title: Intracellular stability of anti-caspase-3 intrabodies determines efficacy in retargeting the antigen.
abstract: Although intracellular antibodies (intrabodies) are being explored as putative therapeutic and research reagents, little is known about the principles that dictate the efficacy of these molecules. In our efforts to address this issue, we generated a panel of five intrabodies, directed against catalytically inactive murine caspase-3, by screening single-chain antibody (Fv) phage display libraries. Here we determined criteria that single-chain Fv fragments must fulfill to act as efficient intrabodies. The affinities of these intrabodies, as measured by surface plasmon resonance, varied approximately 5-fold (50-250 nm). Despite their substantial sequence similarity, only two of the five intrabodies were able to significantly accumulate intracellularly. These disparities in intracellular expression levels were reflected by differences in the stability of the purified protein species when analyzed by urea denaturation studies. We observed varied efficiencies in retargeting the antigen murine caspase-3, from the cytosol to the nucleus, mediated by intrabodies tagged with an SV40 nuclear localization signal. Our results demonstrate that the intrinsic stability of the intrabody, rather than its affinity for the antigen, dictates its intracellular efficacy.
authors: Pfizer Central Research, Groton, Connecticut 06340, USA.
authors: A
authors: A
authors: Rajpal
authors: T G
authors: TG
authors: Turi
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015374:Biosensing Techniques; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015723:Gene Library; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D014162:Transfection
chemical_list: D017931:DNA Primers; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases