Conjugates of antibody to carcinoembryonic antigen (murine monoclonal antibody and affinity-purified heteroantibody) lined to the A chain of ricin are potent and specific cytotoxins for human colorectal adenocarcinoma cells. Several classes of agents, including UV inactivated adenovirus and carboxylic ionophores, produce specific and dramatic enhancement of anti-CEA immunotoxin effect on colorectal cancel cell lines. We propose to explore the effect of these potentiating agents on immunotoxin internalization and cytotoxic effect. Immunotoxin internalization will be studied by flow cytometric, and cell fractionation techniques. Effects of different chemical linkages, toxic A chains, and antibody fragments will be examined. By these methods we hope to better understand the process of immunotoxin effect and produce more rapidly acting and potent anti-tumor immunotoxins. We will also evaluate the anti-tumor effect of anti-CEA immunoconjugates in a well-characterized human tumor model in genetically athymic mide, the subrenal capsule assay. The tumoricidal capacity of immunoconjugates against human tumor colorectal xenografts will be examined in this system, and in a 6 day assay in immunocompetent mice. Effect of ionophores on the in vivo cytotoxicity of conjugates will be examined. Biodistribution studies indicate rapid immunotoxin clearance from the blood after intravenous injection in tumor-bearing animals. Immunotoxins show decreased tumor localization and increased hepatic clearance, in comparison to unconjugated antibody. This rapid clearance may be related to specific clearance by liver macrophages of immunotoxin, dictated by mannose and N-acetylglucosamine on ricin A chain. Liver non-parenchymal cells have specific receptors which bind and catabolize glycoproteins terminating in these carbohydrate residues. We propose to study the biodistribution and tumoricidal effect of immunoconjugates constructed with natural (abrin) or chemically modified (ricin) aglyco-A chains. We will determine if removal of carbohydrates from A chain improves the pharmacology, tumor localization, and tumoricidal ability of such aglycoimmunoconjugates. By these studies we hope to gain a better understanding of the mechanism of immunotoxin effect on tumor cells, and to alter the pharmacologic properties of immunotoxins to improve their in vivo tumor localization and tumoricidal effect.
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