TARGETING VECTORS FOR INTRACELLULAR IMMUNIZATION

We define intracellular immunization as the inhibition or inactivation of the function of a molecule by the ectopic intracellular expression of antibody binding domains which recognise the molecule. Such recomb inant antibodies can be directed to different compartments of eukaryot ic cells by means of previously defined targeting signals, thus permit ing the study of any molecule in any cellular compartment for which an antibody is available. For this purpose, we have created a set of vec tors based on the VHExpress vector described [Persic, L., Roberts, A., Wilton, J., Cattaneo, A., Bradbury, A. and Hoogenboom, H.R. (1997) An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries. Gene 187, 000-000], which has been modified to express scFvs (single chain fragments) linked to specific targeting signals. These permit the loca lisation of scFvs to different intracellular compartments: the endopla smic reticulum (scFvE-er), the nucleus (scFvE-nuclear), the mitochondr ia (scFvE-mit), the cytoplasm (scFvE-cyto), and as secreted proteins ( scFvE-sec). The function of these vectors has been assessed by the imm unofluorescence of COS cells transiently transfected with constructs c ontaining the alpha D11 scFv.