IN-VITRO EVALUATION OF DNA-DNA HYBRIDIZATION AS A 2-STEP APPROACH IN RADIOIMMUNOTHERAPY OF CANCER

The specific delivery of radioisotopes to a tumor at minimal radiation of normal tissue is the ultimate aim of radioimmunotherapy. In this r espect a two step pretargeting regimen generally leads to an improved tumor to normal tissue uptake ratio compared to direct administration of radioimmunoconjugates. In this paper, in vitro studies are describe d in which the specific hybridization of complementary DNA fragments i s the recognition mechanism in a pretargeting regimen comprising tumor cell saturation with a monoclonal antibody (MoAb)-oligonucleotide con jugate, followed by administration of the radiolabeled complementary o ligonucleotide. Complementary oligodeoxynucleotides (15-mers; melting temperature, 68 degrees C) were prepared on a DNA synthesizer. The 5'- end was derivatized with a functional group for labeling with iodine, and the 3'-end was substituted with an amino function suitable for con jugation to an antibody (or attachment of a biotin residue). Both term inal modifications ensure stability of the oligonucleotides against ex onucleases because the unconjugated form is stable for 24 h and the co njugated form is stable for several days when incubated in human plasm a at 37 degrees C. Antibody-DNA conjugates were prepared by introducti on of sulfhydryl groups into the oligonucleotide, followed by conjugat ion to maleimide-substituted MoAbs. Typically, 3 oligonucleotides were conjugated to an IgG, and 4-6 were conjugated to an IgM with preserva tion of immunoreactivity. Histochemistry on fresh frozen sections of b reast cancer tissue demonstrated qualitatively the specificity of our two-step procedure. In vitro experiments with human tumor cell lines a nd tumor-specific MoAbs showed that, after saturation with tumor speci fic MoAb-DNA conjugates, quantitative hybridization of the tumor cell bound oligonucleotides occurred at a 30-fold excess of the labeled com plementary oligonucleotide: hybridization was complete after 30 min of incubation. No reaction was observed with an irrelevant MoAb-DNA conj ugate. The oligonucleotide was neither taken up by tumor cells or endo thelial cells nor hybridized to a significant extent with human genomi c DNA. hese data indicate the feasibility of this two-step approach in radioimmunotherapy.