Diverting a protein from its cellular location by intracellular antibodies- The case of p21Ras

We describe the use of phage libraries to derive new antibodies against p21 Ras to be used for intracellular expression in mammalian cells. A panel of single-chain antibody fragments, binding to Ras, were analyzed and characte rized for their capacity to interfere in vitro with (a) the intrinsic GTPas e activity of Ras and (b) the binding of Ras to its effector Raf, and were found not to neutralize its function, according to these biochemical criter ia. When expressed intracellularly in mouse 3T3 K-Ras transformed cells all the anti-aas single-chain variable fragments (scFv) tested inhibited cell proliferation, as assessed by bromodeoxyuridine incorporation. Double immun ofluorescence analysis of transfected cells using confocal microscopy confi rmed that anti-Ras antibody fragments colocalize with endogenous Ras, at su bcellular locations where the protein Ras is not normally found. These data suggest that the ability of phage-derived anti-Ras scFv fragments to inhib it the function of Ras in vivo is a rather general and frequent property an d that the range of antibodies that can be successfully used for intracellu lar inhibition studies is much greater than anticipated, exploiting the mod e of action of diverting protein traffic.