GIPR antibody, fusion protein with FGF21, pharmaceutical composition thereof, and use thereof

Abstract

The present invention provides a GIPR antibody, a fusion protein with FGF21, a pharmaceutical composition thereof, and uses thereof. It also provides a method for treating, preventing, or ameliorating one or more symptoms of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, obesity, or type 2 diabetes using a GIPR antibody and a fusion protein with FGF21.

Description

[Technical Field]

[0001] The present invention provides a fusion protein of a GIPR antibody and FGF21, and a pharmaceutical composition thereof. The present invention further provides a method for treating, preventing, or ameliorating one or more symptoms of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, obesity, or type 2 diabetes using the fusion protein of a GIPR antibody and FGF21. [Background technology]

[0002] Gastric inhibitory polypeptide (GIP) is a polypeptide hormone secreted by intestinal K cells after feeding. It is a 42-amino acid polypeptide. GIP is involved in the physiological process of stimulating insulin secretion by activating the gastric inhibitory polypeptide receptor (GIPR) on the surface of pancreatic β cells (Tseng et al., 1996, J. Clin. Invest. 98:2440-2445; Ravn et al., 2013, J. Biol. Chem. 288:19760-72). GIPR is widely distributed in the pancreas, skeletal, cardiac, stomach, intestinal, and adipose tissues (Peter et al., 2013, J. Biol. Chem. 288:19760-72). This diverse distribution suggests that the GIP / GIPR pathway has many biological effects beyond blood glucose regulation. Experimental evidence indicates that the GIP / GIPR signaling pathway is closely related to fat metabolism, at least in these tissues (Yip et al., 2000, Life Sci. 66:91-103). Experimental data also show that circulating GIP levels are elevated in obese or diabetic patients (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; Vilsboll et al., 2003, J. Clin. Endocrinol. Metab. 88:2706-2713).

[0003] Fibroblast growth factor 21 (FGF21) is an endogenous protein synthesized in the liver. It is a member of the FGF family and plays an important role in regulating glucose and lipid metabolism (Schlein et al., 2016, Cell Metabolism. 23:441-453; Habegger et al., 2013, Diabetes. 62:1453-1463). FGF21 exerts its physiological functions by activating FGF21-specific receptors in the liver, adipocytes, pancreatic β cells, muscle tissue, and the hypothalamus (Kharitonenkov et al., 2005, J. Clin. Invest. 115:1627-1635; Dunshe et al., 2016, J. Biol. Chem. 291:5986-5996). FGF21 can activate a receptor complex in the cell membrane, which contains β-Klotho and one of the FGF receptors, such as 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). The concentration of endogenous FGF21 in adipose tissue is approximately 100 pg / mL in healthy individuals and can be 10-fold elevated in patients with obesity or non-alcoholic steatohepatitis, indicating the presence of FGF21 resistance in these patients. Dietary changes can also affect the circulating levels of FGF21 in the human body. High intake of fructose or alcohol causes a rapid increase in plasma FGF21 levels, and prolonged restriction of protein intake or excessive carbohydrate intake similarly increases FGF21 levels.

[0004] The present invention synergizes the fat-reducing effect of a GIPR antibody with the hepatitis-reducing effect and fibrosis and lipoprotein-improving effect of FGF21, and uses these synergistic effects to treat non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, obesity, and type 2 diabetes. The present invention provides a fusion protein drug for treating patients suffering from one or more of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, obesity, and type 2 diabetes. Summary of the Invention

[0005] The present invention provides antibodies capable of specifically binding to GIPR, which are antagonists of GIPR.

[0006] The present invention further provides antibodies capable of specifically binding to GIPR, the antibodies comprising one, two, three, four, five, or six amino acid sequences, each independently selected from the amino acid sequences set forth below. 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 sequence: 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.

[0007] The present invention further provides an FGF21 fusion protein comprising an antibody capable of specifically binding to GIPR and one, two, three, four, five, six, seven, or eight FGF21 fragments, in which the amino terminus of the FGF21 fragment is linked to the carboxy terminus of the light chain or heavy chain of the GIPR antibody via a peptide linker sequence (linker).

[0008] The present invention provides an FGF21 fusion protein comprising an antibody capable of specifically binding to GIPR and one, two, three, or four FGF21 fragments, in which the amino terminus of the FGF21 fragment is linked to the carboxy terminus of the light chain or heavy chain of the GIPR antibody via a peptide linker sequence (linker).

[0009] The present invention provides an FGF21 fusion protein comprising an antibody capable of specifically binding to GIPR and one or two FGF21 fragments, in which the amino terminus of the FGF21 fragment is linked to the carboxy terminus of the light chain or heavy chain of the GIPR antibody via a peptide linker sequence (linker).

[0010] The present invention provides an FGF21 fusion protein comprising a GIPR antibody and one or two FGF21 fragments, in which the amino terminus of the FGF21 fragment is linked to the carboxy terminus of the light chain of the GIPR antibody via a peptide linker sequence (linker) (N'-R-Linker-FGF21-C') or the amino terminus of the FGF21 fragment is linked to the carboxy terminus of the heavy chain of the GIPR antibody (N'-R-Linker-FGF21-C') (wherein N' represents the amino terminus of the polypeptide chain of the fusion protein, C' represents the carboxy terminus of the polypeptide chain of the fusion protein, 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).

[0011] The present invention provides polynucleotides encoding the GIPR antibodies described in the present invention.

[0012] The present invention provides a polynucleotide encoding a fusion protein of a GIPR antibody described in the present invention and FGF21.

[0013] The present invention provides a vector comprising a polynucleotide encoding a GIPR antibody described in the present invention.

[0014] The present invention provides a vector comprising a polynucleotide encoding a fusion protein of a GIPR antibody described in the present invention and FGF21.

[0015] The present invention provides a host cell comprising a vector described in the present invention.

[0016] The present invention provides a pharmaceutical composition comprising a GIPR antibody described in the present invention and a pharmaceutically acceptable carrier.

[0017] The present invention provides a pharmaceutical composition comprising a fusion protein of a GIPR antibody and FGF21 described in the present invention and a pharmaceutically acceptable carrier.

[0018] The present invention provides the use of a GIPR antibody according to the present invention in the preparation of a medicament for treating, preventing or ameliorating non-alcoholic steatohepatitis-related diseases.

[0019] The present invention provides use of a fusion protein of a GIPR antibody and FGF21 described in the present invention in the preparation of a medicament for treating, preventing or ameliorating non-alcoholic steatohepatitis-related diseases.

[0020] The present invention provides the use of a GIPR antibody according to the present invention in the preparation of a medicament for treating, preventing or ameliorating type 2 diabetes.

[0021] The present invention provides the use of a fusion protein of a GIPR antibody and FGF21 described in the present invention in the preparation of a medicament for treating, preventing or ameliorating type 2 diabetes.

[0022] The invention provides the use of a GIPR antibody according to the invention in the preparation of a medicament for weight loss or for treating, preventing or ameliorating obesity and obesity-related disorders.

[0023] The present invention provides the use of a fusion protein of a GIPR antibody and FGF21 as described in the present invention in the preparation of a medicament for weight loss or for treating, preventing or ameliorating obesity and obesity-related diseases.

[0024] The present invention provides the use of a GIPR antibody described in the present invention in the preparation of a medicament for simultaneously treating, preventing or ameliorating two or more pathologies of non-alcoholic steatohepatitis-related disease, obesity or type 2 diabetes.

[0025] The present invention provides use of a fusion protein of a GIPR antibody and FGF21 described in the present invention in the preparation of a medicament for simultaneously treating, preventing, or ameliorating two or more pathological conditions of non-alcoholic steatohepatitis-related disease, obesity, or type 2 diabetes.

[0026] The present invention provides a method for treating, preventing, or ameliorating one or more symptoms of non-alcoholic steatohepatitis-associated disease, comprising administering to a subject a therapeutically effective amount of a GIPR antibody described in the present invention.

[0027] The present invention provides a method for treating, preventing, or ameliorating one or more symptoms of non-alcoholic steatohepatitis-associated disease, comprising administering to a subject a therapeutically effective amount of a fusion protein of a GIPR antibody and FGF21 described in the present invention.

[0028] The present invention provides a method for treating, preventing, or ameliorating one or more symptoms of obesity, comprising administering to a subject a therapeutically effective amount of a GIPR antibody described in the present invention.

[0029] The present invention provides a method 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 described in the present invention.

[0030] The present invention provides a method for treating, preventing, or ameliorating one or more symptoms of type 2 diabetes, comprising administering to a subject a therapeutically effective amount of a GIPR antibody described in the present invention.

[0031] The present invention provides a method for treating, preventing, or ameliorating one or more symptoms of type 2 diabetes, comprising administering to a subject a therapeutically effective amount of a fusion protein of a GIPR antibody and FGF21 described in the present invention. [Brief explanation of the drawings]

[0032] [Figure 1] FIG. 1 shows the activation curve of the Fc-FGF21 fusion protein Fc-linker-FGF21 (including SEQ ID NO: 79, SEQ ID NO: 82, SEQ ID NO: 85, and SEQ ID NO: 86) that activates the hFGFR1c / hKLB signaling pathway, as detected by reporter gene experiments. Figure 1(a) shows that the EC50 was 18.61 nM for Fc-FGF21(RGE), 5.57 nM for Fc-FGF21(SEQ ID NO: 79), and 41.40 nM for Fc-FGF21(SEQ ID NO: 82), and Figure 1(b) shows that the EC50 was 8.13 nM for Fc-FGF21(RGE), 156.50 nM for Fc-FGF21(SEQ ID NO: 85), and 31.44 nM for Fc-FGF21(SEQ ID NO: 86), respectively. [Figure 2]Figure 2 shows the concentration-inhibitory curves of the GIPR antibody / FGF21 fusion proteins V1W1-Linker-FGF21 (SEQ ID NO: 89) and V1W2-Linker-FGF21 (containing SEQ ID NO: 89) for antagonizing the GIP-activated hGIPR signaling pathway, as detected in reporter gene experiments. The IC50 values ​​were 6.50 nM for LH, 91.14 nM for V1W1-FGF21 (SEQ: 89), and 15.30 nM for V1W2-FGF21 (SEQ: 89), respectively. [Figure 3] Figure 3 shows the activation curves of the GIPR antibody / FGF21 fusion proteins V1W1-Linker-FGF21 (containing SEQ ID NO: 89) and V1W2-Linker-FGF21 (containing SEQ ID NO: 89) that activate the hFGFR1C / hKLB signaling pathway, as detected in reporter gene experiments. The EC50 values ​​were 11.65 nM for Fc-FGF21(RGE), 16.44 nM for V1W1-FGF21 (SEQ ID NO: 89), and 37.80 nM for V1W2-FGF21 (SEQ ID NO: 89), respectively. [Figure 4] Figure 4 shows the time course curves of mouse body weight (a, c) and weight change rate (b, d) in high-fat diet-induced obese C57BL / 6 mice treated with high, medium, and low doses of the hGIPR antibody / FGF21 fusion protein V1W1-Linker-FGF21 (containing SEQ ID NO: 89) and V1W2-Linker-FGF21 (containing SEQ ID NO: 89) within a pharmacodynamic experimental cycle. [Figure 5] Figure 5 shows the OGTT time course curves (a, c) and area under the blood glucose curve values ​​(b, d) in high-fat diet-induced obese mice treated with high, medium, and low doses of the hGIPR antibody / FGF21 fusion proteins V1W1-Linker-FGF21 (containing SEQ ID NO: 89) and V1W2-Linker-FGF21 (containing SEQ ID NO: 89) within a pharmacodynamic experimental cycle. [Figure 6]Figure 6 shows the effects of high, medium, and low doses of the hGIPR antibody / FGF21 fusion protein V1W1-Linker-FGF21 (comprising SEQ ID NO: 89) on serum indices of liver function (a) and serum indices of liver function (b) in high-fat diet-induced obese mice within a pharmacodynamic experimental cycle. [Figure 7] Figure 7 shows the effects of high, medium, and low doses of the hGIPR antibody / FGF21 fusion protein V1W2-Linker-FGF21 (comprising SEQ ID NO: 89) on serum indices of liver function (a) and serum indices of liver function (b) in high-fat diet-induced obese mice within a pharmacodynamic experimental cycle. [Figure 8] Figure 8 shows the activation curves of the GIPR antibody / FGF21 fusion proteins V1W2-Linker-FGF21 (including SEQ: 89), V1W2-Linker-FGF21 (including SEQ ID NO: 115), V1W2-Linker-FGF21 (including SEQ ID NO: 116), V1W2-Linker-FGF21 (including SEQ ID NO: 117), and V1W2-Linker-FGF21 (including SEQ ID NO: 118) that activate the mFGFR1C / hKLB signaling pathway, as detected in reporter gene experiments. The EC50 values ​​were 0.80 nM for V1W2-FGF21 (SEQ: 89), 1.33 nM for V1W2-FGF21 (SEQ: 115), 1.35 nM for V1W2-FGF21 (SEQ: 116), 1.29 nM for V1W2-FGF21 (SEQ: 117), and 1.71 nM for V1W2-FGF21 (SEQ: 118), respectively. [Figure 9a] Figure 9 shows the effects of high, medium, and low doses of the hGIPR antibody / FGF21 fusion protein V1W2-Linker-FGF21 (containing SEQ ID NO: 118) on serum indices of liver function (a), serum indices of liver function (b), and NAS score and fibrosis score (c) in mice with high-fat diet-induced non-alcoholic steatohepatitis within a pharmacodynamic experimental cycle. [Figure 9b] *See (b) above [Figure 9c] *See (c) above [Figure 10-1] FIG. 10 shows mutant sequences of wild-type FGF21 represented by SEQ ID NO:77 to SEQ ID NO:89, SEQ ID NO:115 to SEQ ID NO:118, and SEQ ID NO:122. [Figure 10-2] ※continuation [Figure 11-1] FIG. 11 shows the amino acid or nucleotide sequences represented by 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. [Figure 11-2] ※continuation [Figure 11-3] ※continuation [Figure 11-4] ※continuation [Figure 11-5] ※continuation [Figure 11-6] ※continuation [Figure 11-7] ※continuation DETAILED DESCRIPTION OF THE INVENTION

[0033] definition In the present invention, unless otherwise defined, scientific and technical terms have the meanings understood by those skilled in the art. Generally, the nomenclature and techniques related to pharmacology, biology, biochemistry, cell and tissue culture, biology, molecular biology, immunology, microbiology, genetics, protein nucleic acid chemistry, and hybridization described in the present invention are well known and commonly used in the art.

