Intrabodies pp 61-74 | Cite as

Intracellular Antibodies as Tools to Study ErbB Receptor Tyrosine Kinases

  • Roger R. Beerli
  • Diana Graus-Porta
  • Nancy E. Hynes

Abstract

Intracellular expression of antibodies provides a novel approach for the manipulation of biochemical processes in intact cells. We have used this approach to test two alternative strategies for the inactivation of individual members of the erbB family of receptor tyrosine kinases (RTKs). Epidermal growth factor (EGF) receptor and erbB-2, two receptors whose aberrant expression is frequently involved in human cancer, were chosen as targets. Genes encoding single-chain Fv domains (scFvs) directed to the extracellular portion of these receptors were derived from hybridoma cells producing the corresponding monoclonal antibodies. The scFvs were provided with a signal peptide to direct their synthesis to the secretory compartment of the cell. Intracellular expression of an scFv that competes with EGF was found to inhibit EGF receptor function in an autocrine manner. In contrast, scFvs provided with an additional endoplasmic reticulum (ER) retention signal were retained in the ER and inactivated their target receptor by preventing its appearance on the cell surface. In NIH/3T3 fibroblasts transformed by a mutated, constitutively active erbB-2, scFv-mediated intracellular retention of erbB-2 led to complete reversion of the transformed phenotype. The potential for the targeted inactivation of cell surface proteins by a gene therapy approach has implications for the treatment of many human diseases including cancer and viral infections. Apart from its potential for future medical applications, intracellular antibody expression has a wide range of immediate applications in basic research. For example, the phenotypic knock-out of growth factor receptors by scFv mediated intracellular retention provides an attractive tool for investigating complex receptor-ligand interactions. This is exemplified by our studies of the erbB signaling network with its four related receptors and multiple activating ligands. Analysis of signaling emanating from this receptor family is complicated by the extensive crosstalk which occurs among the individual receptors. We have downregulated cell surface expression of individual erbB receptors via intracellular antibody expression prior to analyzing ligand induced signaling. The results from these experiments have allowed us to draw some important conclusions concerning erbB receptor interplay and to show that erbB-2 is the key member of this family.

Keywords

Agar Tyrosine Heparin Oncol Half Life 

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Copyright information

© Springer-Verlag Berlin Heidelberg 1998

Authors and Affiliations

  • Roger R. Beerli
  • Diana Graus-Porta
  • Nancy E. Hynes

There are no affiliations available

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