The mode of action of methotrexate‐monoclonal antibody conjugates

Abstract

Summary. 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 eniry into ceils and mechanism of action. In this study the uptake and toxic effect of three different Methotrexalemonoclonal 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 ihese MTX-MoAb conjugates gain entry into tumour cells and are processed by different mechanisms, considering the following results: (a) 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; (b) the specific MTX transpori inhibitor, p-chloromercuribenzene sulphonate (pCMS), reduced MTX toxicity but had no effect on specific MTX-MoAb conjugates; (c) the concentraiion of various ions (Ca"*, Mg-' and Mn-") effected the entry of MTX MoAb but had no effect on free MTX. MTX enters by its own carrier mechanism, while MTX-MoAb conjugaies enter by endocytosis wiih release of MTX at ihe lysosomal membrane, demonstrated by the ability of chloroquine and NH4CI (which inhibit lysosomal function) to inhibit the action of MTXMoAb 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.

The use of monoclonal antibody conjugates for the diagnosis and treatment of cancer

“…With this in mind, spacers between drug and Mab, cleavable at low pH or by lysosomal enzymes, have been developed wbich are active inside but not outside cells (16,17). From our studies and others it is clear that lysosomal degradation is necessary for activity of methotrexate and chlorambucil conjugates (18,19,20) and probably for several other drugs.…”

The effect of an anti-membrane antibody-methotrexate conjugate on the human prostatic tumour line PC3

“…Specific antibody-methotrexate conjugates have been reported to enter the cell as a consequence of antibody binding and subsequent endocytosis (Smyth et al, 1987) rather than via the active carrier-mediated transport system used for methotrexate or by the non-specific uptake of non-immune conjugates. Assessments of the cytotoxic effectiveness of the antibody-methotrexate conjugates in vitro, using measurements of both 5"Cr release from pre-loaded cells (Figure 4) and cell population parameters (Figure 2) (Kulkarni et al, 1981;Lee & Hwang, 1979;Tai et al, 1979) and in some cases an enhanced effect has been observed over the conjugate (Hurwitz et al, 1978).…”

The cellular uptake and cytotoxicity of chlorambucil‐monoclonal autibody conjugates

“…DISCUSSION CBL-MoAb conjugates were one of the first drug-MoAb conjugates described wherein the drug is rendered more toxic after coupling to the antibody; many other drugs, while gaining in specificity, lose activity after coupling to MoAbs (16; 17). It was therefore of particular interest to compare the mode of cell entry and processing of CBL-MoAb conjugates with free CBL -especially as the mode of action of MTX-MoAb conjugates had been determined (5). Given that previous studies have described the internalisation and lysosomal degradation of drug-MoAb conjugates (6), and considering the data presented here, it is likely that CBLMoAb conjugates are processed by tumour cells by endocytosis and lysosomal degrada- tion ofthe drug-MoAb conjugate.…”

“…MTX-MoAb conjugates were discovered to be 5-10 times less toxic than MTX to tumour cells and were demonstrated to enter cells by receptor-mediated endocytosis (5). It appeared likely that a threshold level of MTX incorporation per molecule of MoAb was required before cell death resulted, as some molecules of hound MTX may be sterically hindered to the extent that they cannot bind the dihydrofolate reductase enzyme.…”

“…depending on whether CBL and antibody are linked and upon the nature of that linkage. In this study the toxic actions of free CBL and three covalent CBL-MoAb conjugates are characterised using a set of parameters which had previously demonstrated differences in the cellular processing of Methotrexate (MTX) and MTX-MoAb conjugates (5).…”