[0034] Standard one-letter or three-letter abbreviations are used to indicate polynucleotide and polypeptide sequences. Polypeptide sequences are written with the first amino acid residue (N') having the amino group in the left-most position and the last amino acid residue (C') having the carboxy group in the right-most position, e.g., the sequences of FGF21 fragments of the present invention: 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 designated with the 5' end of the upstream strand on the left and the 3' end on the right. Specific portions of a polypeptide may be designated by amino acid residue number, e.g., amino acids 80-130, or by the actual residues at that site, e.g., Lys80-Lys130. A particular polypeptide or polynucleotide sequence can also be described by interpreting differences from a reference sequence.

[0035] The terms "peptide," "polypeptide," and "protein" all refer to molecules comprising two or more amino acids joined together by peptide bonds. These terms encompass, for example, naturally occurring proteins, artificial proteins, and polypeptide analogs of protein sequences (e.g., muteins, variants, and fusion proteins), as well as proteins that are post-translationally or covalently or non-covalently modified. A peptide, polypeptide, or protein can be monomeric or polymeric.

[0036] The term "polypeptide fragment" refers to a polypeptide that has an amino- and / or carboxy-terminal deletion relative to the corresponding full-length protein. Fragments 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 in length. Fragments can be, for example, up to 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 in length. A fragment can further comprise one or more additional amino acids at one or both ends, and can be, for example, an amino acid sequence derived from a different naturally occurring protein (e.g., an Fc or leucine zipper domain) or an artificial amino acid sequence (e.g., an artificial linker sequence).

[0037] The polypeptides of the present invention include polypeptides modified in any manner for any reason, such as (1) reduced susceptibility to proteolysis, (2) reduced susceptibility to oxidation, (3) altered affinity for forming protein complexes, (4) altered binding affinity, and (5) conferring or modifying other physicochemical or functional properties. Analogs include mutant proteins of polypeptides. For example, one or more amino acid substitutions (e.g., conservative amino acid substitutions) may be made in the native sequence (e.g., in a portion of the polypeptide other than the domain that forms intramolecular contacts). A "conservative amino acid substitution" is one that does not significantly alter the structural characteristics of the parent sequence (e.g., the substituted amino acid does not disrupt a helix present in the parent sequence or other secondary structure types necessary for the characterization or function of the parent sequence).

[0038] A "variant" of a polypeptide includes an amino acid sequence in which one or more amino acid residues have been inserted, deleted, and / or substituted relative to another polypeptide sequence. Variants of the present invention include protein variants, antibody variants, and antibody fusion protein variants. In an embodiment of the present invention, the FGF21 portion contained in the antibody fusion protein used has several different variants. For example, SEQ ID NOs:77 to 89, 115 to 118, and 122 describe mutant sequences of wild-type FGF21 in which one or more amino acid residues have been inserted, deleted, and / or substituted relative to the wild-type FGF21 while maintaining equivalent biological activity and protein structure. These FGF21 mutants, when fused to the antibodies described below, form "variants" of wild-type FGF21 fusion proteins; for example, SEQ ID NOs:104 to 106, 119 to 121, and 123 to 125 set forth the heavy chain sequences of such mutant FGF21 fusion proteins.

[0039] A "derivative" of a polypeptide is a polypeptide that has been chemically modified by other chemical moieties, such as polyethylene glycol, conjugation to albumin (eg, human serum albumin), phosphorylation, and glycosylation.

[0040] Unless otherwise specified, the term "antibody" includes antibodies comprising two full-length heavy chains and two full-length light chains, and derivatives, variants, fragments and muteins thereof, examples of which are set forth below.

[0041] The term "antibody" refers to a protein comprising an antigen-binding portion and, optionally, a scaffold or framework portion that facilitates binding of the antigen to the antibody. Examples of antibodies include complete antibodies, antibody fragments (e.g., the antigen-binding portion of an antibody), antibody derivatives, antibody analogs, and antibody variants, as well as fusion proteins thereof. Antibodies may comprise, for example, alternative protein scaffolds or artificial scaffolds grafted with CDRs or CDR derivatives. Scaffolds include, but are not limited to, antibody-derived scaffolds incorporated to stabilize the three-dimensional structure of the antibody, and fully synthetic scaffolds, including, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins 53:121-129; Roque et al., 2004, Biotechnol. Prog. 20:639-654. Furthermore, the antibody may be a peptide mimetic ("PAM"), or a scaffold comprising an antibody mimetic that uses fibronectin as a scaffold.

[0042] An antibody may have the structure of, for example, a native immunoglobulin. An "immunoglobulin" is a tetrameric molecule. In native immunoglobulins, each tetramer is composed of two pairs of identical polypeptide chains, a "light" chain (approximately 25 kDa) and a "heavy" chain (approximately 50-70 kDa). The amino terminus of each chain contains a variable domain of approximately 100-110 amino acids, primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human antibody light chains are classified as kappa and lambda light chains. Heavy chains are classified as μ, delta, alpha, or epsilon, which define antigen isotypes, e.g., 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 approximately 12 or more amino acids, and the heavy chains further contain a "D" region of approximately 10 or more amino acids. See Basic Immunology Ch. 7 (Paul, 2nd ed., Raven Press, 1989). The variable regions of each light / heavy chain pair form the antibody binding site, such that an intact immunoglobulin has two binding sites.

[0043] Native immunoglobulin chains exhibit the same basic structure of relatively conserved framework regions (FRs), also called complementarity-determining regions or CDRs, connected by three hypervariable regions. From the N-terminus to the C-terminus, both light and heavy chains contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments for each domain are consistent with the definition in Kabat et al., "Sequences of Proteins of Immunological Interest," 5th ed., U.S. Dept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991.

[0044] Unless otherwise specified, the term "antibody" refers to an intact immunoglobulin or an antigen-binding portion capable of specifically binding in competition with an intact antibody. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical digestion of intact antibodies. Antigen-binding portions specifically include Fab, Fab', F(ab'), Fv, domain antibodies (dAbs), fragments containing the complementarity-determining regions (CDRs), single-chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and portions of polypeptides containing at least sufficient immunoglobulin to confer specific antigen binding to the polypeptide.

[0045] Fab fragments are V L , V H , C L , and C H1 A F(ab')2 fragment is a monovalent fragment having two Fab fragments linked by a disulfide bond at the hinge region, and an Fv fragment is a bivalent fragment having two Fab fragments linked by a disulfide bond at the hinge region. H and V L domain, and the dAb fragment has a V H Domain, V L Domain or V H or V Land antigen-binding fragments of the domain (U.S. Patent Nos. US 6,846,634 and US 6,696,245; U.S. Patent Application Publication Nos. US 2005 / 0202512, US 2004 / 0202995, US 2004 / 0038291, US 2004 / 0009507, and US 2003 / 0039958; Ward et al., 1989, Nature 341:544-546).

[0046] Single-chain antibodies (scFv) are V L and V H A fusion protein is a protein in which regions are joined by a linker (e.g., a synthetic sequence of amino acid residues) to form a contiguous protein, where the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen-binding site (see, e.g., Bird et al., 1988, Science 242:423-26, and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83).

[0047] Diabodies are bivalent antibodies containing two polypeptide chains, each of which has a V domain connected by a linker that is too short to allow pairing of the two domains on the same chain. H and V L Diabodies contain three and four polypeptide chains, each of which allows pairing 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). ​​When the two polypeptide chains of a diabody are identical, the resulting diabody has identical antigen-binding sites. Polypeptide chains with different sequences can be used to create diabodies with different antigen-binding sites. Similarly, triabodies and tetrabodies are antibodies that contain three and four polypeptide chains, respectively, forming three and four antigen-binding sites, which may be the same or different.

[0048] In the present invention, the complementarity-determining regions (CDRs) and framework regions (FRs) of a given antibody are identified using the method described by Kabat et al. in Sequences of Proteins of Immunological Interest, 5th ed., U.S. Dept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991. One or more CDRs can be incorporated into a molecule, either covalently or noncovalently, to form an antibody. An antibody can incorporate CDR(s) from a larger polypeptide chain. The CDR(s) can be covalently linked to another polypeptide chain or can be incorporated noncovalently into the CDR(s). The CDRs enable an antibody to specifically bind to a particular antigen of interest.

[0049] An antibody can have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or different. For example, a native human immunoglobulin typically has two identical binding sites, while a "bispecific" or "bifunctional" antibody has two different binding sites.

[0050] The term "murine-derived antibody" includes antibodies having one or more variable and constant regions derived from murine immunoglobulin sequences.

[0051] The term "humanized antibody" refers to an antibody that is created by grafting sequences of the complementarity determining regions of a mouse antibody molecule onto the framework of a human antibody variable region.

[0052] The terms "antigen-binding domain," "antigen-binding region," or "antigen-binding site" refer to the portion of an antibody that contains the amino acid residues that interact with an antigen and contribute to the antibody's specificity and affinity for the antigen. For antibodies that specifically bind to their antigen, this includes at least a portion of at least one of their CDR domains.

[0053] The term "epitope" is the portion of a molecule that binds to (e.g., by) an antibody. An epitope can include noncontiguous portions of a molecule (e.g., in a polypeptide, amino acid residues that are not contiguous in the primary sequence of the polypeptide but are sufficiently close to each other in the tertiary and quaternary structure of the polypeptide to be bound by an antibody).

[0054] The "percent identity" of two polynucleotide or two polypeptide sequences is determined using the GAP computer program (part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) with default parameters comparing sequences.

[0055] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably throughout and can 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, a nucleic acid molecule of the invention comprises a contiguous open reading frame encoding an antibody provided by the invention, or a fragment, derivative, mutein, or variant thereof.

[0056] Two single-stranded nucleotides are "complementary" to each other if their sequences can be antiparallel, with each nucleotide in one polynucleotide being opposite to its complementary nucleotide in the other polynucleotide, with no gaps introduced and no unpaired nucleotides found at the 5' or 3' end of each sequence. A polynucleotide is "complementary" to another polynucleotide if the two polynucleotides can hybridize to each other under moderately stringent conditions. Thus, a polynucleotide can be complementary to another polynucleotide, but not its complementary sequence.

[0057] The term "vector" refers to a nucleic acid that can be used to introduce another nucleic acid linked to it into a cell. One type of vector is a "plasmid," which refers to a linear or circular double-stranded DNA molecule that can be ligated to additional nucleic acid segments. 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. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors containing a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the host cell genome upon introduction into a host cell and are thereby replicated along with the host genome. An "expression vector" is a type of vector that can direct the expression of a selected polynucleotide.

[0058] A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression of the nucleotide sequence (e.g., the level, time, or location of expression). A "regulatory sequence" is a nucleic acid that affects the expression (e.g., the level, time, or location of expression) of a nucleic acid to which it is operably linked. A regulatory sequence may, for example, act directly on the nucleic acid being regulated or through one or more other molecules (e.g., polynucleotides that bind 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.

[0059] The term "host cell" refers to a cell for expressing a nucleic acid, such as a nucleic acid provided by the present invention. A host cell can be a prokaryote, such as E. coli, or a eukaryote, such as a unicellular eukaryote (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 that can then be expressed in the host cell. The phrase "recombinant host cell" can be used to refer to a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell can also be a cell that contains a nucleic acid but does not express that nucleic acid at a desired level unless a control sequence is introduced into the host cell so that it is operably linked to the nucleic acid. It should be understood that the term "host cell" refers not only to the particular subject cell but also to the progeny or potential progeny of that cell. Due to certain modifications that occur in succeeding generations, such as mutations or environmental influences, such progeny may actually differ from the parent cell and still be included within the scope of the term as used herein. Gastric inhibitory polypeptide receptor

[0060] Gastric inhibitory polypeptide receptor (GIPR) belongs to the type B family of seven-transmembrane G protein-coupled receptors. The receptor is coupled to one or more intracellular signaling pathways by heterotrimeric guanine nucleotide-binding proteins (G proteins) (Drucker et al., 2006, Cell Metab. 3:153-65). Research has shown that GIPR is mainly expressed on the surface of pancreatic beta cells and adipocytes (Ravn et al., 2013, J. Biol. Chem. 288:19760-72) and is involved in both glucose and lipid metabolism in humans, and is therefore closely related to diabetes, obesity, and related diseases (Skaw et al., 2016, Diabetes Obes. Metab. 18:847-854). In the present invention, "human GIPR" and "hGIPR" both refer to human-derived gastric inhibitory polypeptide receptors and can be used interchangeably. As used herein, "mouse GIPR" and "mGIPR" both refer to gastric inhibitory polypeptide receptor derived from mouse, and may also be used interchangeably.

[0061] In one embodiment, the antibody provided by the present invention is an antibody that specifically binds to human GIPR. In another embodiment, the antibody provided by the present invention is an antibody that specifically binds to GIPR in the cell membrane, and this antibody can inhibit or block the transmission of GIP signaling in these cells. In another embodiment, the antibody provided by the present invention is an antibody that specifically binds to human GIPR and can bind to GIPR of other species (e.g., monkey or mouse) and block GIP signaling in these species. In yet another embodiment, the antibody provided by the present invention is a mouse-derived antibody that can bind to human GIPR and GIPR of other species (e.g., monkey).

[0062] In one embodiment, the amino acid and polynucleotide sequences of GIPR are shown below, with sequence data obtained from the National Center for Biotechnology Information's GeneBank database and the European Bioinformatics Institute's Uniprot database. Homo sapiens polynucleotide, accession number: S79852; Homo sapiens amino acids, accession number: AAB35419.2. Gastric inhibitory polypeptide receptor antibodies (GIPR antibodies)

[0063] In one embodiment, the present invention provides a GIPR antibody. In another embodiment, the GIPR antibody provided by the present invention is a complete GIPR antibody. In another embodiment, the GIPR antibody provided by the present invention is a fragment of a GIPR antibody. In another embodiment, the GIPR antibody provided by the present invention is a derivative of a GIPR antibody. In another embodiment, the GIPR antibody provided by the present invention is a mutein of a GIPR antibody. In yet another embodiment, the GIPR antibody provided by the present invention is a variant of a GIPR antibody.

[0064] In one embodiment, the GIPR antibodies provided by the invention comprise one, two, three, four, five, or six amino acid sequences, each independently selected from the amino acid sequences set forth below. 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 sequence: SEQ ID NO: 3, 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.

[0065] Table 1 shows the amino acid sequences of the light chain CDRs of the GIPR antibodies provided by the invention, and the corresponding polynucleotide coding sequences. Table 2 shows the amino acid sequences of the heavy chain CDRs of the GIPR antibodies provided by the invention, and the corresponding polynucleotide coding sequences.

