Anti-GLP-1R antibodies and uses thereof

Abstract

The present disclosure relates to antibodies or antigen-binding fragments thereof that specifically bind to GLP-1R, and uses thereof. The antibody or the antigen binding fragment thereof can antagonize the activation effect of endogenous or exogenous GLP-1 on GLP-1R, and can be used for diagnosis, prevention and / or treatment of GLP-1R-related or hypoglycemia-related diseases or symptoms.

Description

Technical Field

[0001] The present disclosure relates to the field of antibodies, and in particular to anti-glucagon-like peptide 1 receptor (GLP-1R) monoclonal antibodies and fragments thereof, and the use of the antibodies or fragments thereof for the diagnosis, prevention and / or treatment of GLP-1R-related diseases and hypoglycemia-related diseases. Background Art

[0002] As obesity becomes an increasingly serious problem worldwide, bariatric surgery, as one of the most effective means of weight loss, is being used by more and more obese patients. In the past decade, the number of patients undergoing bariatric surgery has increased by 50%. Post-bariatric hypoglycemia (PBH), the most serious complication of bariatric surgery, is estimated to affect 10-30% of patients. Post-bariatric hypoglycemia generally occurs 1-3 hours after a meal and becomes more severe with increased carbohydrate intake, causing dizziness, epilepsy, confusion and even death, which seriously affects the patient's ability to live independently. In Roux-en-Y gastric bypass patients, the level of glucagon-like peptide 1 (GLP-1) in the blood after a meal can be as high as 10 times that of normal people, and can reach more than 50 times in patients with hypoglycemia. The increase in the level of glucagon-like peptide 1 will enhance the secretion of insulin, leading to hypoglycemia. So far, there is no effective treatment for post-bariatric hypoglycemia. Avexitide (exendin 9-39) from Eiger Biopharma is a peptide glucagon-like peptide 1 receptor (GLP-1R) antagonist that has shown efficacy in reversing hypoglycemia in clinical practice. This peptide is currently in Phase III clinical research. Since the half-life of Avexitide is very short (~30 minutes), patients need to inject it subcutaneously once or twice a day. This high-frequency dosing method will lead to poor compliance in patients with long-term chronic diseases.

[0003] Currently, Avexitide is the only GLP-1R antagonist in the clinical stage, but it has disadvantages such as short half-life and frequent dosing, and cannot meet the diverse treatment needs of patients for GLP-1R-related diseases. It is urgent to develop a long-acting GLP-1R antagonist, which can greatly reduce the frequency of dosing and improve patient compliance and experience. Summary of the invention

[0004] The present disclosure provides a new monoclonal antibody and its antigen-binding fragment, encoding nucleic acid, vector, cell and composition. The monoclonal antibody and its antigen-binding fragment disclosed in the present disclosure have the characteristics of strong specificity, long half-life, significant in vivo and in vitro efficacy, etc. At the same time, the present disclosure provides a method for treating and / or preventing GLP-1R related diseases, and the use of drugs for treating and / or preventing GLP-1R related diseases.

[0005] In one aspect, the present disclosure provides a monoclonal antibody or an antigen-binding fragment thereof, wherein the amino acid sequences of heavy chain complementary determining regions (HCDR) 1, HCDR2 and HCDR3 of the monoclonal antibody or the antigen-binding fragment thereof are SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and the amino acid sequences of light chain complementary determining regions (LCDR) 1, LCDR2 and LCDR3 are SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; preferably, the amino acid sequence of the heavy chain variable region (HCVR) of the monoclonal antibody or the antigen-binding fragment thereof is SEQ ID NO: 1, and the amino acid sequence of the light chain variable region (LCVR) is SEQ ID NO: 5; more preferably, the heavy chain amino acid sequence of the monoclonal antibody or the antigen-binding fragment thereof is SEQ ID NO: 9, and the light chain amino acid sequence is SEQ ID NO: 10.

[0006] In one aspect, the present disclosure provides a monoclonal antibody or an antigen-binding fragment thereof, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the monoclonal antibody or the antigen-binding fragment thereof are SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 22, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 24, respectively; preferably, the amino acid sequence of HCVR of the monoclonal antibody or the antigen-binding fragment thereof is SEQ ID NO: 21, and the amino acid sequence of LCVR is SEQ ID NO: 23; more preferably, the heavy chain amino acid sequence of the monoclonal antibody or the antigen-binding fragment thereof is SEQ ID NO: 25 or SEQ ID NO: 27, and the light chain amino acid sequence is SEQ ID NO: 26.

[0007] In one aspect, the present disclosure provides a monoclonal antibody or an antigen-binding fragment thereof that can specifically bind to a glucagon-like peptide 1 receptor (GLP-1R). The amino acid sequence of the monoclonal antibody or the antigen-binding fragment thereof has one or more amino acid conservative substitutions in total on the HCDR compared to the HCDR sequence defined by SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4; preferably, the amino acid conservative substitutions are 1, 2 or 3 in total; preferably, the amino acid conservative substitutions are all located on HCDR3; further preferably, the amino acid conservative substitutions are 1 in total and located on HCDR3; the amino acid sequence of the monoclonal antibody or the antigen-binding fragment thereof has one or more amino acid conservative substitutions in total on the LCDR compared to the LCDR sequence defined by SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8; preferably, the amino acid conservative substitutions are 1, 2 or 3 in total; preferably, the amino acid conservative substitutions are all located on LCDR3; further preferably, the amino acid conservative substitutions are 1 in total and located on LCDR3.

[0008] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises: a HCVR having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to or with SEQ ID NO: 1 or 21; and / or a LCVR having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to or with SEQ ID NO: 5 or 23. In a preferred embodiment, the monoclonal antibody or antigen-binding fragment thereof comprises the HCVR of SEQ ID NO: 1 and the LCVR of SEQ ID NO: 5. In another preferred embodiment, the monoclonal antibody or antigen-binding fragment thereof comprises the HCVR of SEQ ID NO: 21 and the LCVR of SEQ ID NO: 23.

[0009] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof is a fully human antibody.

[0010] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof is a full-length IgG antibody; preferably, the monoclonal antibody or antigen-binding fragment thereof contains a human antibody constant region; more preferably, the monoclonal antibody or antigen-binding fragment thereof contains a human antibody variable region and a human antibody constant region.

[0011] In some embodiments, the heavy chain of the monoclonal antibody or its antigen-binding fragment contains a constant region selected from the following: the constant region of human IgG1, IgG2, IgG3 and IgG4 or their conventional variants; preferably, the light chain of the monoclonal antibody or its antigen-binding fragment contains a constant region selected from the following: the constant region of human κ chain, λ chain or their conventional variants.

[0012] In some embodiments, the monoclonal antibody or its antigen-binding fragment has human GLP-1R specificity; preferably, the monoclonal antibody or its antigen-binding fragment can antagonize the activation of human GLP-1R by GLP-1; more preferably, the monoclonal antibody or its antigen-binding fragment does not have mouse GLP-1R specificity.

[0013] In some embodiments, the monoclonal antibody or its antigen-binding fragment is an antagonist of the GLP-1 receptor, which can antagonize the activation of the GLP-1 receptor by GLP-1. In the present disclosure, antibody binding experiments or verifications refer to experiments that verify the interaction between antibodies and specific antigen molecules, or the effect or activity of such interaction on specific effects under different treatment conditions. The experiments include, but are not limited to, experiments to verify the binding ability / activity of antibodies to specific forms of antigens, experiments to verify the antagonistic ability / activity of antibodies to specific antigens and other molecules, etc. Based on the above verification purposes, any achievable verification method known to those skilled in the art can be adopted, including but not limited to enzyme-linked immunosorbent assay (ELISA), flow cytometry assay, surface plasmon resonance assay, cell reporter gene assay, etc.

