GIPR blocking type antibody and antibody conjugate thereof

Abstract

Provided are a blocking type GIPR antibody, an antigen-binding fragment thereof, an antibody conjugate thereof, and use thereof in preparing a medicament. The GIPR antibody and the antigen-binding fragment thereof have a relatively high affinity for GIPR protein and bind to the GIPR protein expressed on the cell surface, blocking the cell-activating activity of GIP. The GIPR antibody has relatively good cellular internalization activity. Pharmacokinetic research shows that the GIPR antibody and the antigen-binding fragment thereof exhibit relatively good stability in mice, with an AUC0-t up to 15,033.01 hour × μg / mL. The antibody conjugate prepared by adopting the GIPR antibody shows stable and lasting activity in reducing the weight of laboratory animal models and therefore has wide application prospects.

Description

GIPR blocking antibodies and antibody conjugates Technical Field

[0001] The present invention relates to the field of biomedicine or biopharmaceutical technology, and in particular to a blocking antibody and an antibody conjugate thereof for glucose-dependent insulinotropic polypeptide receptor (GIPR), and to the use of the GIPR blocking antibody and the antibody-drug conjugate thereof in preparing a drug. Background Art

[0002] Incretins originate from the response of intestinal endocrine cells to food intake. Incretins mainly include glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1), which are produced by K cells in the proximal intestine and L cells in the distal intestine, respectively.

[0003] Glucagon-like peptide 1 (GLP-1) binds to receptors on pancreatic islet cells and stimulates insulin secretion, thereby lowering blood sugar. It can also reduce food intake and delay gastric emptying, helping to control weight. GIP promotes fat storage and glucose-dependent insulin secretion, complementing the effects of GLP-1 receptor agonists, increasing insulin secretion during hyperglycemia and stimulating glucagon release during hypoglycemia.

[0004] Glucose-dependent insulinotropic peptide (GIP) promotes adipocyte formation both in vitro and in vivo. Free GIP levels are elevated in obese mice and humans, suggesting a positive correlation between GIP and obesogenicity.

[0005] The glucose-dependent insulinotropic polypeptide receptor (GIPR) is a receptor for glucose-dependent insulinotropic polypeptide (GIP). Studies have shown that knocking out the GIPR gene in obese mice leads to weight loss. A global genomic association study also showed that people with a genotype that reduces GIPR activity have a lower body mass index (BMI), suggesting that GIPR inhibitors may be effective weight loss drugs.

[0006] In current research, products targeting GIP, GLP-1, or GIPR have demonstrated remarkable efficacy in weight loss and diabetes improvement, and research on these targets continues to advance. Therefore, the development of GIPR-blocking antibodies and the construction of dual-target conjugates targeting GLP-1 and GIPR by conjugating anti-GIPR antibodies to GLP-1 are of great significance for the development of weight loss and diabetes treatment drugs.

[0007] Summary of the Invention

[0008] The present invention provides GIPR antibodies or antigen-binding fragments thereof, and antibody conjugates thereof, as well as their use in treating and / or improving metabolic abnormalities and pharmaceutical applications.

[0009] In a first aspect, the present invention provides a GIPR antibody or an antigen-binding fragment thereof, comprising three light chain complementary determining regions and three heavy chain complementary determining regions, wherein the three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO: 3, LCDR2 set forth in SEQ ID NO: 4, and LCDR3 set forth in SEQ ID NO: 5, and the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO: 6, HCDR2 set forth in SEQ ID NO: 7, and HCDR3 set forth in SEQ ID NO: 8;

[0010] Alternatively, the three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 shown in SEQ ID NO:11, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:13, and the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 shown in SEQ ID NO:14, HCDR2 shown in SEQ ID NO:15, and HCDR3 shown in SEQ ID NO:16.

[0011] Furthermore, the three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 shown in SEQ ID NO:40, LCDR2 shown in SEQ ID NO:41, and LCDR3 shown in SEQ ID NO:42, and the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 shown in SEQ ID NO:43, HCDR2 shown in SEQ ID NO:44, and HCDR3 shown in SEQ ID NO:45;

[0012] Furthermore, the three light chain complementary determining regions of the antibody or its antigen-binding fragment comprise LCDR1 shown in SEQ ID NO:48, LCDR2 shown in SEQ ID NO:49, and LCDR3 shown in SEQ ID NO:50, and the three heavy chain complementary determining regions of the antibody or its antigen-binding fragment comprise HCDR1 shown in SEQ ID NO:51, HCDR2 shown in SEQ ID NO:52, and HCDR3 shown in SEQ ID NO:53.

[0013] Furthermore, the antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2;

[0014] Alternatively, the antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:9, and a heavy chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:10.

[0015] Furthermore, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 33, and / or a light chain constant region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 34.

[0016] Furthermore, the antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 38, and a heavy chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 39;

[0017] Furthermore, the antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 46, and a heavy chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 47. Furthermore, the antibody or antigen-binding fragment thereof is a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody or an antibody fragment thereof.

[0018] Furthermore, the antibody fragment is a Fab fragment, a Fab' fragment or a F(ab')2 fragment.

[0019] In a second aspect, the present invention provides a nucleic acid encoding the anti-GIPR antibody or antigen-binding fragment thereof according to the present invention.

[0020] In a third aspect, the present invention provides a cell comprising a nucleic acid encoding the anti-GIPR antibody or antigen-binding fragment thereof of the present invention.

[0021] A fourth aspect of the present invention provides a GIPR antibody conjugate, comprising:

[0022] (a) an anti-GIPR antibody or an antigen-binding fragment thereof according to the present invention, and

[0023] (b) a coupling moiety coupled to the antibody portion, wherein the coupling moiety is selected from one or more of a detectable label, a chemical drug, a toxin, a radionuclide, and a short peptide;

[0024] Furthermore, the anti-GIPR antibody or antigen-binding fragment thereof is a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody or an antibody fragment thereof;

[0025] Furthermore, the short peptide is a GLP-1 receptor agonist;

[0026] Furthermore, the GLP-1 receptor agonist is a GLP-1 analog polypeptide;

[0027] Furthermore, the GLP-1 analog is selected from a sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in any one of SEQ ID NO:35, SEQ ID NO:34 or SEQ ID NO:35.