[0066] [Table 1-1] [Table 1-2]

[0067] [Table 2]

[0068] In one embodiment, an antibody provided by the invention comprises a sequence that differs from one of the CDR amino acid sequences shown in Tables 1 and 2 by 5, 4, 3, 2, or 1 amino acid addition, substitution, and / or deletion. In other embodiments, an antibody provided by the invention comprises a sequence that differs from one of the CDR amino acid sequences shown in Tables 1 and 2 by 4, 3, 2, or 1 amino acid addition, substitution, and / or deletion.

[0069] In other embodiments, the antibodies provided by the invention comprise a sequence that differs from one of the CDR amino acid sequences shown in Tables 1 and 2 by three, two, or one amino acid addition, substitution, and / or deletion.

[0070] In other embodiments, the antibodies provided by the invention comprise a sequence that differs from one of the CDR amino acid sequences shown in Tables 1 and 2 by two or one amino acid addition, substitution and / or deletion.

[0071] In yet other embodiments, the antibodies provided by the invention comprise a sequence that differs from one of the CDR amino acid sequences shown in Tables 1 and 2 by one amino acid addition, substitution, and / or deletion.

[0072] In one embodiment, the GIPR antibodies provided by the invention comprise one or two amino acid sequences, each independently selected from the amino acid sequences set forth below. 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, and b. Heavy chain CDR1 amino acid sequence: SEQ ID NO: 12.

[0073] In other embodiments, the GIPR antibodies provided by the invention comprise one or two amino acid sequences, each independently selected from the amino acid sequences set forth 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.

[0074] In other embodiments, the GIPR antibodies provided by the invention comprise one or two amino acid sequences, each independently selected from the amino acid sequences set forth below. a. Light chain CDR3 amino acid sequences: SEQ ID NO: 3, SEQ ID NO: 4, and b. Heavy chain CDR3 amino acid sequence: SEQ ID NO: 14.

[0075] In other embodiments, the GIPR antibodies provided by the invention comprise one, two, three, or four amino acid sequences, each independently selected from the amino acid sequences set forth below. 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. Heavy chain CDR1 amino acid sequence: SEQ ID NO: 12, c. Light chain CDR2 amino acid sequence: YAS, SEQ ID NO: 2, and d. Heavy chain CDR2 amino acid sequence: SEQ ID NO: 13.

[0076] In other embodiments, the GIPR antibodies provided by the invention comprise one, two, three, or four amino acid sequences, each independently selected from the amino acid sequences set forth below. 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. Heavy chain CDR1 amino acid sequence: SEQ ID NO: 12, c. Light chain CDR3 amino acid sequences: SEQ ID NO: 3, SEQ ID NO: 4, and d. Heavy chain CDR3 amino acid sequence: SEQ ID NO: 14.

[0077] In yet other embodiments, the GIPR antibodies provided by the invention comprise one, two, three, or four amino acid sequences, each independently selected from the amino acid sequences set forth below. a. Light chain CDR2 amino acid sequence: YAS, SEQ ID NO: 2, b. Heavy chain CDR2 amino acid sequence: SEQ ID NO: 13, c. Light chain CDR3 amino acid sequences: SEQ ID NO: 3, SEQ ID NO: 4, and d. Heavy chain CDR3 amino acid sequence: SEQ ID NO: 14.

[0078] In one embodiment, the GIPR antibodies provided by the invention comprise one, two, or three amino acid sequences, each independently selected from the amino acid sequences set forth below. 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.

[0079] In other embodiments, the GIPR antibodies provided by the invention comprise one, two, or three amino acid sequences, each independently selected from the amino acid sequences set forth below. SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14.

[0080] In one embodiment, the GIPR antibody provided by the present invention comprises a combination of light chain and heavy chain CDR1 amino acid sequences independently selected from the list below. 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.

[0081] In other embodiments, the GIPR antibodies provided by the present invention comprise a combination of light chain and heavy chain CDR2 amino acid sequences independently selected from the list below. YAS and SEQ ID NO: 13, and SEQ ID NO: 2 and SEQ ID NO: 13.

[0082] In yet other embodiments, the GIPR antibodies provided by the present invention comprise a combination of light chain and heavy chain CDR3 amino acid sequences independently selected from the list below. SEQ ID NO: 3 and SEQ ID NO: 14, SEQ ID NO: 4 and SEQ ID NO: 14.

[0083] In one embodiment, the GIPR antibody provided by the present invention comprises: a. A combination of light chain and heavy chain CDR1 amino acid sequences independently selected from the following list: 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 b. A combination of light chain and heavy chain CDR2 amino acid sequences independently selected from the following list: YAS and SEQ ID NO: 13, and SEQ ID NO: 2 and SEQ ID NO: 13. Includes:

[0084] In other embodiments, the GIPR antibodies provided by the invention are a. A combination of light chain and heavy chain CDR1 amino acid sequences independently selected from the following list: 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 b. A combination of light chain and heavy chain CDR3 amino acid sequences independently selected from the following list: SEQ ID NO: 3 and SEQ ID NO: 14, SEQ ID NO: 4 and SEQ ID NO: 14. Includes:

[0085] In other embodiments, the GIPR antibodies provided by the invention are a. A combination of light chain and heavy chain CDR2 amino acid sequences independently selected from the following list: YAS and SEQ ID NO: 13, and SEQ ID NO: 2 and SEQ ID NO: 13, and b. A combination of light chain and heavy chain CDR3 amino acid sequences independently selected from the following list: SEQ ID NO: 3 and SEQ ID NO: 14, SEQ ID NO: 4 and SEQ ID NO: 14. Includes:

[0086] In yet other embodiments, the GIPR antibodies provided by the present invention are a. A combination of light chain and heavy chain CDR1 amino acid sequences independently selected from the following list: 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; b. A combination of light chain and heavy chain CDR2 amino acid sequences independently selected from the following list: YAS and SEQ ID NO: 13, and SEQ ID NO: 2 and SEQ ID NO: 13, and c. A combination of light chain and heavy chain CDR3 amino acid sequences independently selected from the following list: SEQ ID NO: 3 and SEQ ID NO: 14, SEQ ID NO: 4 and SEQ ID NO: 14. Includes:

[0087] In one embodiment, the GIPR antibody provided by the present invention comprises: a. Light chain and heavy chain CDR1, CDR2, and CDR3 amino acid sequence combinations: 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; b. Light chain and heavy chain CDR1, CDR2, and CDR3 amino acid sequence combinations: 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; c. Combinations of light chain 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; d. Light chain and heavy chain CDR1, CDR2, and CDR3 amino acid sequence combinations: 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; e. Combinations of light chain 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; f. Light chain and heavy chain CDR1, CDR2, and CDR3 amino acid sequence combinations: 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; g. A combination of light chain 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 h. Light chain and heavy chain CDR1, CDR2, and CDR3 amino acid sequence combinations: 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; i. Light chain and heavy chain CDR1, CDR2, and CDR3 amino acid sequence combinations: 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 Includes:

[0088] In one embodiment, the GIPR antibodies provided by the invention comprise one or two amino acid sequences, each independently selected from the amino acid sequences set forth below. a. light chain variable domain amino acid sequences: 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 amino acid sequences at least 80%, at least 85%, at least 90%, or at least 95% identical to any of these sequences; and b. Heavy chain variable domain amino acid sequence: SEQ ID NO: 38, and amino acid sequences at least 80%, at least 85%, at least 90%, or at least 95% identical thereto.

[0089] In other embodiments, the polynucleotide coding sequence of a GIPR antibody provided by the invention comprises one or two polynucleotide coding sequences, each polynucleotide coding sequence independently selected from the polynucleotide sequences set forth below. a. light chain variable domain polynucleotide coding sequences: 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 polynucleotide coding sequences at least 80%, at least 85%, at least 90%, or at least 95% identical to any of these sequences; and b. Heavy chain variable domain polynucleotide coding sequence: SEQ ID NO:48, and polynucleotide coding sequences at least 80%, at least 85%, at least 90%, or at least 95% identical thereto.

[0090] In one embodiment, the GIPR antibodies provided by the invention comprise an amino acid sequence independently selected from the list below. 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.

[0091] In other embodiments, the GIPR antibodies provided by the invention comprise an amino acid sequence independently selected from the list below. SEQ ID NO: 38.

[0092] In one embodiment, the GIPR antibody provided by the present invention comprises a combination of light and heavy chain variable domain amino acid sequences independently selected from the list below. 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.

[0093] In one embodiment, the GIPR antibodies provided by the invention comprise an amino acid sequence independently selected from the list below. 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.

[0094] In other embodiments, the GIPR antibodies provided by the invention comprise a combination of light and heavy chain variable domain amino acid sequences independently selected from the list below. 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).

[0095] The GIPR antibodies provided by the present invention may be designated by the symbol "LxHy," where "x" corresponds to the code number of the light chain variable region sequence and "y" corresponds to the code number of the heavy chain variable region sequence. For example, L2H2 refers to a complete 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).

[0096] In one embodiment, the GIPR antibodies provided by the invention comprise one or two amino acid sequences, each independently selected from the amino acid sequences set forth below. a. Light chain constant region amino acid sequence: SEQ ID NO: 49, and b. Heavy chain constant region amino acid sequences: SEQ ID NO: 50, SEQ ID NO: 51.

[0097] In one embodiment, the GIPR antibodies provided by the present invention comprise one or two amino acid sequences, each independently selected from the combinations of light chain and heavy chain constant region amino acid sequences shown below. SEQ ID NO: 49 and SEQ ID NO: 50, SEQ ID NO: 49 and SEQ ID NO: 51.

[0098] In one embodiment, the GIPR antibody provided by the present invention comprises the amino acid sequences of light and heavy chain CDRs and FRs (frameworks) set forth in the present invention. The amino acid sequences of the FRs are included in the light or heavy chain variable domain amino acid sequence and are not separately set forth. In one embodiment, the antibody comprises the light chain CDR1 sequence set forth in the present invention. In another embodiment, the antibody comprises the light chain CDR2 sequence set forth in the present invention. In another embodiment, the antibody comprises the light chain CDR3 sequence set forth in the present invention. In another embodiment, the antibody comprises the heavy chain CDR1 sequence set forth in the present invention. In another embodiment, the antibody comprises the heavy chain CDR2 sequence set forth in the present invention. In another embodiment, the antibody comprises the heavy chain CDR3 sequence set forth in the present invention. In another embodiment, the antibody comprises the light chain FR1 sequence set forth in the present invention. In another embodiment, the antibody comprises the light chain FR2 sequence set forth in the present invention. In another embodiment, the antibody comprises the light chain FR3 sequence set forth in the present invention. In another embodiment, the antibody comprises the light chain FR4 sequence set forth in the present invention. In another embodiment, the antibody comprises the heavy chain FR1 sequence set forth in the present invention. In other embodiments, the antibody comprises a heavy chain FR2 sequence of the invention. In other embodiments, the antibody comprises a heavy chain FR3 sequence of the invention. In yet other embodiments, the antibody comprises a heavy chain FR4 sequence of the invention.

[0099] In one embodiment, the light chain CDR3 sequence of the antibody differs by no more than 6, 5, 4, 3, 2, or 1 amino acid addition, substitution, and / or deletion from one of the light chain CDR3 amino acid sequences SEQ ID NO: 3, SEQ ID NO: 4 set forth in the present invention. In other embodiments, the antibody further comprises a combination of 1, 2, 3, 4, 5, or 6 of the light and heavy chain CDR sequences set forth in the present invention.

[0100] In one embodiment, the GIPR antibodies provided by the present invention comprise a light chain variable domain amino acid sequence selected from the 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 set forth in the present invention. 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 difference, and each sequence difference is independently a deletion, insertion, or substitution of an amino acid residue. In other embodiments, 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 other embodiments, the light chain variable domain polynucleotide coding sequence of the GIPR antibody is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the polynucleotide coding 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 other embodiments, the light chain variable domain polynucleotide coding sequence of the GIPR antibody comprises a polynucleotide sequence that hybridizes under moderate conditions to the complementary sequence of the polynucleotide coding sequence of the light chain variable domain of one of 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 yet other embodiments, the light chain variable domain polynucleotide coding sequence of the GIPR antibody comprises a polynucleotide sequence that hybridizes under stringent conditions to the complementary sequence of the polynucleotide coding 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).

[0101] In one embodiment, the GIPR antibodies provided by the present invention comprise a heavy chain variable domain amino acid sequence selected from the H1 (SEQ ID NO:38) heavy chain variable domain sequences set forth in the present invention.

[0102] In one embodiment, the antibody provided by the invention is a polypeptide comprising a sequence identical to that 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.

[0103] In one embodiment, the antibody provided by the present invention 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.

[0104] The antibodies provided by the present invention 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 alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region, such as a mouse alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region. In one embodiment, the light or heavy chain constant region is a fragment, derivative, mutant, or mutein of a native constant region.

[0105] In one embodiment, the antibody provided by the present invention 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: Human constant light chain kappa domain amino acid sequence: (SEQ ID NO: 49).

[0106] In one embodiment, the antibody provided by the invention further comprises a human heavy chain constant domain or a fragment thereof.

[0107] The heavy chain constant region amino acid sequence is as follows: Human heavy chain constant region amino acid sequence (hIgG2): (SEQ ID NO: 50), and Human heavy chain constant region amino acid sequence (hIgG4): (SEQ ID NO: 51).

[0108] In one embodiment, a GIPR antibody provided by the present invention comprises a light chain domain amino acid sequence selected from V1 (SEQ ID NO: 103) 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 difference, where each sequence difference is independently a deletion, insertion, or substitution of an amino acid residue. In other embodiments, 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 other embodiments, the polynucleotide coding sequence for 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 coding sequence for one of V1 and V2. In other embodiments, the polynucleotide coding sequence for the light chain domain of the GIPR antibody comprises a polynucleotide sequence that hybridizes under moderate conditions to the complementary sequence of the polynucleotide coding sequence for the light chain domain of V1. In yet other embodiments, the polynucleotide coding sequence for the light chain domain of the GIPR antibody comprises a polynucleotide sequence that hybridizes under stringent conditions to the complementary sequence of the polynucleotide coding sequence for the light chain domain of V1.

[0109] In one embodiment, the GIPR antibody provided by the present invention comprises a heavy chain domain amino acid sequence selected from W1 (SEQ ID NO: 111) and W2 (SEQ ID NO: 112) set forth 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 difference, where each sequence difference is independently a deletion, insertion, or substitution of an amino acid residue. In another embodiment, the heavy chain domain amino acid sequence of the GIPR antibody is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the heavy chain domain sequence of one of W1 and W2. In other embodiments, the polynucleotide coding sequence for the heavy chain domain of the GIPR antibody is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the polynucleotide coding sequence for one of W1 and W2. In other embodiments, the polynucleotide coding sequence for the heavy chain domain of the GIPR antibody comprises a polynucleotide sequence that hybridizes under moderately stringent conditions to the complementary sequence of the polynucleotide coding sequence for the heavy chain domain of one of W1 and W2. In one embodiment, the polynucleotide coding sequence for the heavy chain domain of the GIPR antibody comprises a polynucleotide sequence that hybridizes under stringent conditions to the complementary sequence of the polynucleotide coding sequence for the heavy chain domain of one of W1 and W2.