[0014] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof has one or more characteristics selected from the group consisting of:

[0015] (1) According to the ELISA method, the EC50 value of binding to the hGLP-1R extracellular domain (NP_002053.3, aa24-145) is less than or equal to 70 pM; preferably, the EC50 value of binding to the hGLP-1R extracellular domain (NP_002053.3, aa24-145) is less than or equal to 50 pM; further preferably, the EC50 value of binding to the hGLP-1R extracellular domain (NP_002053.3, aa24-145) is less than or equal to 20 pM;

[0016] (2) the EC50 value of binding to the cell membrane protein hGLP-1R is less than or equal to 50 nM as determined by flow cytometry; preferably, the EC50 value of binding to the cell membrane protein hGLP-1R is less than or equal to 30 nM; further preferably, the EC50 value of binding to the cell membrane protein hGLP-1R is less than or equal to 15 nM; and

[0017] (3) According to the hGLP-1R-CRE-Luciferase-HEK293 reporter gene assay, the IC50 value for antagonizing the activity of exogenous 2.5 ng / mL GLP-1 is less than or equal to 100 nM; preferably, the IC50 value for antagonizing the activity of exogenous 2.5 ng / mL GLP-1 is less than or equal to 50 nM; further preferably, the IC50 value for antagonizing the activity of exogenous 2.5 ng / mL GLP-1 is less than or equal to 20 nM.

[0018] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof has a long half-life in vivo. For example, when the monoclonal antibody or antigen-binding fragment thereof is injected subcutaneously in a GLP-1R humanized mouse, the plasma drug protein molecule concentration half-life is greater than 150 hours, preferably greater than 200 hours.

[0019] In one aspect, the present disclosure provides a monoclonal antibody or an antigen-binding fragment thereof, which competitively binds to GLP-1R with any of the aforementioned monoclonal antibodies or antigen-binding fragments thereof.

[0020] In one aspect, the present disclosure also provides a nucleic acid encoding any of the aforementioned monoclonal antibodies or antigen-binding fragments thereof. In one embodiment, the monoclonal antibody or antigen-binding fragment thereof encoded by the nucleic acid molecule comprises HCDR1 with an amino acid sequence of SEQ ID NO: 2, HCDR2 with an amino acid sequence of SEQ ID NO: 3, and HCDR3 with an amino acid sequence of SEQ ID NO: 4; and / or LCDR1 with an amino acid sequence of SEQ ID NO: 6, LCDR2 with an amino acid sequence of SEQ ID NO: 7, and LCDR3 with an amino acid sequence of SEQ ID NO: 8. In another embodiment, the monoclonal antibody or antigen-binding fragment thereof encoded by the nucleic acid comprises HCDR1 with an amino acid sequence of SEQ ID NO: 2, HCDR2 with an amino acid sequence of SEQ ID NO: 3, and HCDR3 with an amino acid sequence of SEQ ID NO: 22; and / or LCDR1 with an amino acid sequence of SEQ ID NO: 6, LCDR2 with an amino acid sequence of SEQ ID NO: 7, and LCDR3 with an amino acid sequence of SEQ ID NO: 24.

[0021] The nucleic acid may be a polynucleotide including a coding sequence, or a polynucleotide including additional coding and / or non-coding sequences. The nucleic acid disclosed herein may be in the form of DNA or RNA. DNA forms include, but are not limited to, cDNA, genomic DNA, or artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof disclosed herein includes, but is not limited to, a coding sequence encoding only the structure of a mature monoclonal antibody or its antigen-binding fragment; a coding sequence encoding a mature monoclonal antibody or its antigen-binding fragment and various additional coding sequences; a coding sequence encoding the structure of a mature monoclonal antibody or its antigen-binding fragment (and optional additional coding sequences) and non-coding sequences, etc.

[0022] The present disclosure also relates to nucleic acids that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% sequence identity between the two sequences. The present disclosure particularly relates to nucleic acids that can hybridize to the nucleic acids described in the present disclosure under stringent conditions. In the present disclosure, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the sequence identity between the two sequences is at least 90%, more preferably 95%. In addition, the polypeptide encoded by the hybridizable nucleic acid molecule has the same biological function and activity as the mature polypeptide.

[0023] The full-length nucleic acid sequence of the antibody disclosed herein or its fragments can usually be obtained by PCR amplification, recombination or chemical synthesis. Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombination. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods. The biomolecules (nucleic acids, monoclonal antibodies and antigen-binding fragments thereof, multivalent monoclonal antibodies, etc.) involved in the present disclosure include biomolecules in isolated form.

[0024] In one aspect, the present disclosure further provides a vector containing a nucleic acid encoding any of the above-mentioned monoclonal antibodies or antigen-binding fragments thereof. The vector includes, but is not limited to: a viral vector, such as an adenoviral vector, a retroviral vector, an adeno-associated viral vector; and a non-viral vector, such as a plasmid, a transposon vector. Preferably, the vector is an expression vector, preferably a plasmid vector, further preferably a eukaryotic expression vector, and more preferably a CHO cell expression vector, such as pcDNA3.4.

[0025] On the one hand, the present disclosure also provides a cell for expressing a monoclonal antibody or an antigen-binding fragment thereof as described above, the cell containing any of the monoclonal antibodies or antigen-binding fragments thereof as described above, a nucleic acid or a vector encoding any of the monoclonal antibodies or antigen-binding fragments thereof as described above. Preferably, the cell is a host cell containing the above-mentioned expression vector or nucleic acid. In one aspect of the present disclosure, the host cell includes but is not limited to mammalian cells, insect cells, plant cells, fungal cells, prokaryotic cells, etc. Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc. Preferably, the host cell provided by the present disclosure for expressing an antibody or antigen-binding fragment thereof that binds to the GLP-1 receptor is a CHO cell.

[0026] Transformation of host cells with DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be performed using electroporation. When the host is a eukaryotic organism, DNA transfection methods such as calcium phosphate coprecipitation, conventional mechanical methods (such as microinjection, electroporation), and liposome packaging can be used.

[0027] In the process of expressing the protein encoded by the nucleic acid of the present disclosure, conventional culture methods can be used. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the promoter selected is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0028] The monoclonal antibodies and antigen-binding fragments thereof in the above-mentioned methods can be expressed intracellularly, on the cell membrane, or secreted outside the cell. If necessary, the monoclonal antibodies and antigen-binding fragments thereof can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

[0029] In one aspect, the present disclosure also provides a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof, nucleic acid, vector or cell described in any of the aforementioned aspects. Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. More preferably, the pharmaceutically acceptable excipient comprises one or more of the following: a pharmaceutically acceptable solvent, a dispersant, an additive, a plasticizer, etc. Generally, the monoclonal antibody or antigen-binding fragment thereof of the present disclosure can be formulated in a non-toxic, inert and pharmaceutically acceptable carrier medium. The formulated pharmaceutical composition can be administered by conventional routes, including but not limited to: subcutaneous, intraperitoneal, intravenous, intramuscular or topical administration.

[0030] In one aspect, the present disclosure further provides a kit comprising any of the monoclonal antibodies or antigen-binding fragments thereof, nucleic acids and / or cells described above.

[0031] In some embodiments, the kit further comprises a detection reagent for detecting anti-GLP-1R antibody response, a negative control, and a positive control.

[0032] The monoclonal antibody or antigen-binding fragment thereof disclosed in the present invention has a wide range of biological and clinical application values, and its application involves multiple fields such as diagnosis or treatment of diseases related to GLP-1R, basic medical research, biological research, etc. A preferred application is for clinical diagnosis and treatment related to GLP-1R.

