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Long-acting recombinant GLP1-Fc-CD47 protein as well as preparation method and application thereof

An A-B-C-D, fusion protein technology, applied in the field of medicine, can solve the problem of low frequency of administration

Active Publication Date: 2020-03-13
ZHEJIANG PALOALTO PHARMA TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The lowest dosing frequency among currently marketed drugs is once a week

Method used

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  • Long-acting recombinant GLP1-Fc-CD47 protein as well as preparation method and application thereof
  • Long-acting recombinant GLP1-Fc-CD47 protein as well as preparation method and application thereof
  • Long-acting recombinant GLP1-Fc-CD47 protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0178] Example 1: Preparation of GLP-1-Fc recombinant protein

[0179] Mammalian cell expression vector (Bacmam) was selected as the vector, and 8 histidines (8His) were selected as the tag protein (Tag) in series with two maltose binding protein (MBP) and sumo protein. The DNA encoding the recombinant protein was artificially synthesized, and its optimized sequence was shown in SEQ ID NO.:16-20, and the gene fragment was cloned into Bacmam- 8His-2MBP-sumo.

[0180] The plasmid was transformed into DH10Bac competent cells, and after blue-white screening, bacmid DNA was extracted, transfected into insect cells to obtain the P0 generation virus, and the virus was further amplified in insect cells to the required volume. The amplified virus was transfected into mammalian cells for expression for 4 days.

[0181] Four days after the virus transfected the mammalian cells, the supernatant was collected, centrifuged at 2000rpm for 20min, and the supernatant was slowly loaded on the...

Embodiment 2

[0182] Example 2: Modification of the GLP-1 part of the GLP-1-Fc recombinant fusion protein and evaluation of cell activity

[0183] There are two main active forms of naturally occurring GLP-1: GLP-1(7-36)NH 2 And GLP-1(7-37) (SEQID NO.: 1), DPPIV enzyme acts between the 7th histidine (His) and the 8th alanine (Ala). A common strategy for prolonging GLP-1 drugs is to mutate the 8th alanine (Ala) to glycine (Gly) (SEQ ID NO.: 2) to resist degradation by DPPIV enzymes.

[0184] Studies have shown that after the 22nd glycine (Gly) of GLP-1 is mutated to glutamic acid (Glu) and the 36th arginine (Arg) is mutated to glycine (Gly), the activity of GLP-1 increases, and GLP-1 The risk of the fusion protein causing an immune response in the body is reduced (US 2007 / 0036806 A1). The GLP-1 portion of the marketed drug dulaglutide introduces mutations at positions 8, 22, and 36, respectively (SEQ ID NO.: 3).

[0185] In this embodiment, the 34th lysine (Lys) of GLP-1 is further mutate...

Embodiment 3

[0189] Example 3: Introduction of CD47 signal into GLP-1-Fc recombinant fusion protein

[0190] The Fc part of the recombinant protein GLP-1-Fc in the present invention is from human IgG4, including the hinge region, CH2 and CH3 domains. Although the ADCC effect induced by IgG4 is weak, it still has potential cytotoxicity.

[0191] The present invention introduces two mutations F234A and L235A in the Fc part of natural human IgG4, which can further effectively reduce the ADCC effect. In addition, the present invention also introduces the amino acid mutation S228P, which can effectively reduce the formation of GLP1-Fc monomers and increase the homogeneity and stability of the recombinant fusion protein.

[0192] Rodriguez and other researchers designed the smallest polypeptide (self) by computer to simulate the function of human CD47 to reduce macrophage clearance (self sequence is shown in SEQ ID NO.: 10). The self polypeptide was introduced into the N-terminus and C-terminu...

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Abstract

The invention relates to a long-acting recombinant GLP-1-Fc fusion protein containing a CD47 signal as well as preparation and an application of the long-acting recombinant GLP-1-Fc fusion protein. Specifically, the invention relates to the brand-new combined fusion protein, which comprises a GLP-1 polypeptide, an Fc fragment and a CD47 signal polypeptide. The fusion protein is found to exist in atetramer form for the first time and is more stable than similar dimer Fc fusion proteins, and the half-life period in vivo is effectively prolonged. The GLP-1 and Fc fragments are mutated, so that the biological activity of GLP-1 is improved, the antibody-dependent cell-mediated cytotoxicity (ADCC) effect is reduced, and the potential cytotoxicity and immunogenicity are reduced. Moreover, the CD47 signal polypeptide in the fusion protein is applied to the fusion protein for the first time, so that the elimination of macrophages can be reduced, and the administration frequency in a human bodycan be reduced.

Description

technical field [0001] The invention relates to the field of medicine, in particular to long-acting recombinant GLP-1-Fc fusion protein containing CD47 signal and its preparation and application. Background technique [0002] Glucagon-like peptide-1 (GLP-1) is an incretin secreted by intestinal L cells, and GLP-1 exerts physiological effects by acting on the GLP-1 receptor (GLP-1R). Receptors belong to the G protein-coupled receptor family, which activate cAMP through G protein, increase the level of intracellular cAMP, and activate protein kinase A, leading to an intracellular cascade reaction. Under normal physiological conditions, its main physiological function is to stimulate pancreatic β-cells to secrete insulin, inhibit pancreatic α-cells to secrete glucagon, suppress appetite, and delay gastric emptying. Most importantly, the insulin-stimulating effect of GLP-1 depends on in blood sugar concentration. In patients with non-insulin-dependent diabetes mellitus (type I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K38/26A61K47/68A61P3/10A61P3/04
CPCC07K14/70503C07K14/605A61K47/6811A61P3/10A61P3/04C07K2319/30A61K38/00A61K47/68C12N15/62C07K2319/31C07K2319/02
Inventor 席志坚徐华强王改红
Owner ZHEJIANG PALOALTO PHARMA TECH CO LTD
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