Hypoallergenic hybrid proteins of major group 1 and 2 mite allergens for use in the treatment of allergies

Abstract

The present invention refers to recombinant AND molecules coding to hybrids polypeptides of different allergens from D. pteronyssinus useful for the prevention and treatment of allergies, particularly allergies caused by mites. Specifically, the invention describes hybrid proteins composed of fragments of allergens Derp p 1 y Derp p 2 with hypoallergenic characteristics and maintain their immunogenic capacity, being particularly useful for the treatment of allergy. The invention also describes the production methods of these polypeptides in heterologous expression systems. Besides, the invention describes efficient purification methods of these hybrid proteins.

1 . A polypeptide with reduced allergenic activity comprising fragments of the amino acid sequences from the group 1 and group 2 mite allergens in which the sequences lack one or more IgE antibody binding epitopes, said fragments being at least 50 amino acid residues in length. 
     
     
         2 . A polypeptide according to  claim 1  in which at least one of the IgE antibody binding epitopes in the group 2 allergen sequence has been deleted by elimination of at least one disulphide bridge. 
     
     
         3 . A polypeptide according to  claim 2  in which at least one disulphide bridge has been deleted by substitution of at least one of the cysteine residues forming the disulphide bridge with an alternative amino acid residue. 
     
     
         4 . A polypeptide according to  claim 3  in which at least one of the cysteine residues at the following positions of the full length mature group 2 allergen protein has been substituted: 8 and 119. 
     
     
         5 . A polypeptide according to  claim 4  in which at least one of the substituted cysteine residues has been substituted with a serine residue. 
     
     
         6 . A polypeptide according to  claim 2  in which at least one disulphide bridge has been deleted by disruption of the sequence of the group 2 allergen by insertion of an additional amino acid sequence. 
     
     
         7 . A polypeptide according to  claim 6  in which the amino acid sequence of the group 2 allergen is disrupted by insertion of a fragment of the amino acid sequence of the group 1 allergen. 
     
     
         8 . A polypeptide according to  claim 7  in which the fragment of the amino acid sequence of the group 1 allergen is the fragment of residues 5 to 222 of the mature group 1 allergen. 
     
     
         9 . A polypeptide according to  claim 1 , characterised in that the group 1 mite allergen is the  D. pteronyssinus  allergen, Der p 1, and the group 2 mite allergen is the  D. pteronyssinus  allergen, Der p 2. 
     
     
         10 . A polypeptide comprising an amino acid sequence of at least 70% homology to SEQ ID NO:2. 
     
     
         11 . A polypeptide comprising an amino acid sequence of at least 70% homology to SEQ ID NO:4. 
     
     
         12 . A polynucleotide comprising the nucleotide sequence coding for the polypeptide according to  claim 1 . 
     
     
         13 . A polynucleotide comprising a nucleotide sequence of at least 70% homology to SEQ ID NO:1. 
     
     
         14 . A polynucleotide comprising a nucleotide sequence of at least 70% homology to SEQ ID NO:3. 
     
     
         15 . An expression vector comprising the polynucleotide sequence of  claim 12 . 
     
     
         16 . A host cell transformed with an expression vector according to  claim 14 . 
     
     
         17 . A host cell according to  claim 16 , said cell being a eukaryotic cell. 
     
     
         18 . A host cell according to  claim 16 , said cell being a prokaryotic cell. 
     
     
         19 . A host cell according to  claim 18 , which belongs to the genus  E. coli.    
     
     
         20 . A method for producing a polypeptide, comprising culturing a host cell of  claim 16  and isolating and purifying the polypeptide produced by the host cell. 
     
     
         21 . A polypeptide according to  claim 1 , or expression vector of  claim 15  for use in the treatment or prevention of allergy. 
     
     
         22 . A use of polypeptide of  claim 1 , or the expression vector of  claim 15  for the preparation of a composition for use in the treatment or prevention of allergy. 
     
     
         23 . A pharmaceutical preparation comprising the polypeptide of  claim 1  or expression vector of  claim 15  and optionally, a pharmacologically acceptable excipient or adjuvant. 
     
     
         24 . A method of treating or preventing an allergy comprising administering a pharmaceutical preparation as claimed in  claim 23  to an individual in need thereof. 
     
     
         25 . A pharmaceutical preparation according to  claim 21  in the form of a freeze-dried, laminated, solution, suspension or emulsion. 
     
     
         26 . A pharmaceutical preparation according to  claim 21  for subcutaneous, sublingual, oral, nasal, rectal, topical, inhaled or parenteral administration. 
     
     
         27 . A polynucleotide comprising a nucleotide sequence encoding SEQ ID NO:2 or a polypeptide having at least 95% homology to SEQ ID NO:2. 
     
     
         28 . The polynucleotide of  claim 27 , comprising a nucleotide sequence encoding SEQ ID NO:2. 
     
     
         29 . The polynucleotide of  claim 27 , comprising a nucleotide sequence designated as SEQ ID NO:1. 
     
     
         30 . An expression vector comprising a polynucleotide of  claim 27 . 
     
     
         31 . The expression vector of  claim 30 , comprising a polynucleotide of  claim 29 . 
     
     
         32 . A host cell transformed with the expression vector of  claim 30 . 
     
     
         33 . The host cell of  claim 32 , said cell being a eukaryotic cell. 
     
     
         34 . The host cell of  claim 32 , said cell being a prokaryotic cell. 
     
     
         35 . The host cell of  claim 34 , wherein said cell is of the genus  E. coli.    
     
     
         36 . A method for producing a polypeptide comprising an amino acid sequence designated as SEQ ID NO:2; said method comprising culturing the host cell of  claim 32 , and isolating and purifying said polypeptide from the culture. 
     
     
         37 . A method for prophylaxis or treatment of an allergic reaction to mite allergen, comprising administering to a patient at risk of or suffering from said reaction, a therapeutically effective amount of the expression vector of  claim 30 . 
     
     
         38 . A polypeptide with reduced allergenic activity comprising fragments of the amino acid sequences from the group 1 and group 2 mite allergens in which the sequences lack one or more IgE antibody binding epitopes, said fragments being at least 50 amino acid residues in length, wherein at least one of the IgE antibody binding epitopes in the group 2 allergen sequence has been deleted by substitution of at least one of the cysteine residues at the positions 8 and 119 of the full length mature group 2 allergen protein with a serine residue. 
     
     
         39 . A method for prophylaxis or treatment of an allergic reaction to mite allergen, comprising administering to a patient at risk of or suffering from said reaction, a therapeutically effective amount of the polypeptide of  claim 38 . 
     
     
         40 . A polynucleotide comprising a nucleotide sequence encoding a polypeptide of  claim 38 . 
     
     
         41 . An expression vector comprising a polynucleotide of  claim 40 . 
     
     
         42 . A method for prophylaxis or treatment of an allergic reaction to mite allergen, comprising administering to a patient at risk of or suffering from said reaction, a therapeutically effective amount of the expression vector of  claim 41 .