Production of functionalized single-chain Fv antibody fragments binding tothe ED-B domain of the B-isoform of fibronectin in Pichia pastoris

The Pichia pastoris expression system was used to produce functionalized si ngle-chain antibody fragments (scFv) directed against the ED-B domain of th e B-fibronectin (B-Fn) isoform which was found to be present only in newly formed blood vessels during tumor angiogenesis, Therefore, scFv antibody fr agments recognizing the ED-B domain are potential markers for angiogenesis, We constructed four functionalized scFv antibody fragments for direct labe ling with radioactive molecules or toxins or for attachment to liposomes se rving as carriers for cytotoxic or antiangiogenic compounds. The C-termini of the scFv antibody fragments contain 1-3 cysteine residues that are separ ated by a hydrophilic linker (GGSSGGSSGS) from the binding domain and are a ccessible for site-specific functionalization with thiol-reactive reagents. Plasmid expression, culture conditions, and purification were optimized in l-L cultures. The scFv antibody fragments were purified by anion exchange chromatography, The yields were 5-20 mg/L culture medium. The large-scale p roduction of one scFv antibody fragment in a 3.7-L fermenter gave a yield o f 60 mg, The reactivity of the cyteines was demonstrated by labeling with t he thiol-reactive fluorescent dye ABD-F, The four scFv antibody fragments b ound specifically to ED-B-modified Sepharose and binding was further confir med by immunofluorescence on cell cultures using ED-B-positive human Caco-2 tumor cells. Furthermore, we could demonstrate specific binding of scFv-mo dified liposomes to ED-B-positive tumor cells. Our results indicate that th e P, pastoris expression system is useful for the large-scale production of cysteine-functionalized alpha -ED-B scFv antibody fragments. (C) 2001 Acad emic Press.