[0033] On the one hand, the present disclosure also provides a method for treating, preventing or ameliorating at least one symptom or sign of a GLP-1R-related disease or condition, the method comprising administering to a subject a therapeutically effective amount of a monoclonal antibody or antigen-binding fragment thereof of the present disclosure. In some embodiments, the GLP-1R-related disease or condition is a GLP-1R over-activation-related disease; preferably hypoglycemia; most preferably hypoglycemia after bariatric surgery. In some embodiments, the pharmaceutical composition of the present disclosure is administered in combination with a second therapeutic agent.

[0034] On the one hand, the present disclosure also provides a method for treating, preventing or improving at least one symptom or sign of a hypoglycemia-related disease or condition, the method comprising administering to a subject a monoclonal antibody or antigen-binding fragment thereof of the present disclosure comprising a therapeutically effective amount. In some embodiments, the hypoglycemia-related disease or condition is preferably a neurological injury or developmental complication caused by hypoglycemia or congenital hyperinsulinemia (HI) (the HI type includes but is not limited to a transient form associated with perinatal stress, a monogenic form due to a monogenic defect, and a syndrome-related form, such as Beckwith-Wiedemann syndrome). In some embodiments, the symptom or sign is preferably hypoglycemia, brain damage, developmental delay, feeding disorder, learning disability and / or epilepsy. In some embodiments, the pharmaceutical composition of the present disclosure is administered in combination with a second therapeutic agent.

[0035] In one aspect, the present disclosure provides the use of any of the aforementioned monoclonal antibodies or antigen-binding fragments thereof, nucleic acids, vectors, cells in the preparation of a drug for preventing, treating or ameliorating a GLP-1R-related or hypoglycemia-related disease or condition and / or in the preparation of a kit for diagnosing or detecting the related disease or condition.

[0036] According to one aspect of the present disclosure, the monoclonal antibody or antigen-binding fragment provided by the present disclosure has one or more of the following advantages: it has a GLP-1R antagonistic effect, can inhibit the activation of GLP-1 by GLP-1, is particularly specific for human GLP-1R, has a long half-life in vivo, has a significant GLP-1 antagonistic effect, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1A The graph shows the relationship between the absorbance at 450 nm and the Log concentration (nM) of the antibody in the ELISA method for detecting the binding ability of the P16288 or P16289 antibody to the hGLP-1R extracellular domain.

[0038] Figure 1B The graph shows the relationship between the mean fluorescence intensity of the antibody and the Log concentration (nM) in an experiment using flow cytometry to detect the binding ability of P16288 or P16289 antibody and negative control antibody IgG1 (P02183) to full-length hGLP-1R expressed on cells.

[0039] Figure 2 The relationship between the inhibition rate of P16288 or P16289 antibody on GLP-1 activation of human GLP-1 receptor and the Log concentration (nM) of the antibody is shown.

[0040] Figure 3A The relationship between the inhibition rate of P16288 or P16289 antibody on GLP-1 activation of human GLP-1 receptor and the Log concentration (μM) of the antibody is shown.

[0041] Figure 3B The relationship between the inhibition rate of the P16288 or P16289 antibody on the activation of the monkey GLP-1 receptor by GLP-1 and the Log concentration (μM) of the antibody is shown.

[0042] Figure 3C The relationship between the inhibition rate of GLP-1 activation of mouse GLP-1 receptor by P16288 or P16289 antibody or Avexitide and the Log concentration (μM) of the antibody is shown.

[0043] Figure 4A The graph shows the changes in blood glucose from 0 to 180 minutes after GLP-1 + glucose treatment on Day 1 in different treatment groups in an intraperitoneal glucose tolerance test (IPGTT) of human GLP-1 receptor mice.

[0044] Figure 4B The graph shows the blood glucose area under the drug-time curve from 0 to 180 minutes after GLP-1+glucose treatment on Day 1 in the intraperitoneal glucose tolerance test of human GLP-1 receptor mice.

[0045] Figure 4C The graph shows the changes in blood glucose from 0 to 180 minutes after GLP-1+glucose treatment in different treatment groups on Day 8 in an intraperitoneal glucose tolerance test of human GLP-1 receptor mice.

[0046] Figure 4D The graph shows the blood glucose area under the curve from 0 to 180 minutes after GLP-1+glucose treatment in different treatment groups on Day 8 in the intraperitoneal glucose tolerance test of human GLP-1 receptor mice.

[0047] Figure 4E The graph shows the changes in blood glucose from 0 to 180 minutes after GLP-1+glucose treatment on Day 15 in different treatment groups in an intraperitoneal glucose tolerance test of human GLP-1 receptor mice.

[0048] Figure 4F The graph shows the blood glucose area under the drug-time curve from 0 to 180 minutes after GLP-1+glucose treatment on Day 15 in the intraperitoneal glucose tolerance test of human GLP-1 receptor mice.

[0049] Figure 5A The graph shows the changes in blood glucose from 0 to 240 minutes after intragastric administration of glucose solution on Day 1 in different treatment groups in an oral glucose tolerance test of human GLP-1 receptor mice.

[0050] Figure 5B The graph shows the blood glucose area under the curve from 0 to 240 minutes after oral gavage of glucose solution in different treatment groups in the oral glucose tolerance test of human GLP-1 receptor mice on Day 1.

[0051] Figure 5C The graph shows the changes in blood glucose from 0 to 180 minutes after intragastric administration of glucose solution to different treatment groups in an oral glucose tolerance test of human GLP-1 receptor mice on Day 8.

[0052] Figure 5D The graph shows the blood glucose area under the curve from 0 to 180 minutes after oral gavage of glucose solution in different treatment groups in the oral glucose tolerance test of human GLP-1 receptor mice on Day 8.

[0053] Fig. 6A The graph shows the changes in blood glucose from 0 to 180 minutes after treatment with GLP-1+glucose solution in different treatment groups in an intraperitoneal glucose tolerance test of C57BL / 6 mice.

[0054] Figure 6B The graph shows the blood glucose area under the drug-time curve from 0 to 180 minutes after GLP-1+glucose solution treatment in different treatment groups in an intraperitoneal glucose tolerance test of C57BL / 6 mice.

[0055] Fig. 7A The graph shows the changes in blood glucose from 0 to 270 minutes after intragastric administration of glucose solution in different treatment groups in an oral glucose tolerance test of C57BL / 6 mice.

[0056] Figure 7B The graph shows the blood glucose area under the curve from 0 to 270 minutes after oral administration of glucose solution in different treatment groups in an oral glucose tolerance test of C57BL / 6 mice.

[0057] Figure 8 The pharmacokinetic properties of a single subcutaneous administration of anti-GLP-1R antibody P16288 or P16289 in SD rats are shown.

[0058] Fig. 9 The relationship between the inhibition rate of GLP-1 activation of human GLP-1 receptor by avexitide and P16888 and P16891 antibodies and the Log concentration (μM) of the antibodies is shown.

[0059] Fig.10 The relationship between the inhibition rate of avexitide and P16888 and P16891 antibodies on GLP-1 activation of monkey GLP-1 receptor and the Log concentration (μM) of the antibodies is shown.

[0060] Fig.11A The graph shows the changes in blood glucose from 0 to 180 minutes after GLP-1+glucose treatment on Day 1 in different treatment groups in an intraperitoneal glucose tolerance test of human GLP-1 receptor mice.

[0061] Fig. 11B The graph shows the blood glucose area under the drug-time curve from 0 to 180 minutes after GLP-1+glucose treatment on Day 1 in the intraperitoneal glucose tolerance test of human GLP-1 receptor mice.

[0062] Fig. 12A The figure shows the changes in blood glucose from 0 to 180 minutes after GLP-1+glucose treatment in different treatment groups in the intraperitoneal glucose tolerance test of human GLP-1 receptor mice on Day 1.

[0063] Fig. 12B The graph shows the changes in blood glucose from 0 to 180 minutes after GLP-1 + glucose treatment in different treatment groups on Day 8 in an intraperitoneal glucose tolerance test of human GLP-1 receptor mice.