[0028] The GIPR antibody conjugate further comprises (c) a linker structure. Preferably, the linker structure (c) is selected from: bromoacetyl (abbreviated as BrAc), or the structure shown in structure (I) or the structure shown in structure (II);

[0029] The structure (I) is as follows:

[0030] The structure shown in the structure (II) is as follows:

[0031] Furthermore, the structure of the GIPR antibody conjugate is shown below:

[0032] Ab-(LP)n;

[0033] Wherein, Ab is the GIPR antibody or antigen-binding fragment thereof provided in the present application;

[0034] L is the joint structure;

[0035] P is a GLP-1 analog peptide;

[0036] The subscript n is the DAR (Drug-to-Antibody Ratio) value, n is 0-2, preferably n is 1 or 2, and more preferably n is 2.

[0037] Further, the Ab comprises three light chain complementary determining regions and three heavy chain complementary determining regions, the three light chain complementary determining regions are LCDR1 shown in SEQ ID NO: 3, LCDR2 shown in SEQ ID NO: 4, and LCDR3 shown in SEQ ID NO: 5, and the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof are HCDR1 shown in SEQ ID NO: 6, HCDR2 shown in SEQ ID NO: 7, and HCDR3 shown in SEQ ID NO: 8;

[0038] Alternatively, the three light chain complementary determining regions of the antibody or antigen-binding fragment thereof are LCDR1 shown in SEQ ID NO:11, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:13, and the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof are HCDR1 shown in SEQ ID NO:14, HCDR2 shown in SEQ ID NO:15, and HCDR3 shown in SEQ ID NO:16;

[0039] or the three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO:40, LCDR2 set forth in SEQ ID NO:41, and LCDR3 set forth in SEQ ID NO:42, and the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO:43, HCDR2 set forth in SEQ ID NO:44, and HCDR3 set forth in SEQ ID NO:45;

[0040] Or the three light chain complementary determining regions of the antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:48, LCDR2 shown in SEQ ID NO:49, and LCDR3 shown in SEQ ID NO:50, and the three heavy chain complementary determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:51, HCDR2 shown in SEQ ID NO:52, and HCDR3 shown in SEQ ID NO:53.

[0041] Furthermore, the antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2;

[0042] or the antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 9, and a heavy chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 10;

[0043] or the antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 38, and a heavy chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 39;

[0044] Alternatively, the antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:46, and a heavy chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:47.

[0045] Furthermore, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 33, and / or a light chain constant region having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 34.

[0046] Furthermore, the L is selected from:

[0047] Bromoacetyl (abbreviated as BrAc), or the structure represented by structure (I) or the structure represented by structure (II);

[0048] The structure (I) is as follows: The structure (II) is as follows:

[0049] Furthermore, P is selected from a sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in any one of SEQ ID NO:35, SEQ ID NO:36 or SEQ ID NO:37.

[0050] Furthermore, the amino acid at position 272, 339 or 400 of the heavy chain constant region sequence of the Ab is a coupling site.

[0051] Furthermore, the heavy chain constant region sequence of the Ab is as shown in SEQ ID NO: 33, and position 272, 339 or 400 of the sequence is mutated to Cys as a coupling site.

[0052] In a preferred embodiment, the Ab in the GIPR antibody conjugate Ab-(LP)n structure is a GIPR antibody, comprising a heavy chain constant region as shown in SEQ ID NO: 33, a light chain constant region as shown in SEQ ID NO: 34, a heavy chain variable region as shown in SEQ ID NO: 39, and a light chain constant region as shown in SEQ ID NO: 38; the amino acid at position 339 of the heavy chain constant region sequence SEQ ID NO: 33 is mutated to Cys as a coupling site for coupling to the (LP) structure; the L is the structure shown in structure (II), and the structure (II) is as follows: The P is the sequence shown in SEQ ID NO: 35; and n is 2.

[0053] A fifth aspect of the present invention provides a pharmaceutical composition comprising the anti-GIPR antibody or antigen-binding fragment thereof provided by the present invention, or a nucleic acid encoding the anti-GIPR antibody or antigen-binding fragment thereof provided by the present invention, or a cell comprising a nucleic acid encoding the anti-GIPR antibody or antigen-binding fragment thereof provided by the present invention, or a GIPR antibody conjugate provided by the present invention; optionally, further comprising a pharmaceutically acceptable excipient.

[0054] A sixth aspect of the present invention provides a method for treating or ameliorating metabolic disorders and / or diseases associated with metabolic disorders, the method comprising administering to a patient an effective dose of an anti-GIPR antibody or antigen-binding fragment thereof provided by the present invention, or a nucleic acid encoding the anti-GIPR antibody or antigen-binding fragment thereof provided by the present invention, or a cell comprising a nucleic acid encoding the anti-GIPR antibody or antigen-binding fragment thereof provided by the present invention, or a GIPR antibody conjugate provided by the present invention, or a pharmaceutical composition provided by the present invention;

[0055] Furthermore, the metabolic abnormalities include obesity, type 2 diabetes (T2DM), and non-alcoholic fatty liver disease (NAFLD); the diseases related to metabolic abnormalities include obstructive sleep apnea syndrome, chronic renal failure, heart failure, peripheral vascular disease, osteoarthritis, and cardiovascular disease (unspecified). The seventh aspect of the present invention provides a pharmaceutical use, which is the use of the anti-GIPR antibody or antigen-binding fragment thereof provided by the present invention, or the nucleic acid encoding the anti-GIPR antibody or antigen-binding fragment thereof provided by the present invention, or the cell containing the nucleic acid encoding the anti-GIPR antibody or antigen-binding fragment thereof provided by the present invention, or the GIPR antibody conjugate of the present invention, or the pharmaceutical composition of the present invention for the preparation of a drug for treating or improving metabolic abnormalities and / or diseases related to metabolic abnormalities.

[0056] An eighth aspect of the present invention provides a nucleic acid construct comprising a nucleic acid encoding the anti-GIPR antibody or antigen-binding fragment thereof of the present invention, and / or a nucleic acid encoding a GLP-1 analog polypeptide.

