Methods and compositions based on shiga toxin type 1 protein

Abstract

The invention is based on the discovery of the epitope in the Stx1 protein for the 13C4 antibody. The invention features non-full length Stx1 polypeptides that include the epitope for the 13C4 monoclonal antibody epitope. The invention also features methods of producing anti-Stx1 antibodies specific for the 13C4 epitope of the Stx1 protein. Additionally, the invention features methods for treating a subject having, or at risk of developing, a Shiga toxin associated disease (e.g., hemolytic uremia syndrome and diseases associated with E. coli and S. dysenteriae infection) with a polypeptide that includes the 13C4 epitope or with an anti-Stx1 antibody developed using the methods of the invention. Furthermore, the invention features the detection of Stx1 in a sample using the antibodies developed using the methods of the invention.

What is claimed is: 
     
         1 . A method for producing an anti-Stx1 antibody that specifically binds to the 13C4 epitope of Shiga toxin type 1 (Stx1) protein, said method comprising the steps of:
 a) immunizing a mammal with a polypeptide comprising at least one amino acid sequence selected from the sequences set forth in SEQ ID NOs: 1, 2, and 3, wherein said polypeptide does not comprise full length Stx1; and   b) purifying said anti-Stx1 antibody that specifically binds to the 13C4 epitope of Stx1 protein from a tissue of said mammal or from a hybridoma made using said tissue.   
     
     
         2 . The method of  claim 1 , wherein said anti-Stx1 antibody that specifically binds to the 13C4 epitope of Stx1 protein does not bind Stx2. 
     
     
         3 . The method of  claim 1 , further comprising the step of:
 (c) screening said antibody against Stx1 and Stx2 in an in vitro neutralization assay, wherein an antibody that neutralizes at least 50% of the cytotoxic effect of Stx1 is an anti-Stx1 antibody that specifically binds to the 13C4 epitope of Stx1 protein.   
     
     
         4 . The method of  claim 1 , wherein said polypeptide comprises at least two of the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3. 
     
     
         5 . The method of  claim 1 , wherein said polypeptide comprises an amino acid sequence substantially identical to the amino acid sequence set forth in SEQ ID NO: 4. 
     
     
         6 . The method of  claim 5 , wherein said polypeptide consists of the amino acid sequence of SEQ ID NO: 4. 
     
     
         7 . The method of  claim 1 , wherein step (a) further comprises using an adjuvant. 
     
     
         8 . The method of  claim 1 , wherein said antibody is a polyclonal antibody or fragment thereof. 
     
     
         9 . The method of  claim 1 , wherein said antibody is a monoclonal antibody or fragment thereof. 
     
     
         10 . The method of  claim 1 , wherein said antibody is a modified antibody. 
     
     
         11 . The method of  claim 10 , wherein said antibody is a chimeric or humanized antibody. 
     
     
         12 . An anti-Stx1 antibody, or fragment thereof, that specifically binds to the 13C4 epitope of Shiga toxin type 1 (Stx1) protein comprising at least one of the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, wherein said antibody inhibits binding of a 13C4 antibody (ATCC CRL-1794), chimeric 13C4 antibody, or humanized 13C4 antibody to said Stx1 protein. 
     
     
         13 . The antibody of  claim 12 , wherein said antibody specifically binds to an epitope in said polypeptide that comprises the sequences set forth in SEQ ID NOs: 1, 2, and 3. 
     
     
         14 . The antibody of  claim 13 , wherein said epitope comprises the sequences set forth in SEQ ID NOs: 1, 2, and 3. 
     
     
         15 . The antibody of  claim 13 , wherein said epitope is a conformational epitope. 
     
     
         16 . The antibody, or fragment thereof, of  claim 12 , wherein said antibody is a monoclonal antibody. 
     
     
         17 . The antibody, or fragment thereof, of  claim 12 , wherein said antibody is humanized. 
     
     
         18 . The antibody, or fragment thereof, of  claim 12 , wherein said antibody is a polyclonal antibody. 
     
     
         19 . The antibody, or fragment thereof, of  claim 12 , wherein said antibody inhibits binding between Stx1 and a 13C4 antibody, wherein said 13C4 antibody has a K d  of 0.50 nM or lower. 
     
     
         20 . The antibody, or fragment thereof of  claim 19 , wherein said 13C4 antibody is a mouse, humanized, or chimeric 13C4 antibody. 
     
     
         21 . The antibody or fragment thereof of  claim 12 , wherein said antibody is IgG, IgM, IgE, IgD, or IgA. 
     
     
         22 . The antibody or fragment thereof of  claim 12 , wherein said antibody is a Fab or Fv fragment. 
     
     
         23 . A composition comprising the antibody, or fragment thereof, of  claim 12 , and a pharmaceutically acceptable carrier. 
     
     
         24 . A hybridoma cell line that produces the antibody of  claim 12 . 
     
     
         25 . The antibody of  claim 12 , wherein said antibody inhibits binding of Stx1 to Eukaryotic receptor globotriaosyl ceramide. 
     
     
         26 . A method of detecting Stx1 in a biological sample, said method comprising the steps of:
 (a) contacting said biological sample with the antibody, or fragment thereof, of  claim 12  under conditions allowing for complex formation between said antibody and Stx1; and   (b) detecting said complex in said biological sample.   
     
     
         27 . The method of  claim 26 , wherein said biological sample is from a tissue, cell, cell extract, bodily fluid, or a biopsy. 
     
     
         28 . The method of  claim 26 , wherein said complex is detected using ELISA, RIA, western blotting, immunoprecipitation, or flow cytometry. 
     
     
         29 . A method of diagnosing a Shiga toxin associated disease in subject, said method comprising the steps of:
 (a) obtaining a biological sample from said subject;   (b) contacting said biological sample with the antibody, or fragment thereof, of  claim 12  under conditions allowing for complex formation between said antibody and Stx1; and   (c) detecting said complex in said biological sample;   wherein the presence of Stx1 in said sample diagnoses the subject with a Shiga toxin associated disease.   
     
     
         30 . The method of  claim 29 , wherein said Shiga toxin associated disease is hemolytic uremic syndrome. 
     
     
         31 . The method of  claim 29 , wherein said Shiga toxin associated disease is  E. coli  or  S. dysenteriae  infection. 
     
     
         32 . An immunological test kit for detecting a Shiga toxin associated disease, said kit comprising the antibody or antigen binding portion of  claim 12  and a means for detecting said antibody. 
     
     
         33 . A method of treating or preventing a Shiga toxin associated disease in a subject, said method comprising administering to said subject a pharmaceutically effective dose of the antibody, or fragment thereof, of  claim 12 . 
     
     
         34 . The method of  claim 33 , wherein said Shiga toxin associated disease is hemolytic uremic syndrome. 
     
     
         35 . The method of  claim 33 , wherein said Shiga toxin associated disease is associated with  E. coli  or  S. dysenteriae  infection.