Evaluation in vitro of adriamycin immunoconjugates synthesized using an acid-sensitive hydrazone linker.

A novel method for linking Adriamycin (ADM) to monoclonal antibodies is described in which the 13-keto position of the anthracycline is used as the attachment site to the linker arm. A new ADM acylhydrazone derivative, Adriamycin 13-[3-(2-pyridyldithio)propionyl]hydrazone hydrochloride, which contains a pyridyl-protected disulfide, was synthesized and used for conjugation to monoclonal antibodies (MAbs) that were thiolated with N-succinimidyl 3-(pyridyldithiol)propionate or 2-iminothiolane. This resulted in formation of a linker between MAb and drug that contained a disulfide bond. Conjugation conditions were optimized to yield conjugates with high ADM:MAb molar ratios. The final immunoconjugate yields were found to decrease as the ADM:MAb molar ratio of the conjugates increased. Stability studies indicated that ADM was released from the immunoconjugates at mildly acidic pHs ranging from 4.5-6.5. Treatment of immunoconjugates with mild reducing agent dithiothreitol resulted in release of an acylhydrazone derivative of ADM. Flow-cytometric studies showed that the binding activity of various MAbs following conjugation to ADM was preserved at ADM:MAb molar ratios up to 10. Antibody-directed cytotoxicity was demonstrated under several assay conditions using combinations of antigen-positive and antigen-negative cells and binding and nonbinding immunoconjugates. In several experiments, ADM immunoconjugates were more potent than equivalent amounts of unconjugated ADM.

Immunohistochemical and biochemical characterization of antisera raised to human tumor nucleoli.

Antinucleolar antisera were raised in rabbits, goats and sheep to nucleoli isolated from three human tumor cell lines. The antisera were shown to cross-react by immunofluorescence with human tumor cell lines originating from different organs and with frozen sections from a wide variety of human malignant and non-malignant tissues. Tumor versus normal tissue discrimination by several antisera was significantly improved by treatment of frozen tissues with a buffered glutaraldehyde/Triton X-100 solution prior to immunofluorescent staining. The molecular specificity of these antisera was determined by immunostaining electrotransfer nitrocellulose strips following SDS-PAGE of nucleolar preparations and nuclear extracts. Although different immunostaining patterns were obtained for individual antinucleolar antisera, nucleolar proteins of molecular weight 120, 100, 94, 68, 54, 38, 33, and 32 kDa were the most often recognized by antisera raised in the three different species. G187 antiserum strongly reacted with 100, 94, and 38 kDa proteins from freshly obtained leukemic specimens. The Immunoreactivity of the 100, 94, and 38 kDa proteins was unaffected by glutaraldehyde/Triton X-100 treatment when immunostained with antisera that demonstrated the greatest tumor specificity on sections treated with glutaraldehyde/Triton X-100. These three nucleolar proteins may be more highly associated with nucleoli of malignant cells than with nucleoli of normal cells.