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Double-target fusion protein containing immune globulin Fc portion

A fusion protein, FGF21 technology, applied in peptide/protein components, medical preparations containing active ingredients, fusion polypeptides, etc., can solve problems such as half-life extension

Active Publication Date: 2018-09-25
SUNSHINE LAKE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reason is that FGF21 will be rapidly degraded by proteases in vivo, and its tendency to form aggregates in vitro will also lead to immunogenicity, which is not conducive to the extension of half-life

Method used

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  • Double-target fusion protein containing immune globulin Fc portion
  • Double-target fusion protein containing immune globulin Fc portion
  • Double-target fusion protein containing immune globulin Fc portion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0188] Embodiment 1: the construction of carrier

[0189] Using the method of molecular cloning, the vector of the fusion protein was constructed, and the fusion protein is shown in Table 1.

[0190] Table 1

[0191]

[0192]

[0193] The nucleotide sequence encoding dulaglutide and S1F1 fusion protein was obtained through chemical synthesis by entrusting GenScript Biotechnology Co., Ltd., and the vector encoding D1F1 fusion protein was synthesized by designing primers PCR template dulaglutide and S1F1 fusion protein The sequence was obtained to obtain fragments GLP-1 and Fc-FGF21, and the fragments were connected by SOE-PCR technology to obtain GLP-1-Fc-FGF21, which is the nucleotide sequence encoding the D1F1 mutant fusion protein.

[0194] Nucleotide sequences encoding D1F2, D2F1, D3F1, D4F1, D5F1, D6F1 and D7F1 fusion proteins were obtained through chemical synthesis entrusted to Suzhou Jinweizhi Biotechnology Co., Ltd.

[0195] At 37°C, the nucleotide sequence and...

Embodiment 2

[0197] Example 2: Transfection of vector encoding dulaglutide and expression in cells

[0198] After recovery and culture of CHOK1SV GS-KO (Lonza) host cells with CD CHO medium (gibco), when the cell density was about 8x 10 5 Cells / mL were harvested for transfection. Transfected cells about 1x10 7 Cell, about 40 μg of vector, was transfected by electroporation (Bio-Rad, Gene pulSXcell). Cells were cultured in 20mL CD CHO medium after electric shock. On the second day of culture, the cells were collected by centrifugation at 200 g for 10 min, and resuspended in 20 mL of CD CHO medium with MSX (sigma) added to a final concentration of 50 μM. When the cell density is about 0.6x10 6 cell / mL, the obtained mixed clones were subcultured with CD CHO medium, and the subcultured cell density was about 0.2x10 6 cell / mL. When the cell survival rate was about 90%, the cell culture fluid was collected.

Embodiment 3

[0199] Example 3: Vectors encoding S1F1 and D1F1, D1F2, D2F1, D3F1, D4F1, D5F1, D6F1, D7F1 fusion proteins were transfected and expressed in cells

[0200] HEK293F host cells (Invitrogen, Freestyle 293F) were revived and cultured with 293Expression Medium medium (Invitrogen), when the cell density was about 1×10 6 Cells / mL were harvested for transfection. Transfected cells about 3x10 7 cell, the carrier is about 37.5μg, using FreeStyle TM MAX Reagent transfection kit was used. After transfection, the cells were cultured in 30 mL of 293Expression Medium. On the second day of cultivation, start to use geneticin (merck) to screen the transformants. According to the growth of the cells, replace the selection medium every 3 to 5 days. After about 14 days of selection, resistant clones can be seen, which can be carried out. Expand cultivation. The cell passage density is about 0.5x10 6 cell / mL, the obtained mixed clones were subcultured with 293Expression Medium medium. When ...

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Abstract

The invention relates to a fusion protein, in particular to a double-target fusion protein containing an immune globulin Fc portion. The double-target fusion protein comprises a first polypeptide, a second polypeptide and a third polypeptide. The first polypeptide contains GLP-1 or other analogues, the second polypeptide contains an IgG4Fc portion, and the third polypeptide contains FGF21 or a variant thereof. The first polypeptide is connected with the second polypeptide through a first joint, and the third polypeptide is connected with the second polypeptide through a second joint. The fusion protein has mutation sites different from those of existing disclosed infusion proteins, is high in stability and remarkably prolonged in half-life period and gives play to synergistic effects in blood glucose reducing, lipid lowering, weight losing and the like.

Description

technical field [0001] The present invention relates to a fusion protein comprising fibroblast growth factor 21 (FGF21) and glucagon like peptide-1 (GLP-1), which comprises immunoglobulin Fc Partly, the fusion protein can be used to treat metabolic diseases. technical background [0002] There are currently three categories of drugs for the treatment of diabetes, oral small molecule drugs, insulin and GLP-1 receptor agonist drugs. Long-term use of small molecule drugs has obvious side effects, and the control of blood sugar in the late stage of diabetes is not ideal. Insulin requires multiple (at least one) injections per day, and is prone to hypoglycemia due to individual dose differences. A single GLP-1 receptor agonist is not a first-line drug, and has limited curative effect on cardiovascular and other complications caused by metabolic abnormalities in diabetes. [0003] Glucagon like peptide-1 (GLP-1) is an incretin secreted by small intestinal L cells, which can sti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K38/17A61P1/16A61P3/04A61P3/10
CPCA61K47/6811A61P1/16A61P3/04A61P3/10C07K14/50C07K14/605C07K2319/30A61K38/00C12N15/62C12N15/63
Inventor 陈超林树珊李玉陈小锋刘亮付正
Owner SUNSHINE LAKE PHARM CO LTD
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