title: 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.
authors.affiliation: Department of Pharmacology, The University of Michigan, Ann Arbor 48109-0582, USA.
authors.forename: D A
authors.initials: DA
authors.lastname: Hamstra
authors.forename: M
authors.initials: M
authors.lastname: Pagé
authors.forename: J
authors.initials: J
authors.lastname: Maybaum
authors.forename: A
authors.initials: A
authors.lastname: Rehemtulla
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D002268:Carboxypeptidases / Q000502:physiology*; D043422:Carboxypeptidases A; D002294:Carcinoma, Squamous Cell / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D006801:Humans; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D011355:Prodrugs / Q000494:pharmacology*; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; D011355:Prodrugs; C091716:methotrexate-alpha-phenylalanine; D010649:Phenylalanine; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D014357:Trypsin; D008727:Methotrexate