IMMUNO-BASED RETARGETED ENDOPEPTIDASE ACTIVITY ASSAYS

• US 20190324041A1
• Filed: 07/08/2019
• Published: 10/24/2019
• 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
• Granted Patent: US 10,900,967 B2
• Time in Patent Office: Days
• 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 a retargeted endopeptidase pharmaceutical wherein the activity has been determined by the methods disclosed.

Claims

1. A method of detecting retargeted endopeptidase activity, the method comprising the steps of:

a) treating a cell from an established cell line with a sample comprising a retargeted endopeptidase, wherein the cell from an established cell line is susceptible to retargeted endopeptidase activity by a retargeted endopeptidase;

b) isolating from the treated cell line a SNAP-25 component comprising a SNAP-25 cleavage product having a carboxyl-terminus at the P1 residue of the BoNT/A cleavage site scissile bond;

c) contacting the SNAP-25 component with an α

-SNAP-25 antibody, wherein the α

-SNAP-25 antibody binds an epitope comprising a carboxyl-terminus at the P1 residue of the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product; and

d) detecting the presence of an antibody-antigen complex comprising the α

-SNAP-25 antibody and the SNAP-25 cleavage product;

wherein detection by the antibody-antigen complex is indicative of retargeted endopeptidase activity, wherein the α

-SNAP-25 antibody binds an epitope comprising a carboxyl-terminus at the P1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product and wherein the isolated α

-SNAP-25 antibody has a V_H CDR1 of SEQ ID NO;

93, a V_H CDR2 of SEQ ID NO;

96, a V_H CDR3 of SEQ ID NO;

100, a V_L CDR1 of SEQ ID NO;

105, a V_L CDR2 of SEQ ID NO;

110, and a V_L CDR3 of SEQ ID NO;

115.

2. The method of claim 1, wherein the α

-SNAP-25 antibody is linked to a solid phase support.

3. The method of claim 2, comprising a step of fixing the SNAP-25 component to said solid phase support.

4. The method of claim 1, wherein the cell is susceptible to retargeted endopeptidase activity by about 500 nM or less, by about 400 nM or less, by about 300 nM or less, by about 200 nM or less, by about 100 nM or less of a retargeted endopeptidase.

5. The method of claim 1, wherein the cell can uptake about 500 nM or less, by about 400 nM or less, by about 300 nM or less, by about 200 nM or less, by about 100 nM or less of retargeted endopeptidase.

6. The method of claim 1, wherein the sample comprises about 100 ng or less, about 10 ng or less, about 1 ng or less, 100 fg or less, 10 fg or less, or 1 fg or less of a retargeted endopeptidase.

7. The method of claim 1, wherein the sample comprises about 100 nM or less, about 10 nM or less, about 1 nM or less, about 0.5 nM or less, or about 0.1 nM or less, of a retargeted endopeptidase.

8. The method of claim 1, wherein the presence of an antibody-antigen complex is detected by an immuno-blot analysis, an immunoprecipitation analysis, an ELISA, or a sandwich ELISA.

9. The method of claim 1, wherein the α

-SNAP-25 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×

10¹ M<sup>−

1</sup> s<sup>−

1</sup>; and

wherein the α

-SNAP-25 antibody has an equilibrium disassociation constant for the epitope of less than 0.450 nM.

10. An isolated monoclonal α

-SNAP-25 antibody which has a V_H CDR1 of SEQ ID NO;

93, a V_H CDR2 of SEQ ID NO;

96, a V_H CDR3 of SEQ ID NO;

100, a V_L CDR1 of SEQ ID NO;

105, a V_L CDR2 of SEQ ID NO;

110, and a V_L CDR3 of SEQ ID NO;

115.