[0057] The ninth aspect of the present invention provides a vector comprising a nucleic acid encoding the anti-GIPR antibody or antigen-binding fragment thereof of the present invention, and / or a nucleic acid encoding a GLP-1 analog polypeptide, and optionally, the vector contains other elements required for expression.

[0058] Furthermore, the metabolic abnormalities include obesity, type 2 diabetes mellitus (T2DM), and non-alcoholic fatty liver disease (NAFLD); the diseases associated with metabolic abnormalities include obstructive sleep apnea syndrome, chronic renal failure, heart failure, peripheral vascular disease, osteoarthritis, and cardiovascular disease (unspecified).

[0059] The GIPR blocking antibodies and antibody conjugates provided by the invention have one or more of the following advantages:

[0060] 1. The present invention provides GIPR-blocking antibodies or antigen-binding fragments thereof that have good affinity and binding activity to hGIPR-Fc protein, mGIPR-Fc protein, and CHOK1-hGIPR cells expressing GIPR;

[0061] 2. The GIPR blocking antibodies or antigen-binding fragments thereof provided by the present invention showed superior ability to block GIP from activating CHOK1-GIPR / GLP-1R cells compared to the control group;

[0062] 3. The GIPR blocking antibodies or antigen-binding fragments thereof provided by the present invention showed internalization activity consistent with that of the control group;

[0063] 4. The GIPR blocking antibodies or antigen-binding fragments thereof provided by the present invention show a lower aggregation risk than the control group, which is more conducive to the development of high-concentration preparations;

[0064] 5. The GIPR blocking antibody or antigen-binding fragment thereof provided by the present invention exhibited good pharmacokinetic activity in mice and good in vivo stability in mice, with AUC 0-t Maximum 15033.01hour×μg / mL;

[0065] 6. The GIPR-blocking antibody conjugates provided by the present invention exhibit low hydrophobicity. Therefore, the antibody conjugates constructed in this application are less likely to aggregate at high concentrations and are more suitable for the development of high-concentration subcutaneous preparations.

[0066] 7. The GIPR blocking antibody conjugates provided by the present invention all exhibited excellent weight-reducing activity in mice, with a weight-reduction ratio of 22.35%-26.54% on day 17. Furthermore, the body weight remained lower than that of the control group for approximately 70 days after drug withdrawal, demonstrating sustained weight-reducing activity and broad application prospects.

[0067] 8. In terms of pharmacokinetics (PK), the GIPR antibody conjugate of the present invention showed higher pharmacokinetic parameters than the control group, such as AUC0-t (area under the total concentration-time curve of the drug in vivo), indicating that the GIPR antibody conjugate of the present invention has better stability in vivo and less polypeptide shedding. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] FIG1 shows the mouse serum antibody titers in Example 1;

[0069] FIG2 shows the ELISA binding activity of the GIPR antibody in Example 3 to the hGIPR-Fc protein;

[0070] FIG3 shows the ELISA binding activity of the GIPR antibody and the mGIPR-Fc protein in Example 3;

[0071] Figures 4A-4D show the binding activity of the GIPR antibody of Example 3 to CHOK1-GIPR / GLP-1R cells;

[0072] Figures 5A-5B show that the GIPR antibody in Example 3 blocks the activity of GIP in activating CHOK1-GIPR / GLP-1R cells;

[0073] Figures 6A-6B show the internalization activity of the GIPR antibody of Example 3 on CHOK1-GIPR / GLP-1R cells;

[0074] FIG7 shows the pharmacokinetic curve of the GIPR antibody in mice in Example 4;

[0075] FIG8 shows the SEC spectrum of the CE947 coupling product in Example 5;

[0076] FIG9 is a bar graph showing the body weight suppression rate after administration of GIPR-GLP-1 in Example 6;

[0077] Figure 10 shows the weight-loss activity of the coupling products at different sites when L1-P1 were coupled in Example 6;

[0078] Figure 11 shows the GLP-1R activation curves of the GIPR antibody conjugate (a) and the GLP-1 polypeptide (b) in Example 7;

[0079] Figure 12 is a weight loss curve diagram of Example 8;

[0080] FIG13 is a pharmacokinetic curve of the GIPR antibody conjugate of Example 9 in hGIPR-DIO mice. DETAILED DESCRIPTION

[0081] The present invention will be further described below in conjunction with specific examples. The described embodiments are some embodiments of the present invention, rather than all embodiments. It should be understood that the following examples are provided to provide a complete disclosure and description of how to utilize the methods and compositions of the present invention to those skilled in the art, and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

[0082] Example 1. Generation of blocking GIPR antibodies

[0083] 1.1 Mouse immunization method

[0084] The mice used in the immunization experiment were fully human antibody transgenic mice (Boan self-developed), mice were immunized with two immunogens: (1) Fc-fused GIPR protein (GIPR corresponding to Uniprot: P48546) and (2) GIPR-overexpressing CHOK1 cells (GenScript, M00486). The GIPR-Fc protein was obtained by Shandong Boan. All mice were immunized by subcutaneous injection in the abdomen and multiple injections in the groin. The immunization doses of GIPR-Fc protein and CHOK1 / GIPR cells were 20 μg / mouse and 5×10 6 cells / mouse. The first immunization used antigen emulsified in Freund's complete adjuvant, and subsequent immunizations used antigen emulsified in Freund's incomplete adjuvant. Four immunizations and one booster immunization were performed. Three days after the booster immunization, mice were euthanized, and spleens were harvested to prepare single-cell suspensions, which were then used to construct phage libraries. Mouse serum titer test results are shown in Figure 1.

[0085] 1.2 Establishment of phage library

[0086] Mice were sacrificed, and spleens were removed by dissection. Splenocytes were obtained by grinding and fragmentation, frozen, and RNA was extracted and reverse-transcribed to obtain cDNA. Phage libraries were constructed according to conventional methods. One phage library was established for each mouse. The library capacity data for the constructed phage libraries are shown in Table 1.

[0087] Table 1 Capacity of the constructed phage library

[0088] 1.3 Screening in three ways

[0089] 1.3.1 Plate screening: Coat plates with GIPR-Fc protein. The next day, add the phage library and incubate for 2 hours. Wash 4-10 times and elute specifically bound phage with elution buffer.