[0110] In another embodiment, the antibody provided by the present invention 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).

[0111] In one embodiment, the GIPR antibody provided by the present invention is selected from a murine-derived 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') 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.

[0112] In one embodiment, the GIPR antibody provided by the present invention is a GIPR monoclonal antibody.

[0113] In other embodiments, the GIPR antibodies provided by the present invention are monoclonal antibodies comprising a combination of amino acid sequences selected from the list below. 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.

[0114] In one embodiment, the GIPR antibody provided by the present invention is a mouse-derived GIPR antibody. In another embodiment, the GIPR antibody provided by the present invention is a humanized GIPR antibody.

[0115] In one embodiment, the IC of the GIPR antibody provided by the present invention that reduces human GIP signaling 50 The value is about 1 nM to about 200 nM or about 1 nM to about 100 nM.

[0116] Antibodies and antibody fragments In one embodiment, the antibodies provided by the present invention are whole antibodies (including polyclonal, monoclonal, chimeric, humanized, or human antibodies having full-length heavy and / or light chains). In another embodiment, the antibodies provided by the present invention are antibody fragments, such as F(ab')2, Fab, Fab', Fv, Fc, or Fd fragments, single-domain antibodies, single-chain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, or bis-scFvs (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology 23:1126-1136). In another embodiment, the antibodies provided by the present invention comprise antibody polypeptides disclosed in U.S. Patent No. 6,703,199, including fibronectin polypeptide monoclonal antibodies. In yet other embodiments, the antibodies provided by the present invention comprise the single-chain polypeptides disclosed in US Patent Publication 2005 / 0238646.

[0117] In one embodiment, the variable regions of the genes expressing the monoclonal antibody of interest in a hybridoma are amplified using nucleotide primers. These primers can be synthesized by one skilled in the art or purchased commercially. Mouse and human variable region primers can be purchased commercially. Ha , V Hb , V Hc , V Hd , C H1 , V L , and C L These primers are the primers for the IMMUNOZAP TM H or IMMUNOZAP TM These vectors can then be introduced into E. coli, yeast, or mammalian-based systems for expression. H and V LLarge amounts of single-chain proteins containing fusions of the domains can be produced using these methods (see Bird et al., 1988, Science 242:423-426).

[0118] It should be understood by those skilled in the art that certain proteins, such as antibodies, can undergo various post-translational 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 alterations in glycosylation, methionine oxidation, diketopiperidine formation, aspartate isomerization, and asparagine deamidation. Carboxy-terminal basic residues (e.g., lysine or arginine) of antibodies can be lost through frequent modification by carboxypeptidases (see Harris, 1995, Journal of Chromatography 705:129-134).

[0119] Murine monoclonal antibodies can be produced using conventional hybridoma cell-based methods. Monoclonal antibodies can be isolated and purified by a variety of established techniques, including affinity chromatography using protein A-Sepharose, size-exclusion chromatography, and ion-exchange chromatography (see, e.g., Coligan, pp. 2.7.1-2.7.12 and 2.9.1-2.9.3; Baines et al., "Purification of Immunoglobulin G (IgG)," Methods in Molecular Biology, Vol. 10, pp. 79-104 (The Humana Press, Inc., 1992)). Monoclonal antibodies can be purified by affinity chromatography using an appropriate ligand selected based on specific characteristics of the antibody (e.g., heavy or light chain isotype, binding specificity, etc.). Examples of suitable ligands for affinity chromatography include protein A, protein G, anti-constant region (light or heavy chain) antibodies, anti-idiotypic antibodies, and FGF21 binding proteins or fragments or variants thereof.

[0120] By modifying and affinity maturing molecules using the complementarity-determining regions (CDRs) in the center of the antibody binding site, antibodies with increased affinity, such as those for c-erbB-2, can be obtained (Schier et al., 1996, J. Mol. Biol. 263:551-567). Thus, such techniques are useful for preparing antibodies against human GIPR.

[0121] Antibodies against human GIPR can be used, for example, in assays to detect the presence of human GIPR, either in vitro or in vivo.

[0122] Antibodies can also be prepared by any conventional technique. For example, antibodies can be purified from cells that naturally express them (e.g., antibodies can be purified from hybridomas that produce them) or can be produced in recombinant expression systems using any technique well known in the art. See, for example, "Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses," Kennet et al., Plenum Press (1980), and "Antibodies: A Laboratory Manual," Harlow and Land, Cold Spring Harbor Laboratory Press (1988). This is described below in the section on nucleic acids.

[0123] Antibodies can be prepared by any known technique and screened for desired properties. Some techniques involve isolating nucleic acid encoding the polypeptide chain (or portion thereof) of the relevant antibody (e.g., an anti-GIPR antibody) and manipulating the nucleic acid by recombinant DNA technology. The nucleic acid can be fused to another relevant nucleic acid or modified (e.g., by mutagenesis or other conventional techniques) to add, delete, or substitute one or more amino acid residues.

[0124] If it is necessary to improve the affinity of an antibody of the present invention comprising one or more of the above CDRs, this can be achieved by several affinity maturation protocols, including maintenance of the CDRs (Yang et al., 1995, J. Mol. Biol. 254: 392-403), chain shuffling (Marks et al., 1992, Bio / Technology 10:779-783), use of mutant strains of E. coli (Low et al., 1996, J. Mol. Biol. 250:350-368), DNA rearrangement (Patten et al., 1997, Curr. Opin. Biotechnol. 8:724-733), phage display (Thompson et al., 1996, J. Mol. Biol. 256:7-88) and further PCR techniques (Crameri et al., 1998, Nature 391:288-291). All of these methods or affinity maturation are described in Vaughan et al., 1998, Nature Biotechnology 16:535-539.

[0125] In one embodiment, the antibody provided by the present invention is an anti-GIPR fragment. This fragment may contain sequences derived entirely from the antibody or 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.

[0126] Single-chain antibodies can be formed by linking the heavy and light chain variable domains (Fv regions) with an amino acid bridge (a short peptide linker) to generate a single polypeptide chain. Such single-chain Fvs (scFvs) consist of two variable domain polypeptides (V L and V HThese have been prepared by fusion DNA encoding a peptide linker between DNAs encoding the variable domains. The resulting polypeptide can fold back on itself to form an antigen-binding monomer, or it can form a multimer (e.g., a dimer, trimer, or tetramer) 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). Various V L and V H By combining the containing polypeptides, multimeric scFvs that bind to various epitopes can be formed (Kriangkum et al., 2001, Biomol. Eng. 18:31-40). Techniques 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 by the present invention, including, but not limited to, scFvs containing the variable domain combination L1H1, are encompassed by the present invention.

[0127] Antigen-binding fragments derived from antibodies can also be obtained according to conventional methods, for example, by proteolysis of antibodies, e.g., pepsin or papain digestion of whole antibodies. For example, antibody fragments can be generated by enzymatic cleavage of antibodies with pepsin, which produces an SS fragment called F(ab')2. This fragment can be further cleaved using a thiol reducing agent to produce a 3.5S Fab' monovalent fragment. Alternatively, the cleavage reaction can be performed using a blocking group for the sulfhydryl groups resulting from cleavage of disulfide bonds. Alternatively, enzymatic cleavage with papain directly produces two monovalent Fab fragments and one 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., John Wiley & Sons, 2003), pp. 2.8, 1-2.8.10 and 2.10A.1-2.10A.5. Other methods for cleaving antibodies, such as separating heavy chains to form monovalent light-heavy chain fragments (Fd), other cleavage of fragments, or other enzymatic, chemical, or genetic techniques, can also be used, so long as the fragment binds to the antigen recognized by the intact antibody.

[0128] Another form of antibody fragment is a peptide comprising one or more complementarity-determining regions (CDRs) of an antibody. CDRs can be obtained by constructing a polypeptide encoding the relevant CDRs. Such polypeptides can be prepared, for example, by using the polymerase chain reaction to synthesize the variable region using mRNA or antibody-producing cells as a template (see, e.g., 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., p. 166 (Cambridge University Press, 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," Monoclonal Antibodies: Principles and Applications, Birch et al., p. 137 (Wiley-Liss, Inc., 1995)). The antibody fragment may further comprise at least one variable domain of an antibody described in the present invention. Thus, for example, a V region domain may comprise 1×10 -7 Monomers and V that can bind independently to GIPR with an affinity equal to or greater than M H or V L It can be a domain.

[0129] The variable region domain may be any naturally occurring variable domain or a genetically engineered form thereof. Genetically engineered forms refer to variable region domains generated using recombinant DNA techniques. Such genetically engineered forms include those generated from a specific antibody variable region, for example, by insertions, deletions, or changes in the amino acid sequence of the specific antibody. Specific examples include genetically engineered variable region domains containing one CDR and, optionally, one or more framework amino acids from one antibody, and the remainder of the variable region domain from another antibody.

[0130] The variable region domain may be covalently linked at the C-terminal amino acid to at least one other antibody domain or fragment thereof. Thus, for example, a V present in the variable region domain may be covalently linked to at least one other antibody domain or fragment thereof. H The domain is immunoglobulin C H1 domain or a fragment thereof. L The domain is C K Thus, for example, an antibody can be a Fab fragment, in which the antigen-binding domains are linked at their C-termini, respectively, to a C domain or fragment thereof. H1 and C K V covalently linked to the domain H and V L Includes domain. C H1 The domain may be extended with additional amino acids to produce, for example, a hinge region or part of a hinge region domain as found in an Fab' fragment, or to produce a region of an antibody C H2 and C H3 Further domains such as the domain may arise. Antibody Derivatives and Variants

[0131] The nucleotide sequence encoding the amino acid sequences L1 and H can be modified, for example, by random mutagenesis or by site-directed mutagenesis (e.g., oligonucleotide-directed site-directed mutagenesis) to generate modified polynucleotides containing one or more specific nucleotide substitutions, deletions, or insertions relative to the non-mutated polynucleotide. Examples of techniques for making such modifications are described in Walder et al., 1986, Gene 42:133; Bauer et al., 1985, Gene 37:73; Craik, 1985, BioTechniques 3:12-19; ​​Smith et al., 1981, Genetic Engineering: Principles and Methods, Plenum Press, and U.S. Patent Nos. 4,518,584 and 4,737,462. These and other methods can be used to generate derivatives of anti-GIPR antibodies that have desirable properties, such as improved affinity, avidity, or specificity for GIPR, improved in vivo or in vitro stability, or reduced in vivo side effects, relative to the non-derivatized antibody.

[0132] Other derivatives of anti-GIPR antibodies in the field of the present invention include covalent or aggregative conjugates of anti-GIPR antibodies or fragments thereof with other proteins or polypeptides, for example, by expressing a recombinant fusion protein containing a heterologous polypeptide fused to the N- or C-terminus of the anti-GIPR antibody polypeptide. For example, the conjugated peptide can be a heterologous signal (or leader) polypeptide, such as the yeast α-factor leader or an epitope tag. Antibody-containing fusion proteins may also contain a peptide (e.g., polyhistidine) added to facilitate antibody purification or identification. The antibody can also be linked to a FLAG peptide, as described in Hopp et al., 1988, Bio / Technology 6:1204, and U.S. Patent No. 5,011,912. The FLAG peptide is highly antigenic and provides an epitope that is reversibly bound by a specific monoclonal antibody (mAb), allowing for rapid assay and easy purification of the expressed recombinant protein. Reagents useful for preparing fusion proteins in which a FLAG peptide is fused to a given polypeptide are commercially available (Sigma-Aldrich, St. Louis, MO). In other embodiments, oligomers containing one or more antibodies can be used as GIPR antagonists or higher-order oligomers. The oligomers can be in the form of covalently or non-covalently linked dimers, trimers, or higher-order oligomers. Oligomers containing two or more antibodies can be used, and one example is a homodimer. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, etc.

[0133] One embodiment relates to oligomers comprising multiple antibodies linked via covalent or non-covalent interactions between peptide moieties fused to the antibodies. Such peptides can be peptide linkers (spacers) or peptides with properties that promote oligomerization. Leucine zippers and certain polypeptides derived from antibodies are peptides that can promote antibody oligomerization, as described in more detail below.

[0134] In certain embodiments, the oligomer comprises two to four antibodies. The antibodies of the oligomer can be in any of the forms described above, such as variants or fragments. Preferably, the oligomer comprises an antibody with GIPR binding activity.

[0135] In one embodiment, oligomers are prepared using immunoglobulin-derived polypeptides. Preparation of fusion proteins comprising heterologous polypeptides fused to various portions of antibody-derived polypeptides (including Fc domains) is 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 of the present invention relates to a dimer comprising two fusion proteins generated by fusing the GIP-binding fragment of an anti-GIPR antibody with the Fc region of the antibody. Dimers can be produced, for example, by inserting a fusion gene encoding the fusion protein into an appropriate expression vector, expressing the fusion gene in a host cell transformed with the recombinant expression vector, allowing the expressed fusion protein to assemble like an antibody molecule, and then immediately forming a dimer through interchain disulfide bonds between the Fc portions.

[0136] As used herein, the term "Fc polypeptide" includes native and mutant forms of polypeptides derived from the Fc region of an antibody. Also included are truncated forms of such polypeptides containing the hinge region that promotes dimerization. Fusion proteins containing the Fc portion (and oligomers formed therefrom) offer the advantage of easy purification by affinity chromatography on Protein A or Protein G columns.

[0137] 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 that of the native Fc sequence shown 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. The mutein exhibits reduced affinity for Fc receptors. In other embodiments, the heavy and / or light chains of an anti-GIPR antibody can be replaced with the variable portions of the heavy and / or light chains of an antibody.

[0138] Alternatively, the oligomer is a fusion protein comprising multiple antibodies with or without linker peptides (spacer peptides). Suitable linker peptides are described in US Pat. Nos. 4,751,180 and 4,935,233.

