Several monoclonal antibodies (MAbs), reactive with tumor-associated antigens, selectively persist on tumor sites in vivo for many days. If biotinylated, such highly specific tags on tumor cells could become targets for radioactive avidin, administered after suitable intervals. The proposed strategy is based on a number of assumptions concerning the ability of avidin t o preserve i t s biological properties in heterologous in vivo environments, on i t s lack of toxicity and on i t s biodistribution. A preliminary study has been carried out in rabbits, using biotinylated nitrocellulose and polystyrene targets. The results of this study indicate that in rabbits I ) avidin can be administered i.v. and i.p. without adverse reactions, 2) it does not show any preferential localization, 3) it is eliminated with a biological half-life of 24 hr, 4) i t s biological properties are not impaired by in vivo conditions, since it accumulates at biotinylated targets only, 5) CEA-bearing targets can be biotinylated in vivo by biotin-labelled anti-CEA MAbs and 6) the biotin-avidin chain can be further extended in vivo since bound avidin is still able t o bind biotinylated radioactive proteins.Several MAbs used in immunoscintigraphy of human tumors persist a long time on the surface of tumors, giving the best localization ratios at 72-96 hr after their administration (e.g., Buraggi et al., 1987). A much slower loss of labelled antibodies from tumor sites as compared to non-specific sites has been documented in both animals and humans (Colcher et al., 1984(Colcher et al., , 1987. Surgical specimens removed 10-20 days after administration still bear evidence of antibody with very high selectivity for tumor cells. Although these antibodies represent a very minute fraction of the injected dose, they can effectively tag the tumor if they are labelled with long-lived isotopes, such as 1251, a property that has been exploited to develop radio-immuno-guided surgery (Martin et al., 1985).Our purpose was to develop a method to post-label anti-tumor MAbs in vivo, exploiting their differential persistence at tumor sites. Our strategy is to biotinylate MAbs, to inject them cold and to leave an appropriate interval before the administration of radioactive avidin. The aim of the study is to evaluate if the very high affinity and specificity of the avidin-biotin reaction (Green, 1975) can be exploited in vivo in laboratory animals, as it has been widely exploited in vitro in a variety of diagnostic applications.
MATERIAL AND METHODS
Material and animalsImmuno-affinity-purified CEA from an hepatic metastasis, MAb F023C5 (reactive with a protein epitope of CEA) and F023C5-coated beads were from SORIN Biomedica. Pure hen egg avidin and biotinyl-eaminocaproic acid Nhydroxysuccinimide ester and MAb L16B8 (reactive with hen egg lysozyme) were from Societh Prodotti Antibiotici. Na13'I (specific activity 16 Ci/mol) and ' "InC13 were purchased from ORIS/CEA, Saclay (France) and Amersham (UK), respectively. Outbred rabbits were used for all experiments.
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