[0064] Fig. 12C The graph shows the changes in blood glucose from 0 to 180 minutes after GLP-1+glucose treatment on Day 15 in different treatment groups in an intraperitoneal glucose tolerance test of human GLP-1 receptor mice.

[0065] Fig.12D The graph shows the changes in blood glucose from 0 to 180 minutes after GLP-1+glucose treatment on Day 22 in different treatment groups in an intraperitoneal glucose tolerance test of human GLP-1 receptor mice.

[0066] Fig.13 Shown are the pharmacokinetic properties of the P16888 antibody in humanized GLP-1R mice.

[0067] Fig.14 The pharmacokinetic properties of P16888 and P16891 antibodies in cynomolgus monkeys are shown. DETAILED DESCRIPTION

[0068] The present disclosure may be better understood with reference to the following examples. However, it should be understood that the following examples are for illustrative purposes only and should not be construed as limiting the scope of protection of the present disclosure in any way.

[0069] The term "GLP-1R" refers to the glucagon-like peptide 1 receptor. Glucagon-like peptide 1 (GLP-1) is a 31 amino acid peptide hormone released by intestinal L cells after nutrient consumption. Binding of GLP-1 to GLP-1R enhances glucose-induced insulin secretion from pancreatic β cells, increases insulin expression, inhibits β cell apoptosis, promotes β cell neogenesis, reduces glucagon secretion, delays gastric emptying, promotes satiety, and improves peripheral glucose disposal.

[0070] The term "antibody" as used herein is intended to include immunoglobulin molecules (i.e., "complete antibody molecules") comprising four polypeptide chains (two heavy (H) chains and two light (L) chains) interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM) and / or antigen-binding fragments thereof. Each heavy chain comprises a heavy chain variable region ("HCVR" or "V H ”) and the heavy chain constant region (comprising domain C H 1. C H 2 and C H 3). Each light chain comprises a light chain variable region ("LCVR" or "V L ”) and the light chain constant region (C L ). H and V L The V region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L It is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments, the FRs of the antibody (or its antigen-binding fragment) may be identical to human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on parallel analysis of two or more CDRs.

[0071] As used herein, the term "antigen-binding fragment" of an antibody includes any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptide, protein or glycoprotein that specifically binds to an antigen to form a complex. As used herein, an "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to bind to GLP1-R.

[0072] The term "fully human antibody" as used herein is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human FR sequences. The term includes antibodies produced recombinantly in non-human mammals or cells of non-human mammals. The term is not intended to include antibodies isolated from or produced in human subjects.

[0073] As used herein, the term "conservative amino acid substitution" refers to an amino acid substitution in which an amino acid residue is replaced by another amino acid residue whose side chain (R group) has similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other due to conservative substitutions, the percentage or degree of similarity may be adjusted upward to correct for the conservatism of the substitution. The means for making this adjustment are well known to those skilled in the art. (See, e.g., Pearson, 1994, Methods Mol. Biol. 24:307-331). Examples of groups of amino acids having side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992, Science 256: 1443-45). A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0074] Glossary

[0075] IC 50 : Half maximal inhibitory concentration.

[0076] EC 50 : Half maximal effect concentration.

[0077] T 1 / 2 : Terminal elimination half-life.

[0078] T max : Time of maximum plasma concentration after administration.

[0079] C max : Maximum plasma concentration after administration.

[0080] AUC: Area under the drug-time curve

[0081] AUC 0-infobs : Based on the last measured concentration (obs), the dosing time is extrapolated to infinite AUC.

[0082] MRT 0-infobs : The average residence time from time 0 to infinity based on the last measured concentration (obs).

[0083] Example 1. Preparation of anti-GLP-1R antibodies

[0084] The human antibody natural library is a human antibody Fab phage library constructed by Sanyou Biopharmaceuticals (Shanghai) Co., Ltd., using immunotubes, magnetic bead screening instrument Kingfisher magnetic bead purification system (Thermo Scientific), and human GLP-1 receptor (hGLP-1R) extracellular domain (NP_002053.3, 24-145aa) recombinant protein for solid phase, liquid phase and hGLP-1R-HEK293 cells (GenScript) cross-screening to screen human antibodies, and the specific Fab antibodies against the antigen extracellular domain and cells are enriched by trypsin and glycine hydrochloride elution respectively.

[0085] The enrichment of different output sets was detected by ELISA and FACS, and monoclones were further selected for binding experimental verification. Finally, the Fab sequence with binding activity was used to construct a full-length antibody, and the screened Fab sequence was connected to the IgG4 (S228P) constant region sequence and constructed into the eukaryotic expression vector pcDNA3.4 (Shanghai Baiying Biotechnology Co., Ltd.), and then based on the CHO cell expression system, the full-length antibody was obtained by transfection, incubation, and finally purified by Protein A affinity chromatography. Activity verification was performed as described in Example 2, and the results showed that P16288 and P16289 had strong binding ability with the hGLP-1R extracellular domain, but in the FACS investigation of the binding of antibodies and receptors on cells, it was found that P16288 had a strong binding with the hGLP-1R on the cell, but P16289 had a very weak binding with the receptor.

[0086] Based on the structure of P16288, the light chain CDR region was mutated to D92E, and the heavy chain CDR region was mutated to D97E to obtain the new antibody P16888. Further, the YTE mutation was added to the Fc region of the P16888 antibody to obtain the P16891 antibody. Then, based on the CHO cell expression system, the full-length antibody was obtained through transfection, incubation, and finally purified by Protein A affinity chromatography.

[0087] Table 1 shows the amino acid sequences of the light chain, heavy chain and CDR of the anti-GLP-1R antibodies P16288, P16289, P16888 and P16891.

[0088] Table 1

[0089]

[0090]

[0091] Example 2 Antibody and Antigen Binding

[0092] 2.1. ELISA method to study antibody and antigen binding

[0093] The hGLP-1R extracellular domain (NP_002053.3, aa 24-145) was expressed as a recombinant protein, and then the ELISA plate was coated with 10 μg / mL of hGLP-1R recombinant protein in PBS and incubated overnight at 4°C. The coating solution was then removed and washed with PBST (PBS + 0.05% Tween). The plate was then blocked with 1% BSA, washed with PBST, and 6.7, 2.2, 0.44, 0.089, 0.018, 0.0036, 0.00071 or 0 nM of antibody (P16288 or P16289) was added and incubated at room temperature for 60 min. After washing with PBST, horseradish peroxidase-labeled anti-human Fc antibody was added and incubated for 60 min. The cells were then washed with PBST, TMB substrate was added for color development, and finally 0.5 M H2SO4 was used to terminate the color development, and the absorbance was measured at 450 nm using an enzyme reader. GraphPadPrism 9 was used to plot the four parameters and calculate the EC 50 value.

[0094] Table 2

[0095] P16288 <![CDATA[EC 50 =13.0pM]]> P16289 <![CDATA[EC 50 =4.8pM]]>

[0096] The results are as follows Figure 1A As shown in Table 2, both P16288 and P16289 antibodies showed the ability to bind to the extracellular domain of hGLP-1R.

[0097] 2.2. Flow cytometry to study antibody-antigen binding

[0098] HEK293 cells overexpressing hGLP-1R (GenScript Biotech Co., Ltd.) were washed with FACS buffer, and then incubated at 4°C for 60 min with 137.0, 45.7, 15.2, 5.1, 1.69, 0.56, 0.19, 0.018 nM antibodies (P16288 or P16289, and negative control IgG1 (Sanyou Biopharmaceuticals (Shanghai) Co., Ltd.), centrifuged, washed, and then PE-labeled anti-human IgG Fc (Abcam, ab98596) was added and incubated at 4°C for 30 min. After centrifugation and washing, the cells were resuspended in FACS buffer, and the fluorescence intensity (MFI) was detected by flow cytometry. GraphPad Prism 9 was used to plot four parameters and calculate EC50 value.

