US 6,642,011 B2 · Human protease and use of such protease for pharmaceutical applications and for reducing the allergenicity of non-human proteins

Abstract

The present invention relates to a method of producing novel improved protein mutant which produce low allergenic response in humans compared to the parent of that mutant. Specifically, the present invention comprises neutralizing or reducing the allergenicity of a protein by introducing therein as replacement or modification of an epitope on such protein a sequence from human subtilisin.

Drawings

Drawings are temporarily unavailable. Open the PDF at USPTO

Drawings

Claims15

Dependent claims

A method for producing a mutant protease having reduced allergenicity comprising the steps of:

a) obtaining a naturally-occurring protease having subtilisin activity and preparing fragments of said naturally-occurring protease having subtilisin activity;
b) contacting said fragments of said naturally-occurring protease with a first solution comprising naïve human CD4+ or CD8+ T-cells and dendritic cells, wherein said dendritic cells have been differentiated;
c) identifying an epitope region of said naturally-occurring protease, wherein said identifying comprises measuring the ability of said fragments of said naturally-occurring protease epitope region to stimulate proliferation of said naïve human CD4+ or CD8+ T-cells;
d) replacing said epitope region identified in step c) with an analogous epitope region in the amino acid sequence set forth in SEQ ID NO:6, to produce said mutant protease;
e) preparing fragments of said mutant protease;
f) contacting said fragments of said mutant protease with a second solution comprising naïve human CD4+ or CD8+ T-cells and dendritic cells, wherein said dendritic cells have been differentiated;
and
g) measuring the ability of said fragments of said mutant protease to stimulate proliferation of said naïve human CD4+ or CD8+ T-cells.

5 more elements

Dependencies are inferred from the claim text.

Description13,945 words

Summary2,246 words

#A. Field of the Invention

#The present invention relates to a human protein sequence which can be used in several applications. Specifically, the novel human protein sequence can be used to design proteins which produce lower allergenic response in humans exposed to such proteins through the use of a predictive assay.

#B. State of the Art

Brief description of the drawings8 figures

#FIGS. 1A-C illustrates the DNA (SEQ ID:NO 1) and amino acid (SEQ ID:NO 2) sequence for Bacillus amyloliquefaciens subtilisin (BPN′) and a partial restriction map of this gene.

#FIG. 2 illustrates the conserved amino acid residues among subtilisins from Bacillus amyloliquefaciens (SEQ ID NO: 2), and Bacillus lentus (wild-type)(SEQ ID:NO 3).

#FIGS. 3A and 3B illustrate an amino acid sequence alignment of subtilisin type proteases from Bacillus amyloliquefaciens (BPN′) (SEQ ID NO: 2), Bacillus subtilis (SEQ ID NO: 5), Bacillus licheniformis (SEQ ID:NO 4) and Bacillus lentus (SEQ ID NO: 3). The symbol * denotes the absence of specific amino acid residues as compared to subtilisin BPN′.

Detailed description11,474 words

#According to the present invention, a method for reducing the allergenicity of a non-human protein is provided wherein an epitope is identified and replaced with an analogous region within a human subtilisin. In a preferred embodiment the non-human protein is an enzyme, more preferably a protease. In another preferred embodiment, the epitope replaced is a T-cell epitope.

#In another embodiment of the present invention, a method for producing the protein of the invention having reduced allergenicity is provided. Preferably, the mutant protein is prepared by modifying a DNA encoding a precursor protein so that the modified DNA encodes the mutant protein of the invention wherein an epitope is replaced with an analogous region from human subtilisin.

#In yet another embodiment of the invention, DNA sequences encoding the mutant protein, as well as expression vectors containing such DNA sequences and host cells transformed with such vectors are provided, which host cells are preferably capable of expressing such DNA to produce the mutant protein of the invention either intracellularly or extracellularly.

Facts

Application

09/060,854Filed 15 Apr 1998

Published

US 2002/0081703 A127 Jun 2002

Granted

4 Nov 2003

Term ended

15 Apr 2018

Estimated · how

20 years from earliest known nonprovisional domestic filing. Maintenance-fee lapses, terminal disclaimers and statutory extensions have not been checked. The continuity chain may be incomplete; this is not an enforceability determination. Patent term adjustment is unavailable; no adjustment was added.

Classification8 codes

C12N 9/6424
From mammals: serine endopeptidases
A61P 37/00
Drugs for immunological or allergic disorders
A61P 43/00
Drugs for specific purposes, not provided for in groups a61p1/00-a61p41/00
C12N 9/54
Derived from bacteria or Archaea: bacteria being Bacillus

Human protease and use of such protease for pharmaceutical applications and for reducing the allergenicity of non-human proteins

Life of this patent

Human protease and use of such protease for pharmaceutical applications and for reducing the allergenicity of non-human proteins

Life of this patent

Life of this patent

Abstract

Drawings

Drawings

Claims15

Description13,945 words

Drawings

Classification8 codes

Life of this patent

Description13,945 words