Expression of endogenously activated secreted or cell surface carboxypeptidase A sensitizes tumor cells to methotrexate-alpha-peptide prodrugs.

Abstract

Methotrexate (MTX) is one of the most commonly used agents in the treatment of solid malignancies; however, the toxicities of MTX to bone marrow and gastrointestinal tract complicate this therapy. We, therefore, propose a gene-dependent enzyme prodrug therapy to limit these toxicities by localizing the production of MTX to the site of the tumor. The combination of MTX-alpha-peptide prodrugs, which cannot be internalized by the cellular reduced folate carrier, with carboxypeptidase A (CPA), which can remove the blocking peptide, has been demonstrated previously in vitro using antibody-dependent enzyme prodrug therapy. CPA is normally synthesized as a zymogen that is inactive without proteolytic removal of its propeptide by trypsin. Therefore, to adapt this system to gene-dependent enzyme prodrug therapy, a mutant form of CPA was engineered, CPA(ST3), that does not require trypsin-dependent zymogen cleavage but is instead activated by ubiquitously expressed intracellular propeptidases. Purification, peptide sequencing, and kinetic analysis indicated that mature CPA(ST3) is structurally and functionally similar to the trypsin-activated, wild-type enzyme. In addition, CPA(ST3)-expressing tumors cells were sensitized to MTX prodrugs in a dose- and time-dependent manner. To limit diffusion of CPA, a cell surface localized form was generated by constructing a fusion protein between CPA(ST3) and the phosphatidylinositol linkage domain from decay accelerating factor. SDS-PAGE and flow cytometric analysis of infected tumor cells indicated that CPA(DAF) was cell surface localized. Finally, after retroviral transduction, this enzyme/prodrug strategy exhibited a potent bystander effect, even when <10% of the cells were transduced, because extracellular production of MTX sensitized both transduced and nontransduced cells.