 31 and SEQ ID NO: 38, SEQ ID NO: 32 and SEQ ID NO: 38, SEQ ID NO: 33 and SEQ ID NO: 38, SEQ ID NO: 34 and SEQ ID NO: 38, SEQ ID NO: 35 and SEQ ID NO: 38, SEQ ID NO: 36 and SEQ ID NO: 38, and SEQ ID NO: 37 and SEQ ID NO:

38.

16. The antibody of any one of claims 15, wherein the antibody comprises or further comprises an amino acid sequence independently selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:

38.

17. The antibody of any one of claim 16, wherein the antibody comprises a combination of light chain and heavy chain variable region amino acid sequences independently selected from the group consisting of SEQ ID NO: 29 and SEQ ID NO: 38, SEQ ID NO: 30 and SEQ ID NO: 38, SEQ ID NO: 34 and SEQ ID NO: 38, SEQ ID NO: 35 and SEQ ID NO: 38, SEQ ID NO: 36 and SEQ ID NO: 38, and SEQ ID NO: 37 and SEQ ID NO:

38.

18. The antibody of claim 16, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO:

38.

19. The antibody of claim 16, wherein the antibody comprises a combination of the amino acid sequences of SEQ ID NO: 29 and SEQ ID NO:

38.

20. The antibody of any one of claims 1 to 19, wherein the antibody further comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the group consisting of: a. Light chain constant region amino acid sequence: SEQ ID NO: 49; and b. Heavy chain constant region amino acid sequence: SEQ ID NO: 50, SEQ ID NO:

51.

21. The antibody of any one of claims 1 to 20, wherein the antibody is a murine GIPR antibody or a humanized GIPR antibody.

22. The antibody of any one of claims 1 to 21, wherein the antibody is a GIPR monoclonal antibody.

23. The antibody of any one of claims 1 to 22, wherein the antibody is a monoclonal antibody comprising a combination of amino acid sequences selected from the group consisting of SEQ ID NO: 29 and SEQ ID NO: 38, SEQ ID NO: 30 and SEQ ID NO: 38, SEQ ID NO: 31 and SEQ ID NO: 38, SEQ ID NO: 32 and SEQ ID NO: 38, SEQ ID NO: 33 and SEQ ID NO: 38, SEQ ID NO: 34 and SEQ ID NO: 38, SEQ ID NO: 35 and SEQ ID NO: 38, SEQ ID NO: 36 and SEQ ID NO: 38, and SEQ ID NO: 37 and SEQ ID NO:

38.

24. The antibody of any one of claims 1 to 23, wherein the antibody has one or more of the following properties: a. When binding to human GIPR, its Kd is the same as or better than that of the reference GIPR antibody; b. When inhibiting GIP activation of human GIPR, its IC50 is the same as or better than that of the reference GIPR antibody; and c. The GIPR antibody cross-competes with a reference GIPR antibody for binding to human GIPR.

25. The antibody of claim 24, wherein said antibody cross-competes for binding to human GIPR with said reference GIPR antibody.

26. The antibody of claim 24 or 25, wherein the reference GIPR antibody comprises the antibody of any one of claims 1 to 24.

27. The antibody of claim 26, wherein the reference GIPR antibody comprises a combination of a light chain variable domain amino acid sequence of SEQ ID NO: 29 and a heavy chain variable domain amino acid sequence of SEQ ID NO:

38.

28. The antibody according to any one of claims 1 to 27, characterized in that: The antibody is 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 three-chain antibody, a four-chain antibody, a Fab fragment, a F(ab')x fragment, a domain antibody, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

29. The antibody of any one of claims 1 to 28, wherein the antibody has an IC of reducing human GIP signaling. 50 The value is approximately 1 nM to 200 nM or 1 nM to 100 nM.

30. An FGF21 fusion protein, characterized in that: the fusion protein comprises a GIPR antibody and one, two, three, four, five, six, seven, or eight FGF21 fragments; the fusion protein connects the amino terminus of an FGF21 fragment to the carboxyl terminus of a GIPR antibody light chain or a heavy chain via a peptide linker sequence, wherein the FGF21 fragment comprises an amino acid sequence independently selected from one of the following: SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, 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:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, and SEQ ID NO:

122.

