The long-term objective of this project is to harness the lethal potency of plant and bacterial toxins for beneficial use in medicine. To accomplish this goal we have designed a novel mechanism which combines the accurate targeting of antibody-bound toxin to cells with a precise means of triggering its release and full activity once it has reached its intracellular destination. Certain toxic proteins such as diphtheria toxin and Pseudomonas exotoxin, undergo an acid (pH 4-5) dependent conformational change within endosomal compartments inside the cell. This denaturation is critical to the passage of toxin out of these vesicles and into the cytosol where it acts to kill the cell.
The aim of this project is to generate murine and human monoclonal antibodies which will tightly bind toxin at physiological pH levels (pH 6-8) but efficiently release this toxin when it undergoes its conformational transition at pH 4-5. Such antibodies will be covalently linked to a second cell-reactive antibody or receptor ligand to form a hybrid molecule with dual specificity. This hybrid will carry and attach antibody-bound toxin to the surface of only those cells bearing chosen target sites. Toxin will be released in its lethal form when the hybrid-toxin complex is taken into cells and is exposed to the low pH in endosomes. This approach is very flexible since antibodies or ligands to virtually any membrane site can be easily coupled with the toxin-bearing antibody to form a variety of highly specific cytotoxic agents. The binding of hybrid-antibody to toxin would also block the indiscriminate attachment of toxin to cells and prevent the neutralization of hybrid-delivered toxin by any circulating endogenous antibodies. The hybrid-antibody system will be especially useful for delivering toxin fragments or genetically modified toxins with improved characteristics. A monoclonal antibody to the human transferrin receptor has been coupled to an acid-releasable monoclonal antibody directed against diphtheria toxin. This hybrid will serve to evaluate the release strategy since its receptor internalization pathway involves entry into acidified cellular compartments. Ensuing therapeutic studies with this reagent will allow us to judge its in vivo effectiveness. An athymic mouse model of early and advanced stage human malignant mesothelioma will be used to test tumoricidal action and to monitor the biodistribution of hybrid-delivered diphtheria toxin and toxin analogs.
| Raso, V; Brown, M; McGrath, J (1997) Intracellular targeting with low pH-triggered bispecific antibodies. J Biol Chem 272:27623-8 |
| Raso, V; Brown, M; McGrath, J et al. (1997) Antibodies capable of releasing diphtheria toxin in response to the low pH found in endosomes. J Biol Chem 272:27618-22 |
| Recht, L D; Raso, V; Davis, R et al. (1996) Immunotoxin sensitivity of Chinese hamster ovary cells expressing human transferrin receptors with differing internalization rates. Cancer Immunol Immunother 42:357-61 |
| Raso, V (1994) Immunotargeting intracellular compartments. Anal Biochem 222:297-304 |
