The long-term objectives of this project are to target radioactive metals to tumors for diagnosis and therapy of human cancer. This is done through a combination of chemical synthesis and protein engineering, to develop methodology to improve targeting efficiency, to increase the residence times of these metals in the targets, and to decrease their levels in normal tissues. Antibodies have been conjugated to metal chelates for radioimmunotherapy and studied extensively in recent years, and are in clinical trials at various stages. Recent data from our collaboration indicate that radioimmunoconjugates have efficacy comparable to conventional antineoplastic drugs, and work in synergy with them (but without severe side effects). The systemic use of antineoplastic drugs is associated with undesirable side effects including toxicity to normal cells, which effectively traverse cell membranes and become widely distributed through the body. Polymers and other macromolecules do not traverse membranes; however, they may be selectively accumulated in the interstitial space of a tumor, since tumors typically do not possess an efficient lymphatic drainage system. Such targeted, non-penetrating molecules have less toxicity to normal tissue. The residence of macromolecules in tumors may be prolonged if they become anchored to immobile elements, such as polymorphic epithelial mucin, the secreted product of the MUCI gene, or HLA-DR, a long-lived cell surface protein. The reagents of choice for this anchoring reaction are monoclonal antibodies and their derivatives. The main focus of this proposal is to take advantage of our increased knowledge of the detailed features of antibody-antigen structures to develop reagents that bind to their targets with extreme tenacity. This will be carried out first with an antibody-antigen complex where experiments can be evaluated directly by comparison with the crystal structure. We will extend this methodology to another antibody-antigen pair to eliminate the use of streptavidin and biotin in pretargeted antibody protocols. And we will explore its broader use to improve the binding of engineered antibody fragments to their antigenic targets. In a second project, we will follow up our recent results with combinatorial peptide libraries for development of cleavable peptide linkers to select those which remain uncleaved in circulation and at the tumor target, but are readily cleaved intracellularly and clear the liver, kidney, and other non-target sites. The linkers developed should be of considerable use to other researchers in the area of drug delivery. Finally, we will develop pretargeted enzyme-activated binding of radiopharmaceuticals to tumor cell membranes, by enzymatic conversion of circulating hydrophilic metal chelates to hydrophobic membrane binders.
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