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

Abstract

Antibody-directed enzyme prodrug therapy (ADEPT) has the potential of greatly enhancing antitumor selectivity of cancer therapy by synthesizing chemotherapeutic agents selectively at tumor sites. This therapy is based upon targeting a prodrug-activating enzyme to a tumor by attaching the enzyme to a tumor-selective antibody and dosing the enzyme-antibody conjugate systemically. After the enzyme-antibody conjugate is localized to the tumor, the prodrug is then also dosed systemically, and the previously targeted enzyme converts it to the active drug selectively at the tumor. Unfortunately, most enzymes capable of this specific, tumor site generation of drugs are foreign to the human body and as such are expected to raise an immune response when injected, which will limit their repeated administration. We reasoned that with the power of crystallography, molecular modeling and site-directed mutagenesis, this problem could be addressed through the development of a human enzyme that is capable of catalyzing a reaction that is otherwise not carried out in the human body. This would then allow use of prodrugs that are otherwise stable in vivo but that are substrates for a tumor-targeted mutant human enzyme. We report here the first test of this concept using the human enzyme carboxypeptidase A1 (hCPA1) and prodrugs of methotrexate (MTX). Based upon a computer model of the human enzyme built from the well known crystal structure of bovine carboxypeptidase A, we have designed and synthesized novel bulky phenylalanine-and tyrosine-based prodrugs of MTX that are metabolically stable in vivo and are not substrates for wild type human carboxypeptidases A. Two of these analogs are MTX-␣-3-cyclobutylphenylalanine and MTX-␣-3-cyclopentyltyrosine. Also based upon the computer model, we have designed and produced a mutant of human carboxypeptidase A1, changed at position 268 from the wild type threonine to a glycine (hCPA1-T268G). This novel enzyme is capable of using the in vivo stable prodrugs, which are not substrates for the wild type hCPA1, as efficiently as the wild type hCPA1 uses its best sub-

Novel Integrin-Targeted Binding-Triggered Drug Delivery System for Methotrexate

“…The MTX-α amino acid prodrugs reported in literature utilized CPA as the specific enzyme for activation, since it can cleave the terminal amino acid with a free α-carboxylic acid group (COOH) (4). Prodrugs of the current study differ from MTX-α amino acid prodrugs in two ways and hence cannot use CPA enzyme to activate the prodrug.…”

“…These prodrugs were made by allowing glutamic acid residue, α-COOH of MTX, to react with an additional amino acid. When phenylalanine (MTX-α-Phe) is used as the additional amino acid, it was shown to be a better substrate for CPA among all other single amino acid conjugated prodrugs (4). The bulkier structure of the labile link in MTX-α-Phe prodrug made it insensitive to the endogenous carboxypeptidase enzyme and hence metabolically more stable in the plasma.…”

“…Most improvements for tumor-targeted enzyme-based prodrug activation delivery methods have been focused on optimizing specificity (K cat /K M ) and reducing the half-life in order to reduce prodrug activation away from the tumor environment (4,5). In addition, introducing clearing agents to expedite the removal of residual enzyme antibody conjugate (E-Ab) from blood, restricting the enzyme expression within the tumor cell, and using humanized antibodies have alleviated these problems to some extent (1,3,6,7).…”

Antibody‐guided enzyme therapy of cancer producing cyanide results in necrosis of targeted cells

“…Current prodrug activation strategies rely on the regeneration of conventional chemotherapeutic drugs such as methotrexate and doxorubicin. 9 Tumour cells that have already built up resistance to such drugs are unlikely to be susceptible to the same drugs, even if generated at higher concentrations in situ by targetable systems.…”

Prodrug-activating systems in suicide gene therapy

“…Black et al (15) applied these techniques to obtain mutants of HSV-TK with improved kinetic parameters for the prodrugs GCV and ACV. Similarly, Smith and colleagues (16) performed site-directed mutagenesis on carboxypeptidase A to improve the efficiency of this enzyme toward specific substrates that, by design, are less prone to interfere with other human peptidases (16).…”