[0099] Table 3

[0100] P16288 <h2 style=";text-align:left;direction:ltr"><![CDATA[EC <h2 style=";text-align:left;direction:ltr"> 50 <h2 style=";text-align:left;direction:ltr"> <9.9nM]]><h2 style=";text-align:left;direction:ltr"> P16289 <![CDATA[EC 50 >137nM]]>

[0101] The results are as follows Figure 1B As shown in Table 3, P16288 showed strong binding ability to the full-length hGLP-1R expressed on cells, but P16289 bound only weakly.

[0102] Therefore, P16288, but not P16289, has the ability to bind to both the extracellular domain of hGLP-1R and the membrane proteins on the cell.

[0103] Example 3 Cellular Activity Experiment of Antibody Antagonism to hGLP-1 Receptor

[0104] The hGLP-1R-CRE-Luciferase-HEK293 reporter gene experiment was used to investigate the effects of the antibody and its dose on the activity of natural GLP-1 (7-37aa, R&D Systems, cat no. 5374 / 1).

[0105] hGLP-1R-overexpressing HEK293 cells were transfected with a plasmid containing multiple copies of a cAMP response element (CRE)-driven luciferase expression cassette to obtain a hGLP-1R-CRE-Luciferase-HEK293 cell line with a stably transfected luciferase-expressing plasmid.

[0106] In a 96-well plate, add 50 μL hGLP-1R-CRE-Luciferase-HEK293 (6*10 5 cells / mL), then add 10 3-fold dilution gradients of P16288 such as 5240nM, 1747nM, 582nM, 10 3-fold dilution gradients of P16289 such as 2825nM, 942nM, 313nM, and an equal volume of culture medium (minimum inhibition control group and maximum inhibition control group). Incubate in a 37°C, 5% CO2 incubator for 30 minutes, then add GLP-1 to the minimum inhibition control group and antibody treatment group, the final concentration of GLP-1 is 2.5ng / mL, and at the same time add an equal volume of culture medium to the maximum inhibition control group, and continue to incubate overnight. Add 100μL Right-Glo reagent (Promega), tap the plate gently to facilitate solution mixing, and read the chemiluminescence value with an enzyme reader after three minutes. The maximum inhibition control group without GLP-1 treatment is defined as 100% inhibition rate, and the minimum inhibition control group treated with 2.5ng / mL GLP-1 is defined as 0% inhibition rate. Four-parameter plotting and IC calculation using GraphPad Prism 950 value.

[0107] Table 4

[0108] P16288 <![CDATA[IC 50 =13.4nM]]> P16289 <h2 style=";text-align:left;direction:ltr"><![CDATA[IC <h2 style=";text-align:left;direction:ltr"> 50 <h2 style=";text-align:left;direction:ltr"> >2825nM]]><h2 style=";text-align:left;direction:ltr">

[0109] In experiments where GLP-1 was incubated with cells alone, GLP-1 could activate hGLP-1R-mediated reporter gene expression in a dose-dependent manner. Figure 2 The results in Table 4 show that in the presence of antibody P16288, the activity of natural GLP-1 is antagonized in a dose-dependent manner, while P16289, which binds weakly to hGLP-1R on the cell surface, cannot effectively antagonize the activity of natural GLP-1.

[0110] Example 4 Study on the antagonistic effect of antibodies on GLP-1R of different species

[0111] HEK293 cells (ATCC, cat no.CRL-1573) were transfected with GLP-1R_pcDNA3.1 / G418(+) plasmids containing mRNAs for transcribing human (NM_002062.5), monkey (JN033215.1), and mouse (NM_021332.2) GLP-1R polypeptides. 48 hours after transfection, 10 3-fold serial dilutions of P16288 (10.2, 3.4, 1.13 μM) and 10 3-fold serial dilutions of P16289 antibodies (5.5, 1.83, 0.61 μM), or an equal volume of culture medium (minimum inhibition control group and maximum inhibition control group) were added, and the cells were incubated at 37°C for 30 min, and then 1 ng / mL GLP-1 (for human and monkey receptors) or 6 ng / mL GLP-1 (for mouse receptor) was added to the minimum inhibition control group and antibody treatment group, and an equal volume of culture medium was added to the maximum inhibition control group, and incubated at 37°C for 120 min. Finally, the cAMP level was detected using a cAMP HTRF kit (Cisbio, 62AM4PEB). The maximum inhibition control group without GLP-1 treatment was defined as 100% inhibition, and the minimum inhibition control group treated with GLP-1 was defined as 0% inhibition. GraphPad Prism 9 was used to plot four parameters and calculate IC 50 value.

[0112] Table 5

[0113]

[0114] The results are as follows Figure 3A-C and Table 5, the experimental results show that P16288 can antagonize human and monkey GLP-1R at the same level for the GLP-1R activation of GLP-1 molecules, but has no antagonistic activity for mouse GLP-1R. The same method was used to detect the effect of avexitide on GLP-1 activation in mouse GLP-1R-expressing HEK293, and the results showed that avexitide antagonized GLP-1 activation of mouse GLP-1R.

[0115] Example 5 Study of the Antibody in the Intraperitoneal Glucose Tolerance Test (IPGTT) Experiment in Humanized GLP-1R Mice (Addition of Exogenous GLP-1)

[0116] Female humanized GLP-1R mice (25-28g, Shanghai South Model Organisms Technology Co., Ltd.) aged 10 weeks were divided into 5 groups, with 5 experimental animals in each group, and each group was treated according to the scheme in Table 6. The antibody treatment group was subcutaneously injected with antibody drugs, while the other groups were subcutaneously injected with vehicle PBS solution once a week for 2 consecutive weeks, and the IPGTT experiment was performed on the second day after administration. The drug protein was subcutaneously administered for the first time on Day 0, and the second time on Day 7. The mice fasted for 18 hours after drug administration, and only fasted on Day 14. After fasting was completed (Day 1, Day 8 and Day 15), the experimental animals were given exogenous GLP-1 (7-37, R&D System, cat no.5374 / 1) and glucose solution at a standard dose of 25.0nmol / kg and 13.9mmol / kg, respectively, by intraperitoneal injection.

[0117] Table 6

[0118]

[0119] Blood glucose testing and statistical methods:

[0120] The blood glucose level of the experimental animals was measured by blood glucose meter at 0 min before sugar administration and 15 min, 30 min, 60 min, 90 min, 120 min, and 180 min after sugar administration. The area under the blood glucose curve (AUC) of each animal from 0 to 180 min was calculated using GraphPad Prism 9, and the blood glucose level and AUC of each group were displayed as mean ± standard error (SEM) to compare the differences between groups. The statistical difference was analyzed by One-way ANOVA. Compared with the GLP-1 alone treatment group, *, p<0.05; **, p<0.01; , ***, p<0.001; ****, p<0.0001; ns, no statistical difference; because the significant marks in the line graph overlap and are difficult to distinguish, they are not shown.

[0121] The results are as follows Figure 4A -F, in humanized GLP-1R mice, the addition of 25nmol / kg GLP-1 can effectively reduce glucose levels. After administration of 100 and 200nmol / kg P16288 antibodies, they can effectively inhibit the reduction of blood glucose caused by exogenous GLP-1. In contrast, P16289 cannot change the regulation of blood glucose by GLP-1.

[0122] P16288 can regulate blood sugar for more than 8 days, which means that P16288 has a long half-life. In contrast, according to WO2021 / 231366A1, Avexitide (exendin-9) only shows a blood sugar control effect of a few hours due to its short half-life.

