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US-12624080-B2 - Multi-receptor (GLP-1 receptor, GIP receptor, and Gcg receptor) agonist protein

US12624080B2US 12624080 B2US12624080 B2US 12624080B2US-12624080-B2

Abstract

This invention provides a novel protein. Experimental results have shown that the protein has agonist activity at three receptors, namely the glucagon-like peptide 1 (GLP-1) receptor, the glucose-dependent insulinotropic polypeptide (GIP) receptor, and the glucagon (Gcg) receptor, and has both blood glucose lowering and body weight reducing functions.

Inventors

  • Hai Chen
  • Gaoyong Liao
  • Yi Zhang

Assignees

  • XINTRUM PHARMACEUTICALS, LTD.

Dates

Publication Date
20260512
Application Date
20220527
Priority Date
20201210

Claims (9)

  1. 1 . A protein consisting of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
  2. 2 . A body weight reducing agent comprising the protein of claim 1 .
  3. 3 . A method of reducing body weight of a subject, comprising administering to the subject the protein of claim 1 .
  4. 4 . A blood glucose lowering agent comprising the protein of claim 1 .
  5. 5 . A protein consisting of the amino acid sequence of SEQ ID NO:1.
  6. 6 . A protein consisting of the amino acid sequence of SEQ ID NO:2.
  7. 7 . The protein of claim 1 , where the protein is an agonist for the glucagon-like peptide 1 (GLP-1) receptor.
  8. 8 . The protein of claim 1 , where the protein is an agonist for the glucose-dependent insulinotropic polypeptide (GIP) receptor.
  9. 9 . The protein of claim 1 , where the protein is an agonist for the glucagon (Geg) receptor.

Description

TECHNICAL FIELD The present invention relates to the field of biotechnology and more particularly to a multi-receptor agonist protein that is highly effective in reducing body weight and lowering blood glucose. DESCRIPTION OF RELATED ART As is well known in the art, glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon (Gcg) are all incretins related to metabolic disorders such as diabetes and obesity. A glucagon-like peptide 1 receptor agonist (abbreviated as GLP-1RA) acts directly on the pancreas to promote the release of insulin and inhibit the secretion of glucagon. A GLP-1RA also inhibits peristalsis of the stomach, delays gastric emptying, and acts on the central nervous system to suppress appetite. Nevertheless, the foregoing treatment solutions have only limited therapeutic effects; their clinical therapeutic effects fail to satisfy the needs of a patient with a metabolic disorder completely. By incorporating a plurality of metabolic pathways into a single treatment, however, multiple effects may be achieved to improve the existing therapeutic effects. GIP is the major cause of the postprandial incretin effect in normal people and functions differently from GLP-1. Gcg is a gastrointestinal hormone produced in the pancreas, increases blood glucose by stimulating gluconeogenesis and glycogenolysis in the liver, and enhances catabolism and thermogenesis. In the prior art, drugs for treating diabetes and obesity have long been focused on those having an individual therapeutic effect on a single molecular target. Clinically, therefore, the use of concomitant drugs involves only a number of single-action drugs directed to their respective single targets, or a compound preparation of those drugs. Studies have shown that administering a GLP-1RA together with a different gastrointestinal hormone can produce a synergistic effect. It is hence desirable to design and obtain a single molecule that has proper balanced activity at multiple receptors, or more particularly the GLP-1 receptor, the GIP receptor, and the Gcg receptor, in order to activate different signal pathways at the same time and thereby produce a synergistic effect on blood glucose control, body weight reduction, and regulation of the metabolism of body fat, the objective being to bring about such advantages as maximized therapeutic effects, reduced side effects, and stable pharmacokinetic properties. BRIEF SUMMARY OF THE INVENTION One objective of the present invention is to provide a protein that has agonist activity at three receptors—namely the GLP-1 receptor, the GIP receptor, and the Gcg receptor—at the same time, and that can reduce the body weight and lower the blood glucose of a patient with diabetes significantly. The present invention provides a multi-receptor agonist protein (hereinafter also referred to as GGGF1 for short) having the amino acid sequence of SEQ ID NO: 2. Sites 1-39 of the amino acid sequence of the protein GGGF1 constitute an active functional area and have the amino acid sequence of SEQ ID NO: 1, whereas the remaining portion of the amino acid sequence makes up a non-functional area and serves to increase protein stability. The multi-receptor agonist protein exists in the form of a homodimer. The present invention involves constructing the multi-receptor agonist protein in an expression vector, wherein the expression vector is a eukaryotic expression vector and can be introduced into a host cell by transient transfection or stable transfection. The host cell is a mammalian cell, and the mammalian cell is a Chinese hamster ovary (CHO) cell or a human embryonic kidney 293 cell. More specifically, the following research work was conducted for the present invention: 1. A nucleotide sequence corresponding to the amino acid sequence of GGGF1 was designed and synthesized in accordance with the codon usage bias of the CHO cell and was constructed in the pcDNA3.4 vector to produce the expression vector pcDNA3.4-GGGF1. 2. The expression vector was transfected into a CHO cell through a transfection reagent, and after culturing, a cell supernatant in which GGGF1 was expressed was obtained. 3. GGGF1 was separated and purified through Protein A and was tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 4. The bioactivity of GGGF1 was tested with the GLP-1 receptor, the GIP receptor, and the Gcg receptor. 5. A study on the activity of GGGF1 in reducing body weight and lowering blood glucose was conducted on db/db diabetic model mice. The results of multiple experiments have proved that the protein provided by the present invention can: 1. Activate multiple receptors, namely the GLP-1, GIP, and Gcg receptors, simultaneously (see embodiments 2, 3, and 4); 2. Reduce body weight significantly (P<0.05) (see embodiment 5); 3. Lower the non-fasting blood glucose (NFBG) level (P<0.001) and the fasting blood glucose (FBG) level (P<0.0001) extremely significantly (see e