Biological effects of anti-ErbB2 single chain antibodies selected for internalizing function

Two internalizing monovalent single chain antibody fragments (scFv), C6.5 a nd F5, that recognize distinct ErbB2 extracellular domain (ECD) epitopes, a nd their bivalent forms dbC6.5 and FB(scFv')(2), were compared to the growt h-inhibiting anti-ErbB2 antibody Herceptin/trastuzumab, in either its bival ent (Her) or monovalent (4D5Fab') form, for their abilities to induce biolo gical responses in the ErbB2-overexpressing breast cancer cells, SkBr-3. As says compared internalization by receptor-mediated endocytosis, effects on cell cycling and culture growth, and interference with intracellular MAPK a nd PI3K signaling pathways. We found no correlation between ErbB2 epitope a ffinity or valency on degree of antibody-induced endocytosis, since all the scFv were able to internalize better than Her. Unlike Her, neither the mon ovalent or bivalent forms of the internalizing scFv had any sustained effec t on cell growth. Basal levels of MAPK and PI3K signaling in SkBr-3 cells w ere not inhibited by up to 8 h scFv treatment, while decreased MAPK and PI3 K signals were noted within 8 h of Her treatment. In summary, antibody-indu ced ErbB2-mediated endocytosis is not a surrogate marker for resultant biol ogical response, as it shows no correlation with cell cycle, culture prolif eration, or intracellular kinase signal induction by internalizing antibodi es. Thus, the enhanced endocytotic property of scFv like C6.6 and F5 in coj unction with their absence of any growth or signaling impact on ErbB2 overe xpressing cells favors their choice as ErbB2 targeting moieties for intrace llular delivery of novel cancer therapeutics. (C) 2001 Academic Press.