[0090] 1.3.2 Magnetic bead screening: Biotinylate the GIPR-Fc protein according to the general coupling kit procedure, then bind to Thermo magnetic beads. After blocking with blocking buffer, incubate with the phage library for 2 hours. Wash 4-10 times and elute the specifically bound phage with elution buffer.

[0091] 1.3.3 Cell Screening: CHOK1-GIPR cells were harvested and incubated with the phage library for 2 h. Specifically bound phages were then washed 4-10 times and eluted with elution buffer. The clones obtained and their sources are shown in Table 2.

[0092] Table 2 Sources of GIPR antibody clones obtained by screening

[0093] Example 2. Construction and production of GIPR antibodies

[0094] The antibody variable region genes were amplified using conventional molecular biology techniques (PCR, 2× Phanta Max Master Mix, Vazyme, P515-P1-AA). The heavy chain variable region gene was ligated into the vector pCDNA3.4 (Life Technology) containing the nucleic acid sequence of the antibody heavy chain constant region, and the light chain variable region gene was ligated into the vector pCDNA3.4 containing the nucleic acid sequence of the antibody light chain constant region. Plasmids from sequenced positive clones were extracted and co-transfected into HEK293 cells and cultured at 37°C, 8% CO₂, and a 125 rpm shaker. After 7 days of transient expression, the supernatant was purified by Protein A affinity chromatography to obtain the antibody. The antibody concentration was determined by UV280 combined with the theoretical extinction coefficient.

[0095] The variable region sequences of the antibodies in the examples of this application are shown in Table 3, and the heavy chain and light chain constant region sequences are shown in Table 4.

[0096] Positive clones BA191 and CE947 were constructed into IgG1 and sequenced, using the light and heavy chain variable region sequences of antibodies 5G12 and 2G10, referenced from patent application publication number CN110662558A, SEQ ID NOs: 2025, 2182, and SEQ ID NOs: 1958, 2115. The amino acid sequences of the variable regions of each antibody are shown in Table 3 (CDR regions are underlined, and the analysis system was the IMGT system).

[0097] Table 3 Variable region sequences of active clone amino acid sequences

[0098] Table 4 Heavy and light chain constant region sequences

[0099] Example 3. Characterization of GIPR Antibodies

[0100] 3.1 ELISA binding activity of GIPR antibodies to human GIPR-Fc protein

[0101] Experimental and control groups were set up. The experimental group included BA191, CE947, CA360 and CE560, and the control group included 2G10.

[0102] hGIPR-Fc protein was diluted to 0.2 μg / mL in carbonate buffer, with 100 μL / well added, and coated overnight at 4°C. Blocked with 3% nonfat dry milk, 300 μL / well was added, and incubated at 37°C for 1 hour. The intact antibody was serially diluted in PBST (phosphate buffered saline + 0.05% Tween-20) to 0.2 μg / mL in eight four-fold dilutions, with 100 μL / well added, and incubated at 37°C for 1 hour. After washing, goat anti-human IgG (Fab-specific) / HRP secondary antibody (1:40,000 dilution) (Sigma, A0293) was added at 100 μL / well, and incubated at 37°C for 1 hour. After washing, 100 μL of TMB was added to each well. After developing for 10 minutes, the reaction was terminated by adding 50 μL of 2 M H2SO4 to each well, and the OD450 value was read on a multi-functional microplate reader. Figure 2 shows the binding curve of the GIPR antibody to hGIPR-Fc at the protein level.

[0103] As can be seen from Figure 2, compared with the control group 2G10, the antibodies screened in this application have better binding sensitivity to hGIPR-Fc.

[0104] 3.2 ELISA binding activity of GIPR antibodies to mouse GIPR-Fc protein

[0105] mGIPR-Fc protein was diluted to 0.2 μg / mL in carbonate buffer, with 100 μL / well added, and coated overnight at 4°C. Blocked with 3% nonfat dry milk, 300 μL / well was added, and incubated at 37°C for 1 hour. The intact antibody was serially diluted in PBST (phosphate buffered saline + 0.05% Tween-20) to 0.2 μg / mL in eight four-fold dilutions, with 100 μL / well added, and incubated at 37°C for 1 hour. After washing, goat anti-human IgG (Fab-specific) / HRP secondary antibody (1:40,000 dilution) (Sigma, A0293) was added at 100 μL / well, and incubated at 37°C for 1 hour. After washing, 100 μL of TMB was added to each well. After developing for 10 minutes, the reaction was terminated by adding 50 μL of 2 M H2SO4 to each well, and the OD450 value was read on a multi-functional microplate reader. Figure 3 shows the binding curve of the GIPR antibody to mGIPR-Fc at the protein level.

[0106] As can be seen from Figure 3, the antibody CA360 obtained by the screening of this application has strong binding activity with mouse GIPR-Fc, while the other screened antibodies have no significant binding activity with mouse GIPR-Fc protein, and the control group 2G10 has weak binding activity with mouse GIPR-Fc.

[0107] 3.3 Binding activity of GIPR antibodies to CHOK1-GIPR / GLP-1R cells

[0108] The cell binding activity of the GIPR antibody was tested on CHOK1-GIPR / GLP-1R cells. The antibody was diluted with PBS to prepare a 2× antibody solution. 8 concentrations were serially diluted 4-fold starting from 40 μg / mL. 50 μL was added to each well of a 96-well U-bottom plate. After the CHOK1-GIPR / GLP-1R cells were washed with PBS, 50 μL of the antibody solution was added to each well. The cell number was 1×10 5 cells / w, incubated at 4°C for 1 hour. Washed twice with PBS and then added with 100 μL / w Alexa 488-Anti-Human IgG (Jackson, 109-545-008) secondary antibody was used for incubation at 4°C for 1 hour. After washing twice with PBS, the cells were resuspended in 100 μL / w PBS and the MFI value of the FITC channel was measured by flow cytometry (ACEA, NovoCyte).

[0109] The results are shown in Table 5 and Figures 4A-4D. As shown in Figures 4A-4D, BA191, CE947, and CE560 have higher binding activity against CHOK1-GIPR / GLP-1R cells than the control group 2G10; CA360 has similar EC 50 However, the maximum binding value Rmax of CA360 is larger than that of 2G10, indicating that CA360 can bind to more GIPRs on the cell surface.

