Intracellular Stability of Anti-caspase-3 Intrabodies Determines Efficacy in Retargeting the Antigen

Arvind Rajpal, Thomas G. Turi
2001 Journal of Biological Chemistry  
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
more » ... s efficient intrabodies. The affinities of these intrabodies, as measured by surface plasmon resonance, varied ϳ5-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.
doi:10.1074/jbc.m101332200 pmid:11425853 fatcat:oji53xmj2bhjpngulq4k46qgoi