Activation of Methotrexate Prodrugs by Enzyme/Monoclonal Antibody Conjugates

Abstract

One of the major goals in cancer chemotherapy is the development of procedures for the selective delivery of drugs to tumor cells. This strategy, when perfected, will allow drugs to be used at the high concentrations necessary for virtually complete (i. e., > 99%) eradication of the tumor cells without concomitant destruction of normal cells. Monoclonal antibodies targeted to tumor antigens offer considerable promise for effecting the desired selectivity. To date, however, only limited success has been achieved in clinical trials with monoclonals as vehicles for carrying drugs. Two limitations are evident in the use of drug-monoclonal conjugates: (a) Relatively few drug molecules can be linked to each monoclonal without impairing binding activity; and (b) A mechanism is needed to release covalently-bound drug from the antibody. In an effort to overcome these problems, attention has focused recently on the attachment of enzymes rather than drugs to monoclonals, thereby taking advantage of the catalytic power of the enzyme to generate large amounts of the active drugs from inert prodrugs at the tumor site [reviewed by Senter (1)].Methotrexate (MTX), employed extensively in cancer chemotherapy, is well-suited to the prodrug/enzymemonoclonal conjugate strategy. α-Peptides of the drug (i.e., derivatives in which the a-COOH has been linked to an amino acid) are relatively non-toxic, but these derivatives can be hydrolyzed by carboxypep-" Recipient of an EORTC Fellowship. Present address: Institute

Toward Antibody-directed Enzyme Prodrug Therapy with the T268G Mutant of Human Carboxypeptidase A1 and Novel in VivoStable Prodrugs of Methotrexate

“…For this, we synthesized MTXPhe and tested its substrate activity with the human CPA1 and CPA2. As reported for the bovine CPA, MTX-Phe is a good hCPA1 substrate and a fair hCPA2 substrate (21,31). We also confirmed that the prodrug was stable in fetal bovine serumand human serum-based growth media and was approximately 200 times less toxic than MTX in cell culture.…”

“…We reported previously that the compound is a good substrate for both human isozymes, hCPA1 and hCPA2 (31). Thus, an hCPAantibody conjugate should effectively hydrolyze MTX-Phe for human enzyme-based ADEPT as had been shown previously for a bovine CPA-antibody conjugate (21).…”

“…These prodrugs were relatively nontoxic in cell culture but could be activated by bovine CPA or carboxypeptidase B to form MTX. The most successful of these prodrugs was MTX-Phe, which was found to be an excellent substrate for bovine CPA (21). This system was effective under cell culture conditions where the IC 50 of MTX-Phe against L1210 cells changed from 2.2 ϫ 10 Ϫ6 to 6.3 ϫ 10 Ϫ8 M, indistinguishable from MTX itself, in the presence of bovine CPA or a bovine CPA-antibody conjugate (21).…”

“…The most successful of these prodrugs was MTX-Phe, which was found to be an excellent substrate for bovine CPA (21). This system was effective under cell culture conditions where the IC 50 of MTX-Phe against L1210 cells changed from 2.2 ϫ 10 Ϫ6 to 6.3 ϫ 10 Ϫ8 M, indistinguishable from MTX itself, in the presence of bovine CPA or a bovine CPA-antibody conjugate (21). An important positive aspect of this system is its use of MTX with its well known efficacy and toxicity profile (38,39).…”

“…MTX was included in separate wells in all experiments as an internal control for IC 50 reproducibility. (21) reported that MTX-Phe is a good substrate for bovine CPA. Only one pancreatic CPA isozyme has been found in this species; however, in rodents and humans two isozymes exist.…”