Novel bee venom polypeptides and methods of use thereof

Abstract

The present invention provides novel protein named Api m 6. Api m 6 was identified in bee venom and is found in four isoforms, each about 8 kD. The purified Api m 6 can be used to modulate immune responses, e.g., in individuals hypersensitive to bee venom.

What is claimed is:  
     
         1 . A substantially pure polypeptide comprising an amino acid sequence at least 70% identical to the amino acid sequence of SEQ ID NO:1  
     
     
         2 . The polypeptide of  claim 1 , wherein said polypeptide comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 1.  
     
     
         3 . The polypeptide of  claim 1 , wherein said polypeptide comprises the amino acid sequence of SEQ ID NO: 1.  
     
     
         4 . The polypeptide of  claim 1 , wherein said polypeptide comprises an amino acid sequence at least 70% identical to the amino acid sequence of SEQ ID NO: 2.  
     
     
         5 . The polypeptide of  claim 1 , wherein said polypeptide comprises an amino acid sequence at least 70% identical to the amino acid sequence of SEQ ID NO: 3.  
     
     
         6 . The polypeptide of  claim 1 , wherein said polypeptide comprises an amino acid sequence at least 70% identical to the amino acid sequence of SEQ ID NO: 4.  
     
     
         7 . The polypeptide of  claim 1 , wherein said polypeptide is an  Apis mellifera  bee venom protein.  
     
     
         8 . The polypeptide of  claim 1 , wherein said polypeptide is glycosylated.  
     
     
         9 . The polypeptide of  claim 1 , wherein said polypeptide binds to a human IgE antibody.  
     
     
         10 . The polypeptide of  claim 1 , wherein said polypeptide stimulates T-cell proliferation.  
     
     
         11 . The polypeptide of  claim 1 , wherein said polypeptide binds to the monoclonal antibody 5E11 (Accession No.______).  
     
     
         12 . An antibody which binds to the polypeptide of  claim 1 .  
     
     
         13 . The antibody of  claim 12 , wherein said antibody is a monoclonal antibody.  
     
     
         14 . The antibody of  claim 12 , wherein said antibody is a polyclonal antibody.  
     
     
         15 . The antibody of  claim 12 , wherein said antibody is a humanized antibody.  
     
     
         16 . The antibody of  claim 13 , wherein said antibody binds to the same epitope to which the monoclonal antibody produced by the hybridoma 5E11 (Accession No______) binds.  
     
     
         17 . The antibody of  claim 16 , wherein said antibody is the antibody produced by the hybridoma 5E11 (Accession No.______).  
     
     
         18 . A hybridoma producing an antibody which binds to the same epitope to which the monoclonal antibody produced by the 5E11 (Accession No. ______) binds.  
     
     
         19 . The hybridoma of  claim 18 , wherein said hybridoma is hybridoma 5E11 (Accession No. ______).  
     
     
         20 . A composition comprising polypeptide fragments of the Api m 6 protein, wherein said polypeptide fragments are between 6-72 amino acids in length.  
     
     
         21 . The composition of  claim 20 , wherein said polypeptide fragments are between 20-100 amino acids in length.  
     
     
         22 . The composition of  claim 20 , wherein said polypeptide fragments are between 30-70 amino acids in length.  
     
     
         23 . The composition of  claim 20 , wherein said polypeptide fragments are between 40-60 amino acids in length  
     
     
         24 . The composition of  claim 20 , wherein at least one polypeptide in the composition has an amino acid sequence that overlaps by at least 3 amino acids with at least one other polypeptide in the composition  
     
     
         25 . The composition of  claim 20 , wherein the polypeptide fragments of Api m 6 overlap by between 5 and 10 amino acids.  
     
     
         26 . The composition of  claim 20 , wherein the composition comprises of a set of polypeptide fragments that map the total length of the Api m 6 protein.  
     
     
         27 . A pharmaceutical composition comprising the polypeptide of  claim 1  and a pharmaceutically acceptable carrier.  
     
     
         28 . The pharmaceutical composition of  claim 27 , further comprising a second bee venom polypeptide.  
     
     
         29 . The pharmaceutical composition of  claim 28 , wherein said second bee venom polypeptide is selected from the group comprising phospholipase A 2 , hyaluronidase, allergen C, mellitin, adolapin, minimine, acid phosphatase, protease inhibitor, and glycosylated IgE-binding proteins, or analogs or derivatives thereof.  
     
     
         30 . A method of modulating an immune response, said method comprising administering the polypeptide of  claim 1  to a subject in need thereof in an amount sufficient to inhibit an immune reaction by the subject against said polypeptide.  
     
     
         31 . The method of  claim 30 , further comprising administering a second bee venom polypeptide to said subject.  
     
     
         32 . The method of  claim 31 , wherein the second bee venom polypeptide is phospholipase A 2 , hyaluronidase, allergen C, mellitin, adolapin, minimine, acid phosphatase, protease inhibitor, and acid phosphatase, and glycosylated IgE-binding proteins, or analogs or derivatives thereof.  
     
     
         33 . A method of identifying an individual at risk for bee venom hypersensitivity, the method comprising: 
 administering to said individual the polypeptide of  claim 1;  and    measuring an immune response raised against said polypeptide, wherein a detectable immune response indicates that said individual is at risk for bee venom hypersensitivity.    
     
     
         34 . The method of  claim 33 , wherein said polypeptide is administered to said subject intradermally.  
     
     
         35 . The method of  claim 34 , wherein said polypeptide is administered at a concentration of less than about 1 μg/ml.  
     
     
         36 . A method of purifying the polypeptide of  claim 1 , the method comprising: 
 providing a cell expressing the polypeptide of  claim 1;     contacting said cell with an antibody which binds to polypeptide comprising an amino acid sequence at least 70% identical to the amino acid sequence of SEQ ID NO:1 to form a polypeptide-antibody complex;    isolating said antibody-polypeptide complex; and    recovering said polypeptide from said antibody-polypeptide complex, thereby purifying said polypeptide.    
     
     
         37 . A kit comprising, in one or more containers, a substance selected from the group consisting of: an Api m 6 polypeptide, a overlapping polypeptide fragments of an Api m 6 polypeptide, an antibody against said Api m 6 polypeptide.