Immuno-based retargeted endopeptidase activity assays

• US 10,345,306 B2
• Filed: 05/12/2017
• Issued: 07/09/2019
• Est. Priority Date: 03/13/2009
• Status: Active Grant

• Current Assignee: Allergan, Inc. (Canada) (Abbvie Incorporated)
• Original Assignee: Allergan, Inc. (Canada) (Abbvie Incorporated)
• Inventors: Wang, Joanne, Zhu, Hong, Hodges, Dianne D., Fernandez-Salas, Ester
• Primary Examiner(s): Carlson, Karen Cochrane
• Application Number:
  US15/593,727
• Publication Number:
  US 20170248602A1
• Time in Patent Office: 788 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, wherein retargeted endopeptidase refers to a Clostridial toxin having its naturally occurring binding domain replaced with a targeting domain, the targeting domain having selective binding activity for a non-Clostridial toxin receptor, which receptor is present in a neuronal or non-neuronal target cell, the method comprising the steps of:

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

b) isolating from the treated cell 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 linked to a solid phase support,

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,

wherein the α

-SNAP-25 antibody comprises;

a heavy chain variable region comprising an amino acid sequence encoded by a nucleic acid sequence that comprises at least 90% sequence identity with SEQ ID NO;

71, and a light chain variable region comprising an amino acid sequence encoded by a nucleic acid sequence that comprises at least 90% sequence identity with SEQ ID NO;

83;

a heavy chain variable region comprising an amino acid sequence encoded by a nucleic acid sequence that comprises at least 90% sequence identity with SEQ ID NO;

75, and a light chain variable region comprising an amino acid sequence encoded by a nucleic acid sequence that comprises at least 90% sequence identity with EQ ID NO;

87;

a heavy chain variable region comprising an amino acid sequence encoded by a nucleic acid sequence that comprises at least 90% sequence identity with SEQ ID NO;

77, and a light chain variable region comprising an amino acid sequence encoded by a nucleic acid sequence that comprises at least 90% sequence identity with SEQ ID NO;

89;

a heavy chain variable region comprising an amino acid sequence encoded by a nucleic acid sequence that comprises at least 90% sequence identity with SEQ ID NO;

79, and a light chain variable region comprising an amino acid sequence encoded by a nucleic acid sequence that comprises at least 90% sequence identity with SEQ ID NO;

91;

or

a heavy chain variable region comprising an amino acid sequence encoded by a nucleic acid sequence that comprises at least 90% sequence identity with SEQ ID NO;

81, and a light chain variable region comprising an amino acid sequence encoded by a nucleic acid sequence that comprises at least 90% sequence identity with SEQ ID NO;

91;

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.

2. The method of claim 1, wherein the α

-SNAP-25 antibody comprises;

a heavy chain variable region comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO;

71, and a light chain variable region comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO;

83;

a heavy chain variable region comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO;

75, and a light chain variable region comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO;

87;

a heavy chain variable region comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO;

77, and a light chain variable region comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO;

89;

a heavy chain variable region comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO;

79, and a light chain variable region comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO;

91;

or

a heavy chain variable region comprising an amino acid sequence encoded by the nucleic acid sequence SEQ ID NO;

81, and a light chain variable region comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO;

91.

3. The method of claim 1, wherein the α

-SNAP-25 antibody has;

a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

72, and a light chain variable region comprising the amino acid sequence of SEQ ID NO;

84;

a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

76, and a light chain variable region comprising the amino acid sequence of SEQ ID NO;

88;

a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

78, and a light chain variable region comprising the amino acid sequence of SEQ ID NO;

90;

a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

80, and a light chain variable region comprising the amino acid sequence of SEQ ID NO;

92;

or

a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

82, and a light chain variable region comprising the amino acid sequence of SEQ ID NO;

92.

4. The method of claim 1, wherein the α

-SNAP-25 antibody is an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

72 and a light chain variable region comprising the amino acid sequence of SEQ ID NO;

84.