[0139] Another method for preparing oligomeric antibodies involves the use of leucine zippers. Leucine zipper domains are peptides that promote oligomerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., 1988, Science 240:1759) and have since been found in a variety of different proteins. Among the well-known leucine zippers are natural peptides or derivatives thereof that can dimerize or trimerize. An example of a leucine zipper domain suitable for producing soluble oligomeric proteins is described in PCT application WO 94 / 10308, and a leucine zipper derived from pulmonary surfactant protein D (SPD) is described in Hoppe et al., 1994, FEBS Letters 344:191 (incorporated herein by reference). The use of a modified leucine zipper that allows for stable trimerization of a heterologous protein fused thereto is described in Fanslow et al., 1994, Semin. Immunol. 6:267-78. In one method, a recombinant fusion protein comprising an anti-GIPR antibody fragment or derivative fused to a leucine zipper peptide is expressed in a suitable host cell, and the formed soluble oligomeric anti-GIPR antibody fragment or derivative thereof is recovered from the culture supernatant.

[0140] In other embodiments, antibody derivatives may comprise at least one of the CDRs disclosed herein. For example, one or more CDRs may be incorporated into a known antibody framework region (e.g., IgG1, IgG2, etc.) or linked to a suitable vector to increase its half-life. Suitable vectors include, but are not limited to, Fc, albumin, transferrin, etc. These and other suitable vectors are well known in the art. Such CDR-binding peptides may be in the form of a monomer, dimer, tetramer, or other forms. In one embodiment, one or more water-soluble polymers are attached to one or more specific sites of the binding agent, e.g., 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, e.g., 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 include one or more of monomethoxy-polyethylene glycol, dextran, cellulose or other carbohydrate-based polymers, poly(N-vinylpyrrolidone)-polyethylene glycol, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, and 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 serve to improve the therapeutic efficacy 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.

[0141] It should be understood that the antibodies provided by the present invention may have at least one amino acid substitution, so long as the antibody retains its binding specificity. Accordingly, modifications to the antibody structure are within the scope of the present invention. These may include amino acid substitutions, which may be conservative or non-conservative, that do not destroy the antibody's GIPR-binding ability. Conservative amino acid substitutions may include non-natural amino acid residues that are typically incorporated by chemical peptide synthesis rather than synthesis in biological systems. This includes peptidomimetics and other reverse or inverted forms of amino acid moieties. Conservative amino acid substitutions may also include the substitution of a natural amino acid residue with a standard residue, such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Non-conservative substitutions may involve replacing a member of one class of amino acids or amino acid analogs with a member from another class that has different physical properties (e.g., size, polarity, hydrophobicity, charge).

[0142] Furthermore, those skilled in the art can generate test mutants containing single amino acid substitutions at each desired amino acid residue. The mutants can then be screened using activity assays well known to those skilled in the art. Such mutants can be used to collect information about suitable mutants. For example, if it is found that a change to a specific amino acid residue results in destroyed, undesirably reduced, or inappropriate activity, the mutant with such a change can be avoided. In other words, based on the information collected from such routine experiments, those skilled in the art can easily determine amino acids for which further substitutions should be avoided, alone or in combination with other mutations.

[0143] Those skilled in the art can determine suitable variants of the polypeptides described in the present invention using well-known techniques. In certain embodiments, those skilled in the art can identify suitable regions of the molecule that can be changed without destroying activity by targeting regions that are not important for activity. In certain embodiments, those skilled in the art can identify residues and portions of molecules that are conserved among similar polypeptides. In certain embodiments, regions that may be important for biological activity or structure can also be subject to conservative amino acid substitutions without destroying biological activity or adversely affecting polypeptide structure. Furthermore, those skilled in the art can consider structure-function studies to identify residues in similar polypeptides that are important for activity or structure. In light of such comparisons, those skilled in the art can predict the importance of amino acid residues in the protein that correspond to amino acid residues important for activity or structure in similar proteins. Those skilled in the art can select chemically similar amino acid substitutions in place of such predicted important amino acid residues.

[0144] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence relative to the corresponding structure in similar polypeptides. Taking such information into account, one skilled in the art can predict the alignment of amino acid residues of an antibody with respect to its three-dimensional structure. In certain embodiments, one skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Several 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. Furthermore, computer programs are now available to assist in the prediction of secondary structure. For example, two polypeptides or proteins with greater than 30% sequence identity or greater than 40% similarity often have similar structural topologies. The recent growth of the protein structural database (PDB) has provided improved predictability of secondary structure, including the potential number of folds within a polypeptide or protein structure. See Holm et al., 1999, Nucl. Acid. Res. 27:244-247. It has been suggested that there are a limited number of folds in a given polypeptide or protein, and that after a critical number of structures have been determined, structure prediction becomes significantly more accurate (Brenner et al., 1997, Curr. Op. Struct. Biol. 7:369-376).

[0145] Additional 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" (Holm, supra (1999), and Brenner, supra (1997). In certain embodiments, antibody variants include glycosylation variants, in which the number and / or type of glycosylation sites have been altered compared to the amino acid sequence of the parent polypeptide. In certain embodiments, variants contain more or fewer N-linked glycosylation sites than the native protein. Alternatively, removal of such sequences by substitution is removal of existing N-linked carbohydrate chains. Rearrangements of N-linked carbohydrate chains are also provided, in which one or more N-linked glycosylation sites (typically those that occur naturally) are removed. and generating one or more new N-linked sites. Additional preferred antibody variants include cysteine ​​variants, in which one or more cysteine ​​residues are deleted or substituted by another amino acid (e.g., serine) compared to the parent amino acid sequence. Cysteine ​​variants can be useful when antibodies must be refolded into a biologically active conformation, for example, after isolation of insoluble inclusion bodies. Cysteine ​​variants generally have fewer cysteine ​​residues than the native protein, and typically have an even number to minimize interactions resulting from unpaired cysteines.

[0146] Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art when such substitutions are desired. In certain 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 in the present invention.

[0147] According to certain embodiments, preferred amino acid substitutions are those that (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity to form protein complexes, (4) alter binding affinity, and / or (5) confer or modulate other physicochemical or functional properties in such polypeptides. According to certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) can be made in the native sequence (in certain embodiments, in portions of the polypeptide other than the domains that form intermolecular contacts). In certain embodiments, conservative amino acid substitutions typically cannot substantially alter the structural features of the parent sequence (e.g., the replacement amino acid should not disrupt helices present in the parent sequence or other types of secondary structure that characterize the parent sequence). Art-recognized examples of polypeptide secondary and tertiary structure are described in Proteins, Structures and Molecular Principles, Creighton, W.H. Freeman and Company (1984), Introduction to Protein Structure, Branden and Tooze, Garland Publishing (1991), and Thornton et al., 1991, Nature 354:105, each of which is incorporated herein by reference.

[0148] In some embodiments, the antibodies provided by the present invention may be chemically conjugated to a polymer, lipid, or other moiety.

[0149] An antigen-binding agent may comprise at least one of the 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, capable of presenting one or more sequences of amino acids that bind to an antigen (e.g., CDRs, variable regions, etc.) in a localized surface area. Such a structure may be a native polypeptide or polypeptide "fold" (structural motif), or may have one or more modifications, such as addition, deletion, or substitution of amino acids, compared to a native polypeptide or fold. These scaffolds may be derived from polypeptides of any species (or more than one species), including humans, other mammals, other vertebrates, invertebrates, plants, bacteria, or viruses.

[0150] Typically, biocompatible framework structures are based on protein scaffolds or skeletons other than immunoglobulin domains, e.g., those based on fibronectin, ankyrin, lipocalin, neocarzinostatin, cytochrome b, CP1 zinc finger, PST1, coiled coil, LACI-D1, Z domain, and tendamistat domain can be used (see, e.g., Nygren and Uhlen, 1997, Current Opinion in Structural Biology 7:463-469).

[0151] Furthermore, those skilled in the art will recognize that suitable binding agents include portions of these antibodies, such as one or more of the heavy chain CDR1s, CDR2s, and CDR3s and light chain CDR1s, CDR2s, and CDR3s specifically disclosed in the present invention. At least one of the heavy chain CDR1, CDR2, CDR3, CDR1, CDR2, and CDR3 regions may have at least one amino acid substitution, as long as the antibody retains the binding specificity of the unsubstituted CDR. The non-CDR portion of the antibody may be a non-protein molecule, where the binding agent cross-blocks the binding of the antibody disclosed in the present invention to human GIPR and / or inhibits the activity of GIP signaling via the receptor. The non-CDR portion of the antibody may be a non-protein molecule, where the antibody exhibits a binding pattern to human GIP peptides similar to that exhibited by at least one of antibodies L1H1 / L8H1 in a competitive binding assay and / or neutralizes the activity of GIP. The non-CDR portion of the antibody may be composed of amino acids, wherein the antibody is a recombinant binding protein or a synthetic peptide, and the recombinant binding protein cross-blocks the binding of the antibody disclosed herein to human GIPR and / or neutralizes the activity of GIP in vitro or in vivo. 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 binding pattern to human GIPR peptides similar to that exhibited by at least one of antibodies L1H1 / L8H1 in a competitive binding assay and / or neutralizes the activity of GIP. GIPR antibody and FGF21 fusion protein

[0152] In one embodiment, the present invention provides a fusion protein of a GIPR antibody and FGF21, comprising an antibody capable of specifically binding to GIPR and one, two, three, four, five, six, seven, or eight FGF21 fragments, in which the amino terminus of the FGF21 fragment is linked to the carboxy terminus of the light chain or heavy chain of the GIPR antibody via a peptide linker sequence (linker).

[0153] In one embodiment, the present invention provides a fusion protein of a GIPR antibody and FGF21, comprising an antibody capable of specifically binding to GIPR and one, two, three, or four FGF21 fragments, in which the amino terminus of the FGF21 fragment is linked to the carboxy terminus of the light chain or heavy chain of the GIPR antibody via a peptide linker sequence (linker).

[0154] In one embodiment, the present invention provides a fusion protein of a GIPR antibody and FGF21, comprising an antibody capable of specifically binding to GIPR and one or two FGF21 fragments, in which the amino terminus of the FGF21 fragment is linked to the carboxy terminus of the light chain or heavy chain of the GIPR antibody via a peptide linker sequence (linker).

[0155] In another embodiment, the present invention provides an FGF21 fusion protein comprising a GIPR antibody and one or two FGF21 fragments, in which the amino terminus of the FGF21 fragment is linked to the carboxy terminus of the light chain of the GIPR antibody via a peptide linker sequence (linker) (N'-R-Linker-FGF21-C') or the amino terminus of the FGF21 fragment is linked to the carboxy terminus of the heavy chain of the GIPR antibody (N'-R-Linker-FGF21-C') (wherein N' represents the amino terminus of the polypeptide chain of the fusion protein, C' represents the carboxy terminus of the polypeptide chain of the fusion protein, 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).

[0156] In one embodiment, in the FGF21 fusion protein provided by the present invention, each of the FGF21 fragments is 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.

[0157] In one embodiment, in the FGF21 fusion protein provided by the present invention, the sequences of the peptide linkers (linkers) each independently contain 1 to 200 amino acid amines, 2 to 100 amino acid amines, 5 to 50 amino acid amines, 6 to 25 amino acid amines, or 10 to 20 amino acid amines.

[0158] In another embodiment, in the FGF21 fusion protein provided by the present invention, the sequences of the peptide linkers (linkers) are each 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.

[0159] nucleic acid In one aspect, the present invention provides isolated nucleic acid molecules. Nucleic acid molecules include, for example, polynucleotides encoding all or a portion of an antibody, e.g., one or both chains of an antibody or FGF21 fusion protein of the present invention, or fragments, derivatives, muteins, or mutants thereof; polynucleotides sufficient for use as hybridization probes; PCR or sequencing primers for identifying, analyzing, mutating, or amplifying polynucleotides encoding polypeptides; antisense nucleic acids for inhibiting expression of polynucleotides; and complementary sequences thereof. Nucleic acids can be of any length. Nucleic acids can contain, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more nucleotides, and / or can include one or more additional sequences, e.g., regulatory sequences, and / or can be part of a larger nucleic acid, e.g., a vector. Nucleic acids can be single- or double-stranded and can include RNA and / or DNA nucleotides, as well as artificial variants thereof (e.g., peptide nucleic acids).

[0160] Nucleic acids encoding antibody polypeptides (e.g., heavy or light chains, variable domains only, or full length) can be isolated from B cells of mice immunized with a GIPR antigen. Antibody or FGF21 fusion protein nucleic acids can be isolated by conventional methods, such as polymerase chain reaction (PCR).

[0161] The nucleic acid sequences encoding the heavy and light chain variable regions are set forth above. Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, each of the polypeptide sequences disclosed in the present invention is encoded by numerous other nucleic acid sequences. The present invention provides each degenerate nucleotide sequence encoding the antibodies or FGF21 fusion proteins provided by the present invention.

[0162] The present invention further provides 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 include a hybridization buffer of 5x sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), approximately 50% formamide, a pre-wash solution containing 6x SSC, a hybridization temperature of 55°C (or other similar hybridization solutions, such as those containing approximately 50% formamide, hybridization at 42°C), and elution conditions of 60°C in 0.5x SSC, 0.1% SDS. Stringent hybridization conditions include hybridization in 6×SSC at 45° C., followed by one or more washes in 0.1×SSC, 0.2% SDS at 68° C. Moreover, one of skill in the art can manipulate hybridization and / or wash conditions to increase or decrease the stringency of hybridization, with nucleic acids comprising nucleotide sequences that are at least 65, 70, 75, 80, 85, 90, 95, 98, or 99% identical to each other typically remaining hybridized to each other. Basic parameters influencing the selection of hybridization conditions and guidelines for devising suitable conditions are described, for example, in Sambrook, Fritsch, and Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Chapters 9 and 11; Current Protocols in Molecular Biology, 1995, Ausubel et al., John Wiley & Sons, Inc., Sections 2.10 and 6.3-6.4), and can be readily determined by one of skill in the art, for example, based on the length and / or base composition of the DNA.Changes are introduced into a nucleic acid by mutation, resulting in a change in the amino acid sequence of the encoded polypeptide (e.g., an antigen binding protein). Mutations can be introduced using any technique well known in the art. In one embodiment, one or more specific amino acid residues are changed, for example, using a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are changed, for example, using a random mutagenesis protocol. However generated, the mutant polypeptides can be expressed and screened for desired properties.

[0163] Mutations can be introduced into a nucleic acid without significantly altering the biological activity of the encoded polypeptide. For example, nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues can be made. In one embodiment, the nucleotide sequences provided by the present invention for L1-L9 and H1 or FGF21 fusion proteins, or fragments, variants, or derivatives thereof, are mutated so that they encode the amino acid sequences provided by the present invention for L1-L9 and H1, including one or more deletions or substitutions of amino acid residues, resulting in a sequence with two or more different amino acid residues. In another embodiment, mutagenesis inserts amino acids adjacent to one or more amino acid residues provided by the present invention for L1-L9 and H1 or FGF21 fusion proteins, resulting in a sequence with two or more different amino acid residues. Alternatively, one or more mutations can be introduced into a nucleic acid to selectively alter the biological activity (e.g., binding to GIPR) of the encoded polypeptide. For example, mutations can quantitatively or qualitatively alter a biological activity. Examples of quantitative changes include improving, reducing, or eliminating this activity. Examples of qualitative changes include changes in the antigen specificity of an antibody or FGF21 fusion protein.

