Intracellular expression of single chain antibodies reverts ErbB-2 transformation.
We report a novel approach for specific in vivo inactivation of the ErbB-2 receptor tyrosine kinase and suppression of ErbB-2-induced transformation. Genes encoding single chain antibodies that specifically bind to the extracellular domain of human ErbB-2 were constructed and expressed intracellularly in NIH/3T3 fibroblasts transformed by activated ErbB-2. The single chain antibodies are derived from monoclonal antibodies FRP5 and FWP51 (Harwerth, I.M., Wels, W., Marte, B. M., and Hynes, N. E. (1992) J. Biol. Chem. 267, 15160-15167) and are composed of heavy and light chain variable domains connected by a flexible peptide linker. The antibodies were provided with: 1) an N-terminal hydrophobic leader sequence to target their synthesis to the lumen of the endoplasmic reticulum, and 2) a C-terminal retention signal to prevent secretion. When expressed in ErbB-2-transformed cells, the single chain antibodies bound to the receptor and prevented its transit through the endoplasmic reticulum. This resulted in the functional inactivation of the receptor and reversion of the transformed phenotype. This is the first demonstration of a targeted and stable inactivation of a cellular oncoprotein via intracellular antibody expression. The use of such a strategy represents a simple and powerful approach to study the in vivo function of receptors and other cellular proteins.
Beerli
R R
RR
Friedrich Miescher-Institut, Basel, Switzerland.
Wels
W
W
Hynes
N E
NE
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002461:Cell Line, Transformed; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000502:physiology*; D007135:Immunoglobulin Variable Region; D051379:Mice; D008969:Molecular Sequence Data; D010641:Phenotype; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000502:physiology; D013347:Subcellular Fractions / Q000378:metabolism
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D018719:Erb-b2 Receptor Tyrosine Kinases; C055966:immunoglobulin Fv