[0110] Table 5 ECs of GIPR antibodies binding to CHOK1-GIPR / GLP-1R cells 50

[0111] 3.4 Affinity Detection of GIPR Antibodies and Human GIPR Protein

[0112] The binding kinetics of GIPR antibodies to hGIPR-His protein were measured using an Octet 96 instrument based on bio-layer interferometry (BLI). The antibodies were captured using a FAB2G sensor (Fortebio, 18-5125), and the hGIPR-His protein (Novoprotein, C28P) was serially diluted 2-fold with PBST buffer, starting at 50nM, diluted 2-fold in 4 concentration gradients, and set to 0 concentration for binding and dissociation with the probe. The binding constant (ka) and dissociation constant (kd) were calculated using a 1:1 binding binding model, and the equilibrium dissociation constant (KD) was calculated as the ratio kd / ka. The results showed that each candidate antibody was able to bind to the corresponding antigen, and the affinity data are shown in Table 6.

[0113] Table 6 Affinity of GIPR antibodies to hGIPR-His protein

[0114] A smaller KD value indicates a stronger affinity. Table 6 shows that BA191, CE947, CE560, and CA360 have higher affinities for the hGIPR-His protein than the control group 2G10. The Response value represents the binding signal of each antibody to the same antigen concentration. A higher value indicates that the antibody can bind more antigen at steady state. This parameter shows that BA191, CE947, CE560, and CA360 have higher antigen binding signals for hGIPR-His than the control group.

[0115] 3.5 Neutralizing activity of GIPR antibodies at the cellular level

[0116] The cellular neutralization activity of the GIPR antibody was tested using CHOK1-GIPR / GLP-1R cells. Antibodies were diluted using the Stimulation buffer in the cAMP assay kit (purchased from Cisbio, Cat. No. 62AM4PEB) to prepare a 4× antibody solution. Eight concentrations were serially diluted fourfold starting from 80 μg / mL, and 2.5 μL was added to each well of a 384-well plate. After washing the cells with Stimulation buffer, 2.5 μL of the antibody solution was added to each well, adjusting the cell count to 1×10. 4 After incubation at room temperature for 30 minutes, 5 μL of GIP (Hangzhou Zhongpeptide, GIPS-001) was added to a final concentration of 0.1 μg / mL. After incubation at 37°C for 30 minutes, cAMP levels were measured using a cAMP detection kit. The cellular neutralization activity of each GIPR antibody is shown in Figures 5A-5B and Tables 7-8.

[0117] Table 7 IC of GIPR antibodies BA191, CE947 and 2G10 neutralizing GIP-activated CHOK1-GIPR / GLP-1R cells 50

[0118] Table 8 IC values ​​of GIPR antibodies CA360, CE560 and 2G10 in neutralizing GIP-activated CHOK1-GIPR / GLP-1R cells 50

[0119] 5A-Figure 5B and Tables 7-8 show that the experimental group antibodies numbered BA191, CE947, CE560 and CA360 all showed better cellular blocking activity than the control group 2G10.

[0120] 3.6 GIPR Antibody Internalization Activity

[0121] The cellular internalization activity of GIPR antibodies was tested using CHOK1-GIPR / GLP-1R cells as materials. Complete culture medium was prepared, and the antibody was diluted with complete culture medium to make the final concentration of the antibody 6μg / mL; the antibody was labeled with a labeling reagent (Invitrogen, catalog number: Z25611) according to the instructions, and 50μL / well of cells was added and mixed, and then incubated at 37°C for 0h, 2h, 6h, and 22h (the number of cells was 100,000 / well), and then the MFI value of the FITC channel was detected by flow cytometry. Alternatively, the internalization activity of the antibody was detected using the DT3C kit. After labeling the antibody with the DT3C labeling kit (Huamei Bio, CSB-EP360556CQR1) according to the instructions, the labeled antibody was serially diluted with complete culture medium, and incubated with CHOK1-GIPR / GLP-1R cells at 37°C for 3 days, and then the antibody was labeled with a DT3C labeling kit (Huamei Bio, CSB-EP360556CQR1). Cell viability was measured using a kit (Novozymes Catalog No. DD1101-01). The internalization activity of each GIPR antibody at the cellular level is shown in Figures 6A-6B. The figures show that CE947, CE560, and CA360 antibodies exhibited similar internalization activity to the control 2G10 antibody, while the unrelated antibody isotype group exhibited only low internalization activity.

[0122] Example 4. PK study of GIPR antibodies in mice

[0123] In vivo pharmacokinetic studies were conducted using ICR mice. Male ICR mice were purchased from Jinan Pengyue Company. Mice were divided equally according to body weight into four experimental groups, with three mice in each group. Each group received a 10 mg / kg dose via a single injection into the tail vein. For CE947, 0.05 mL of blood was collected from the orbital venous plexus at 10 minutes, 1 hour, 6 hours, 24 hours, 72 hours, 120 hours, 240 hours, 264 hours, 336 hours, and 504 hours after administration, and placed into 1.5 mL EP tubes. For CE560, CA360, and 2G10, 0.05 mL of blood was collected from the orbital venous plexus at 5 minutes, 1 hour, 6 hours, 24 hours, 72 hours, 120 hours, 168 hours, 240 hours, 336 hours, and 504 hours after administration, and placed into 1.5 mL EP tubes. After standing at room temperature for 1 hour, the serum was separated by centrifugation at 8000 rpm for 10 minutes and stored at -80°C until assayed.

[0124] The concentration of samples in mouse serum was determined using ELISA. hGIPR-Fc was coated on an ELISA plate as a capture reagent. After blocking, the standard curve sample, quality control sample, and test sample were added to the ELISA plate and incubated. The sample then bound to the captured antigen to form an antigen-antibody complex that was captured on a 96-well plate. After washing away the free sample, a detection antibody (Goat Anti-Human IgG-Fab-HRP) was added to bind to the antigen-antibody complex captured on the ELISA plate. After washing away the free detection antibody, a substrate was added for color development, and a stop solution was added to terminate the color development reaction. The OD value was read at a wavelength of 450 nm (reference 650 nm). The sample concentration was positively correlated with the depth of the color produced by the final reaction. The pharmacokinetic curves of GIPR antibodies in mice are shown in Figure 7. Table 9 shows the pharmacokinetic parameters of GIPR antibodies CE947, CE560, and CA360 in mice, with 2G10 serving as the control group.

