Mr 25,000 single-chain Fv (scFv) molecules are rapidly eliminated from the circulation of immunodeficient mice, yielding highly specific retention of small quantities of scFv in human tumor xenografts. We postulated that the specific retention of scFv in tumor could be enhanced by engineering significant increases in the affinity of the scFv for its target antigens. Affinity mutants of the human anti-HER2/neu (c-erbB-2) scFv C6.5 were generated by site-directed mutagenesis, which target the same antigenic epitope with a 320-fold range in affinity (3.2 × 10-7 to 1.0 × 10-9m). In vitro, the Kd of each scFv correlated closely with the duration of its retention on the surface of human ovarian carcinoma SK-OV-3 cells overexpressing HER2/neu. In biodistribution studies performed in scid mice bearing established SK-OV-3 tumors, the degree and specificity of tumor localization increased significantly 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 clearance of scFv may blunt the impact of improved affinity on tumor targeting, the distributions were also assayed in the absence of renal clearance (e.g., in mice rendered surgically anephric). In this model, the peak tumor retentions of the two higher affinity scFv approximated that reported previously for IgG targeting the same SK-OV-3 tumors in scid mice with intact kidneys. In contrast, the mutant with the lowest affinity for HER2/neu failed to accumulate in tumor, indicating the presence of an affinity threshold that must be exceeded for active in vivo tumor uptake. These results indicate that affinity can significantly impact the in vivo tumor-specific retention of scFv molecules.

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Supported by National Cancer Institute Grant CA 65559, Department of Defense Grant DAMD17-94-J-4433. National Cancer Institute Grant CA06927, an appropriation from the Commonwealth of Pennsylvania, the Bernard A. and Rebecca S. Bernard Foundation, the Frank Strick Foundation, and the CaPCURE Foundation. This work was initiated with the support of National Cooperative Drug Discovery Group Grant U01 CA51880. Members of this group included: Drs. L. L. Houston (Prism Pharmaceuticals); James S. Huston, John E. McCartney, Mei-Sheng Tai, and Hermann Oppermann (Creative BioMolecules); Drs. Craig Reynolds and George Johnson (National Cancer Institute); Dr. Walter Stafford (Boston Biomedical Research Inst.); Cindy Wong (University of California, San Francisco, CA); Dr. Michael Bookman, Dr. Bruce Giantonio, and Josephine Schultz (Fox Chase Cancer Center); and Gerald Apell (Chiron Corp.).

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