In Antibody-Directed Enzyme-Prodrug Therapy (ADEPT), antibody - enzyme constructs localize to tumor tissue the toxification of non-toxic prodrugs. Recombinant fusion proteins may overcome limitations of chemical conjugates regarding stability and size. As a general model system, we have constructed fusion proteins of an antibody against the tumor antigen A33 with cytosine deaminase (CD), which converts 5-fluorocytosine (5-FC) into cytotoxic 5-fluorouracil (5-FU) . Using a T7 polymerase-based expression system, a plasmid vector was designed to allow cloning of the scFv either preceding or following the enzyme. The fusion proteins were produced in E. coli and purified and renatured from inclusion bodies. Antibody and enzyme activities were confirmed by separate functional assays. To test the complete ADEPT system in vitro, A33-positive cell cultures were incubated with the fusion protein, washed, and cultured for 48h in the presence of 5-PC. While fusion protein or up to I mM 5-PC alone had no effect on cell growth, their sequential combination quantitatively increased median 5-PC toxicity. Preincubation with anti-A33 blocked this effect. A control fusion protein with GFP instead of CD had no effect on 5-PC toxicity. While avidity of the constructs remains to be improved, these data prove the feasibility of scPv-based ADEPT in principle. Currently, this system is transferred using breast-cancer-specific antibodies Fl9 and 3Sl93.