[0125] The pharmacokinetic curves and pharmacokinetic parameters of GIPR antibody in mice show that the tested CE947, CE560 and CA360 antibodies all show good stability in mice. 0-t They can reach 15033.01, 11339.65 and 11533.07hour*μg / mL respectively.

[0126] Table 9 Pharmacokinetic parameters of GIPR antibody in mice

[0127] Example 5. Preparation and Analysis of GIPR Antibody Conjugates

[0128] 5.1 Preparation of GIPR Antibody Conjugates

[0129] Table 10 Linker structures used for coupling

[0130] Table 11 GLP-1 peptide numbers

[0131] Where X is diaminoisobutyl (CAS Registry Number: 62-57-7)

[0132] The seven GLP-1 analog peptide-linker structures commissioned to be synthesized by Hangzhou Zhongpeptide were numbered: L1-P1, L2-P1, L1-P2, L2-P2, L1-P3, L2-P3, and L4-P1.

[0133] The GIPR antibody conjugate was prepared by mutating the amino acid at position 272 or 339 of the heavy chain constant region sequence of the GIPR-blocking antibody prepared in Example 2 (as shown in SEQ ID NO: 33) to Cys for conjugation. The antibody was incubated in 2.5 mM cystamine / 2.5 mM cysteamine 40 mM Tris buffer, pH 8.2, and then incubated again in sodium acetate buffer, pH 5.2, containing sucrose. 3-8 equivalents of TCEP were added for reaction. After completion of the reaction, the solution was changed to sodium phosphate buffer, pH 7.5, containing EDTA, and oxidized with 8-16 equivalents of dehydroascorbic acid (DHAA) for 2 hours. After oxidation, when linkers L1 and L4 were used, samples of GLP-1 analog peptide-linker structures numbered L1-P1, L1-P2, L1-P3, or L4-P1 were directly added for coupling. When linker L2 was used, samples of GLP-1 analog peptide-linker structures numbered L2-P1, L2-P2, or L2-P3 were exchanged with neutral PB buffer. After the coupling reaction, ring opening was performed. The coupled product was purified by SEC using Chromdex 200PG packing (Borgron, AG0083) and then exchanged with pH 6.0 His buffer.

[0134] 5.2 Coupling efficiency of antibody conjugated to linker-GLP-1 at different sites

[0135] The 272-site mutant antibody 2G10-272 and the 339-site mutant antibody 2G10-339 were constructed and produced. 2G10-272 and 2G10-339 were coupled to L1-P2 according to the method of Example 5.1. The coupled products were subjected to SDS-PAGE electrophoresis analysis, and the proportion of aggregates and the coupling efficiency (the proportion of heavy chains that were successfully coupled) were calculated based on the grayscale of the electrophoretogram. The following table shows the aggregate proportion and coupling efficiency of the two-site coupling products. Coupling at the 272 site produced 38% aggregates and a coupling efficiency of 66%. Coupling at the 339 site did not produce aggregates, and the coupling efficiency was also higher than that of the 272 site, at 79%. Therefore, coupling at the 339 site can reduce antibody loss and increase the yield of the final coupled product.

[0136] Table 12 Aggregate ratio and coupling efficiency of 272- and 339-site coupling products

[0137] 5.3 Aggregation Risk at 4°C After Antibody Conjugation with Linker-GLP-1

[0138] CE947, BA191, CE560, and CA360 were conjugated to L1-P1 to obtain antibody-peptide conjugates. The conjugated products were placed at 4°C for 3 days and observed for precipitation. After centrifugation, the protein content in the supernatant before and after placement was detected (OD280 detection), and the reduction ratio of the conjugated products was calculated. The results are shown in Table 13 below. CA360 had the smallest reduction ratio, and no aggregation occurred after placement.

[0139] Table 13 Reduction ratio of supernatant protein after the coupling product was placed at 4°C for 3 days

[0140] 5.4 Analysis of Antibody Conjugates

[0141] The conjugated product was analyzed using hydrophobic interaction HIC to determine the DAR (Drug-to-Antibody Ratio). The DAR represents the number of GLP-1 analog peptide molecules bound to the antibody. The sample was injected onto a TSK-GEL Butyl-NPR 4.6×35 mm, 2.5 μm column and eluted at 0.5 mL / min. Mobile phase A consisted of 20 mM phosphate in 25% isopropanol, pH 7.0. Mobile phase B consisted of 20 mM phosphate in 1.5 M ammonium sulfate, pH 7.0. Detection was performed at 220 nm to determine the conjugated amount.

[0142] Figure 8 shows the SEC profile of the CE947 conjugated product, and Table 14A reports the DAR2 yield of the CE947 conjugated product after SEC purification. The SEC purification profile of the CE947 conjugated product shown in Figure 8 shows that the conjugated product obtained at the 272 conjugation site has a relatively large number of aggregate peaks, while the conjugated product obtained at the 339 site has virtually no or very few aggregate peaks. Therefore, after the conjugated product is purified by SEC to remove aggregates, the DAR2 yield at the 339 conjugation site is significantly higher than that at the 272 site, indicating that for the CE947 antibody, the 339 conjugation site has a higher conjugation efficiency than the 272 site.

[0143] Table 14A DAR2 yield after SEC purification of CE947 coupling product

[0144] Table 14B records the HIC analysis retention timetable of the conjugates after CA360 antibody was conjugated to L1-P1, L2-P1, and L4-P1 at position 339, and 2G10 antibody was conjugated to L1-P2 at position 272 under the same HIC experimental conditions. The HIC retention time of DAR2 after CA360 was conjugated to the three linker-peptides was lower than that of 2G10.

