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igg-like long-acting immune fusion protein and its application

A fusion protein and protein technology, applied in the biological field, can solve the problem of short biological half-life

Active Publication Date: 2020-05-15
BEIJING BIYANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a general disadvantage of these Fc immunofusion proteins is the short biological half-life

Method used

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  • igg-like long-acting immune fusion protein and its application
  • igg-like long-acting immune fusion protein and its application
  • igg-like long-acting immune fusion protein and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1, the construction of high-efficiency expression vector of glutamine synthetase

[0057] 1. Cloning of gene encoding diabetes-associated IgG-like immune fusion protein and construction of expression vector

[0058] 1. Synthesis of dulaglutide-encoding gene and construction of expression vector

[0059] Dulaglutide (Trulicity) is an FDA-approved glucagon-like peptide-1 (GLP-1) receptor agonist and an immune fusion protein used to help normalize blood sugar levels , whose structure is GLP-1-Fc, which belongs to the existing typical Fc immune fusion protein.

[0060] 根据International Nonproprietary Name(INN)数据库中和专利中有关Dulaglutide的氨基酸残基序列“HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(序列52)”委托上海捷瑞生物工程有限公司合成将Dulaglutide的编码基因“CACGGAGAGGGCAC...

Embodiment 2

[0092] Example 2, Screening, expression and purification of IgG-like immune fusion protein expressing cells

[0093] 1. Screening of cells expressing IgG-like immune fusion proteins

[0094] 1. CHO-K1 cells (ATCC, catalog number: CCL-61) were adapted and grown adherently in CD OptiCHO (Invitrogen product, catalog number: 12681) with 10% D-FBS and 4 mM glutamine.

[0095] 2. After 24 hours, according to the instructions of Lipofectin 2000 (Invitrogen product number: 11668-019), transfect the cells with the recombinant vector constructed in Example 1, and carry out in a 10 cm culture dish: wash with serum-free and antibiotic-free IMDM medium for 2 Each time, IMDM 100 μl / well was added each time. The expression plasmid constructed in Example 1 was mixed evenly with the IMDM medium, and the Lipofectin 2000 was mixed evenly with the IMDM medium. After standing for 5 minutes, mix the two. After standing at room temperature for 15 minutes, the Lipofectin 2000CD and expression plas...

Embodiment 3

[0101] Example 3, Biacore 3000 detects the affinity of IgG-like immune fusion protein and its target

[0102] 1. Experimental method

[0103] 1. Fixed

[0104] Dilute GLP-1R, VEGF-A, VEGF-B and TNF-α (all Yiqiao Shenzhou products) to a concentration of 10 μg / ml, and use amino coupling method to covalently immobilize on carboxymethyl-dextrin via primary amine Sugar-coated CM5 chip (General Electric product), fixation buffer 10mM sodium acetate (pH5.0), fixation amount is 1000RU.

[0105] 2. Regeneration conditions

[0106] Regeneration solution: glycine buffer solution with a pH of 1.5-2.0; injection time: 30s; flow rate: 30 μl / min.

[0107] 3. Kinetic analysis

[0108] PBST (recipe: Tween 20 containing 0.005% by volume in PBS) is the Running buffer; use the Kinetic Analysis Wizard mode; dilute Dulaglutide, BY23.2, BY25 and BY19.3 to 600, 300, 150, Concentration gradient of 75, 37.5 and 18.75nM; injection time: 2min, dissociation time: 5min, flow rate: 30μl / min.

[0109] ...

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Abstract

Disclosed are an IgG-like long-acting immune fusion protein and the use thereof. The IgG-like long-acting immune fusion protein consists of an effector molecule and a constant region of an IgG antibody, and the effector molecule is linked to the constant region of the IgG antibody by means of a linker peptide; the effector molecule is a protein capable of exerting physiological functions in the body; and the constant region of the IgG antibody is a structure of the IgG antibody with two heavy chain variable regions and two light chain variable regions removed.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an IgG-like long-acting immune fusion protein and its application. Background technique [0002] Recombinant protein drugs are a very important category of biotechnology drugs, which are generally administered clinically through intravenous and subcutaneous injections. After intravenous and subcutaneous injection, it is often accompanied by protein degradation, resulting in reduced activity and low bioavailability; those with smaller molecular weight (less than 60kD) can even be filtered into the urine through the glomerulus, so the required blood concentration and treatment must be achieved. The effect requires repeated administration, and some need to be injected 2-3 times a day, which not only brings inconvenience to the patient, but also reduces the compliance of the patient, thereby affecting the curative effect. Therefore, it is necessary to develop long-acting recombinant prote...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00A61K38/27A61K38/26A61K38/18A61K38/17A61P5/00A61P35/00A61P3/10A61P37/02
CPCA61K38/00C07K14/435C07K2319/30A61K38/17A61K38/18A61K38/26A61K38/27A61P3/10A61P5/00A61P35/00A61P37/00A61P37/02C07K19/00C12N15/00
Inventor 胡品良何芸邹敬洪伟东宋凌云杨泽荣
Owner BEIJING BIYANG BIOTECH
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