To date, antibody-directed enzyme prodrug therapy (ADEPT) treatment of patients has used enzymes of nonhuman origin. The immunogenicity of these foreign proteins precludes their long-term use for therapy. To produce enzymes of decreased immunogenicity, we will attempt to develop novel approaches using 2 human enzymes. Specifically, we will use human thymidine phosphorylase (hTP), currently a target of prodrug therapy because it is overexpressed in some human tumors, and we hypothesize that by targeting additional hTP to tumors, we will be able to use its prodrug, 5'-deoxy-5-fluorouridine. as a more effective anti-tumor therapeutic. Although the wide-spread expression of human purine nucleoside phosphorylase (hPNP) precludes its direct use as an enzyme for ADEPT we hypothesize that we can produce a mutant with altered substrate specificity that can use adenosine and deoxyadenosine containing prodrugs as substrates. A delivery system in which the same antibody can be used to deliver different molecules makes it possible to readily evaluate the efficacy of many different potential therapeutic proteins. We hypothesize that we can produce a nonimmunogenic universal delivery system comprised of human neutrophil elastase (NE) and its inhibitor (NEI). These form a strong and stable complex and we hypothesize that the NE/NEI interaction can be used to make antibody/enzyme complexes for ADEPT. Specifically, hTP and mutant hPNP will be expressed connected to the 3' end of the NE gene via a flexible linker sequence and NEI will be attached, via a flexible linker sequence, to the 3' end of the heavy chain from an antibody specific for a tumor associated antigen and expressed with the appropriate light chain. Alternatively, it can be fused to smaller antibody fragments such as scFv, Fab, and F(ab2'). If we encounter difficulties with the NE/NEI system, we will use the """"""""S.tag/S.protein"""""""" system. The enzymes and fusion proteins will be evaluated in vitro for their ability to convert prodrugs to cytotoxic agents effective against cultured cancer cells. If efficacy is observed, we will evaluate the proteins in mice. The maximum tolerated dose for the fusion proteins and the prodrugs will be determined. Biodistribution, pharmacokinetics and tumor targeting will be evaluated by traditional techniques using 125I labeled proteins and by high-resolution small animal PET imaging with 124I-labeled proteins. Antibodies specific for human CEA, HER2/neu and TfR will be compared for their ability to target tumors expressing these antigens. Mice bearing tumors will be treated by ADEPT using the most effective antibody/enzyme combination(s). We hypothesize that we will be able to develop a therapeutic approach effective in mice that can readily be applied to the treatment of human malignancy.
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|Afshar, Sepideh; Sawaya, Michael R; Morrison, Sherie L (2009) Structure of a mutant human purine nucleoside phosphorylase with the prodrug, 2-fluoro-2'-deoxyadenosine and the cytotoxic drug, 2-fluoroadenine. Protein Sci 18:1107-14|
|Afshar, Sepideh; Asai, Tsuneaki; Morrison, Sherie L (2009) Humanized ADEPT comprised of an engineered human purine nucleoside phosphorylase and a tumor targeting peptide for treatment of cancer. Mol Cancer Ther 8:185-93|