[0145] Table 14B HIC Analysis Retention Schedule

[0146] Example 6. Weight-reducing activity of GIPR antibody conjugates in DIO wild-type mice

[0147] Male B-DIO mice weighing 40-50 g were purchased from Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd. (Cat. No. 112938). DIO modeling ultra-high-fat diet was purchased from Ruidi Biotechnology (Shenzhen) Co., Ltd. (60 kcal%, Cat. No. D12492). B-DIO mice were maintained in an SPF-grade barrier facility at the animal breeding center of Shandong Boan Biotechnology Co., Ltd. and maintained on a DIO modeling ultra-high-fat diet. After 7 days of adaptive feeding, all mice were divided into 8 5G12 conjugated product experimental groups according to their body weight. According to the different conjugation sites, linkers and GLP-1 analog peptides, they were numbered as follows: 272-L1-P1, 272-L1-P2, 272-L2-P2, 339-L1-P1, 339-L1-P3, 339-L2-P2, and a blank control group (Vehicle). There were 6 mice in each group. Drug administration began on the day of grouping, with a dose of 5 mg / kg, and the administration route was IP, and the administration time was Q2W. Body weight was measured 3, 7, 10, 14, 17, 22, 26, 31, 35, 38, 42, 47, 51, 58, 66, and 80 days after administration. The results were analyzed and expressed as mean ± SEM. Table 15 shows the body weight suppression rate after GIPR-GLP-1 administration. Figure 9 is a bar graph constructed using the values ​​in Table 15. The values ​​in Table 15 and the bar graph in Figure 9 indicate that all seven groups of antibody-peptide conjugates exhibited excellent weight-reducing activity in mice, with weight reduction rates reaching 22.35%-26.54% on day 17. Furthermore, body weight remained lower than that of the control group for approximately 70 days after drug discontinuation, demonstrating sustained weight-reducing activity. Figure 10 is a weight-reduction curve, with arrows indicating dosing. Figure 10 and Table 15 indicate that when both conjugated to L1-P1, the 339-site conjugated product exhibited superior weight-reducing activity compared to the 272-site conjugated product.

[0148] Table 15 Body weight suppression rate after GIPR-GLP-1 administration

[0149] Example 7. GLP-1R agonist activity of GIPR antibody conjugates and GLP-1 polypeptides in vitro

[0150] The cellular level GLP-1R agonist activity of the GIPR antibody conjugate was tested using CHOK1-GIPR / GLP-1R cells as the material. The antibody conjugate and GLP-1 peptide were serially diluted using the Stimulation buffer in the cAMP assay kit (purchased from Cisbio, catalog number: 62AM4PEB). 5 μL per well was added to a 384-well plate. The CHOK1-GIPR / GLP-1R cells were washed once with the Stimulation buffer, and 5 μL per well was added to the antibody. The cell number was adjusted to 5000 cells / w. After incubation at 37°C for 30 minutes, the cAMP content was detected using the cAMP assay kit. Figure 11 shows the GLP-1R activation curves of the GIPR antibody conjugate (a) and the GLP-1 peptide (b), and Table 16 shows the EC values ​​of the activation curves. 50 Values, from activation curves and EC 50 It can be seen that L4-P1 (EC 50 =0.15 nM) showed a slightly weaker 50 =0.10nM) activation activity, which is beneficial to reduce the side effects of the drug in vivo, while the conjugate CA360-339-L4-P1 (EC 50 =0.20 nM) showed superiority over 2G10-272-L1-P2 (EC 50 =0.35 nM), indicating that CA360-339-L4-P1 may have a better weight loss effect in vivo.

[0151] Table 16 GLP-1R activation EC of GIPR antibody conjugates and GLP-1 polypeptides 50 value

[0152] Example 8. Weight-reducing activity of GIPR antibody conjugates in GIPR humanized DIO mice

[0153] hGIPR-DIO mice were purchased from Jicui Pharmaceuticals Co., Ltd., weighing 40-50 g. DIO modeling ultra-high-fat diet was purchased from Ruidi Biotechnology (Shenzhen) Co., Ltd. (60 kcal%, Catalog No. D12492). hGIPR-DIO mice were maintained in an SPF-grade barrier facility at the Animal Breeding Center of Shandong Boan Biotechnology Co., Ltd. and maintained on a DIO modeling ultra-high-fat diet. After 7 days of adaptive feeding, all mice were divided into five experimental groups according to their body weight, namely CA360-339-L1-P1, CA360-339-L2-P1, CA360-339-L4-P1, 2G10-272-L1-P2 and blank control group (Vehicle), with 6 mice in each group. Drug administration began on the day of grouping, with a dose of 5 mg / kg, administered IP, single dose. Body weight was measured 3, 7, 10, 14, 17, 21, 24, 28, 31, 35, 38, 42, 45, 50, 53, 56, 59 and 65 days after administration. The results were expressed as mean ± SEM.

[0154] Figure 12 is a weight loss curve chart, where the arrows indicate drug administration. It can be seen from the figure that CA360-339-L4-P1 has the best weight loss effect. On the 14th day, CA360-339-L4-P1 weight loss (19.60% + 6.76% = 26.36%) began to show a weight loss effect that was better than 2G10-272-L1-P2 (19.33% + 6.76% = 26.09%). Moreover, CA360-339-L4-P1 lost weight (26.77% + 0.07% = 26.84%) after 65 days of administration, which was significantly better than 2G10-272-L1-P2 (26.77% - 15.29% = 11.48%), P < 0.05, showing an excellent and lasting weight loss effect.

[0155] Example 9 PK Study of GIPR Antibody Conjugates in hGIPR-DIO Mice

[0156] In vivo pharmacokinetic studies were conducted using hGIPR-DIO mice purchased from Jicui Pharmaceuticals. Mice were divided equally according to body weight into two experimental groups of three each. The drug was administered at a dose of 10 mg / kg via a single injection through the tail vein. Blood (0.05 mL) was collected from the orbital venous plexus at 10 minutes, 1 hour, 6 hours, 24 hours, 96 hours, 144 hours, 192 hours, 264 hours, and 336 hours after administration. The blood was placed in a 1.5 mL EP tube and allowed to stand at room temperature for 1 hour. The serum was separated and stored at -80°C for testing. The concentration of the samples in mouse serum was determined by ELISA.

