Antibody-directed enzyme prodrug therapy with the T268G mutant of human carboxypeptidase A1: In vitro and in vivo studies with prodrugs of methotrexate and the thymidylate synthase inhibitors GW1031 and GW1843

Antibody-directed enzyme prodrug therapy (ADEPT) is a technique to increase antitumor selectivity in cancer chemotherapy. Our approach to this technol ogy has been to design a mutant of human carboxypeptidase A (hCPA1-T268G) w hich is capable of hydrolyzing in vivo stable prodrugs of MTX and targeting this enzyme to tumors on an Ep-CAM1-specific antibody, ING1. Through the u se of this >99% human enzyme which is capable of catalyzing a completely no nhuman reaction, we hope to increase ADEPT selectivity while decreasing ove rall immunogenicity of the enzyme-antibody conjugate. In the current report , prodrugs of the thymidylate synthase inhibitors GW1031 and GW1843 and the dihydrofolate reductase inhibitor methotrexate were studied for their wild -type and mutant hCPA enzyme hydrolysis, their in vivo stability, and their use in therapy. Prodrugs with high k(cat)/K-m ratios for mutated versus wi ld-type hCPA1 were examined in vitro for their stability in human pancreati c juice, and in vivo for their stability in mouse plasma and tissues. In ad dition, targeting and in vivo enzyme activity studies were performed with a n ING1 antibody conjugate of the mutant enzyme (ING1-hCPA1-T268G). Finally, in vivo therapy studies were performed with LS174T tumors to demonstrate p roof of principle. Results indicate that prodrugs can be synthesized that a re selective and efficient substrates of hCPA1-T268G and not substrates of the endogenous CPA activities; this leads to excellent in vivo stability fo r these compounds. In vivo conjugate targeting studies showed that the anti body-enzyme conjugate was targeted to the tumor and enzyme was initially ac tive in vivo at the site. Unfortunately therapeutic studies did not demonst rate tumor reduction. Experiments to determine reasons for the lack of anti tumor activity showed that the enzyme activity decreased as a result of enz yme instability. The results offer encouragement for additional novel mutan t enzyme improvements and additional in vivo studies on this unique approac h to ADEPT.