[0164] In another aspect, the present invention provides nucleic acid molecules suitable for use as primers or hybridization probes for detecting nucleic acid sequences of the present invention. The nucleic acid molecules of the present invention may comprise only a portion of a nucleic acid sequence encoding a full-length polypeptide of the present invention, for example, a fragment used as a probe or primer or a fragment encoding an active portion of a polypeptide of the present invention (e.g., a GIPR-binding portion).

[0165] Probes based on the sequences of the nucleic acids of the present invention can be used to detect nucleic acids or similar nucleic acids, such as transcripts encoding the polypeptides of the present invention. 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.

[0166] In another aspect, a vector provided by the present invention comprises a nucleic acid encoding a polypeptide of the present invention 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.

[0167] The recombinant expression vector of the invention may comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell. The recombinant expression vector comprises one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed, selected based on the host cell to be used for expression. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell (e.g., the SV40 early gene enhancer, the Rous sarcoma virus promoter, and the cytomegalovirus promoter), those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences; Voss et al., 1986, Trends Biochem. Sci. 11:287; Maniatis et al., 1987, Science 236:1237, incorporated herein by reference in its entirety), and those that direct inducible expression of nucleotide sequences in response to specific treatments or conditions (e.g., the metallothionein promoter in mammalian cells, and tet-responsive and / or streptomycin-responsive promoters in both prokaryotic and eukaryotic systems (see ibid.). It will be understood by those of skill in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention, upon introduction into host cells, can produce proteins or peptides, including fusion proteins or peptides, encoded by the nucleic acids described herein.

[0168] In another aspect, the present invention provides a host cell into which an expression vector of the present invention has been introduced. The host cell can be any prokaryotic or eukaryotic cell. Prokaryotic host cells include gram-negative or gram-positive bacteria, such as Escherichia coli or Bacillus. Higher eukaryotic cells include insect cells, yeast cells, and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells or their derivatives, such as Veggie CHO and related cell lines grown in serum-free medium (see Rasmussen et al., 1998, Cytotechnology 28:31) or the DHFR-deficient CHO strain DXB-11 (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 SEQ ID NO: 6210924), HeLa cells, the BHK (ATCC CRL-10) cell line, the CV1 / EBNA cell line (ATCC CCL-70) derived from the African green monkey kidney cell line CV1 (see McMahan et al., 1991, EMBO J. 10:2821), 293, 293 EBNA, or MSR. Examples of suitable cloning and expression vectors include human embryonic kidney cells such as 293, human epidermal A431 cells, human C010205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro culture of primary tissues, primary transplants, HL-60, U937, HaK, or Jurkat cells. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, 1985).

[0169] Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. For stable transfection of mammalian cells, it is known that only a small proportion of cells may integrate the foreign DNA into their genome, depending on the expression vector and transfection technique used. To identify and select these integrants, a gene encoding a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those that confer resistance to drugs such as G418, hygromycin, and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by drug selection, among other methods (e.g., cells that have integrated the selectable marker gene will survive, while other cells die).

[0170] The transformed cells may be cultured under conditions that promote expression of the polypeptide, and the polypeptide recovered by conventional protein purification procedures. One such purification procedure is described in the Examples below. Polypeptides contemplated for use in the present invention include substantially homogeneous recombinant mammalian GIPR antibody or FGF21 fusion protein polypeptides that are substantially free of contaminating endogenous substances.

[0171] GIPR antibody activity The activity of a GIPR antibody refers to the effect of the antibody provided by the present invention in specifically binding to GIPR, inhibiting or blocking GIP signaling, and subsequently exhibiting a therapeutic biological effect, for example, treating nonalcoholic steatohepatitis, obesity, and / or type 2 diabetes. The terms "reducing the biological activity of GIP signaling" or "inhibiting or blocking the biological activity of GIP signaling" refer to the effect of a GIPR antibody or a fusion protein with FGF21 in binding to GIPR in vivo, thereby inhibiting or blocking downstream cellular responses to GIP. These responses include, but are not limited to, insulinotropic effects, promotion of adiposity, and inhibition of lipolysis. In one embodiment, the mouse-derived or humanized antibody provided by the present invention can specifically bind to human GIPR. Such antibodies include antagonistic or neutralizing antibodies that reduce or neutralize GIP signaling.

[0172] In one embodiment, the IC of the antibody provided by the present invention that reduces human GIP signaling 50 In another embodiment, the IC value of the antibody provided by the present invention for reducing human GIP signaling is 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 IC value of the antibodies provided by the present invention for reducing human GIP signaling is about 1 nM to about 200 nM. 50 In another embodiment, the IC value of the antibodies provided by the present invention that reduces human GIP signaling is about 10 nM to about 100 nM. 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.

[0173] In one embodiment, the GIPR antibodies provided by the present invention have one or more of the following properties when binding to human GIPR: a. Substantially similar K as the reference antibody when binding to human GIPR d Give. b. Substantially similar IC to the reference antibody in inhibiting GIP activation of human GIPR 50 Give. c. Cross-competes with a reference antibody for binding to human GIPR.

[0174] In other embodiments, the GIPR antibody described in the present invention is an antibody having one or more of the following characteristics: a. Provides an IC50 equal to or greater than that of a reference GIPR antibody when inhibiting GIP activation of human GIPR. b. The GIPR antibody cross-competes with a reference GIPR antibody for binding to human GIPR.

[0175] In the present invention, the term "substantially similar" means that the IC of the reference antibody 50 or K d or equivalent to the IC of the reference antibody 50 or K d "Reference antibody" refers to an antibody having a light chain sequence of SEQ ID NO: 29 and a heavy chain sequence of SEQ ID NO: 38, for example. In another embodiment, the reference antibody comprises a GIPR antibody L1H1, L9H1, or a GIPR antibody L2H1. The "reference antibody" refers to an antibody having a light chain sequence of SEQ ID NO: 29 and a heavy chain sequence of SEQ ID NO: 38, for example. The "reference antibody" refers to an antibody having a light chain sequence of SEQ ID NO: 29 and a heavy chain sequence of SEQ ID NO: 38, for example. In another embodiment, the reference antibody comprises a GIPR antibody L1H1, L9H1, or a GIPR antibody L2H1 ... Biological activity of fusion protein of GIPR antibody and FGF21

[0176] The biological activity of a fusion protein of a GIPR antibody and FGF21 includes both the biological activity of FGF21 and the activity of the GIPR antibody. The activity of the GIPR antibody is as described above. "FGF21 biological activity" refers to the biological activity of a fusion protein of a GIPR antibody and FGF21 that binds to and activates the FGF21 receptor in vivo, triggering an intracellular stress response and exhibiting therapeutic effects for nonalcoholic steatohepatitis, obesity, type 2 diabetes, and the like. The intracellular stress response includes, but is not limited to, increased insulin secretion, suppression of glucagon secretion, appetite suppression, weight loss, satiety induction, apoptosis inhibition, induction of pancreatic beta cell proliferation, and pancreatic beta cell differentiation. By combining the biological activities of FGF21 and a GIPR antibody, the FGF21 fusion protein described in the present invention can be used to treat various diseases and disorders associated with FGFRs and GIPRs. Because the fusion proteins exert their biological effects by acting on FGF21R ​​and / or GIPR, the FGF21 fusion proteins described in the present invention can be used to treat subjects suffering from diseases and disorders that would benefit from "increased FGF21R ​​stimulation" or "decreased GIPR stimulation."These subjects are referred to as "subjects in need of FGF21R ​​stimulation therapy" or "subjects in need of reduced GIPR stimulation" and are considered to be at risk for liver steatosis (GIPR, see US 2017 / 0275370 A1), non-alcoholic fatty liver disease (FGF21R, see Debra et al., 2016, Hepatobiliary Surg Nutr. 5:515-518, GIPR, see US 2017 / 0275370 A1), non-alcoholic steatohepatitis (FGF21, see Armstrong et al., 2013, BMJ Open. 3:e003995, GIPR, see US 2017 / 0275370 A1), non-insulin-dependent diabetes mellitus, insulin-dependent diabetes mellitus, stroke (FGF21R, see WO 00 / 16797), myocardial infarction (FGF21R, see WO 00 / 16797), and other conditions. 98 / 08531), obesity (FGF21R, see WO 98 / 19698; GIPR, see Furija et al., 2008; PLoS ONE 3:e3163; US 2017 / 0275370 A1), catabolic changes after surgery (FGF21R, see US 6,006,753), functional dyspepsia and irritable bowel syndrome (FGF21R, see WO 99 / 64060), and also subjects at risk of developing non-insulin dependent diabetes mellitus (see WO 00 / 07617), subjects with impaired glucose tolerance or impaired fasting glucose, subjects who weigh about 25% above normal body weight, and subjects who have had a partial pancreatectomy.

[0177] In one embodiment, changes in the biological activity of a GIPR antibody or FGF21 fusion protein are detected using a reporter gene assay method that quantifies the function of the GIPR antibody or FGF21 fusion protein in inhibiting GIPR in vitro. Pharmaceutical Composition

[0178] In one embodiment, a pharmaceutical composition provided by the invention comprises a GIPR antibody provided by the invention and one or more pharmaceutically acceptable carriers.

[0179] In other embodiments, pharmaceutical compositions provided by the present invention comprise a fusion protein of a GIPR antibody and FGF21 provided by the present invention and one or more pharmaceutically acceptable carriers.

[0180] The term "carrier" as used herein includes carriers, pharmaceutical excipients, or stabilizers that are innocuous to cells or mammals upon exposure thereto at the dosages and concentrations employed. Treatment method

[0181] In one embodiment, the present invention provides a method for treating, preventing, or ameliorating non-alcoholic steatohepatitis, comprising administering to a subject a therapeutically effective amount of a GIPR antibody or a pharmaceutical composition thereof provided by the present invention.

[0182] In another embodiment, the present invention provides 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 by the present invention, or a pharmaceutical composition thereof.

[0183] In another embodiment, the present invention provides a method for treating, preventing, or ameliorating obesity, comprising administering to a subject a therapeutically effective amount of a GIPR antibody provided by the present invention, or a pharmaceutical composition thereof.

[0184] In another embodiment, the present invention provides 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 by the present invention, or a pharmaceutical composition thereof.

[0185] In another embodiment, the present invention provides a method for treating, preventing, or ameliorating type 2 diabetes, comprising administering to a subject a therapeutically effective amount of a GIPR antibody provided by the present invention, or a pharmaceutical composition thereof.

[0186] In another embodiment, the present invention provides 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 by the present invention, or a pharmaceutical composition thereof.

[0187] In any of the uses provided by the present invention, the pharmaceutical composition provided by the present invention is for intravenous or subcutaneous injection.

[0188] As used herein, the term "subject" refers to a mammal, including a human, and is used interchangeably with the term "patient."

[0189] The term "treatment" includes alleviation or prevention of at least one symptom or other aspect of a disease, or reduction in disease severity. The GIPR antibody or fusion protein of a GIPR antibody and FGF21 provided by the present invention need not provide a complete cure or eradicate all symptoms or signs of a disease to be an effective therapeutic agent. As recognized in the relevant art, a therapeutic agent may reduce the severity of a given condition, but need not eliminate all signs of the disease to be effective. Similarly, a prophylactic agent need not completely prevent the onset of a condition to be effective. It is sufficient to merely reduce the impact of the disease (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect) or reduce the likelihood that the disease will occur or worsen in a subject. One embodiment of the present invention relates to a method comprising administering to a patient a GIPR antibody or a fusion protein of a GIPR antibody and FGF21 in an amount and for a time sufficient to induce a sustained improvement over baseline in an indicator reflecting the severity of a particular disease.

[0190] Pharmaceutical compositions of GIPR antibodies or fusion proteins of GIPR antibodies and FGF21 can be administered by any suitable technique, including, but not limited to, parenteral, topical, or inhalation. When injected, the pharmaceutical compositions can be administered, for example, via intraarticular, intravenous, intramuscular, intralesional, intraperitoneal, or subcutaneous routes, by bolus injection or continuous infusion. Local administration at the site of disease or injury, such as transdermal administration and sustained release of implantable formulations, is also contemplated. Delivery by inhalation includes, for example, nasal or oral inhalation, use of a nebulizer, or inhalation of an antibody in aerosol form. Other alternatives include oral formulations, including tablets, syrups, or lozenges.

[0191] Advantageously, the GIPR antibodies or FGF21 fusion proteins provided by the invention are administered as a composition comprising one or more other components, such as a physiologically acceptable carrier, excipient, or diluent. The composition further comprises one or more physiologically active agents, as described below. In many specific embodiments, the composition comprises one, two, three, four, five, or six physiologically active agents in addition to one or more antibodies (e.g., murine-derived or humanized antibodies) or FGF21 fusion proteins provided by the invention.

[0192] In one embodiment, the pharmaceutical composition comprises a murine-derived or humanized antibody or FGF21 fusion protein provided by the present invention, together with one or more substances selected from the group consisting of a buffer at 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 having fewer than 10 amino acids), a protein, an amino acid, a carbohydrate such as dextrin, a chelating agent such as EDTA, glutathione, a stabilizer, and an excipient. Preservatives may also be added in accordance with appropriate industry standards. The composition may be formulated as a lyophilizate using an appropriate excipient solution as a diluent. Suitable ingredients are harmless to recipients at the dosages and concentrations used. Further examples of ingredients that can be used in pharmaceutical formulations are described in Remington's Pharmaceutical Sciences, 16th Edition (1980) and 20th Edition (2000). Mack Publishing Company provides kits for use by physicians containing one or more of the antibodies or FGF21 fusion proteins provided by the present invention and other instructions for use in treating any of the conditions described in the present invention. In one embodiment, the kit comprises a sterile preparation of one or more antibodies or FGF21 fusion proteins in the form of the compositions described above contained in one or more tube-type bottles.

[0193] The dosage and frequency of administration can vary depending on factors such as the route of administration, the particular antibody or FGF21 fusion protein used, the nature and severity of the disease being treated, whether the condition is acute or chronic, and the size and general condition of the subject. Appropriate dosages can be determined by procedures known in the relevant art, for example, in clinical trials, which may include dose escalation studies.

