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CN-115947849-B - Bispecific proteins

CN115947849BCN 115947849 BCN115947849 BCN 115947849BCN-115947849-B

Abstract

The present disclosure provides bispecific proteins. Specifically, the disclosure provides a human GCGR antibody, GLP-1 peptide and mutants thereof, and bispecific protein formed by fusion of the GCGR antibody and the GLP-1 peptide and a preparation method thereof, which can be used for reducing weight and treating diabetes.

Inventors

  • CAO ZHUOXIAO
  • LUO XIAO
  • HE NING
  • HU QIYUE
  • ZHANG LIANSHAN
  • TAO WEIKANG

Assignees

  • 江苏恒瑞医药股份有限公司
  • 上海恒瑞医药有限公司

Dates

Publication Date
20260512
Application Date
20191220
Priority Date
20181221

Claims (20)

  1. 1. A monoclonal antibody against GCGR, or an antigen-binding fragment thereof, comprising a combination of heavy and light chain variable regions selected from the group consisting of: Heavy chain variable regions comprising the HCDR1, HCDR2 and HCDR3 regions as shown in SEQ ID NOS: 20, 21 and 22, respectively, and A light chain variable region comprising the LCDR1, LCDR2 and LCDR3 regions as shown in SEQ ID NOS: 23, 24 and 25, respectively.
  2. 2. The monoclonal antibody or antigen-binding fragment thereof against GCGR according to claim 1, which is a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.
  3. 3. The anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, according to claim 2, comprising a framework region derived from a human antibody, or a framework region variant thereof, wherein: the framework region variants have back mutations of up to 10 amino acids in the light chain framework region and/or heavy chain framework region, respectively, of a human antibody.
  4. 4. The anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, according to claim 2, comprising a light chain variable region and a heavy chain variable region selected from the group consisting of: A heavy chain variable region having a sequence as set forth in SEQ ID NO. 4 or having at least 90% sequence identity to the sequence set forth in SEQ ID NO. 4, and A light chain variable region having a sequence as set forth in SEQ ID NO. 5 or having at least 90% sequence identity to the sequence set forth in SEQ ID NO. 5.
  5. 5. The anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, according to claim 1, wherein said antibody is a full length antibody, further comprising an antibody constant region.
  6. 6. The anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, according to claim 5, wherein said antibody constant regions comprise heavy chain constant regions selected from the group consisting of human IgG1, igG2, igG3, and IgG4, and conventional variants thereof, and light chain constant regions selected from the group consisting of human antibodies kappa and lambda chains, and conventional variants thereof.
  7. 7. The monoclonal antibody or antigen-binding fragment thereof according to claim 6, which comprises the human antibody heavy chain constant region shown in SEQ ID No. 72 and the human antibody light chain constant region shown in SEQ ID No. 73.
  8. 8. The anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, according to claim 5, comprising a combination of heavy and light chains selected from the group consisting of: The heavy chain is shown in SEQ ID NO. 80 or has at least 85% sequence identity thereto, and the light chain is shown in SEQ ID NO. 81 or has at least 85% sequence identity thereto.
  9. 9. The monoclonal antibody or antigen-binding fragment thereof directed against GCGR according to claim 1, wherein said antigen-binding fragment is selected from the group consisting of Fab, fab ', F (ab') 2, single chain antibodies, diabodies, and disulfide stabilized V regions (dsFv).
  10. 10. A bispecific protein comprising a GLP-1 peptide and an anti-GCGR antibody or antigen binding fragment thereof, The carboxy terminus of the GLP-1 peptide is linked to the amino terminus of the heavy chain variable region of the anti-GCGR antibody or antigen-binding fragment thereof by a peptide bond or linker, wherein the anti-GCGR antibody or antigen-binding fragment thereof is the anti-GCGR monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 9.
  11. 11. The bispecific protein of claim 10, wherein: the GLP-1 peptide is a GLP-1 peptide variant shown in SEQ ID NO. 91, wherein the sequence of the GLP-1 peptide variant is shown in SEQ ID NO. 94.
  12. 12. The bispecific protein according to claim 10, comprising a first polypeptide chain comprising the heavy chain of the anti-GCGR monoclonal antibody according to any one of claims 1 to 9 and a second polypeptide chain comprising the light chain of the anti-GCGR monoclonal antibody according to any one of claims 1 to 9, wherein: The first polypeptide chain comprises the polypeptide shown in SEQ ID NO. 109, and The second polypeptide chain comprises the polypeptide shown in SEQ ID NO. 81.
  13. 13. A pharmaceutical composition comprising: A therapeutically effective amount of a monoclonal antibody or antigen-binding fragment thereof against GCGR according to any one of claims 1 to 9, or a bispecific protein according to any one of claims 10 to 12, and One or more pharmaceutically acceptable carriers.
  14. 14. An isolated nucleic acid molecule encoding the anti-GCGR monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the bispecific protein according to any one of claims 10 to 12.
  15. 15. A recombinant vector comprising the isolated nucleic acid molecule of claim 14.
  16. 16. A host cell transformed with the recombinant vector of claim 15, said host cell selected from the group consisting of a prokaryotic cell and a eukaryotic cell.
  17. 17. The host cell of claim 16, which is a eukaryotic cell.
  18. 18. The host cell of claim 16, which is a mammalian cell or an insect cell.
  19. 19. A method of making the anti-GCGR monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the bispecific protein of any one of claims 10 to 12, comprising: Culturing the host cell of claims 16 to 18 to form the anti-GCGR monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 9, or the bispecific protein of any one of claims 10 to 12, and Recovering the monoclonal antibody or antigen binding fragment thereof, or bispecific protein from the culture.
  20. 20. Use of the anti-GCGR monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the bispecific protein according to any one of claims 10 to 12, or the pharmaceutical composition according to claim 13, for the preparation of a medicament for the treatment of metabolic disorders, comprising: administering to a subject a therapeutically effective amount of the anti-GCGR monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the bispecific protein of any one of claims 10 to 12, or the pharmaceutical composition of claim 13; the metabolic disorder is selected from obesity and diabetes.