[0123] Example 6 Study of the Antibody in the Oral Glucose Tolerance Test (OGTT) of Humanized GLP-1R Mice (without Exogenous GLP-1)

[0124] Male humanized GLP-1R mice (25-32g) aged 10 weeks were divided into 4 groups, with 5 experimental animals in each group. The drug treatment groups were subcutaneously injected with 200nmol / kg P16288, 400nmol / kg P16288 or 400nmol / kg P16289 drug protein molecules, and the vehicle group was subcutaneously injected with vehicle PBS solution. The drug protein was subcutaneously administered for the first time on Day 0 and the second time on Day 7. OGTT experiments were performed on Day 1 and Day 8. Mice were gavaged with 25% glucose (w / v) solution at a standard dose of 13.9mmol / kg. There was an 18h fasting period between drug protein administration and glucose solution gavage. The blood glucose level of the experimental animals was detected by a blood glucose meter before (0min) and 15min, 30min, 60min, 90min, 120min, 180min (or 240min) after sugar administration. The area under the blood glucose curve (AUC) of each animal from 0 to 180 min (or 240 min) was calculated using GraphPad Prism 9, and the blood glucose values ​​and AUC of the groups were displayed as mean ± standard error (SEM) to compare the differences between the groups. The statistical difference was analyzed by One-way ANOVA. Compared with the vehicle group, *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, no statistical difference; because the significant marks in the line graph overlapped and were difficult to distinguish, they were not shown.

[0125] The results are as follows Figure 5A-D shows that in the absence of exogenous GLP-1, humanized GLP-1R mice were given oral glucose, and we can see a curve in which blood sugar first rises and then falls. In this blood sugar regulation process, endogenous GLP-1 and insulin play a major role. When mice were injected with P16288 and P16289, it can be seen that P16288 showed an effect of increasing blood sugar after both administrations, while P16289 did not show an effect of increasing blood sugar. This means that P16288 affects the regulation of blood sugar by endogenous GLP-1 by antagonizing the activity of GLP-1 on the hGLP-1R receptor.

[0126] Example 7 Study of Antibodies in IPGTT Experiment of C57BL / 6 Mice (Addition of Exogenous GLP-1)

[0127] Male C57BL / 6 mice (22-26g) aged 7-9 weeks were divided into 5 groups, with 5 experimental animals in each group, and mice in each group were treated according to the dosing regimen shown in Table 6. The antibody drug treatment group was given a single subcutaneous injection of drug protein molecules, and fasted for 18 hours after administration. After fasting, the experimental animals were given exogenous GLP-1 and glucose solution by intraperitoneal injection at a standard dose of 25.0nmol / kg and 13.9mmol / kg, respectively. The blood glucose level of the experimental animals was detected by a blood glucose meter before (0min) and 15min, 30min, 60min, 90min, 120min, and 180min after sugar administration. The area under the blood glucose curve (AUC) of each animal from 0 to 180min was calculated using GraphPad Prism 9, and the group blood glucose value and AUC were displayed as mean ± standard error (SEM) and the differences between groups were compared. Statistical differences were analyzed by One-way ANOVA. Compared with the GLP-1 group alone, *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, no statistical difference; because the significant marks in the line graph overlapped and were difficult to distinguish, they were not shown.

[0128] Since P16288 does not recognize the mouse GLP-1R, the antibody should not show regulation of the receptor and blood glucose in normal C57BL / 6 mice, which will further verify the specificity of the antibody activity. In this glucose tolerance study, GLP-1 can lower blood glucose levels, while Fig. 6A As shown in Figure 2-B, P16288 did not show an antagonistic effect on GLP-1 and thus did not increase blood sugar, which means that the regulation of blood sugar by P16288 is specifically through human GLP-1R.

[0129] Example 8 Study of Antibodies in OGTT Experiment of Normal C57BL / 6 Mice (without Exogenous GLP-1)

[0130] Six-week-old male C57BL / 6 mice (18-21g) were divided into four groups, with five experimental animals in each group. Each group was treated with a single subcutaneous injection of a vehicle PBS solution, 200nmol / kg P16288, 100nmol / kg P16288, and 100nmol / kg P16289, and fasted for 18h after administration. After fasting, the experimental animals were gavaged with a standard dose of 13.9mmol / kg glucose solution. The blood glucose level of the experimental animals was measured by a blood glucose meter before (0min) and 15min, 30min, 60min, 90min, 120min, 180min, and 270min after sugar administration. The area under the blood glucose curve (AUC) of each animal from 0 to 270min was calculated using GraphPad Prism 9, and the group blood glucose value and AUC were displayed as mean ± standard error (SEM) to compare the differences between groups. The statistical difference was analyzed by One-way ANOVA. Compared with the vehicle group, *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, no statistical difference; because the significant marks in the line graph overlapped and were difficult to distinguish, they were not shown.

[0131] like Fig. 7A -B shows that in normal C57BL / 6 mice, the oral glucose tolerance test gave a time-dependent glucose curve; after the animals were injected with P16288, blood glucose showed no statistical difference from the vehicle group. This also means that P16288 regulates blood glucose specifically through human GLP-1R.

[0132] Example 9 Pharmacokinetic study of antibody in SD rats

[0133] Male SD rats aged 6-8 weeks were subcutaneously injected with a single dose of 5 mg / kg of drug protein molecules P16288 or P16289, with 3 animals in each group. Whole blood was collected from the animals 5 minutes (-5min) before administration and 2h, 4h, 24h, 48h, 72h, 96h, 120h, 168h, 192h, 240h, and 264h after administration, and EDTA plasma was prepared. The concentration of drug protein molecules in plasma was detected by ELISA method. The specific detection process is as follows: 96-well plates were coated with 1μg / mL AffiniPure GoatAnti-Human IgG antibody (Jackson lab, cat.no.109-005-098) diluted in PBS and incubated overnight at 4°C. The coating solution was removed and washed with PBST (PBS+0.05% Tween). Then the plate was blocked with 1% BSA, washed with PBST, and the standard (P16288 or P16289) diluted in PBS + 0.1% BSA + 0.1% blank rat plasma was added. The specific antibody concentrations were 1000, 200, 40, 8, 1.6, 0.32, 0.064, 0nM for standard curve drawing. At the same time, plasma samples diluted 1000 times in PBS + 0.1% BSA were added to other wells and incubated at room temperature for 60 minutes. After washing with PBST, horseradish peroxidase-labeled Goat Anti-Human IgG, Fcγfragment specific (Jackson lab, cat. no. 109-035-098) was added and incubated for 60 minutes. Subsequently, it was washed with PBST, TMB substrate was added for color development, and finally the reaction was terminated with 0.5M H2SO4, and the absorbance was measured at 450nm with an enzyme reader. GraphPad Prism 9 was used to plot drug concentration-time curves, and the pharmacokinetic parameters (C max 、T max 、T 1 / 2 The relevant results are shown in Table 7.

[0134] Table 7

[0135]

[0136] The results are as follows Figure 8 As shown in Table 7, in the pharmacokinetic experiment in SD rats, P16288 showed a longer half-life (T 1 / 2 : 99.1h) and better exposure.

