Intracellular and cell surface displayed single-chain diabodies

Intracellularly expressed antibody fragments have found various application s in therapy by virtue of their ability to inhibit the function of cellular proteins or interfere with subcellular trafficking. Bivalent antibody frag ments might further improve this inhibitory potential by increasing the fun ctional affinity and bispecific antibody fragments may also be useful for t he intracellular retargeting of molecules. Here, we have evaluated the func tional expression of intracellular diabodies. A previously constructed secr eted bispecific single-chain diabody directed against carcinoembryonic anti gen and Escherichia coli beta-galactosidase was modified for subcellular ta rgeting to the cell surface membrane, endoplasmic reticulum, mitochondria, cytoplasm, and nucleus. Subcellular localisation was analysed by immunofluo rescence, and the assembly of functional antibodies was analysed by binding of beta-galactosidase to the antibody fragment and subsequent substrate co nversion. Bispecific single-chain diabodies could be directed to all subcel lular compartments analysed. However, functional assembly was only observed for single-chain diabodies retained in the endoplasmic reticulum or displa yed in the cell membrane while no antigen binding activity was seen with di abodies directed to the cytoplasm, nucleus, or mitochondria. The results de monstrate the functional expression of bispecific recombinant antibody frag ments in the secretory pathway and integration into the plasma membrane of mammalian cells. (C) 1999 Elsevier Science B.V. All rights reserved.