INCREASED AFFINITY LEADS TO IMPROVED SELECTIVE TUMOR DELIVERY OF SINGLE-CHAIN FV ANTIBODIES

M-r 25,000 single-chain Fv (scFv) molecules are rapidly eliminated fro m the circulation of immunodeficient mice, yielding highly specific re tention of small quantities of scFv in human tumor xenografts. We post ulated that the specific retention of scFv in tumor could be enhanced by engineering significant increases in the affinity of the scFv for i ts target antigens. Affinity mutants of the human anti-HER2/neu (c-erb B-2) scFv C6.5 were generated by site-directed mutagenesis, which targ et the same antigenic epitope with a 320-fold range in affinity (3.2 x 10(-7) to 1.0 x 10(-9) M). In vitro, the K-d of each scFv correlated closely with the duration of its retention on the surface of human ova rian carcinoma SK-OV-3 cells overexpressing HER2/neu. In biodistributi on studies performed in scid mice bearing established SK-OV-3 tumors, the degree and specificity of tumor localization increased significant ly with increasing affinity. At 24 h after injection, tumor retention of the highest affinity scFv was 7-fold greater than that of a mutant with 320-fold lower affinity for HER2/neu. Because the rapid renal cle arance of scFv may blunt the impact of improved affinity on tumor targ eting, the distributions were also assayed in the absence of renal cle arance (e.g., in mice rendered surgically anephric). In this model, th e peak tumor retentions of the two higher affinity scFv approximated t hat reported previously for IgG targeting the same SK-OV-3 tumors in s cid mice with intact kidneys, In contrast, the mutant with the lowest affinity for HER2/neu failed to accumulate in tumor, indicating the pr esence of an affinity threshold that must be exceeded for active in vi vo tumor uptake. These results indicate that affinity can significantl y impact the in vivo tumor-specific retention of scFv molecules.