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IFNAR2 mutants, their production and use

A mutant and carrier technology, applied in the field of mutant polypeptides, can solve the problems of low bioavailability and no clear correlation of IFNβ bioavailability.

Inactive Publication Date: 2005-06-01
YEDA RES & DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The clearance of IFNβ is rather rapid, so its bioavailability is lower than expected for most cytokines
Although the pharmacodynamics of IFNβ in humans has been assessed, no clear correlation between IFNβ bioavailability and clinical efficacy has been established

Method used

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  • IFNAR2 mutants, their production and use
  • IFNAR2 mutants, their production and use
  • IFNAR2 mutants, their production and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0153] Example 1: Expression and purification of proteins

[0154] E. coli expressed IFNAR2-EC (extracellular domain) and IFNa were purified by ion exchange and size exclusion chromatography as described (Piehler & Schreiber, 1999A). The expression level of the IFNAR2-EC mutant was as high as the wild type. Wild-type glycosylated IFN-[beta] was produced in CHO (as described in EP220574). Determine protein concentration from absorbance at 280 nm (Piehler & Schreiber, 1999A), 1:280 = 18,070 M-1 for IFNα2; 1:280 = 30,050 M-1 for IFN-β; 1:280 = 1:280 for IFNAR2-EC 26,500M-1 (for IFNAR2-EC which is the tryptophan mutant of W102A and W74F, corrected to 1:280=21,100M-1). Protein purity was analyzed by SDS-PAGE under non-reducing conditions.

Embodiment 2

[0155] Example 2: Generation of IFNAR-EC mutants

[0156]High-fidelity polymerases pwo (Boehringer Mannheim) and pfu (Stratagene) were used as template pT72CR2 (Piehler & Schreiber, 1999A) and nucleotide primers containing mutated codons as detailed in (Albeck & Schreiber, 1999). PCR for site-directed mutagenesis. After phosphorylation and ligation, E. coli TG1 cells were transduced with the mutated plasmid. The full sequence of the expressed gene containing the mutation was verified by DNA sequencing (Ausubel et al., Current Protocols in Molecular Biology, Greene Publications and Wiley Interscience, New York, NY, 1987-1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory , Cold Spring Harbor, NY, 1989).

[0157] There are two amino acid residues in the generated mutant: 78-position histidine (H78) and 100-position asparagine (N100) are mutated: A-both become alanine (H78A / N100A), B- to alanine and aspartic acid (H78A / N100D), respect...

Embodiment 3

[0158] Example 3: Thermodynamic and Kinetic Analysis

[0159] All thermodynamic and kinetic data were obtained from label free heterozygous phase assays. The interaction between IFN-β2 and IFNAR2-EC was monitored by reflectance interference spectrophotometry [RifS] under flow-through conditions as described in Piehler & Schreiber, 1999A. This method is similar to Biacore and is used to accurately determine the binding affinity between two proteins. IFNAR2-EC (wild type or mutant) was immobilized by means of immobilized specific antibodies (as described in Piehler & Schreiber, 2001). All assays for IFN-β, IFNα2 and IFNAR2-EC were performed in 50 nM Hepes and 500 mM NaCI, 0.01% TritonX100, pH 7.4. The interaction at 500 mM NaCI was determined to eliminate the non-specific reaction of IFN-[beta] with the surface seen at 150 mM NaCI.

[0160] Association and dissociation kinetics were determined with standard injection protocols and corrected for with blank runs. Dissociatio...

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Abstract

The present invention relates to mutant polypeptides of the beta chain of the type I interferon receptor (MIFNAR2) with enhanced affinity for interferon-beta, which prolong the in vivo action of IFN-beta compared to the wild-type protein.

Description

field of invention [0001] The present invention relates to mutant polypeptides of the beta chain of the type I interferon receptor (MIFNAR2) with enhanced affinity for interferon-beta, which prolong the in vivo action of IFN-beta compared to the wild-type protein. Background of the invention [0002] The types of interferons are divided into type I interferons produced by leukocytes and fibroblasts, and type II interferons which are mitogen-induced or "immune" (Pestka et al., 1987). By analysis of sequence identity and common biological activity, type I interferons include interferon alpha (IFN-α), interferon beta (IFN-beta), and interferon omega (IFN-omega); while type II interferons include Interferon gamma (IFN-gamma). [0003] The IFN-α, IFN-β and IFN-ω genes are clustered on the short arm 25 of chromosome 9 (Lengyl, 1982). There are at least 25 non-allelic IFN-α genes, 6 non-allelic IFN-ω genes, and 1 IFN-β gene. All are believed to have evolved from a common ancestr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/00C12N15/09A61K38/21A61K45/00A61K48/00A61P1/04A61P1/16A61P3/10A61P17/02A61P19/02A61P21/04A61P25/00A61P31/14A61P31/20A61P35/00A61P35/02A61P37/06A61P43/00C07K14/715C07K19/00C12N1/15C12N1/19C12N1/21C12N5/10C12P21/02
CPCA61K38/00C07K14/7156A61P1/04A61P1/16A61P17/02A61P19/02A61P21/04A61P25/00A61P29/00A61P31/12A61P31/14A61P31/20A61P35/00A61P35/02A61P37/02A61P37/06A61P43/00A61P3/10Y02A50/30C07K14/715
Inventor G·斯莱伯
Owner YEDA RES & DEV CO LTD
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