Preclinical evaluation of a humanized NR-LU-10 antibody-streptavidin fusion protein for pretargeted cancer therapy

A humanized single chain F nu antibody fragment specific to the EGP40 antigen was genetically engineered as a streptavidin fusion (scF nu SA) for use in pretargeted radioimmunotherapy. The scF nu SA construct was expresses as a soluble, tetrameric species in the Escherichia coli periplasm at 110-140 mg/liter. The fusion protein was purified from crude lysates by iminobiotin affinity chromatography with an overall yield of 50-60%. Characterization of the purified protein by SDS-PAGE, light scattering, and size exclusion chromatography demonstrated that the fusion protein was tetrameric with a molecular weight of similar to 172,000. Competitive immunoreactivity assays showed a two-fold greater binding to the antigen than the comparable whole antibody. The purified protein had a biotin disassociation rate identical to recomdinant streptavidin and bound an average of three of four possible biotins per molecules. The radiolabeled fusion protein showed a faster blood clearance rate in normal mince than the corresponding whole antibody-streptavidin molecule was demonstrated in nude bearing SW1222 human colon carcinoma xenografts. A single dose of 800 mu Ci of Y-90-DOTA-biotin produced cures in mice with established subcutaneous human small cell lung or colon cancer xenografts.