The mode of action of methotrexate-monoclonal antibody conjugates.

Drug-monoclonal antibody conjugates have been evaluated for their specificity and toxicity towards tumour cells in vitro and in vivo; however, few studies have investigated their mode of entry into cells and mechanism of action. In this study the uptake and toxic effect of three different Methotrexate-monoclonal antibody (MTX-MoAb) conjugates (MTX-anti-transferrin receptor (TFR), MTX-anti-Ly-2.1 and MTX-anti-L3T4) were examined and compared with free MTX. It was concluded that MTX and these MTX-MoAb conjugates gain entry into tumour cells and are processed by different mechanisms, considering the following results: alterations in temperature had a greater effect on the toxicity of MTX-MoAb than on MTX; in addition, MTX and MTX-MoAb had different rates of action on cells; the specific MTX transport inhibitor, p-chloromercuribenzene sulphonate (pCMS), reduced MTX toxicity but had no effect on specific MTX-MoAb conjugates; the concentration of various ions (Ca2+, Mg2+ and Mn2+) effected the entry of MTX-MoAb but had no effect on free MTX. MTX enters by its own carrier mechanism, while MTX-MoAb conjugates enter by endocytosis with release of MTX at the lysosomal membrane, demonstrated by the ability of chloroquine and NH4Cl (which inhibit lysosomal function) to inhibit the action of MTX-MoAb but not MTX. Therefore, these MTX-MoAb conjugates are not degraded at the surface but bind to their receptor and then enter the cell by endocytosis as one entity; the MTX-MoAb conjugates are then degraded within the lysosomes, resulting in the release of free MTX into the cytoplasm where it acts on dihydrofolate reductase (DHFR) to inhibit cell metabolism.