BoNT/A Peptides and Methods of Predicting and Reducing Immunoresistance to Botulinum Toxin Therapy

Abstract

The present invention provides BoNT/A peptides as well as methods of predicting or determining immunoresistance to botulinum toxin therapy in an individual using BoNT/A peptides.

1 . A tolerogizing composition capable of inducing a specific immunological tolerance to a botulinum toxin antigen, said tolerogizing composition comprising a tolerogizing agent conjugated to a BoNT/A peptide;
 wherein said BoNT/A peptide is at most 60 amino acids in length and comprises an amino acid sequence selected from the group consisting of amino acids 449-467 of SEQ ID NO: 1, amino acids 491-509 of SEQ ID NO: 1, amino acids 519-537 of SEQ ID NO: 1, amino acids 533-551 of SEQ ID NO: 1, amino acids 547-565 of SEQ ID NO: 1, amino acids 589-607 of SEQ ID NO: 1, amino acids 631-649 of SEQ ID NO: 1, amino acids 659-677 of SEQ ID NO: 1, amino acids 673-691 of SEQ ID NO: 1, 715-733 of SEQ ID NO: 1, amino acids 743-761 of SEQ ID NO: 1, amino acids 813-831 of SEQ ID NO: 1, amino acids 827-845 of SEQ ID NO: 1, amino acids 869-887 of SEQ ID NO: 1, amino acids 911-929 of SEQ ID NO: 1, amino acids 925-943 of SEQ ID NO: 1, amino acids 981-999 of SEQ ID NO: 1, amino acids 995-1013 of SEQ ID NO: 1, amino acids 1051-1069 of SEQ ID NO: 1, amino acids 1121-1139 of SEQ ID NO: 1, amino acids 1135-1153 of SEQ ID NO: 1, amino acids 1177-1195 of SEQ ID NO: 1, amino acids 1247-1265 of SEQ ID NO: 1, amino acids 1275-1296 of SEQ ID NO: 1, a conservative BoNT/A amino acid sequence variant thereof, and a tolerogenic BoNT/A amino acid sequence fragment thereof;   wherein said conservative BoNT/A amino acid sequence variant comprises 1-4 conservative amino acid substitutions to amino acids 449-467 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 491-509 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 519-537 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 533-551 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 547-565 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 589-607 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 631-649 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 659-677 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 673-691 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 715-733 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 743-761 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 813-831 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 827-845 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 869-887 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 911-929 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 925-943 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 981-999 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 995-1013 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 1051-1069 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 1121-1139 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 1135-1153 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 1177-1195 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 1247-1265 of SEQ ID NO: 1, or 1-4 conservative amino acid substitutions to amino acids 1275-1296 of SEQ ID NO: 1; and   wherein said tolerogenic BoNT/A amino acid sequence fragment comprises at least three consecutive amino acids of 449-467 of SEQ ID NO: 1, at least three consecutive amino acids of 491-509 of SEQ ID NO: 1, at least three consecutive amino acids of 519-537 of SEQ ID NO: 1, at least three consecutive amino acids of 533-551 of SEQ ID NO: 1, at least three consecutive amino acids of 547-565 of SEQ ID NO: 1, at least three consecutive amino acids of 589-607 of SEQ ID NO: 1, at least three consecutive amino acids of 631-649 of SEQ ID NO: 1, at least three consecutive amino acids of 659-677 of SEQ ID NO: 1, at least three consecutive amino acids of 673-691 of SEQ ID NO: 1, at least three consecutive amino acids of 715-733 of SEQ ID NO: 1, at least three consecutive amino acids of 743-761 of SEQ ID NO: 1, at least three consecutive amino acids of 813-831 of SEQ ID NO: 1, at least three consecutive amino acids of 827-845 of SEQ ID NO: 1, at least three consecutive amino acids of 869-887 of SEQ ID NO: 1, at least three consecutive amino acids of 911-929 of SEQ ID NO: 1, at least three consecutive amino acids of 925-943 of SEQ ID NO: 1, at least three consecutive amino acids of 981-999 of SEQ ID NO: 1, at least three consecutive amino acids of 995-1013 of SEQ ID NO: 1, at least three consecutive amino acids of 1051-1069 of SEQ ID NO: 1, at least three consecutive amino acids of 1121-1139 of SEQ ID NO: 1, at least three consecutive amino acids of 1135-1153 of SEQ ID NO: 1, at least three consecutive amino acids of 1177-1195 of SEQ ID NO: 1, at least three consecutive amino acids of 1247-1265 of SEQ ID NO: 1 or at least three consecutive amino acids of 1275-1296 of SEQ ID NO: 1.   
     
