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

Abstract

Antibody-directed enzyme prodrug therapy (ADEPT) is a technique to increase antitumor selectivity in cancer chemotherapy. Our approach to this technology has been to design a mutant of human carboxypeptidase A (hCPA1-T268G) which 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 use of this >99% human enzyme which is capable of catalyzing a completely nonhuman reaction, we hope to increase ADEPT selectivity while decreasing overall 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 kcat/Km ratios for mutated versus wild-type hCPA1 were examined in vitro for their stability in human pancreatic juice, and in vivo for their stability in mouse plasma and tissues. In addition, targeting and in vivo enzyme activity studies were performed with an ING1 antibody conjugate of the mutant enzyme (ING1-hCPA1-T268G). Finally, in vivo therapy studies were performed with LS174T tumors to demonstrate proof of principle. Results indicate that prodrugs can be synthesized that are selective and efficient substrates of hCPA1-T268G and not substrates of the endogenous CPA activities; this leads to excellent in vivo stability for these compounds. In vivo conjugate targeting studies showed that the antibody-enzyme conjugate was targeted to the tumor and enzyme was initially active in vivo at the site. Unfortunately therapeutic studies did not demonstrate tumor reduction. Experiments to determine reasons for the lack of antitumor activity showed that the enzyme activity decreased as a result of enzyme instability. The results offer encouragement for additional novel mutant enzyme improvements and additional in vivo studies on this unique approach to ADEPT.

Modifying an immunogenic epitope on a therapeutic protein: a step towards an improved system for antibody-directed enzyme prodrug therapy (ADEPT)

“…Hence repeated treatment with ADEPT, which is ultimately necessary to achieve effective therapy, has previously only been possible with cyclosporin immunosuppression, which carries the potential of unwanted side effects such as organ toxicity (Sharma et al, 1996). Attempts to overcome this by engineering human carboxypeptidase have been elegant in principal but ineffective in vivo (Wolfe et al, 1999).…”

Metallocarboxypeptidases: Emerging Drug Targets in Biomedicine

“…Excellent in vitro results were obtained, strongly supporting the feasibility of ADEPT using a mutated human enzyme. However, in vivo therapeutic studies did not demonstrate tumor reduction in xenografted mice models, mainly as a result of enzyme instability [125,126]. In spite of its associated immunogenicity, CPG2 has been also largely used in ADEPT.…”

Metallocarboxypeptidases: Emerging Drug Targets in Biomedicine

“…Attempts are being made to overcome the immune response through "humanization" of the antibodies, in which most of the sequence of the mouse monoclonal antibody is replaced with corresponding human antibody sequence. However, in vivo therapeutic studies did not demonstrate tumor reduction in xenografted mice models, mainly as a result of enzyme instability [125,126]. The active site of the human enzyme is changed at one or two residues to produce an almost fully human enzyme capable of efficiently performing a nonhuman reaction.…”