Intracellular stability of anti-caspase-3 intrabodies determines efficacy in retargeting the antigen

Although intracellular antibodies (intrabodies) are being explored as putat ive therapeutic and research reagents, little is known about the principles that dictate the efficacy of these molecules. In our efforts to address th is issue, we generated a panel of five intrabodies, directed against cataly tically inactive murine caspase-3, by screening single-chain antibody (Fv) phage display libraries. Here we determined criteria that single-chain Fv f ragments must fulfill to act as efficient intrabodies. The affinities of th ese intrabodies, as measured by surface plasmon resonance, varied similar t o5-fold (50-250 rim). Despite their substantial sequence similarity, only t wo of the five intrabodies were able to significantly accumulate intracellu larly. These disparities in intracellular expression levels were reflected by differences in the stability of the purified protein species when analyz ed by urea denaturation studies. We observed varied efficiencies in retarge ting the antigen murine caspase-3, from the cytosol to the nucleus, mediate d by intrabodies tagged with an SV40 nuclear localization signal. Our resul ts demonstrate that the intrinsic stability of the intrabody, rather than i ts affinity for the antigen, dictates its intracellular efficacy.