Delivery of Radionuclides to Pretargeted Monoclonal Antibodies Using Dihydrofolate Reductase and Methotrexate in an Affinity System

Abstract

A novel affinity system for a two-phase delivery of radionuclides to tumor cells has been developed. In the first phase, a nontoxic bivalent monoclonal antibody conjugated to an enzyme is targeted to the tumor cells. In the second phase, a radionuclide-derivatized enzyme inhibitor, specific for the enzyme conjugated to the antibody, is administered. The model system selected for this study is the recombinant human enzyme dihydrofolate reductase (rhDHFR) and its high-affinity competitive inhibitor methotrexate (MTX). MTX was labeled with a radionuclide by covalent attachment of diethylenetriaminepentaacetic acid (DTPA) complexed with ¹¹¹In. Using the γ-carboxyl residue of MTX for the attachment of DTPA, binding of the inhibitor to rhDHFR was not affected. The inhibitory activities of nonderivatized MTX and DTPA-MTX were indistinguishable. Human K562 erythroleukemia cells were used to evaluate under in vitro conditions the DHFR-MTX affinity system for the delivery of ¹¹¹In-labeled DTPA-MTX to pretargeted α-transferrin receptor antibody-rhDHFR conjugates (α-TFR-DHFR). The data demonstrate that the delivery of ¹¹¹In is dose dependent and highly specific. Under saturating conditions, binding of ¹¹¹In-DTPA-MTX to α-TFR-DHFR-treated cells was 14-fold higher than to cells treated with nonconjugated α-TFR antibody. Further experiments indicated that the low level of nonspecific binding of ¹¹¹In-DTPA-MTX was comparable to that of ¹¹¹In-DTPA, known for its complete extracellular distribution and rapid clearance through the kidneys. Based on the data of this study, antibody-conjugated rhDHFR and radionuclide-labeled DTPA-MTX complexes provide components for an alternative radioimmunotherapeutic approach that can be expected to result in improved tumor tissue ratios of both the targeting moiety and the radionuclide-labeled derivative as compared to current approaches.