[0194] The antibodies or FGF21 fusion proteins provided by the present invention can be administered, for example, once or twice or more at regular intervals over a predetermined period of time. In certain embodiments, murine-derived or humanized antibodies or FGF21 fusion proteins are administered once for at least one month or longer, for example, for one, two, or three months, or indefinitely. For treating chronic diseases, long-term treatment is generally most effective. However, for treating acute diseases, administration over a shorter period of time, for example, one to six weeks, may be sufficient. Generally, humanized antibodies are administered until the patient shows a medically relevant degree of improvement over baseline for the selected indicator.

[0195] One example of a treatment regimen provided by the present invention involves subcutaneous injection of an antibody or FGF21 fusion protein at an appropriate dosage once per week or more to treat symptoms caused by conditions such as nonalcoholic steatohepatitis, obesity, or type 2 diabetes. The antibody or FGF21 fusion protein may be administered weekly or monthly until the desired results are achieved, e.g., until the patient's symptoms subside. Treatment may be resumed as needed, or a maintenance dose may be administered.

[0196] A patient's hepatic fat fraction (HFF), liver damage marker alanine aminotransferase (ALT), body weight, and blood glucose concentration can be monitored before, during, and / or after treatment with an antibody or FGF21 fusion protein, such as a human antibody or FGF21 fusion protein, to detect changes in these parameters. For some diseases, changes in HFF, ALT, and blood glucose can vary depending on factors such as disease progression. Changes in these levels can be determined using well-known techniques.

[0197] Specific embodiments of the methods and compositions of the present invention include, for example, the use of an antibody or FGF21 fusion protein and one or more GIP antagonists, or two or more antibodies or FGF21 fusion proteins provided by the present invention, or an antibody or FGF21 fusion protein of the present invention and one or more other GIP antagonists. In yet other embodiments, the antibody or FGF21 fusion protein is administered alone or in combination with other agents used to treat the condition from which the patient suffers. Examples of these agents include proteinaceous and non-proteinaceous agents. When multiple agents are administered in combination, dosages should be adjusted accordingly, as is well known in the art. The term "co-administration" in combination therapy is not limited to simultaneous administration and also includes treatment regimens in which an antigen and a protein are administered at least once during a course of administration that also includes administering at least one other therapeutic agent to the patient.

[0198] In another aspect, the present invention provides a method for preparing a medicament for treating nonalcoholic steatohepatitis, obesity, and type 2 diabetes, and related disorders, comprising a mixture of an antibody or FGF21 fusion protein provided by the present invention and a pharmaceutically acceptable excipient for the treatment of disorders related to the above-mentioned disorders. The method for preparing the medicament is as described above.

[0199] The present invention further provides compositions, kits, and methods related to antibodies or FGF21 fusion proteins capable of specifically binding to human GIPR. Nucleic acid molecules, including polynucleotides encoding all or part of a polypeptide that binds to GIPR, and derivatives and fragments thereof, such as nucleic acids encoding all or part of an anti-GIPR antibody, antibody fragment, antibody derivative, or FGF21 fusion protein, are also provided. The present invention further provides vectors and plasmids containing such nucleic acids, as well as cells and cell lines containing such nucleic acids and / or vectors and plasmids. Methods provided by the present invention include, for example, methods for preparing, identifying, or isolating an antibody or FGF21 fusion protein that binds to human GIPR, such as an anti-GIPR antibody or FGF21 fusion protein, methods for determining whether an antibody or FGF21 fusion protein binds to GIPR, and methods for administering an antibody or FGF21 fusion protein that binds to GIPR to an animal model. [Example]

[0200] The technical solution of the present invention will be further understood by the following examples.

[0201] In the present invention, unless otherwise specified, the starting materials and equipment used are commercially available or commonly used in the art. Unless otherwise specified, all methods in the following examples are conventional methods in the art.

[0202] 1. Preparation of immunization antigen CHO-DHFR- cells were seeded in 6-well plates. After 24 hours of culture, the cells in the 6-well plates were transfected with the pTM15 plasmid containing the hGIPR gene (nucleotide sequence: SEQ ID NO: 114, amino acid sequence: SEQ ID NO: 113). Transfection was performed according to the transfection conditions recommended by Invitrogen using Lipofectamine 2000. After 48 hours, the medium was replaced with complete medium containing 300 μg / mL hygromycin, and the medium was changed every 3 days. After approximately 2 weeks of culture, stable clones emerged. Cell colonies were dispersed by digestion and serially subcultured until the cells reached 100% confluence. The constructed stable cell lines were detected by FACS using a V5 tag antibody (Life Technologies), and the cell populations after pressure screening were identified from the FACS detection results. A large amount of hGIPR was expressed in the CHO-DHFR-hGIPR cell membrane after screening. Finally, after subcloning and further identification, three GIPR-expressing stable cell lines were selected. These hGIPR-highly expressing cell lines were used as immunogens to prepare antibodies (see Example 2). Furthermore, in one embodiment, a fusion protein of the hGIPR extracellular domain and hIgG Fc was also used as an immunogen to prepare antibodies. The preparation method was as follows: The gene sequence of the fusion protein of the hGIPR extracellular domain, hIgG2 Fc, and a peptide linker (linker) was subcloned into pTM5 plasmid. High-level transient expression was performed in suspension-cultured HEK293 cells, and the cell supernatant was obtained and purified by affinity chromatography to obtain the hGIPR extracellular domain fusion protein.

[0203] 2. Preparation of antibodies Antibodies against hGIPRs can be produced using any of the following immunogens. For example, in one embodiment, whole cells expressing hGIPRs were used as the immunogen to produce antibodies against hGIPRs. Furthermore, in one embodiment, a fusion protein containing the amino acid sequence of the N-terminal domain of hGIPR and hFc was used as the immunogen to produce antibodies against hGIPRs. The immunogen and aluminum hydroxide adjuvant were homogeneously mixed and subcutaneously injected into BALB / c mice (6-8 weeks old) with weekly booster immunizations. After a total of six immunizations, blood was collected from the tail vein. Serum was isolated by centrifugation, and serum titers were detected by FACS. When an appropriate antibody titer was achieved, the mice were sacrificed by cervical dislocation, and spleen cells were collected under aseptic conditions. SP2 / 0 cells in the logarithmic growth phase were harvested and centrifuged. The cell pellet was resuspended in serum-free medium, centrifuged again, resuspended, and counted. To ensure that the number of SP2 / 0 cells was close to that of splenocytes, splenocytes and SP2 / 0 cells were mixed and washed and centrifuged three times. After removing the cell pellet from the final centrifugation, preheated PEG-1500 was added dropwise. The mixture was then pipetted up and down, and 30 mL of preheated serum-free medium was slowly added to stop the PEG fusion. After another centrifugation, the cell pellet was removed and added to the fusion medium. The splenocytes and feeder layer cells were plated in a 96-well plate, with 100 μL of medium added per well. The fused hybridoma cells and feeder layer cells were co-cultured in a 96-well plate and subjected to HAT (hypoxanthine, methotrexate, and thymidine) screening to remove unfused cells. After 10 days, the supernatant of the hybridoma cells in the culture plate was collected for ELISA detection.

[0204] 3. Antibody screening by ELISA CHO-DHFR-hGIPR cells overexpressing hGIPRs and CHO-DHFR- cells not expressing hGIPRs were seeded into 96-well plates. When the cells reached 90% confluence, the cell culture supernatant was removed. The cells were washed twice with PBS and fixed with 100% methanol at 4°C. Next, 100 μL of freshly prepared 0.6% HO-PBS was added and incubated for 20 min at room temperature. Then, the cells were washed twice with PBS. After blocking with 1% BSA (dissolved in PBS), hybridoma cell supernatant was added and incubated for 90 min at 4°C. After multiple washes, 100 μL of diluted goat anti-mouse Fc-HRP secondary antibody (Sigma-Aldrich) was added per well and incubated for 30 min at 37°C. After washing five times, 100 μL of TMB chromogenic substrate was added per well and incubated at 37°C for 15 minutes. Color development was stopped by adding 50 μL of 2 M H2SO4, and the OD450 value was read. Furthermore, in one embodiment, a fusion protein containing the amino acid sequence of the N-terminal region of hGIPR and hFc was used as a coating antigen to coat a 96-well plate. After blocking with 1% BSA (dissolved in PBS), hybridoma cell supernatant was added and incubated at 4°C for 90 minutes. Subsequent steps were similar to the above-described ELISA method for screening anti-hGIPR monoclonal antibodies. The positive control was serum from immunized mice, and the negative control was cell culture supernatant. After preliminary detection by ELISA, several positive hybridoma cell lines secreting anti-hGIPR antibodies were screened. These hybridoma lines secreting anti-hGIPR antibodies were selected and cloned to obtain cell lines capable of stably secreting anti-hGIPR antibodies. Finally, positive hybridoma cell supernatants were selected for FACS confirmation (see Example 10).

[0205] 4. Cloning and subcloning of antibody genes Antibody-secreting hybridoma cells were harvested, and mRNA from the hybridoma cells was extracted according to the manufacturer's protocol for the QIAGEN mRNA extraction kit. The extracted mRNA was then reverse transcribed into cDNA. The reverse transcription primers were specific for the mouse light and heavy chain constant regions: the heavy chain reverse transcription primer was 5'-TTTGGRGGGAAGATGAAGAC-3', and the light chain reverse transcription primers were 5'-TTAACACTCTCCCCTGTTGAA-3' and 5'-TTAACACTCATTCCTGTTGAA-3'. The RT-PCR reaction conditions were 25°C for 5 min, 50°C for 60 min, and 70°C for 15 min. The reverse-transcribed cDNA was diluted to 500 μL with 0.1 mM TE and placed in an ultrafiltration centrifuge tube (Amicon Ultra-0.5). The tube was centrifuged at 2000 g for 10 min. The filtrate was discarded, and 500 μL of 0.1 mM TE was added. The tube was then centrifuged at 2000 g for 10 min. The filtrate was discarded, and the tube was inverted and placed in a new centrifuge tube. The tube was then centrifuged at 2000 g for 10 min to obtain purified cDNA. Ten μL of the purified cDNA was used as a template. 4 μL of 5x tailing buffer (Promega), 4 μL of 1 mM dATP, and 10 U of terminal transferase (Promega) were added, mixed thoroughly, and incubated at 37°C for 5 min, followed by incubation at 65°C for 5 min. The poly(A)-tailed cDNA was then used as a template for PCR amplification of antibody light and heavy chain variable region genes. All upstream primers were oligo-dT, and the downstream primers for the heavy chain were 5'-TGGACAGGGATCCAGAGTTCC-3' and 5'-TGGACAGGGCTCCATAGTTCC-3'. The downstream primer for the light chain was 5'-ACTCGTCCTTGGTCAACGTG-3'. The PCR reaction conditions were 95°C for 5 min, 95°C for 30 s, 56°C for 30 s, 72°C for 1 min, 40 cycles, and 72°C for 7 min. The PCR products were ligated into the PMD 18-T vector (Takara Bio) for sequencing.PCR primers were designed based on the DNA sequences of the sequenced antibodies to ligate the complete light chain, heavy chain signal peptide and variable domains and mouse IgG1 constant region into the expression vector pTM5.

[0206] 5. Antibody humanization and optimization First, the NCBI database was used to search for humanized antibody germline gene sequences (Ig germline gene sequences) homologous to the light and heavy chain variable region sequences of the screened mouse-derived antibody. The humanized gene sequence with the highest homology, excluding the CDR sequence, was used as a template for CDR grafting to obtain the humanized antibody variable region sequence. Humanized antibody light and heavy chain genes were synthesized and combined with human IgG2 or IgG4 constant region sequences to obtain a fully recombinant humanized antibody sequence. The recombinant antibody was expressed according to Example 8, and its affinity for GIPRs was confirmed using FACS technology in step 12. The antibody with the best affinity was selected. Finally, the variable region sequence of the humanized antibody was further modified by site-directed mutagenesis to improve the antibody's affinity for GIPRs.

[0207] 6. Gene cloning and subcloning of humanized hGIPR antibody The heavy and light chain variable region sequences of the optimized humanized antibody were synthesized by outsourcing. During synthesis, an Nhe1 restriction enzyme site was introduced at the 5' end of the heavy chain variable region and a Sal1 restriction enzyme site was introduced at the 3' end, allowing the complete heavy chain variable region sequence to be ligated into the expression vector pTM5 containing the heavy chain constant region. Similarly, during synthesis, an Nhe1 restriction enzyme site was introduced at the 5' end of the light chain variable region and a Bsiw1 restriction enzyme site was introduced at the 3' end, allowing the complete light chain variable region sequence to be ligated into the expression vector pTM5 containing the light chain constant region.

[0208] 7. Construction of fusion protein of humanized hGIPR antibody and FGF21 The optimized humanized antibody was fused to FGF21 or its derivative sequences at the C-terminus of the heavy chain to form FGF21 fusion proteins (e.g., SEQ ID NOs: 104-106, 119-121, 123-125). The two sequences were linked by a peptide linker sequence (linker). The nucleotide sequence of Linker-FGF21 was synthesized by GenScript Biotech, Inc. The "Linker-FGF21" portion was amplified by PCR using the synthetic gene as a template. Furthermore, the signal peptide-antibody portion of the fusion protein was amplified using the nucleotide sequence of the humanized antibody as a template. Next, the "Linker-FGF21" portion of the fusion protein nucleic acid sequence was linked to the antibody portion by overlap PCR. Nhe1 and Not1 restriction enzyme sites were introduced at both ends of the primers, allowing the complete fusion protein sequence to be ligated into the expression vector pTM5.

[0209] 8. Transient expression of hGIPR antibody, FGF21 fusion protein, and FC-FGF21(RGE) 5×10 5 HEK293 or CHO expression cell lines cultured in suspension were inoculated into a spinner flask at a density of 1 × 10 / mL. After 24 hours of rotational culture at 37°C and 5% CO, the density reached 1 × 10 6 Once the concentration reached 1000kJ / mL, the cells were subjected to transfection. In the transfection process, polyethyleneimine (PEI) was used as a transfection reagent and mixed with DNA. The mixture was incubated statically for 15 minutes before being added to the cell culture. After administering the PEI and DNA mixture, the cells were cultured with a rotation at 37°C and 5% CO2 for 24 hours. Tryptone was then added to the cell culture medium as a necessary additive for expression. Finally, after expression was completed (more than 96 hours), the cell supernatant was collected and subjected to antibody purification and isolation.

