Immuno-based retargeted endopeptidase activity assays

• US 8,455,203 B2
• Filed: 03/12/2010
• Issued: 06/04/2013
• Est. Priority Date: 03/13/2009
• Status: Active Grant

• Current Assignee: Allergan Incorporated (Abbvie Incorporated)
• Original Assignee: Allergan Incorporated (Abbvie Incorporated)
• Inventors: Wang, Joanne, Zhu, Hong, Hodges, Dianne D., Fernandez-Salas, Ester
• Primary Examiner(s): Counts, Gary W
• Application Number:
  US12/723,595
• Publication Number:
  US 20100233741A1
• Time in Patent Office: 1,180 Days
• Field of Search: None
• US Class Current: 435/7.1
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C12N 9/52 derived from bacteria or Archaea
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Y 304/24069 Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin
• CPC Class Codes: G01N 2333/33 from Clostridium (G)
• CPC Class Codes: G01N 2333/47 Assays involving proteins of known structure or function as defined in the subgroups
• CPC Class Codes: G01N 2333/952 derived from bacteria
• CPC Class Codes: G01N 2333/96425 from mammals
• CPC Class Codes: G01N 33/5014 for testing toxicity
• CPC Class Codes: G01N 33/5035 on sub-cellular localization
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

The present specification discloses SNAP-25 immune response inducing compositions, methods of making α-SNAP-25 antibodies that selectively binds to an epitope comprising a SNAP-25 having a carboxyl-terminus at the P₁ residue of the BoNT/A cleavage site scissile bond, α-SNAP-25 antibodies that selectively bind to an epitope comprising a SNAP-25 having a carboxyl-terminus at the P₁ residue of the BoNT/A cleavage site scissile bond, methods of detecting retargeted endopeptidase activity, and methods of detecting neutralizing α-re-targeted endopeptidase antibodies.

Claims

1. A method of detecting the activity of retargeted endopeptidase in a sample, wherein the retargeted endopeptidase comprises an enzymatic domain, comprising

a) contacting a cell with a sample suspected of comprising retargeted endopeptidase;

wherein the cell is from an established cell line expressing a SNAP-25 polypeptide consisting essentially of at least a portion of human SNAP-25 comprising SEQ ID NO;

5 cleavable by the enzymatic domain;

wherein the established cell line is susceptible to intoxication by the retargeted endopeptidase at about or less than 50 nmol per liter culture medium, as indicated by the enzymatic cleavage of the SNAP-25 polypeptide by the enzymatic domain to yield a fragment of said SNAP25 polypeptide comprising the C-terminal amino acid sequence of SEQ ID NO;

38;

b. isolating polypeptides from the cell;

c. contacting the polypeptides with a monoclonal antibody that specifically binds to the peptide of SEQ ID NO;

38;

wherein said antibody specifically binds to an epitope of said fragment of said SNAP25 polypeptide comprising the C-terminal amino acid sequence of SEQ ID NO;

38 with an equilibrium disassociation constant of less than 0.450 nM and wherein said antibody has an association rate constant for an epitope of intact SNAP-25 polypeptide comprising SEQ ID NO;

5 of less than 1×

10¹ M<sup>−

1</sup> s<sup>−

1</sup>; and

,

d. detecting the presence of any antibody-antigen complex comprising the antibody and the fragment of said SNAP-25 polypeptide comprising the C-terminal amino acid sequence of SEQ ID NO;

38;

wherein a higher amount of the antibody-antigen complex detected correlates to a higher amount of activity of the retargeted endopeptidase in the sample.