Fc fusion protein of human granulocyte colony stimulin with enhanced bioactivity

A fusion protein and cell technology, applied in colony-stimulating factor, animal/human protein, cytokine/lymphokine/interferon, etc., can solve problems such as weak complement activity

Inactive Publication Date: 2003-04-16
PHARMAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Human IgG2 does not bind Fc γ R, but

Method used

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  • Fc fusion protein of human granulocyte colony stimulin with enhanced bioactivity
  • Fc fusion protein of human granulocyte colony stimulin with enhanced bioactivity
  • Fc fusion protein of human granulocyte colony stimulin with enhanced bioactivity

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Experimental program
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Effect test

Embodiment Construction

[0018] 1. Construction of encoding hG-CSF-L-vFc γ2 fusion protein gene

[0019] Fusion proteins are constructed from several DNA fragments. Using RNA prepared from human bladder cancer 5637 cell line, genes encoding the leader peptide and mature protein of human G-CSF were obtained by reverse transcription and polymerase chain reaction (PCR). To facilitate cloning, SEQ ID NO: 1 introducing a restriction enzyme endonuclease site (HindIII) was used as a primer for the 5' oligonucleotide. Table 1 lists the oligonucleotide sequences used to clone the hG-CSF-L-vFc fusion protein. The 3' primer (SEQ ID NO: 2) removed the G-CSF terminal codon and introduced a BamHI site. The thus obtained DNA fragment of about 600 bp in length is inserted into the HindIII and BamHI sites of an accepting vector (such as pUC19) to obtain a phGCS plasmid. The sequence of the human G-CSF gene was verified by DNA sequencing.

[0020] The Fc region encoding human IgG2 (Fc γ2 ) genes. The resulting F...

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Abstract

A human hG-CSF-L-VFC fusion protein whose bioactivity is higher than that of rhG-CSF is disclosed. It contains human G-CSF, 20 or less flexible peptide linkers, and human IgG Fc variant which can notbe cracked and can show very little poor by-effect of Fc-mediation. A process for preparing the said fusion protein with high expression level is also disclosed. This hG-CSF-L-VFC fusion protein has longer serum half-life period and higher bioactivity, so improving its pharmacodynamics and effect.

Description

Background of the invention [0001] Granulocyte colony stimulating factor (G-CSF) is a 20 kilodalton glycoprotein that promotes the proliferation of progenitor cells and induces their differentiation into neutrophils. In addition, G-CSF prolongs the survival of mature neutrophils and activates their function. Human G-CSF (hG-CSF) is produced by monocytes, macrophages, fibroblasts and endothelial cells (see, e.g., Moore, Annu. Rev. Immunol., 9: 159-191, 1991; Nicola , Annu. Rev. Biochem., 58:45-77, 1991). The biological effects of G-CSF are mediated by its interaction with the G-CSF receptor (G-CSF-Rc) expressed on the surface of myeloid hematopoietic progenitor cells and cells of the myeloid lineage. Upon binding to G-CSF, the receptor is activated and undergoes homodimerization followed by phosphorylation of the Janus family of tyrosine kinases. Subsequently, a cascade of intracellular signal transductions occurs that increases the number of progenitor cells that mature int...

Claims

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Application Information

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IPC IPC(8): C07K14/535
CPCC07K2319/00C07K14/535C07K2319/30
Inventor 金宜慧孙乃超周若芸
Owner PHARMAB
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