Extended half-life and elevated steady-state level of a single-chain Fv intrabody are critical for specific intracellular retargeting of its antigen,caspase-7

Two single-chain Fv (sFv) antibodies (C8 and H2) specific for Mch3/caspase- 7, a component in the signaling pathway for induction of apoptosis, were ge netically fused to different intracellular targeting signals and analyzed b y expression in mammalian cells. Immunofluorescence microscopy confirmed th at these anti-caspase-7 intrabodies were expressed in the cellular compartm ents dictated by their C-terminal trafficking signals. Cytosolic caspase-7 was successfully retargeted to different subcellular compartments by specif ic intrabodies through direct association of antigen with intrabody. Seques tration of caspase-7 in nuclei had a significant biological impact in that the expression of a nuclear-targeted anti-caspase-7 intrabody in a stable J urkat cell Line markedly inhibited staurosporine-induced apoptosis. The cri teria for choosing an optimal intrabody were also evaluated in this study. A gene dosage titration study demonstrated that the C8 intrabody was more p otent in retargeting of caspase-7 than the H2 intrabody, even though the H2 sFv had a higher affinity for caspase-7 than the C8. Pulse-chase experimen ts and western blot analysis revealed that the anti-caspase-7 C8 sFv intrab odies exhibited a long half-life (>8 h) and high steady-state levels of pro tein accumulation, while the H2 intrabodies had a half-life of 2 h and less protein at steady state. These results suggest that the choice of sFv as a n intrabody depends critically on the intracellular sFv protein having an e xtended half-life and elevated steady-state level, Thus, extended half-life must be considered together with sFv antibody specificity and affinity whe n choosing an optimal sFv intrabody for functional studies of cellular prot eins. (C) 1999 Elsevier Science B.V. All rights reserved.