31. An FGF21 fusion protein, characterized in that: the fusion protein comprises a GIPR antibody according to any one of claims 1 to 29 and one, two, three, four, five, six, seven, or eight FGF21 fragments; the fusion protein connects the amino terminus of an FGF21 fragment to the carboxyl terminus of the light chain or heavy chain of the GIPR antibody via a peptide linker sequence.

32. The fusion protein of claim 30 or 31, wherein the fusion protein comprises a GIPR antibody and one, two, three, or four FGF21 fragments; the fusion protein connects the amino terminus of an FGF21 fragment to the carboxyl terminus of the light chain or heavy chain of the GIPR antibody via a peptide linker sequence.

33. The fusion protein of claims 30 to 32, wherein the fusion protein comprises a GIPR antibody and one or two FGF21 fragments; the fusion protein connects the amino terminus of an FGF21 fragment to the carboxyl terminus of the light chain or heavy chain of the GIPR antibody via a peptide linker sequence.

34. The fusion protein of claims 30 to 33, wherein the GIPR antibody, FGF21 fragment, and peptide linker sequence are fused by one of the following methods to form the fusion protein: The amino terminus of an FGF21 fragment and the carboxyl terminus of the GIPR antibody light chain were connected via a peptide linker sequence: N'-R-Linker-FGF21-C'; The amino terminus of an FGF21 fragment and the carboxyl terminus of the GIPR antibody heavy chain were connected via a peptide linker sequence: N'-R-Linker-FGF21-C'; Wherein: N' represents the amino terminus of the polypeptide chain, C' represents the carboxyl terminus of the polypeptide chain, FGF21 represents the FGF21 fragment, R is the amino acid sequence of the light chain or heavy chain of the GIPR antibody according to any one of claims 1 to 29, and Linker represents a peptide linker sequence.

35. The FGF21 fusion protein of any one of claims 30 to 34, wherein the peptide linker sequence comprises a full-length, partial, or repeated amino acid sequence independently selected from one of the following: SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO:

68.

36. The FGF21 fusion protein of any one of claims 31 to 34, wherein the FGF21 fragment comprises an amino acid sequence independently selected from one of the following: SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, 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:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, and SEQ ID NO:

122.

37. The FGF21 fusion protein of any one of claims 30 to 36, wherein the FGF21 fragment comprises SEQ ID NO: 89 or SEQ ID NO:

118.

38. The fusion protein of any one of claims 30 to 37, wherein the fusion protein comprises a GIPR antibody, two FGF21 fragments, and a peptide linker sequence therebetween, wherein the fusion protein sequence comprises an amino acid sequence independently selected from one of the following: a. Light chain amino acid sequence: SEQ ID NO: 103 b. Amino acid sequences of modified heavy chains (heavy chain, peptide linker and FGF21 analog): SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 124 and SEQ ID NO:

125.

39. A polynucleotide encoding the GIPR antibody of any one of claims 1 to 29 or the FGF21 fusion protein of any one of claims 30 to 37.

40. A vector comprising the polynucleotide of claim 39. A host cell comprising the vector of claim 40.

42. A pharmaceutical composition comprising the GIPR antibody of any one of claims 1 to 29 or the FGF21 fusion protein of any one of claims 30 to 38 in admixture with a pharmaceutically acceptable carrier.

43. Use of a pharmaceutical composition comprising the GIPR antibody according to any one of claims 30 to 38 or the FGF21 fusion protein according to any one of claims 30 to 37 in the preparation of a medicament for preventing or treating non-alcoholic fatty liver disease.

44. Use of a pharmaceutical composition comprising the GIPR antibody according to any one of claims 30 to 38 or the FGF21 fusion protein according to any one of claims 30 to 37 in the preparation of a medicament for preventing or treating obesity and obesity-related disorders.

45. Use of a pharmaceutical composition comprising the GIPR antibody according to any one of claims 30 to 38 or the FGF21 fusion protein according to any one of claims 30 to 37 in the preparation of a medicament for preventing or treating type 2 diabetes.

46. ​​Use of a pharmaceutical composition comprising the GIPR antibody of any one of claims 30 to 38 or the FGF21 fusion protein of any one of claims 30 to 37 in the preparation of a medicament for simultaneously preventing or treating two or more of non-alcoholic fatty liver disease, obesity or type 2 diabetes.

47. The use according to any one of claims 43 to 46, wherein the pharmaceutical composition is for intravenous or subcutaneous injection.

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