Immuno-based retargeted endopeptidase activity assays
First Claim
Patent Images
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;
ora 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.
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Abstract
The present specification discloses a retargeted endopeptidase pharmaceutical wherein the activity has been determined by the methods disclosed.
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Citations
17 Claims
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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:
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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;
ora 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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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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;
ora 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. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17)
- -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;
Specification