Construction and targeting of a 5F11-scFv-methioninase fusion protein for antibody-directed enzyme pro-drug therapy (ADEPT) against neuroblastoma

Abstract

3043 Background: Recombinant methioninase (METase) depletes methionine and inhibits tumor growth in preclinical models. 5F11 single-chain variable fragment (scFv) can target fusion proteins (e.g. streptavidin (SA), J. Nucl. Med., 45:867, 2004) preferentially to GD2- positive human neuroblastoma (NB). METase targeted to tumors may release methylselenol from the prodrug selenomethionine for tumor therapy. Methods: 5F11-scFv gene fused upstream of METase in pKK-223–3 to form 5F11-scFv-METase. Fusion protein produced in E. coli and purified by chromatography and endotoxin removal retained enzyme activity. Binding of 5F11-scFv-METase to tumor cells was assayed by indirect immunofluorescence and binding kinetics to GD2 analyzed by surface plasmon resonance. Cytotoxicity against human NB cell lines LAN-1, NMB-7 and SK-N-ER was tested using proliferation inhibition and apoptosis assays. Targeting to NB xenografts was studied by biodistribution using 125I-anti-idiotype 1G8 as the secondary tracking antibody. Results: 5F11-scFv-METase was purified to 84% and 94.1% homogeneity by SDS-PAGE and HPLC, respectively. Enzyme activity was 3.6 units/mg protein. Its avidity (KD=1.72×10-7M) as a dimer compared favorably with the monomeric 5F11-scFv (KD=1.07×10- 7M) and tetrameric 5F11-scFv-SA (KD=2.62×10-9M). Immunofluorescent staining of 5F11-scFv-METase was comparable to 5F11-scFv-SA for LAN-1 and NMB-7. In the presence of 20 μM selenomethionine, IC50 of 5F11-scFv-METase was 0.03, 0.02 and 0.02 units/ml for LAN-1, NMB-7 and SK-N-ER, respectively. When treated with 0.3 units/ml of 5F11-scFv-METase and 20 μM selenomethionine, apoptosis was induced in SK-N-ER by 8 hours, peaking at 24 hours. In biodistribution studies, tumor uptake in LAN-1 xenograft averaged 4.07±0.6% injected dose per gram at 48 hours. Conclusions: 5F11-scFv-METase fusion protein retains enzyme activity and immunoreactivity. It targets to tumors in vivo and activates the pro-drug selenomethionine to effect tumor cytotoxicity. Its potential for tumor-selective methionine depletion as well as in ADEPT applications deserves further studies. No significant financial relationships to disclose.