In an effort to develop a gene-dependent
enzyme/
prodrug therapy (GDEPT) for
tumor-specific delivery of
methotrexate (MTX) we have chosen to construct mutant forms of
carboxypeptidase A1 (CPA) that circumvent the requirement for
trypsin-dependent activation. The basis of this strategy is that
methotrexate-alpha-
peptides are inefficient substrates for the
reduced folate carrier (RFC) and hence cannot be internalized by cells. However, the blocking
amino acid can be cleaved by CPA to liberate MTX, which is then internalized by the RFC, resulting in inhibition of
dihydrofolate reductase and cytotoxicity. A battery of mutant CPAs was generated, in which the putative
trypsin cleavage sites in the propeptide were mutated to the consensus recognition sequence for mammalian
subtilisin-like propeptidases. These mutant forms of CPA were evaluated for expression, activation, and catalytic activity by transiently transfecting them into COS-1 cells both in the absence and in the presence of cotransfected propeptidases. CPA95 was identified as the most efficiently cleaved mutant, and further studies of this mutant indicated that the endogenously activated
enzyme had kinetic parameters identical to those of the
trypsin-activated wild-type
protein. In addition, endogenously activated CPA95 could effectively sensitize cells to
MTX-Phe in culture, decreasing the IC50 of
MTX-Phe from 25- to 250-fold in
squamous cell carcinoma cells expressing active CPA as compared with the parental lines.