Development of methotrexate α-peptides as prodrugs for activation by enzyme-monoclonal antibody conjugates

Abstract

Selective delivery of lethal concentrations of drugs to tumors, allowing the latter to be eradicated without damage to other tissues, continues to be a major goal in cancer chemotherapy. Prodrugs (i.e. drugs that have been derivatized to prevent uptake into cells or interaction with targets), activated by enzyme-monoclonal antibody conjugates positioned at tumor sites, offer promise for achieving this objective. Methotrexate α-peptides (derivatives in which an amino acid is linked to the α-carboxyl group of the glutamate moiety) are ideal prodrugs, since they are not transported into cells and can be converted to the parent drug by carboxypeptidases. The l,l-diastereomer of MTX-α-Phe, synthesized in good yield by treatment of the p-nitrophenyl ester of 4-amino-4-deoxy-10-methylpteroic acid with Glu-α-Phe, was hydrolyzed readily by carboxypeptidase A (CP-A). Conjugate was prepared by derivatizing the enzyme and monoclonal antibody KS1/4 with linkers containing maleimide and sulfhydryl groups, respectively; interaction of these groups to form a stable thioether bond joined the proteins. When administered in vitro to UCLA-P3 human lung adenocarcinoma cells (ca. 5 × 10 4 antibody binding sites/cell) that had been pre-treated with the conjugate (whose antibody KS1/4 is targeted to these cells), and excess conjugate removed by extensive washing, MTX-Phe (ID 50 = 6.3 × 10 −8 m) approached the toxicity of MTX (ID 50 = 4.5 × 10 −8 m) In the absence of conjugate, MTX-Phe was much less toxic (ID 50 = 2.2 × 10 −6 m).