[0157] The total antibody concentration is determined by coating hGIPR-Fc on an ELISA plate as a capture reagent. After blocking, the standard curve sample, quality control sample, and test sample are added to the plate and incubated. The sample binds to the capture antigen, forming an antigen-antibody complex that is captured on the 96-well plate. After washing away the free sample, the detection antibody (Goat Anti-Human IgG-Fab-HRP) is added to bind to the antigen-antibody complex captured on the plate. After washing away the free detection antibody, the substrate is added for color development. The color reaction is terminated by adding the stop solution. The OD value is read at a wavelength of 450nm (reference 650nm). The sample concentration is positively correlated with the intensity of the final color produced by the reaction.

[0158] The concentration of conjugates still containing peptides is determined by coating hGIPR-Fc on an ELISA plate as a capture reagent. After blocking, standard curve samples, quality control samples, and test samples are added to the plate and incubated. The samples bind to the capture antigen, forming an antigen-antibody complex that is captured on the 96-well plate. After washing away the free sample, a detection antibody (Goat Anti-Human GLP-1-HRP) is added to bind to the antigen-antibody complex captured on the plate. After washing away the free detection antibody, a substrate is added for color development. The color reaction is terminated by adding a stop solution. The OD value is read at a wavelength of 450 nm (reference 650 nm). The sample concentration is positively correlated with the intensity of the final reaction color.

[0159] The pharmacokinetic curves of the GIPR antibody conjugates in hGIPR-DIO mice are shown in Figure 13. Table 20 shows the pharmacokinetic parameters of the GIPR antibody conjugates CA360-339-L4-P1 and 2G10-272-L1-P2 in mice. The pharmacokinetic curves and pharmacokinetic parameters show that the stability of the total antibodies of the tested CA360-339-L4-P1 and 2G10-272-L1-P2 antibody conjugates in transgenic mice is basically the same, with AUC 0-t 23152.24 and 23538.48 respectively, but the stability of CA360-339-L4-P1 conjugate is higher than that of 2G10-272-L1-P2, AUC 0-t They are 16963.40 and 15514.44, respectively, indicating that the CA360-339-L4-P1 conjugate has less polypeptide shedding in transgenic mice.

[0160] Table 17 PK parameters of GIPR antibody conjugates in hGIPR-DIO mice

Claims

1. A GIPR antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises three light chain complementary determining regions and three heavy chain complementary determining regions, the three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 as shown in SEQ ID NO:40, LCDR2 as shown in SEQ ID NO:41, and LCDR3 as shown in SEQ ID NO:42, and the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 as shown in SEQ ID NO:43, HCDR2 as shown in SEQ ID NO:44, and HCDR3 as shown in SEQ ID NO:45; The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof include LCDR1 shown in SEQ ID NO:3, LCDR2 shown in SEQ ID NO:4, and LCDR3 shown in SEQ ID NO:5, and the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof include HCDR1 shown in SEQ ID NO:6, HCDR2 shown in SEQ ID NO:7, and HCDR3 shown in SEQ ID NO:8; The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof include LCDR1 shown in SEQ ID NO:11, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:13, and the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof include HCDR1 shown in SEQ ID NO:14, HCDR2 shown in SEQ ID NO:15, and HCDR3 shown in SEQ ID NO:16; Or the three light chain complementary determining regions of the antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:48, LCDR2 shown in SEQ ID NO:49, and LCDR3 shown in SEQ ID NO:50, and the three heavy chain complementary determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:51, HCDR2 shown in SEQ ID NO:52, and HCDR3 shown in SEQ ID NO:

53.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:38, and a heavy chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:39; The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and a heavy chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 2; The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:9, and a heavy chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:10; Alternatively, the antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:46, and a heavy chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

47.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain constant region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:33, and / or comprises a light chain constant region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

34.

4. A nucleic acid encoding the anti-GIPR antibody or antigen-binding fragment thereof according to any one of claims 1 to 3. A cell comprising the nucleic acid of claim 4 .

6. A GIPR antibody conjugate, characterized in that: The structure of the GIPR antibody conjugate is shown below: Ab-(LP)n; wherein Ab is an anti-GIPR antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3; L is the joint structure; P is a conjugated moiety coupled to the GIPR antibody or antigen-binding fragment thereof; The subscript n is the DAR (Drug-to-Antibody Ratio) value, n is 0-2, preferably n is 1 or 2, and more preferably n is 2; The P is selected from: one or more of a detectable marker, a chemical drug, a toxin, a radionuclide and a short peptide; Preferably, the P is a GLP-1 analog polypeptide; Preferably, the GLP-1 receptor agonist is a GLP-1 analogue; Preferably, the GLP-1 analog is selected from a sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in any one of SEQ ID NO:35, SEQ ID NO:36 or SEQ ID NO:

37.

7. The GIPR antibody conjugate according to claim 6, characterized in that The joint structure is selected from: a) bromoacetyl; b) the structure represented by structure (I); or c) The structure shown in structure (II); The structure (I) is as follows: The structure shown in the structure (II) is as follows: Preferably, the amino acid at position 272, 339 or 400 of the heavy chain constant region sequence of the Ab is a coupling site; More preferably, the heavy chain constant region sequence of the Ab is as shown in SEQ ID NO: 33, and position 272, 339 or 400 of the sequence shown in SEQ ID NO: 33 is mutated to Cys as a coupling site.

8. A pharmaceutical composition comprising the anti-GIPR antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the nucleic acid according to claim 4, or the cell according to claim 5, or the GIPR antibody conjugate according to any one of claims 6 or 7; Optionally, it also contains pharmaceutically acceptable excipients.

9. Use of the anti-GIPR antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the nucleic acid according to claim 4, or the cell according to claim 5, or the GIPR antibody conjugate according to any one of claims 6 or 7, or the pharmaceutical composition according to claim 8 in the preparation of a medicament for treating or ameliorating metabolic disorders and diseases associated with metabolic disorders.

10. The method according to claim 9, characterized in that The metabolic abnormalities include obesity, type 2 diabetes, and non-alcoholic fatty liver disease; the diseases associated with metabolic abnormalities include obstructive sleep apnea syndrome, chronic renal failure, heart failure, peripheral vascular disease, osteoarthritis, and cardiovascular disease.

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