     
         2 . The tolerogizing composition of  claim 1 , wherein said BoNT/A peptide comprises an amino acid sequence selected from the group consisting of amino acids 449-467 of SEQ ID NO: 1, amino acids 491-509 of SEQ ID NO: 1, amino acids 519-537 of SEQ ID NO: 1, amino acids 533-551 of SEQ ID NO: 1, amino acids 547-565 of SEQ ID NO: 1, amino acids 589-607 of SEQ ID NO: 1, amino acids 631-649 of SEQ ID NO: 1, amino acids 659-677 of SEQ ID NO: 1, amino acids 673-691 of SEQ ID NO: 1, 715-733 of SEQ ID NO: 1, amino acids 743-761 of SEQ ID NO: 1, amino acids 813-831 of SEQ ID NO: 1, amino acids 827-845 of SEQ ID NO: 1, amino acids 869-887 of SEQ ID NO: 1, amino acids 911-929 of SEQ ID NO: 1, amino acids 925-943 of SEQ ID NO: 1, amino acids 981-999 of SEQ ID NO: 1, amino acids 995-1013 of SEQ ID NO: 1, amino acids 1051-1069 of SEQ ID NO: 1, amino acids 1121-1139 of SEQ ID NO: 1, amino acids 1135-1153 of SEQ ID NO: 1, amino acids 1177-1195 of SEQ ID NO: 1, amino acids 1247-1265 of SEQ ID NO: 1, amino acids 1275-1296 of SEQ ID NO: 1. 
     
     
         3 . The tolerogizing composition of  claim 1 , wherein said BoNT/A peptide comprises an amino acid sequence selected from the group consisting of amino acids 547-565 of SEQ ID NO: 1, amino acids 589-607 of SEQ ID NO: 1, amino acids 659-677 of SEQ ID NO: 1, amino acids 743-761 of SEQ ID NO: 1, amino acids 869-887 of SEQ ID NO: 1, amino acids 925-943 of SEQ ID NO: 1, amino acids 981-999 of SEQ ID NO: 1, amino acids 995-1013 of SEQ ID NO: 1, amino acids 1051-1069 of SEQ ID NO: 1, amino acids 1121-1139 of SEQ ID NO: 1, amino acids 1135-1153 of SEQ ID NO: 1, amino acids 1177-1195 of SEQ ID NO: 1, amino acids 1275-1296 of SEQ ID NO: 1, a conservative BoNT/A amino acid sequence variant thereof, and a tolerogenic BoNT/A amino acid sequence fragment thereof;
 wherein said conservative BoNT/A amino acid sequence variant comprises 1-4 conservative amino acid substitutions to amino acids 547-565 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 589-607 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 659-677 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 743-761 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 869-887 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 925-943 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 981-999 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 995-1013 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 1051-1069 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 1121-1139 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 1135-1153 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 1177-1195 of SEQ ID NO: 1, or 1-4 conservative amino acid substitutions to amino acids 1275-1296 of SEQ ID NO: 1; and   wherein said tolerogenic BoNT/A amino acid sequence fragment comprises at least three consecutive amino acids of 547-565 of SEQ ID NO: 1, at least three consecutive amino acids of 589-607 of SEQ ID NO: 1, at least three consecutive amino acids of 659-677 of SEQ ID NO: 1, at least three consecutive amino acids of 743-761 of SEQ ID NO: 1, at least three consecutive amino acids of 869-887 of SEQ ID NO: 1, at least three consecutive amino acids of 925-943 of SEQ ID NO: 1, at least three consecutive amino acids of 981-999 of SEQ ID NO: 1, at least three consecutive amino acids of 995-1013 of SEQ ID NO: 1, at least three consecutive amino acids of 1051-1069 of SEQ ID NO: 1, at least three consecutive amino acids of 1121-1139 of SEQ ID NO: 1, at least three consecutive amino acids of 1135-1153 of SEQ ID NO: 1, at least three consecutive amino acids of 1177-1195 of SEQ ID NO: 1 or at least three consecutive amino acids of 1275-1296 of SEQ ID NO: 1.   
     