[0210] 9. Purification and isolation of hGIPR antibody, FGF21 fusion protein, and FC-FGF21(RGE) The collected cell supernatant from Example 8 was centrifuged at high speed (8000 rpm) to remove cells and cell debris, and then filtered through a 0.22 μm membrane filter for clarification. The clarified supernatant was subjected to purification. The purification process was completed by chromatography. The supernatant was first passed through a Protein A / G affinity chromatography column. During this time, the antibodies contained in the supernatant bound to the ligands of the Protein A / G affinity chromatography column and remained on the column. Next, the chromatography column was washed with a low pH (below 3.0) elution buffer to dissociate the antibodies bound to the chromatography column. The collected antibody eluate was rapidly neutralized with 1 M Tris-HCl. The obtained antibody eluate was dialyzed and then replaced with PBS or another buffer system.

[0211] 10. Construction and detection of stable cell lines expressing the β-Klotho / FGFR1c receptor complex Commercially available CHO-DHFR cells were seeded into 6-well plates and cultured for 24 h. Then, the cells in the 6-well plates were transfected with a plasmid containing the V5-hKLB gene and a plasmid containing Flag-FGFR1c. The medium was replaced before transfection, and transfection was performed using Lipofectamine 2000 according to the transfection conditions recommended by Invitrogen. Forty-eight hours after transfection, the medium was replaced with complete medium containing 10 nM methotrexate (MTX). The medium was replaced every 3 days for 2 weeks until stable clones appeared. Cell colonies were dispersed by digestion. After growing to 50% confluence, pressure screening was performed using gradually increasing concentrations of MTX (until the MTX concentration reached 10 μM). The constructed stable cell lines were analyzed by FACS. Cell populations after pressure screening were identified using antibodies against the N'-terminal V5 tag of KLB protein (FITC-anti-V5, Invitrogen; Flag-anti-V5, Invitrogen). After screening with 10 μM MTX, high levels of hKLB were expressed in the CHO-DHFR-V5-hKLB cell membrane. Finally, two highly KLB-expressing stable cell lines were obtained through subcloning and characterization.

[0212] 11. Reporter gene experiments to detect the biological activity of hGIPR antibody / FGF21 fusion protein in activating the hFGFR1c / hKLB signaling pathway in vitro hFGFR1c-β-KLB-SRE-luciferase co-expressing CHO-DHFR cells were seeded at 20,000 cells / well into a 96-well cell culture plate and cultured overnight at 37°C and 5% CO2. The following day, the culture supernatant was removed. The cell surface was washed twice with serum-free medium to remove residual liquid. 100 μL of purified antibody or FGF21 diluted in serum-free medium was added and incubated for 4 hours at 37°C. After stimulation, 100 μL of Bright Glo chemiluminescent substrate (Promega) was added. Finally, the cell lysate was transferred to a white 96-well plate and the relative fluorescence intensity was read using a SpectraMax L microplate reader (Molecular Devices) (results are shown in Figure 1).

[0213] 12. Reporter gene experiments to detect the biological activity of hGIPR antibody / FGF21 fusion proteins V1W1 and V1W2 in antagonizing GIPR in vitro hGIPR-CRE-luciferase co-expressing CHO-DHFR cells were seeded at 30,000 cells / well into a 96-well cell culture plate and cultured overnight in a 37°C, 5% CO2 incubator. The following day, the cell supernatant was removed. The cell surface was washed twice with serum-free medium to remove residual liquid. 50 μL of GIP diluted in serum-free medium and 50 μL of gradient-diluted hGIPR antibody or hGIPR antibody / FGF21 fusion protein in serum-free medium were added per well and incubated at 37°C for 4 hours. After stimulation, 100 μL of Bright Glo chemiluminescent substrate (Promega) was added. Finally, the cell lysate was transferred to a white 96-well plate and the relative fluorescence intensity was read using a SpectraMax L microplate reader (Molecular Devices) (results are shown in Figure 2).

[0214] 13. Reporter gene experiments to detect the biological activity of hGIPR antibody / FGF21 fusion proteins V1W1 and V1W2 in activating hβ-Klotho / hFGF1Rc in vitro CHO-DHFR cells co-expressing hFGFR1c-β-KLB-SRE-luciferase or mFGFR1c-β-KLB-SRE-luciferase were seeded at 20,000 cells / well into 96-well cell culture plates and cultured overnight at 37°C. The next day, the culture supernatant was removed, the cell surface was washed twice with serum-free medium, and the residual liquid was removed. 100 μL of purified antibody or FGF21 diluted in serum-free medium was added and incubated for 4 hours at 37°C. After stimulation, 100 μL of Bright Glo chemiluminescent substrate (Promega) was added. Finally, the cell lysates were transferred to a white 96-well plate and the relative fluorescence intensity was read using a SpectraMax L microplate reader (Molecular Devices) (Figures 3 and 8).

[0215] 14. Pharmacodynamic evaluation of the in vivo efficacy of hGIPR antibody / FGF21 fusion protein in high-fat diet-induced C57BL / 6 obese mice. A 60% high-fat diet-induced obesity model (DIO mice) was established in C57BL / 6 mice. After purchasing the mice, they were fed a normal diet for one week. A certain number of mice were randomly selected as a normal control group fed with regular mouse chow, and the remaining animals were fed a high-fat diet. The mice were continuously fed for 8 weeks (wk), and their body weight and food intake were assessed once a week. Mice were then randomly assigned to receive a high-fat diet and given V1W1-FGF21 (SEQ:89) at 1 mg / kg, V1W1-FGF21 (SEQ:89) at 3 mg / kg, V1W1-FGF21 (SEQ:89) at 10 mg / kg, V1W2-FGF21 (SEQ:89) at 1 mg / kg, V1W2-FGF21 (SEQ:89) at 3 mg / kg, and V1W2-FGF21 (SEQ:89) at 10 mg / kg, as well as a positive drug control and model group. The drugs were injected subcutaneously every 3 days for 4 weeks. The normal control group received no drug, and the model group received the same amount of blank formulation. Data on mouse weight, food intake, and behavioral observations were collected during the experimental period. Fasting blood glucose levels were measured on days 0, 7, 14, 21, and 28 of the experiment. An oral glucose tolerance test (OGTT) was performed on days 14 and 28 of the experiment. On the day before the final day of the experiment, animals were fasted for 12 hours (water was allowed ad libitum), and orbital blood was collected to isolate serum. The animals were then euthanized, and serum ALT, AST, HDL-C, LDL-C, TC, and TG were detected (the results are shown in Figures 4, 5, 6, and 7).

[0216] 15. Pharmacodynamic evaluation of the in vivo efficacy of hGIPR antibody / FGF21 fusion protein in C57BL / 6 mice with choline-deficient, L-amino acid-defined, high-fat diet-induced nonalcoholic steatohepatitis. A nonalcoholic steatohepatitis model was established using C57BL / 6 mice fed a choline-deficient, L-amino acid-defined, high-fat diet. After purchasing, mice were fed a normal diet for one week. A certain number of mice were randomly selected as a normal control group fed with normal mouse chow, while the remaining mice were fed a choline-deficient, L-amino acid-defined, high-fat diet. After continuous feeding for six weeks, mice in the choline-deficient, L-amino acid-defined, high-fat diet group were randomly divided into V1W2-FGF21 (SEQ:118) group (1 mg / kg), V1W2-FGF21 (SEQ:118) group (3 mg / kg), V1W2-FGF21 (SEQ:118) group (10 mg / kg), a positive drug control group, and a model group based on serum triglyceride and triglyceride levels. Drugs were injected subcutaneously every three days for 8 weeks. The normal control group received no drug, while the model group received the same amount of blank formulation. During the experimental period, data on mouse weight, food intake, and behavioral observations were collected. At week 8 of the experiment, blood was collected from the mice, and serum was separated for the detection of ALT, AST, TG, and TC. Livers were collected and weighed, and 95–105 mg of liver tissue was taken for the detection of TC and TG. The liver was removed from the same lobe, fixed in 4% paraformaldehyde, and subjected to HE and Sirius Red staining (results are shown in Figure 9).

[0217] The above embodiments are intended to fully disclose and describe 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. Modifications obvious to those skilled in the art are within the scope of the claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference, as if each was specifically and individually indicated to be incorporated by reference.

Claims

1. An antibody capable of specifically binding to human GIPR, said antibody comprising one, two, three, four, five, or six amino acid sequences, each amino acid sequence independently selected from the amino acid sequences set forth below: 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 sequence: 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, each amino acid sequence independently selected from the amino acid sequences set forth below: 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, and b. Heavy chain CDR1 amino acid sequence: SEQ ID NO:

12.

3. 3. The antibody of claim 1 or 2, wherein the antibody comprises, or further comprises, one or two amino acid sequences, each independently selected from the amino acid sequences set forth 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, each amino acid sequence independently selected from the amino acid sequences set forth below: a. Light chain CDR3 amino acid sequences: 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, each amino acid sequence independently selected from the amino acid sequences set forth below: 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, each amino acid sequence independently selected from the amino acid sequences set forth below: 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 one combination of light chain and heavy chain CDR1 amino acid sequences independently selected from the following list: 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 one combination of light chain and heavy chain CDR2 amino acid sequences independently selected from the following list: 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 one combination of light chain and heavy chain CDR3 amino acid sequences independently selected from the following list: SEQ ID NO: 3 and SEQ ID NO: 14, and SEQ ID NO: 4 and SEQ ID NO:

14.

10. The antibody (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 The antibody according to any one of claims 1 to 9, comprising:

11. The antibody of any one of claims 1 to 10, wherein the antibody comprises one or two amino acid sequences, each amino acid sequence independently selected from the amino acid sequences set forth below: a. light chain variable domain amino acid sequences: 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 amino acid sequences at least 80%, at least 85%, at least 90%, or at least 95% identical to any of these sequences; and b. Heavy chain variable domain amino acid sequence: SEQ ID NO: 38, and amino acid sequences at least 80%, at least 85%, at least 90%, or at least 95% identical thereto.

12. 12. The antibody of any one of claims 1 to 11, wherein the antibody polynucleotide coding sequence comprises one or two polynucleotide sequences, each polynucleotide sequence independently selected from the polynucleotide sequences set forth below: a. light chain variable domain polynucleotide coding sequences: 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 polynucleotide sequences at least 80%, at least 85%, at least 90%, or at least 95% identical to any of these sequences; and b. Heavy chain variable domain polynucleotide coding 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 one of the amino acid sequences independently selected from the following list: 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 one amino acid sequence independently selected from the list below: SEQ ID NO:

38.

15. The antibody of any one of claims 1 to 14, wherein the antibody comprises one combination of light chain and heavy chain variable region amino acid sequences independently selected from the following list: 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. No:

38.

16. The antibody of any one of claims 15, wherein the antibody comprises, or further comprises, one of the amino acid sequences independently selected from the following list: 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 claims 16, wherein the antibody comprises one combination of light chain and heavy chain variable region amino acid sequences independently selected from the following list: 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 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, each amino acid sequence independently selected from the amino acid sequences set forth below: 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 according to any one of claims 1 to 20, wherein the antibody is a mouse-derived GIPR antibody or a humanized GIPR antibody.

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

23. The antibody according to any one of claims 1 to 22, which is a monoclonal antibody comprising one combination of amino acid sequences selected from the following list: 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. has a Kd for binding to human GIPR that is equal to or greater than that of a reference GIPR antibody; b. has an IC50 that is equal to or greater than that of a reference GIPR antibody when inhibiting GIP activation of human GIPR; and c) The GIPR antibody cross-competes with a reference GIPR antibody for binding to human GIPR.

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

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

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

38.

28. The antibody of any one of claims 1 to 27, wherein the antibody is a mouse-derived 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 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.

29. IC of the antibody that reduces human GIP signaling ５０ The antibody of any one of claims 1 to 28, wherein the value is between about 1 nM and 200 nM or between 1 nM and 100 nM.

30. An FGF21 fusion protein comprising one GIPR antibody and one, two, three, four, five, six, seven, or eight FGF21 fragments, wherein the amino terminus of the FGF21 fragment is linked to the carboxy terminus of the light chain or heavy chain of the GIPR antibody via a peptide linker sequence (linker), and the FGF21 fragment has the structural feature of comprising one amino acid sequence independently selected from the following sequences: 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 comprising a GIPR antibody according to any one of claims 1 to 29 and one, two, three, four, five, six, seven, or eight FGF21 fragments, wherein the fusion protein has the structural feature that the amino terminus of the FGF21 fragment is linked to the carboxy terminus of the light chain or heavy chain of the GIPR antibody via a peptide linker sequence (linker).

32. The fusion protein of claim 30 or 31, wherein the fusion protein comprises one GIPR antibody and one, two, three, or four FGF21 fragments, and in the fusion protein, the amino terminus of the FGF21 fragment is linked to the carboxy terminus of the light chain or heavy chain of the GIPR antibody via a peptide linker sequence (linker).

33. The fusion protein according to claims 30 to 32, comprising one GIPR antibody and one or two FGF21 fragments, wherein the amino terminus of the FGF21 fragment is linked to the carboxy terminus of the light chain or heavy chain of the GIPR antibody via a peptide linker sequence (linker).

34. The fusion protein of claims 30 to 33, wherein the GIPR antibody, FGF21 fragment, and peptide linker sequence (linker) are fused to form the fusion protein by one of the following methods: The amino terminus of the FGF21 fragment and the carboxy terminus of the light chain of the GIPR antibody are linked via a peptide linker sequence (linker): N'-R-Linker-FGF21-C', The amino terminus of the FGF21 fragment and the carboxy terminus of the heavy chain of the GIPR antibody are linked via a peptide linker sequence (linker): N'-R-Linker-FGF21-C', wherein N' represents the amino terminus of the polypeptide chain, C' represents the carboxy terminus of the polypeptide chain, FGF21 represents an FGF21 fragment, R represents the amino acid sequence of the light chain or heavy chain of the GIPR antibody described in 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 (linker) comprises one of a full-length, partial, or repeated amino acid sequence independently selected from the following sequences: SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO:

68.

36. The FGF21 fusion protein of any one of claims 31 to 34, wherein the FGF21 fragment comprises one of the amino acid sequences independently selected from the following sequences: 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:

119. 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, comprising one GIPR antibody, two FGF21 fragments, and a peptide linker sequence between the GIPR antibody and the FGF21 fragment, wherein the sequence of the fusion protein comprises one of the amino acid sequences independently selected from the following sequences: 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.

41. A host cell comprising the vector of claim 40.

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

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

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

45. Use of a pharmaceutical composition comprising a GIPR antibody described in any one of claims 30 to 38 or an FGF21 fusion protein described in 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 a GIPR antibody described in any one of claims 30 to 38 or an FGF21 fusion protein described in any one of claims 30 to 37 in the preparation of a drug for simultaneously preventing or treating two or more pathological conditions of non-alcoholic fatty liver-related 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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