Description

Bispecific proteins The application is a divisional application of a Chinese patent application with the application number of 2019800701999. X, the application date of 2019, 12 months and 20 days, and the name of 'bispecific protein'. The present application claims priority from patent application 201811573634.0 filed on day 21 of 12 in 2018 and patent application 201811606887.3 filed on day 27 of 12 in 2018, both of which are incorporated herein by reference. Technical Field The disclosure relates to human GCGR antibodies, GLP-1 peptides and mutants thereof, bispecific proteins formed by fusion of GCGR antibodies and GLP-1 peptides, and preparation methods and applications thereof. Background The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art. Diabetes mellitus (diabetes mellitus, DM) is a metabolic disease characterized by hyperglycemia, which is manifested as a defect in insulin secretion and/or dysfunction of insulin action, the onset of which is primarily the result of insulin co-action with glucagon. GLP-1 is one of the most important hormones affecting insulin secretion, and is derived from preproinsulin together with glucagon (Glucagon). Preproinsulin consists of about 158 amino acids and is cleaved at various sites into different peptide chains. GLP-1 having biological activity in humans mainly comprises two forms of GLP-1 (7-36) amide and GLP-1 (7-37). GLP-1 is secreted by L cells of the small intestine to promote insulin secretion mainly in a glucose concentration-dependent manner, protect islet beta cells and inhibit glucagon secretion to reduce blood glucose levels in the body. GLP-1 also has the effects of inhibiting gastric emptying and reducing appetite. The composition can be used for treating type II diabetes and obesity clinically. The half-life of natural active GLP-1 in the organism is very short (less than 2 minutes), and the natural active GLP-1 is easily degraded by DPPIV enzyme in the organism and has no clinical use value. Extending half-life has been the main direction of GLP-1 drug development, and many GLP-1 agonists such as duloxetine (Dulaglutide) and cable Ma Lutai (Semaglutide) are currently marketed. Although GLP-1 has been fully confirmed in curative effect, there are also a number of side effects, mainly manifested by gastrointestinal symptoms, hypoglycemia, pancreatitis, kidney injury, and the like. Glucagon, as opposed to insulin, acts primarily to raise blood glucose in the body. Glucagon is a 29 amino acid peptide secreted by islet alpha cells that, upon binding to the receptor GCGR on the liver cell membrane, accelerates glycogenolysis, lipolysis and gluconeogenesis by activating the downstream cAMP/PKA pathway, leading to an increase in blood glucose. The study shows that GCGR gene knockout mice show a series of phenotypes such as GLP-1 elevation, hepatic glucose output reduction, lipid metabolism increase, appetite reduction and the like. GCGR is one of the most popular targets for diabetes treatment, but currently development of antagonistic drugs against GCGR is slow. REMD Biotherapeutics company REMD-477, currently the leading GCGR monoclonal antibody drug, is in clinical stage two. The prior art has disclosed GCGR antibodies in patent CN101589062A、CN101983208A、CN102482350A、CN103314011A、CN105189560A、CN107614695A、US20180273629A1、WO2013059531A1 et al, but still remains to provide new and efficient GCGR antibodies and methods of treatment for diabetes. Disclosure of Invention The present disclosure provides a monoclonal antibody or antigen binding fragment thereof directed against GCGR. The antibody or antigen binding fragment thereof has the ability to bind to human GCGR (or an epitope comprised thereof). In some embodiments, the anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, comprises a combination of heavy and light chain variable regions selected from the group consisting of a) or b) below: a) The heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 regions shown as SEQ ID NOS: 48, 49 and 50, respectively, and the light chain variable region comprises the LCDR1, LCDR2 and LCDR3 regions shown as SEQ ID NOS: 51, 52 and 53, respectively, or B) The heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 regions shown in SEQ ID NOS: 38, 39, and 54, respectively, and the light chain variable region comprises the LCDR1, LCDR2, and LCDR3 regions shown in SEQ ID NOS: 55, 56, and 57, respectively. In some embodiments, the anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, comprises any combination of heavy and light chain variable regions selected from the group consisting of i) to vi) below: i) The heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 regions as shown in SEQ ID NOs 14, 15 and 16 respectively, and the light chain variable region comprises the LCDR1, LCDR2 and LCDR3 regions as shown in SEQ ID NOs 1