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 antig en 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 iminobioti n 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 m olecular 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 t o recomdinant streptavidin and bound an average of three of four possible b iotins per molecules. The radiolabeled fusion protein showed a faster blood clearance rate in normal mince than the corresponding whole antibody-strep tavidin molecule was demonstrated in nude bearing SW1222 human colon carcin oma xenografts. A single dose of 800 mu Ci of Y-90-DOTA-biotin produced cur es in mice with established subcutaneous human small cell lung or colon can cer xenografts.