[0137] Example 10 Study on the antagonistic effect of antibodies on human or monkey GLP-1R

[0138] (1) Cellular activity assay of antibody antagonism to GLP-1 activated human receptors

[0139] In a 96-well plate, add 50 μL hGLP-1R-CRE-Luciferase-HEK293 (6*10 5 cells / mL), then add 10 3-fold dilution gradients of P16888 (3425nM, 1142nM, 381nM), 10 3-fold dilution gradients of P16891 (3973nM, 1324nM, 441nM), 10 3-fold dilution gradients of Avexitide (14706nM, 4902nM, 1634nM), and an equal volume of culture medium (minimum inhibition control group and maximum inhibition control group). Incubate in a 37°C, 5% CO2 incubator for 30 minutes, then add GLP-1 to the minimum inhibition control group and antibody treatment group, with a final concentration of GLP-1 of 2.5ng / mL, and add an equal volume of culture medium to the maximum inhibition control group, and continue incubation overnight. Add 100μL of Bright-Glo reagent (Promega), tap the plate gently to facilitate solution mixing, and read the chemiluminescence value with an enzyme reader three minutes later. The maximum inhibition control group without GLP-1 treatment was defined as 100% inhibition rate, and the minimum inhibition control group treated with 2.5 ng / mL GLP-1 was defined as 0% inhibition rate. GraphPad Prism 9 was used to plot four parameters and calculate IC50 values.

[0140] The results are as follows Fig. 9 As shown in Table 8. The experimental results show that P16888 and P16891 have antagonistic effects on human GLP-1R activation induced by GLP-1 molecules, and the inhibitory activity is higher than that of Avexitide.

[0141] Table 8

[0142] P16888 <![CDATA[IC 50 =17.4nM]]> P16891 <![CDATA[IC 50 =17.2nM]]> Avexitide <h2 style=";text-align:left;direction:ltr"><![CDATA[IC <h2 style=";text-align:left;direction:ltr"> 50 <h2 style=";text-align:left;direction:ltr"> <260.3nM]]><h2 style=";text-align:left;direction:ltr">

[0143] (2) Cellular activity assay of the antibody's antagonism to GLP-1-activated monkey receptors

[0144] In a 96-well plate, add 50 μL monkey GLP-1R (GenBank: JN033215.1)-CRE-Luciferase-HEK293 (6*10 5cells / mL), then add 10 3-fold dilution gradients of P16888 (3425nM, 1142nM, 381nM), 10 3-fold dilution gradients of P16891 (3973nM, 1324nM, 441nM), 10 3-fold dilution gradients of Avexitide (14706nM, 4902nM, 1634nM), and an equal volume of culture medium (minimum inhibition control group and maximum inhibition control group). Incubate in a 37°C, 5% CO2 incubator for 30 minutes, then add GLP-1 at a final concentration of 2.5ng / mL to the minimum inhibition control group and antibody treatment group, and add an equal volume of culture medium to the maximum inhibition control group, and continue to incubate overnight. Add 100μL Bright-Glo reagent (Promega), tap the plate gently to facilitate solution mixing, and read the chemiluminescence value with an enzyme reader after three minutes. The maximum inhibition control group without GLP-1 treatment was defined as 100% inhibition rate, and the minimum inhibition control group treated with 2.5 ng / mL GLP-1 was defined as 0% inhibition rate. GraphPad Prism9 was used to draw four-parameter graphs and calculate IC50 values.

[0145] The results are as follows Fig.10 As shown in Table 9. The experimental results show that P16888 and P16891 have antagonistic effects on the activation of monkey GLP-1R induced by GLP-1 molecules, and the inhibitory activity is higher than that of Avexitide.

[0146] Table 9

[0147] P16888 <h2 style=";text-align:left;direction:ltr"><![CDATA[IC <h2 style=";text-align:left;direction:ltr"> 50 <h2 style=";text-align:left;direction:ltr"> <15.3nM]]><h2 style=";text-align:left;direction:ltr"> P16891 <![CDATA[IC 50 =13.8nM]]> Avexitide <![CDATA[IC 50 =2118nM]]>

[0148] Example 11 Study of Antibodies in Humanized GLP-1R Mouse IPGTT Experiment (Addition of Exogenous GLP-1)

[0149] Female humanized GLP-1R mice (25-28g) aged 10 weeks were divided into 5 groups, with 5 experimental animals in each group, and each group was treated according to the scheme in Table 10. The antibody treatment group was given antibody drugs by subcutaneous injection, and the IPGTT experiment was performed the next day after administration. The drug protein was subcutaneously administered on Day 0, and the mice were fasted for 18 hours after drug protein administration. After fasting (Day 1), the experimental animals were given exogenous GLP-1 and glucose solution by intraperitoneal injection at a standard dose of 25.0nmol / kg and 13.9mmol / kg, respectively. The blood glucose level of the experimental animals was detected by a blood glucose meter before (0min) and 15min, 30min, 60min, 90min, 120min, and 180min after sugar administration. The area under the blood glucose curve (AUC) of each animal was calculated using GraphPad Prism 9, and the group blood glucose value and AUC were displayed as mean ± standard error (SEM) and the differences between groups were compared. The statistical differences were analyzed by One-way ANOVA. Compared with the GLP-1 alone treatment group, *, p < 0.05, **, p < 0.01; ***, p < 0.001, ns, no statistical difference.

[0150] Table 10

[0151]

[0152]

[0153] The results are as follows Fig.11A and 11B As shown, P16888 exhibited a dose-dependent effect of increasing blood glucose. 200 nmol / kg P16888 antibody could increase the blood glucose area to the vehicle control group after only one administration.

[0154] Example 12 Study on the persistence of the efficacy of the P16888 antibody in the IPGTT experiment of humanized GLP-1R mice (addition of exogenous GLP-1)

[0155] A method similar to that in Example 11 was used to select 10-week-old female humanized GLP1-R mice (25-28g), with 5 experimental animals in each group. The antibody-treated group was given a drug protein molecule P16888 by subcutaneous injection once, and IPGTT experiments were performed on the 2nd, 9th, 16th and 23rd days after administration. The first subcutaneous administration of drug protein was recorded as Day 0. After the mice were given drug protein or fasted for 18 hours before IPGTT, after fasting (Day 1, Day 8, Day 15, Day 22), the experimental animals were given exogenous GLP-1 (human, R&D Systems) and glucose solution at a standard dose of 25.0nmol / kg and 13.9mmol / kg by intraperitoneal injection, respectively. The blood glucose level of the experimental animals was detected by a blood glucose meter before sugar administration (0min) and 30min, 60min, 90min, 120min, and 180min after sugar administration. The area under the blood glucose curve (AUC) of each animal was calculated using GraphPad Prism 9, and the blood glucose values ​​and AUC of each group were displayed as mean ± standard error (SEM) to compare the differences among groups.

[0156] The results are as follows Fig. 12A -D, in the IPGTT efficacy experiment of humanized GLP-1R mice, P16888 showed a long-lasting effect of increasing blood sugar. After a single subcutaneous injection of the drug, the effect of increasing blood sugar continued 15 days after administration (compared with the GLP-1 treatment group, the P value of the blood sugar area under the drug-time curve measured in each treatment group on the corresponding days was less than 0.001), indicating that the antibody has a long-lasting effect.

[0157] Example 13 Pharmacokinetic study of antibody in humanized GLP1-R mice

[0158] The reference antibody P16444 was used for comparison with P16888 of the present disclosure. The sequence of P16444 can be found in mAb36986 in WO 2021 / 231366A1.

[0159] Female humanized GLP-1R mice (25-28 g) aged about 10 weeks were subcutaneously injected with a single dose of 5 mg / kg (34 nmol / kg) of drug protein molecules P16888 or P16444) (n=4). Whole blood was collected from the animals before administration (-5 min) and 2h, 4h, 24h, 48h, 72h, 96h, 120h, 168h, 216h, 264h, 312h, and 360h after administration, and plasma samples were prepared. The concentration of drug protein molecules in plasma was detected by ELISA, and the drug concentration-time curve was drawn using GraphPad Prism 9, and the pharmacokinetic parameters (C max 、T max 、T1 / 2 , AUC, MRT) were calculated.

[0160] Table 11

[0161]

[0162]

[0163] from Fig.13 From the results in Table 11, it can be found that P16888 has a half-life of 289.4 h, which is much longer than the reported half-life of avexitide: ~30 min (Gasbjerg LS, et al..Diabetes, Obesity&Metabolism. 2021 Nov; 23(11):2419-2436.), and longer than the reference antibody P16444.