     
         4 . The tolerogizing composition of  claim 3 , wherein said BoNT/A peptide comprises an amino acid sequence selected from the group consisting of amino acids 547-565 of SEQ ID NO: 1, amino acids 589-607 of SEQ ID NO: 1, amino acids 659-677 of SEQ ID NO: 1, amino acids 743-761 of SEQ ID NO: 1, amino acids 869-887 of SEQ ID NO: 1, amino acids 925-943 of SEQ ID NO: 1, amino acids 981-999 of SEQ ID NO: 1, amino acids 995-1013 of SEQ ID NO: 1, amino acids 1051-1069 of SEQ ID NO: 1, amino acids 1121-1139 of SEQ ID NO: 1, amino acids 1135-1153 of SEQ ID NO: 1, amino acids 1177-1195 of SEQ ID NO: 1, amino acids 1275-1296 of SEQ ID NO: 1. 
     
     
         5 . The tolerogizing composition of  claim 1 , wherein said BoNT/A peptide comprises an amino acid sequence selected from the group consisting of amino acids 547-565 of SEQ ID NO: 1, amino acids 869-887 of SEQ ID NO: 1, amino acids 925-943 of SEQ ID NO: 1, amino acids 981-999 of SEQ ID NO: 1, amino acids 995-1013 of SEQ ID NO: 1, amino acids 1051-1069 of SEQ ID NO: 1, amino acids 1121-1139 of SEQ ID NO: 1, amino acids 1275-1296 of SEQ ID NO: 1, a conservative BoNT/A amino acid sequence variant thereof, and a tolerogenic BoNT/A amino acid sequence fragment thereof; wherein said conservative BoNT/A amino acid sequence variant comprises 1-4 conservative amino acid substitutions to amino acids 547-565 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 869-887 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 925-943 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 981-999 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 995-1013 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 1051-1069 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 1121-1139 of SEQ ID NO: 1, or 1-4 conservative amino acid substitutions to amino acids 1275-1296 of SEQ ID NO: 1; and
 wherein said tolerogenic BoNT/A amino acid sequence fragment comprises at least three consecutive amino acids of 547-565 of SEQ ID NO: 1, at least three consecutive amino acids of 869-887 of SEQ ID NO: 1, at least three consecutive amino acids of 925-943 of SEQ ID NO: 1, at least three consecutive amino acids of 981-999 of SEQ ID NO: 1, at least three consecutive amino acids of 995-1013 of SEQ ID NO: 1, at least three consecutive amino acids of 1051-1069 of SEQ ID NO: 1, at least three consecutive amino acids of 1121-1139 of SEQ ID NO: 1 or at least three consecutive amino acids of 1275-1296 of SEQ ID NO: 1.   
     
     
         6 . The tolerogizing composition of  claim 5 , wherein said BoNT/A peptide comprises an amino acid sequence selected from the group consisting of amino acids 547-565 of SEQ ID NO: 1, amino acids 869-887 of SEQ ID NO: 1, amino acids 925-943 of SEQ ID NO: 1, amino acids 981-999 of SEQ ID NO: 1, amino acids 995-1013 of SEQ ID NO: 1, amino acids 1051-1069 of SEQ ID NO: 1, amino acids 1121-1139 of SEQ ID NO: 1, amino acids 1275-1296 of SEQ ID NO: 1. 
     
     
         7 . The tolerogizing composition of  claim 1 , wherein said BoNT/A peptide comprises an amino acid sequence selected from the group consisting of amino acids 981-999 of SEQ ID NO: 1, amino acids 1051-1069 of SEQ ID NO: 1, amino acids 1121-1139 of SEQ ID NO: 1 and amino acids 1275-1296 of SEQ ID NO: 1. 
     
     
         8 . The tolerogizing composition of  claim 1 , wherein said BoNT/A peptide comprises the amino acid sequence of amino acids 981-999 of SEQ ID NO: 1, a conservative BoNT/A amino acid sequence variant thereof, and an immunoreactive BoNT/A amino acid sequence fragment thereof;
 wherein said conservative BoNT/A amino acid sequence variant comprises 1-4 conservative amino acid substitutions to amino acids 981-999 of SEQ ID NO: 1; and   wherein said tolerogenic BoNT/A amino acid sequence fragment comprises at least three consecutive amino acids of 981-999 of SEQ ID NO: 1.   
     
