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The mode of action of methotrexate-monoclonal antibody conjugates
Abstract:
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.
Insights
Methotrexate-monoclonal antibody (MTX-MoAb) conjugates enter tumor cells via endocytosis, distinct from free Methotrexate (MTX) entry. This difference impacts their toxicity and cellular processing, offering insights into targeted cancer therapy.
Area of Science:
- Oncology
- Pharmacology
- Cell Biology
Background:
- Drug-monoclonal antibody conjugates are explored for cancer therapy.
- Limited understanding exists regarding their cellular uptake and mechanisms of action.
- Investigating cellular entry pathways is crucial for optimizing drug delivery and efficacy.
Purpose of the Study:
- To compare the cellular uptake and toxic effects of Methotrexate-monoclonal antibody (MTX-MoAb) conjugates with free Methotrexate (MTX).
- To elucidate the distinct mechanisms by which MTX and MTX-MoAb conjugates enter tumor cells.
- To evaluate the influence of environmental factors and inhibitors on the cellular processing of MTX and MTX-MoAb conjugates.
Main Methods:
- Comparison of toxicity and cellular uptake of three MTX-MoAb conjugates (MTX-anti-TFR, MTX-anti-Ly-2.1, MTX-anti-L3T4) versus free MTX.
- Assessment of temperature effects on conjugate and drug toxicity.
- Evaluation of transport inhibitor (pCMS) effects on MTX and MTX-MoAb uptake.
- Investigation of ion concentration effects (Ca2+, Mg2+, Mn2+) on cellular entry.
- Analysis of lysosomal function inhibitors (chloroquine, NH4Cl) on MTX and MTX-MoAb activity.
Main Results:
- MTX-MoAb conjugates exhibited temperature-dependent toxicity, differing from MTX.
- Specific MTX transport inhibitors affected MTX but not MTX-MoAb conjugates.
- Ion concentrations influenced MTX-MoAb entry, not free MTX entry.
- Lysosomal inhibitors blocked MTX-MoAb action but not MTX action.
- MTX enters via its carrier, while MTX-MoAb conjugates are internalized by endocytosis as intact entities.
Conclusions:
- MTX-MoAb conjugates and free MTX utilize different cellular entry and processing mechanisms.
- MTX-MoAb conjugates are internalized via endocytosis, releasing MTX within lysosomes.
- MTX-MoAb conjugates are degraded in lysosomes, releasing MTX into the cytoplasm to inhibit dihydrofolate reductase (DHFR).