Development of a Novel Enzyme/Prodrug Strategy for Gene Therapy of Breast Cancer
Abstract
We developed and tested a novel enzyme/prodrug strategy for cancer gene therapy using Carboxypeptidase A (CPA) to enzymatically convert the prodrug methotrexate-Alpha-phenylalanine (MTX-Phe) to methotrexate (MTX). CPA is normally synthesized as a zymogen that requires proteolytic removal of its pro-peptide by trypsin in order to gain catalytic activity. In Specific Aim I in order to use CPA as a therapeutic gene we engineered a battery of mutant forms of CPA which were designed to be activated by normal cellular secretory processes. We found that the best mutant, termed CPA(sub ST3), was secreted from tumor cells in an active form and biochemical analysis revealed that it had similar activity and substrate specificity to the wild-type enzyme. In Specific Aim II we produced recombinant retroviruses coding for this mutant form of CPA and demonstrated that tumor cells infected with these viruses were potently sensitized to MTX-Phe and that there was a significant 'bystander effect' even when less than 10% of the tumor cells were expressing CPA. We were unable to carry-out in vivo analysis of the MTX-Phe based system due to the unexpected instability of MTX-Phe in vivo. However, we did synthesize an active-site mutant of CPA(sub ST3) to convert the stable prodrug MTX-3-cyclopentyl-tyrosine.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jun 01, 2000
- Accession Number
- ADA391121
Entities
People
- Alnawaz Rehemtulla
- Daniel Hamstra
Organizations
- University of Michigan