Therapeutic Monoclonal Antibodies that Neutralize Botulinum Neurotoxins

Abstract

This invention provides antibodies that specifically bind to and neutralize botulinum neurotoxin type A (BoNT/A) and the epitopes bound by those antibodies. The antibodies and derivatives thereof and/or other antibodies that specifically bind to the neutralizing epitopes provided herein can be used to neutralize botulinum neurotoxin and are therefore also useful in the treatment of botulism.

1 - 83 . (canceled) 
     
     
         84 . An isolated antibody that specifically binds Botulinum neurotoxin (BoNT), or an antigen-binding fragment thereof, wherein said antibody, or an antigen-binding fragment thereof, comprises:
 a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:81;   b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:83;   c) a VL CDR3 comprising the amino acid sequence of SEQ ID NO:113; and   d) VH CDRs comprising:
 i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:25; 
 ii) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:27; and 
 ii) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:57; 
   or
 i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:29; 
 ii) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:31; and 
 ii) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:60. 
   
     
     
         85 . The isolated antibody, or an antigen-binding fragment thereof, of  claim 84 , wherein said antibody comprises:
 a) a VL FR1 comprising the amino acid sequence of SEQ ID NO:80;   b) a VL FR2 comprising the amino acid sequence of SEQ ID NO:82;   c) a VL FR3 comprising the amino acid sequence of SEQ ID NO:112; and   d) a VL FR4 comprising the amino acid sequence of SEQ ID NO:114;   
     
     
         86 . The isolated antibody, or an antigen-binding fragment thereof, of  claim 84 , wherein said antibody comprises:
 a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:25;   b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:27; and   c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:57.   
     
     
         87 . The isolated antibody, or an antigen-binding fragment thereof, of  claim 86 , wherein said antibody comprises:
 a) a VH FR1 comprising the amino acid sequence of SEQ ID NO:24;   b) a VH FR2 comprising the amino acid sequence of SEQ ID NO:26;   c) a VH FR3 comprising the amino acid sequence of SEQ ID NO:56; and   d) a VH FR4 comprising the amino acid sequence of SEQ ID NO:58;   
     
     
         88 . The isolated antibody, or an antigen-binding fragment thereof, of  claim 84 , wherein said antibody comprises:
 a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:29;   b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:31; and   c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:60.   
     
     
         89 . The isolated antibody, or an antigen-binding fragment thereof, of  claim 88 , wherein said antibody comprises:
 a) a VH FR1 comprising the amino acid sequence of SEQ ID NO:28;   b) a VH FR2 comprising the amino acid sequence of SEQ ID NO:30;   c) a VH FR3 comprising the amino acid sequence of SEQ ID NO:59; and   d) a VH FR4 comprising the amino acid sequence of SEQ ID NO:61;   
     
     
         90 . The antibody, or an antigen-binding fragment thereof, of  claim 84 , wherein said antibody is a single chain Fv (scFv). 
     
     
         91 . The antibody, or an antigen-binding fragment thereof, of  claim 84 , wherein said antibody is an IgG. 
     
     
         92 . The antibody, or an antigen-binding fragment thereof, of  claim 84 , wherein said antigen-binding fragment is a Fab. 
     
     
         93 . The antibody, or an antigen-binding fragment thereof, of  claim 84 , wherein said antigen-binding fragment is a (Fab′)2. 
     
     
         94 . The antibody, or an antigen-binding fragment thereof, of  claim 84 , wherein said antibody is a (scFv′)2. 
     
     
         95 . A composition comprising at least two different antibodies, or antigen-binding fragments thereof, for a Botulinum neurotoxin serotype, wherein a first antibody, or an antigen-binding fragment thereof, of said two different antibodies, or antigen-binding fragments, comprises:
 a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:81;   b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:83;   c) a VL CDR3 comprising the amino acid sequence of SEQ ID NO:113; and   d) VH CDRs comprising:
 i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:25; 
 ii) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:27; and 
 ii) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:57. 
   or
 i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:29; 
 ii) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:31; and 
 ii) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:60. 
   
     
     
         96 . The composition of  claim 95 , wherein said first antibody comprises:
 a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:25;   b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:27; and   c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:57.   
     
     
         97 . The composition of  claim 95 , wherein said first antibody comprises:
 a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:29;   b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:31; and   c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:60.   
     
     
         98 . The composition of  claim 95 , wherein a second antibody, or an antigen-binding fragment thereof, of said two different antibodies, or antigen-binding fragments thereof, is selected from the group consisting of RAZ1, CR1, CR2, ING1, ING2, 1D11, 2G11, 3D12, AND 5G4. 
     
     
         99 . A method of binding Botulinum neurotoxin (BoNT) in a mammal, said method comprising administering to said mammal an antibody, or an antigen-binding fragment thereof, comprising:
 a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:81;   b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:83;   c) a VL CDR3 comprising the amino acid sequence of SEQ ID NO:113; and   d) VH CDRs comprising:
 i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:25; 
 ii) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:27; and 
 ii) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:57. 
   or
 i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:29; 
 ii) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:31; and 
 ii) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:60. 
   
     
     
         100 . The method of  claim 99 , wherein said antibody comprises:
 a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:25;   b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:27; and   c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:57.   
     
     
         101 . The method of  claim 99 , wherein said antibody comprises:
 a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:29;   b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:31; and   c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:60.   
     
     
         102 . The method of  claim 99 , further comprising administering a second antibody selected from the group consisting of RAZ1, CR1, CR2, ING1, ING2, 1D11, 2G11, 3D12, AND 5G4. 
     
     
         103 . A kit comprising:
 a composition according to any of  claims 95 ,  96 ,  97  and  98 ; and   instructional materials teaching the use of said composition to neutralize a Botulinum neurotoxin.   
     
     
         104 . A method of detecting a Botulinum neurotoxin (BoNT) in a biological sample, the method comprising:
 a) contacting the biological sample with the isolated antibody, or an antigen-binding fragment thereof, of  claim 84 ; and   b) detecting binding, if any, of the antibody to an epitope present in the sample.