| Abstract
| - Antibody-directed enzyme prodrug therapy (ADEPT) is a technique to increase antitumor selectivityin cancer chemotherapy. Our approach to this technology has been to design a mutant of humancarboxypeptidase A (hCPA1-T268G) which is capable of hydrolyzing in vivo stable prodrugs of MTXand targeting this enzyme to tumors on an Ep-CAM1-specific antibody, ING1. Through the use ofthis >99% human enzyme which is capable of catalyzing a completely nonhuman reaction, we hopeto increase ADEPT selectivity while decreasing overall immunogenicity of the enzyme−antibodyconjugate. In the current report, prodrugs of the thymidylate synthase inhibitors GW1031 and GW1843and the dihydrofolate reductase inhibitor methotrexate were studied for their wild-type and mutanthCPA enzyme hydrolysis, their in vivo stability, and their use in therapy. Prodrugs with high kcat/Kmratios for mutated versus wild-type hCPA1 were examined in vitro for their stability in humanpancreatic juice, and in vivo for their stability in mouse plasma and tissues. In addition, targetingand in vivo enzyme activity studies were performed with an ING1 antibody conjugate of the mutantenzyme (ING1-hCPA1-T268G). Finally, in vivo therapy studies were performed with LS174T tumorsto demonstrate proof of principle. Results indicate that prodrugs can be synthesized that are selectiveand efficient substrates of hCPA1-T268G and not substrates of the endogenous CPA activities; thisleads to excellent in vivo stability for these compounds. In vivo conjugate targeting studies showedthat the antibody−enzyme conjugate was targeted to the tumor and enzyme was initially active invivo at the site. Unfortunately therapeutic studies did not demonstrate tumor reduction. Experimentsto determine reasons for the lack of antitumor activity showed that the enzyme activity decreased asa result of enzyme instability. The results offer encouragement for additional novel mutant enzymeimprovements and additional in vivo studies on this unique approach to ADEPT.
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