     
         9 . The tolerogizing composition of  claim 1 , wherein said BoNT/A peptide comprises the amino acid sequence of amino acids 1051-1069 of SEQ ID NO: 1, a conservative BoNT/A amino acid sequence variant thereof, and an immunoreactive BoNT/A amino acid sequence fragment thereof;
 wherein said conservative BoNT/A amino acid sequence variant comprises 1-4 conservative amino acid substitutions to amino acids 1051-1069 of SEQ ID NO: 1; and   wherein said tolerogenic BoNT/A amino acid sequence fragment comprises at least three consecutive amino acids of 1051-1069 of SEQ ID NO: 1.   
     
     
         10 . The tolerogizing composition of  claim 1 , wherein said BoNT/A peptide comprises the amino acid sequence of amino acids 1121-1139 of SEQ ID NO: 1, a conservative BoNT/A amino acid sequence variant thereof, and an immunoreactive BoNT/A amino acid sequence fragment thereof;
 wherein said conservative BoNT/A amino acid sequence variant comprises 1-4 conservative amino acid substitutions to amino acids 1121-1139 of SEQ ID NO: 1; and   wherein said tolerogenic BoNT/A amino acid sequence fragment comprises at least three consecutive amino acids of 1121-1139 of SEQ ID NO: 1.   
     
     
         11 . The tolerogizing composition of  claim 1 , wherein said BoNT/A peptide comprises the amino acid sequence of amino acids 1275-1296 of SEQ ID NO: 1, a conservative BoNT/A amino acid sequence variant thereof, and an immunoreactive BoNT/A amino acid sequence fragment thereof;
 wherein said conservative BoNT/A amino acid sequence variant comprises 1-4 conservative amino acid substitutions to amino acids 1275-1296 of SEQ ID NO: 1; and   wherein said tolerogenic BoNT/A amino acid sequence fragment comprises at least three consecutive amino acids of 1275-1296 of SEQ ID NO: 1.   
     
     
         12 . The tolerogizing composition of  claim 1 , wherein said BoNT/A peptide is at most 40 amino acids in length. 
     
     
         13 . The tolerogizing composition of  claim 1 , wherein said BoNT/A peptide is at most 25 amino acids in length. 
     
     
         14 . The tolerogizing composition of  claim 1 , wherein said tolerogizing agent is a polyethylene glycol. 
     
     
         15 . The tolerogizing composition of  claim 1 , wherein said tolerogizing agent is a monomethoxypolyethylene glycol. 
     
     
         16 . The tolerogizing composition of  claim 1 , wherein said tolerogizing agent is a polyvinyl alcohol. 
     
     
         17 . A tolerogizing method of preventing or reducing immunoresistance to botulinum toxin therapy in an individual, the method comprising the step of administering to said individual a tolerogizing composition according to  claim 1 ; wherein administration of said tolerogizing composition decreases an immunological response to a botulinum toxin antigen. 
     
     
         18 . The tolerogizing method of  claim 17 , wherein said tolerogizing composition is administered to said individual prior to receiving botulinum toxin therapy, during a course of botulinum toxin therapy, or after onset of immunoresistance to botulinum toxin therapy. 
     
     
         19 . The tolerogizing method of  claim 17 , wherein said individual is at increased risk for immunoresistance to a botulinum toxin therapy. 
     
     
         20 . The tolerogizing method of  claim 17 , wherein said botulinum toxin antigen is a botulinum toxin type A antigen. 
     
     
         21 . The tolerogizing method of  claim 17 , wherein said decreased immunological response is a decrease in a T-cell response. 
     
     
         22 . The tolerogizing method of  claim 21 , wherein said decrease T-cell response is a decrease in T-cell proliferation or a decrease in T-cell cytokine secretion. 
     
     
         23 . The tolerogizing method of  claim 17 , wherein said decreased immunological response is a decrease in an anti-botulinum toxin antibody titer. 
     
     
         24 . The tolerogizing method of  claim 23 , wherein said anti-botulinum toxin antibody titer is an anti-botulinum toxin type A antibody titer. 
     
     
         25 . The tolerogizing method of  claim 17 , wherein said tolerogizing composition is prepared as a pharmaceutical composition.