[0164] Example 14 Pharmacokinetic study of antibodies in cynomolgus monkeys

[0165] Male cynomolgus monkeys aged 3 to 4 years were given a single subcutaneous injection of 1 mg / kg drug protein molecules (P16888 or P16891) (n=2-3). Whole blood was collected from the animals before administration (-5 min) and 0.5h, 4h, 8h, 24h, 48h, 72h, 96h, 120h, 168h, 240h, 336h, 504h, 672h, 840h, 1008h, and 1344h after administration, and plasma samples were prepared. The concentration of drug protein molecules in plasma was detected by ELISA, and the drug concentration-time curve was drawn using GraphPad Prism 9, and the pharmacokinetic parameters (C max 、T max 、T 1 / 2 , AUC, MRT) were calculated.

[0166] Table 12

[0167]

[0168] from Fig.14 From the results in Table 12, it can be found that P16888 has a half-life of 330 h, while antibody P16891 has a longer half-life of up to 529 hours.

[0169] Example 15 Antibody Thermal Stability Study

[0170] 1 mg / mL P16888 antibody was prepared in PBS, pH 5.5 or pH 7.4 solution, and then incubated at 40°C, and samples were taken at Day 0, Day 10, Day 20 and Day 30. The samples at each time point were measured by cell activity and SEC-HPLC. The cell activity was detected by hGLP-1R-CRE-Luciferase-HEK293 reporter gene assay, and the experimental method is shown in Example 3.

[0171] Table 13

[0172]

[0173] The experimental results are shown in Table 13. The P16888 antibody exhibited good stability under the conditions of pH 5.5, 40°C and pH 7.4, 40°C.

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the monoclonal antibody or the antigen-binding fragment thereof are SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody or the antigen-binding fragment thereof is SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is SEQ ID NO: 5; more preferably, the heavy chain amino acid sequence of the monoclonal antibody or the antigen-binding fragment thereof is SEQ ID NO: 9, and the light chain amino acid sequence is SEQ ID NO:

10.

2. A monoclonal antibody or an antigen-binding fragment thereof, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the monoclonal antibody or the antigen-binding fragment thereof are SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 22, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 24, respectively; preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody or the antigen-binding fragment thereof is SEQ ID NO: 21, and the amino acid sequence of the light chain variable region is SEQ ID NO: 23; more preferably, the heavy chain amino acid sequence of the monoclonal antibody or the antigen-binding fragment thereof is SEQ ID NO: 25 or SEQ ID NO: 27, and the light chain amino acid sequence is SEQ ID NO:

26.

3. A monoclonal antibody or an antigen-binding fragment thereof that specifically binds to a glucagon-like peptide 1 receptor (GLP-1R), wherein: The amino acid sequence of the monoclonal antibody or antigen-binding fragment thereof has one or more conservative amino acid substitutions in the HCDR region compared to the HCDR region sequence defined by SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4; preferably, the conservative amino acid substitutions total 1, 2 or 3; preferably, the conservative amino acid substitutions are all located in HCDR3; further preferably, the conservative amino acid substitutions total 1 and are located in HCDR3; The amino acid sequence of the monoclonal antibody or its antigen-binding fragment has one or more conservative amino acid substitutions in the LCDR region compared to the LCDR region sequence defined by SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8; preferably, the conservative amino acid substitutions total 1, 2 or 3; preferably, the conservative amino acid substitutions are all located in LCDR3; further preferably, the conservative amino acid substitutions total 1 and are located in LCDR3.

4. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the monoclonal antibody or antigen-binding fragment thereof comprises: A heavy chain variable region as shown in SEQ ID NO: 1 or 21 or having at least 90% sequence identity to SEQ ID NO: 1 or 21; and / or A light chain variable region as set forth in SEQ ID NO: 5 or 23 or having at least 90% sequence identity to that set forth in SEQ ID NO: 5 or 23.

5. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the monoclonal antibody or antigen-binding fragment thereof is a full-length IgG antibody; preferably, the monoclonal antibody or antigen-binding fragment thereof contains a human antibody constant region; more preferably, the monoclonal antibody or antigen-binding fragment thereof contains a human antibody variable region and a human antibody constant region.

6. The monoclonal antibody or antigen-binding fragment thereof according to claim 5, wherein the heavy chain of the monoclonal antibody or antigen-binding fragment thereof contains a constant region selected from the following: the constant region of human IgG1, IgG2, IgG3 and IgG4 or their conventional variants; preferably, the light chain of the monoclonal antibody or antigen-binding fragment thereof contains a constant region selected from the following: the constant region of human κ chain, λ chain or their conventional variants.

7. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is a fully human antibody.

8. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the monoclonal antibody or antigen-binding fragment thereof has human GLP-1R specificity; preferably, the monoclonal antibody or antigen-binding fragment thereof can antagonize the activation of human GLP-1R by GLP-1; more preferably, the monoclonal antibody or antigen-binding fragment thereof does not have mouse GLP-1R specificity.

9. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the monoclonal antibody or antigen-binding fragment thereof has one or more characteristics selected from the following: (1) The EC50 value of binding to the hGLP-1R extracellular domain (NP_002053.3, aa24-145) is less than or equal to 70 pM as determined by the ELISA method; (2) having an EC50 value of less than or equal to 50 nM for binding to the cell membrane protein hGLP-1R as determined by flow cytometry; and (3) According to the hGLP-1R-CRE-Luciferase-HEK293 reporter gene assay, the IC50 value for antagonizing the activity of exogenous 2.5 ng / mL GLP-1 was less than or equal to 100 nM.

10. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the half-life of the plasma drug protein molecule concentration is greater than 150 hours when the monoclonal antibody or antigen-binding fragment thereof is injected subcutaneously in mice once.

11. A monoclonal antibody or an antigen-binding fragment thereof, which competitively binds to GLP-1R with the monoclonal antibody or the antigen-binding fragment thereof according to any one of claims 1 to 10.

12. A nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 11. A vector comprising the nucleic acid according to claim 12. 14 . A cell comprising the monoclonal antibody or antigen-binding fragment thereof according to claim 1 , the nucleic acid according to claim 12 , and / or the vector according to claim 13 .

15. A method for preparing the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, comprising: (1) constructing a polynucleotide sequence encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 into an expression vector; (2) transforming, transfecting or transducing the vector obtained in (1) into an expression system to obtain an expression product; (3) Isolate and purify the expression product obtained in (2). 16 . A pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof according to claim 1 and a pharmaceutically acceptable carrier.

17. A method for treating, preventing or ameliorating at least one symptom or sign of a GLP-1R-related disease or condition or a hypoglycemia-related disease or condition, the method comprising administering to a subject a therapeutically effective amount of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

18. The method according to claim 17, wherein the GLP-1R related disease or disorder is a GLP-1R over-activation related disease or disorder; preferably hypoglycemia; most preferably, post-bariatric hypoglycemia.

19. The method of claim 17, wherein the hypoglycemia-related disease or disorder is neurological damage or developmental complications caused by hypoglycemia or congenital hyperinsulinemia (HI).

20. The method of claim 19, wherein the symptoms or signs of the hypoglycemia-related disease or disorder are hypoglycemia, brain damage, developmental delay, feeding disorder, learning disability and / or epilepsy.

21. The method of any one of claims 17 to 20, wherein the monoclonal antibody or antigen-binding fragment thereof is administered subcutaneously, intraperitoneally, intravenously, intramuscularly or topically.

22. The method of any one of claims 17-21, wherein the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-11 is administered in combination with a second therapeutic agent.

23. A kit comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the nucleic acid according to claim 12, the vector according to claim 13 and / or the cell according to claim 14.

Images