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Fc-gamma receptor mutants

A mutant and receptor technology, applied in the direction of antibody mimics/stents, allergic diseases, drug combinations, etc., can solve the problems of prolonging serum half-life, short half-life, short residence time, etc.

Pending Publication Date: 2020-05-19
KOOKMIN UNIV IND ACAD COOP FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, many peptide drugs that have pharmacological effects but are limited in clinical application due to short in vivo half-lives and short residence times can be fused with FcγR mutants to greatly extend serum half-lives

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0147] Example 1. Construction of FcγRIIa mutant library

[0148] Based on the pMopac12-NlpA-FcγRIIa-FLAG vector, the FcγRIIa gene introducing random mutations was prepared using MJ#160 and MJ#161 primers so that about 0.2% of random mutations occurred in FcγRIIa. In addition, target inserts incorporating various amino acids were prepared by introducing a degenerate codon NNK at the IgG Fc binding site of FcγRIIa using MJ#160, MJ#161, p788, p789, p790, p791, p792, and p793 primers (Table 1). The prepared two inserts were treated with SfiI (New England Biolab) restriction endonuclease and ligated to the vector. Then, by transforming into Escherichia coli (E.coli) Jude1 ((F'[Tn10(Tet r )proAB + lacI q Δ(lacZ)M15]mcrAΔ(mrr-hsdRMS-mcrBC)Φ80dlacZΔM15ΔlacX74 deoR recA1 araD139Δ(ara leu)7697 galUgalKrpsLendA1nupG) to construct FcγRIIa mutant library (library size: 2.2×10 9 )( figure 1 ).

[0149] [Table 1]

[0150]

[0151]

[0152]

Embodiment 2

[0153] Example 2. Screening of FcγRIIa mutant library by bacterial culture and flow cytometry

[0154] Culture 1 mL of the established FcγRIIa mutant library cells in Super Broth (TB) medium containing 2 wt / vol% glucose and chloramphenicol (40 μg / mL) at 37°C for 4 hours with shaking at 250 rpm . The cultured pool cells were inoculated into TB medium at 1:100, and then cultured to OD at 37°C while shaking at 250 rpm 600 0.6. Then, after incubation at 25° C. for 20 minutes for cooling, 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) was added to induce expression. After the culture was completed, the cells were recovered, and the OD 600 Normalization Centrifuge at 14000 rpm for 1 min. After collection, cells were resuspended by adding 1 mL of 10 mM Tris-HCl (pH 8.0), and centrifuged for 1 min. This washing process was repeated twice. After resuspension in 1 mL of STE [0.5M sucrose, 10 mM Tris-HCl, 10 mM EDTA (pH 8.0)], the outer cell membrane was removed by spinning at ...

Embodiment 3

[0155] Example 3: Cloning, expression and purification of isolated SH2A40 mutants for expression in mammalian cells

[0156] To express the screened SH2A40 mutants in HEK293F cells, pMAZ-FcγRIIa (wild type) and pMAZ-FcγRIIa mutant (SH2A40) were prepared by PCR amplification of the gene using Vent polymerase and MJ#162 and MJ#163 primers, and then Ligation was performed by restriction enzyme treatment with BssHII and XbaI (New England Biolab). The gene cloned into the mammalian cell expression vector pMAZ was transfected into HEK293F cells and expressed transiently at a scale of 300 mL. After the incubation was complete, the cells were removed by centrifugation at 2000 rpm for 10 minutes, and the supernatant was taken and equilibrated with 25xPBS. The resulting solution was filtered through a 0.2-μm bottle top filter (Merck Millipore). After adding 1 mL of a Ni-NTA agarose (Qiagen) slurry equilibrated with PBS, the solution was stirred at 4°C for 16 hours before being poured ...

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Abstract

The present invention relates to a polypeptide comprising an Fc-[gamma] receptor mutant. The Fc-[gamma] receptor mutant of the present invention is optimized by replacing a part of an amino acid sequence of an Fc-[gamma] receptor with a different amino acid sequence, so as to have an excellent selective binding ability to immunoglobulins, thereby being effectively useful for uses of increasing thein vivo half-life of drugs, detecting and purifying immunoglobulins, and inhibiting organ transplantation reactions or preventing or treating autoimmune diseases.

Description

technical field [0001] The present disclosure relates to Fcγ receptor mutants with improved binding capacity to IgG antibodies. Background technique [0002] Fcy receptors (FcyRI, FcyRIIa, FcyRIIb, FcyRIIIa) expressed in human immune cells bind to the Fc region (lower hinge region and upper CH2 region) of IgG antibodies. Among these Fcγ receptors, FcγRI has the highest affinity for IgG in blood. However, there are difficulties in medical or industrial applications due to very poor thermal stability and low expression levels. [0003] Meanwhile, FcγRIIa is a transmembrane protein expressed on the surface of various immune cells such as macrophages, monocytes, neutrophils, and the like. It is relatively low affinity for IgG (K D = about 10 -6 ) receptors. FcγRIIa binds to the Fc region (lower hinge region and upper CH2 region) of IgG antibodies and activates immune cells through intracellular signaling mechanisms. [0004] If soluble Fc-γ receptors that exist in the extr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/735A61K47/68G01N33/68A61K38/00
CPCG01N33/6857G01N33/6845G01N2500/04A61K38/1774C07K14/70535A61P37/06C07K16/22C07K2319/31C07K2317/94C07K2317/622C07K2319/24C07K2319/21C07K2319/50C07K2319/43A61K38/00C12N5/00G01N33/53C07K16/46C12N15/00
Inventor 郑相泽曹美京高翔焕黄宝拿
Owner KOOKMIN UNIV IND ACAD COOP FOUND
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