Immuno-based botulinum toxin serotype a activity assays

Abstract

The present specification discloses SNAP-25 compositions, methods of making α-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the P 1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product, α-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the P 1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product, methods of detecting BoNT/A activity, and methods of detecting neutralizing α-BoNT/A antibodies.

1 . An isolated α-SNAP-25 antibody, comprising a heavy chain variable region comprising complementary determining regions (CDRs) comprising the amino acid sequences of at least one of SEQ ID NOs: 93, 96, and 100 and a light chain variable region comprising CDRs comprising the amino acid sequences of at least one of SEQ ID NOs: 105, 110 and 115. 
     
     
         2 . The antibody of  claim 1 , wherein the antibody specifically binds to an epitope comprising a carboxyl-terminus glutamine from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product. 
     
     
         3 . The antibody of  claim 2 , wherein the epitope comprises SEQ ID NO:38. 
     
     
         4 . The antibody of  claim 2 , wherein the antibody binds to the epitope with an equilibrium disassociation constant of less than 0.450 nM. 
     
     
         5 . The antibody of  claim 2 , wherein the antibody has an association rate constant for an epitope not comprising a carboxyl-terminus glutamine of the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product of less than 1×10 1  M −1  s −1 . 
     
     
         6 . The antibody of  claim 2 , wherein the epitope is SEQ ID NO: 32. 
     
     
         7 . The antibody of  claim 1 , for use in a method for detecting BoNT/A activity in a SiMa or Neuro-2a cell line expressing a SNAP-25 comprising a BoNT/A cleavage site. 
     
     
         8 . The antibody of  claim 7 , wherein the SiMa or Neuro-2a cell line is susceptible to intoxication by about 50 pM or less of a BoNT/A. 
     
     
         9 . The antibody of  claim 7 , wherein the Neuro-2a cell line has been genetically engineered to express a non-naturally occurring SNAP-25. 
     
     
         10 - 11 . (canceled) 
     
     
         12 . An isolated α-SNAP-25 antibody, comprising a heavy chain variable region comprising complementary determining regions (CDRs) comprising the amino acid sequences of SEQ ID NO: 95 (CDR1), SEQ ID NO: 99 (CDR2), and SEQ ID NO: 101 (CDR3) and a light chain variable region comprising CDRs comprising the amino acid sequences of SEQ ID NO: 103 (CDR1), SEQ ID NO: 108 (CDR2), and SEQ ID NO: 113 (CDR3). 
     
     
         13 . The antibody of  claim 12 , wherein the antibody specifically binds to an epitope comprising a carboxyl-terminus glutamine from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product. 
     
     
         14 . The antibody of  claim 13 , wherein the epitope comprises SEQ ID NO:38. 
     
     
         15 . The antibody of  claim 13 , wherein the antibody binds to the epitope with an equilibrium disassociation constant of less than 0.450 nM. 
     
     
         16 . The antibody of  claim 13 , wherein the antibody has an association rate constant for an epitope not comprising a carboxyl-terminus glutamine of the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product of less than 1×10 1  M −1  s −1 . 
     
     
         17 . The antibody of  claim 12 , wherein the epitope is SEQ ID NO: 32. 
     
     
         18 . The antibody of  claim 12 , for use in a method for detecting BoNT/A activity in a SiMa or Neuro-2a cell line expressing a SNAP-25 comprising a BoNT/A cleavage site. 
     
     
         19 . The antibody of  claim 18 , wherein the SiMa or Neuro-2a cell line is susceptible to intoxication by about 50 pM or less of a BoNT/A. 
     
     
         20 . The antibody of  claim 18 , wherein the Neuro-2a cell line has been genetically engineered to express a non-naturally occurring SNAP-25. 
     
     
         21 . A non-animal based process for manufacturing an injectable botulinum toxin type A (BoNT/A) pharmaceutical product, comprising:
 a. obtaining an aliquot of a formulated BoNT/A pharmaceutical composition, said composition comprising:   (i) a botulinum toxin type A; and   (ii) a non-naturally occurring polypeptide comprising the HIV TAT protein basic region, comprising the amino acid sequence RKKRRQRRR;   b. diluting one or more samples of the aliquot;   c. contacting the diluted samples with differentiated Neuro-2a cells susceptible to intoxication by about 10 pM or less of BoNT/A; and   d. determining suitability of the BoNT/A pharmaceutical composition for release as an injectable BoNT/A pharmaceutical product.   
     
     
         22 . A product produced by the process of  claim 21 .