Identification and modification of immunodominant epitopes in polypeptides

Abstract

The invention provides for methods of modifying immunodominant epitopes on polypeptides, preferably polypeptides intended for therapeutic use. Knowledge of immunodominant epitopes prior to clinical use of polypeptides would be useful to design and engineer less immunogenic molecules. The invention provides for methods of identifying immunodominant epitopes and modifying an immunodominant epitope to reduce the immune response to the polypeptide while still retaining a substantial therapeutic activity of the polypeptide. The modified polypeptides are useful therapeutically.

1 - 22 . (cancelled)  
     
     
         23 . A modified therapeutic polypeptide having a modification only in an immunodominant epitope, wherein the modification reduces the immune response to the polypeptide while retaining a substantial therapeutic activity of the polypeptide.  
     
     
         24 . A modified human recombinant thrombopoietin comprising an amino acid sequence of amino acids 1 to 311 of FIG. 7.  
     
     
         25 . A modified human recombinant thrombopoietin comprising an amino acid sequence of amino acids 1 to 317 of FIG. 7.  
     
     
         26 . A modified polypeptide according to  claim 23 , wherein the polypeptide is human recombinant thrombopoietin with at least one modification of amino acids 312 to 332.  
     
     
         27 . A modified polypeptide according to  claim 26 , wherein the modification is in amino acids 318 to 332.  
     
     
         28 . A modified polypeptide according to  claim 23 , wherein the modification is a deletion, substitution or insertion of at least one amino acid in the immunodominant epitope.  
     
     
         29 . A modified polypeptide according to  claim 23 , wherein the modification is a chemical modification of at least one amino acid in the immunodominant epitope, wherein the chemical modification is N-glycosylation or pegylation.  
     
     
         30 . A method of modifying a nucleic acid encoding a modified polypeptide comprising: 
 a) identifying at least one immunodominant epitope in the polypeptide;    b) providing an isolated nucleic acid sequence encoding the polypeptide; and    c) modifying the isolated nucleic acid to encode a modified polypeptide wherein the modified polypeptide has at least one change in the immunodominant epitope and wherein the change reduces an immune response to the polypeptide while still retaining a substantial therapeutic activity of the polypeptide.    
     
     
         31 . The method according to  claim 30 , further comprising transforming a host cell with the modified isolated nucleic acid.  
     
     
         32 . An isolated nucleic acid encoding a modified human thrombopoietin whereby the modified thrombopoietin has a modification only of amino acids 312 to 332.  
     
     
         33 . A pharmaceutical composition comprising the modified polypeptide of  claim 23  and a pharmaceutically acceptable carrier.  
     
     
         34 . A method of treating thrombocytopenia by administering a modified recombinant human thrombopoietin, wherein the human recombinant thrombopoietin has at least one modification only of amino acids 312-332, wherein the modification reduces an immune response to human thrombopoietin while still retaining a substantial therapeutic activity of human thrombopoietin.  
     
     
         35 . A modified native thrombopoietin comprising a modification in an immunodominant epitope of thrombopoeitin, wherein the modification reduces the immune response to thrombopoietin while retaining a substantial therapeutic activity of thrombopoietin.  
     
     
         36 . The modified thrombopoietin of  claim 35 , wherein the immunodominant epitope has an amino acid sequence of amino acids 312 to 332 of native thrombopoietin (SEQ ID NO: 2).  
     
     
         37 . The modified thrombopoietin of  claim 35 , wherein the epitope has an amino acid sequence of amino acids 318 to 332 of native thrombopoietin (SEQ ID NO: 1).  
     
     
         38 . The modified thrombopoietin of  claim 35 , wherein the modification comprises a deletion of one or more of the amino acids of the immunodominant epitope.  
     
     
         39 . The modified thrombopoietin of  claim 38 , wherein the modification consists of a deleting the amino acid sequence of SEQ ID NO: 1.  
     
     
         40 . The modified thrombopoietin of  claim 36 , wherein the modification comprises an amino acid substitution of an amino acid in the immunodominant epitope.  
     
     
         41 . The modified thrombopoietin of  claim 35 , wherein the modification comprises an insertion of at least one amino acid in the immunodominant epitope.  
     
     
         42 . The modified thrombopoietin of  claim 36 , wherein the modification comprises N-glycosylation or N-pegylation of at least one amino acid in the immunodominant epitope.  
     
     
         43 . A composition comprising a modified thrombopoietin of  claim 35  and a physiologically acceptable carrier.  
     
     
         44 . An isolated nucleic acid encoding a modified thrombopoietin according to  claim 35 .  
     
     
         45 . A vector comprising an isolated nucleic acid of  claim 44 .  
     
     
         46 . A host cell comprising a vector of  claim 45 .  
     
     
         47 . A method for producing a modified thrombopoietin comprising culturing a host cell of  claim 46  under conditions suitable for production of a modified thrombopoietin, and recovering the modified thrombopoietin from the cell culture.  
     
     
         48 . A method of treating thrombocytopenia comprising administering an effective amount of a modified thrombopoietin of  claim 35  to a subject.