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Construction, Binding Properties, Metabolism, and Tumor Targeting of a Single-Chain Fv Derived from the Pancarcinoma Monoclonal Antibody CC49

Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 [D. E. M., T. Y., J. S.]; Genex Corporation, Gaithersburg, Maryland 20877 [D. R. F., M. A. J. F., S. W. D., J. F. W., M. W.]; and Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland 20892 [P. S.]

CC49 is a "second generation" monoclonal antibody to B72.3, which reacts with the pancarcinoma antigen TAG-72. CC49 has been shown to efficiently target human colon carcinoma xenografts and is currently being evaluated in both diagnostic and therapeutic clinical trials. We describe here the construction and characterization of a recombinant single-chain Fv (sFv) of CC49. The sFv was shown to be a M_r 27,000 homogeneous entity which could be efficiently radiolabeled with ¹²⁵I or ¹³¹I. Comparative direct binding studies and competition radioimmunoassays using CC49 intact IgG, F(ab')₂, Fab', and sFv revealed that the monomeric CC49 Fab' and sFv had relative binding affinites 8-fold lower than the dimeric F(ab')₂ and intact IgG. Nonetheless, the ¹³¹I-labeled sFv was shown to bind biopsies of TAG-72-expressing tumors. Metabolism studies in mice, using radiolabeled CC49 IgG, F(ab')₂, Fab', and sFv, demonstrated an extremely rapid plasma and whole body clearance for the sFv. CC49 sFv plasma pharmacokinetic studies in rhesus monkeys also showed a very rapid plasma clearance (T of 3.9 min and Tß of 4.2 h). Tumor targeting studies with all four radiolabeled Ig CC49 forms, using the LS-174T human colon carcinoma xenograft model, revealed a much lower percentage injected dose/g tumor binding for the CC49 monomeric sFv and Fab' as compared to the dimeric F(ab')₂ and intact IgG. However, tumor:normal tissue ratios (radiolocalization indices) for the sFv were comparable to or greater than those of the other Ig forms. High kidney uptake with ¹²⁵I-labeled Fab' and F(ab')₂ was not seen with ¹²⁵I-sFv. Gamma scanning studies also showed that ¹³¹I-CC49 sFv could efficiently localize tumors. The CC49 sFv may thus have utility in diagnostic and perhaps therapeutic applications for a range of human carcinomas.

¹ To whom requests for reprints should be addressed, at Laboratory of Tumor Immunology and Biology, National Cancer Institute, NIH, Building 10, Room 8B07, Bethesda, MD 20892.

Received 5/ 6/91. Accepted 9/24/91.