5. The method of claim 1, wherein the α

-SNAP-25 antibody is an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

76 and a light chain variable region comprising the amino acid sequence of SEQ ID NO;

88.

6. The method of claim 1, wherein the α

-SNAP-25 antibody is an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

78 and a light chain variable region comprising the amino acid sequence of SEQ ID NO;

90.

7. The method of claim 1, wherein the α

-SNAP-25 antibody is an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

80 or SEQ ID NO;

82 and a light chain variable region comprising the amino acid sequence of SEQ ID NO;

92.

8. The method of claim 1, wherein the SNAP-25 cleavage product is SNAP-25₁₉₇.

9. The method of claim 1, wherein the presence of an antibody-antigen complex is detected using a sandwich ELISA.

10. An α

-SNAP-25 antibody that 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;

wherein the α

-SNAP-25 antibody comprises;

a heavy chain variable region comprising an amino acid sequence encoded by a nucleic acid sequence that comprises at least 90% sequence identity with SEQ ID NO;

71, and a light chain variable region comprising an amino acid sequence encoded by a nucleic acid sequence that comprises at least 90% sequence identity with SEQ ID NO;

83;

a heavy chain variable region comprising an amino acid sequence encoded by a nucleic acid sequence that comprises at least 90% sequence identity with SEQ ID NO;

77, and a light chain variable region comprising an amino acid sequence encoded by a nucleic acid sequence that comprises at least 90% sequence identity with SEQ ID NO;

89;

a heavy chain variable region comprising an amino acid sequence encoded by a nucleic acid sequence that comprises at least 90% sequence identity with SEQ ID NO;

79, and a light chain variable region comprising an amino acid sequence encoded by a nucleic acid sequence that comprises at least 90% sequence identity with SEQ ID NO;

91;

or

a heavy chain variable region comprising an amino acid sequence encoded by a nucleic acid sequence that comprises at least 90% sequence identity with SEQ ID NO;

81, and a light chain variable region comprising an amino acid sequence encoded by a nucleic acid sequence that comprises at least 90% sequence identity with SEQ ID NO;

91.

11. The antibody of claim 10, wherein the α

-SNAP-25 antibody comprises;

a heavy chain variable region comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO;

71, and a light chain variable region comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO;

83;

a heavy chain variable region comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO;

77, and a light chain variable region comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO;

89;

a heavy chain variable region comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO;

79, and a light chain variable region comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO;

91;

or

a heavy chain variable region comprising an amino acid sequence encoded by the nucleic acid sequence SEQ ID NO;

81, and a light chain variable region comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO;

91.

12. The antibody of claim 10, wherein the α

-SNAP-25 antibody comprises;

a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

72, and a light chain variable region comprising the amino acid sequence of SEQ ID NO;

84;

a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

78, and a light chain variable region comprising the amino acid sequence of SEQ ID NO;

90;

a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

80, and a light chain variable region comprising the amino acid sequence of SEQ ID NO;

92;

or

a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

82, and a light chain variable region comprising the amino acid sequence of SEQ ID NO;

92.

13. The antibody of claim 10, wherein the α

-SNAP-25 antibody is an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

72 and a light chain variable region comprising the amino acid sequence of SEQ ID NO;

84.

14. The antibody of claim 10, wherein the α

-SNAP-25 antibody is an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

80 and a light chain variable region comprising the amino acid sequence of SEQ ID NO;

92.

15. The antibody of claim 10, wherein the α

-SNAP-25 antibody is an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

78 and a light chain variable region comprising the amino acid sequence of SEQ ID NO;

90.

16. The antibody of claim 10, wherein the α

-SNAP-25 antibody is an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

82 and a light chain variable region comprising the amino acid sequence of SEQ ID NO;

92.

17. The antibody of claim 10, wherein the SNAP-25 cleavage product is SNAP -25₁₉₇.