Target-selective cytotoxicity of methotrexate conjugated with monoclonal anti-MM46 antibody

Abstract

In studies on antitumor antibody-cytotoxic drug conjugates as potential tumor-selective cytotoxic agents, methotrexate (MTX) was conjugated via its active ester derivative with a murine monoclonal antibody (aMM46) to a mouse mammary tumor antigen (MM antigen) on syngeneic, ascitic C3H/He mouse mammary tumor MM46 cells. The conjugate retained full antibody activity, as assayed by complement-dependent cytolysis. The target-selective cytotoxicity of aMM46-MTX was verified by the observations that this conjugate showed greater cytotoxicity than the corresponding normal mouse immunoglobulin (nIg) conjugate to MM46 cells, neither aMM46 nor nIg being cytotoxic, and that it showed less cytotoxicity to MM antigen negative mouse mammary tumor MM48 cells than to MM46 cells, its cytotoxicity to MM48 cells being similar to that of the nIg conjugate. From the results of assays of cell binding and uptake of 131I-labeled aMM46 and aMM46-3H-MTX, aMM46 and aMM46-MTX were internalized after their binding to MM46 cell surface antigen. Leupeptin, an inhibitor of the lysosomal endopeptidase cathepsin, decreased the cytotoxicity of aMM46-MTX, supporting the involvement of lysosomal degradation of the conjugate in its action.

Effects of methotrexate-carcinoembryonic-antigen-antibody immunoconjugates on GW-39 human tumors in nude mice

“…Therefore, conjugates with an increased level of MTX substitution, through the use of an intermediate carrier, appear to be superior to the direct conjugates. Similar results wem observed when MTX was directly or indirectly (through the HSA carrier) conjugated with an aMM46 antibody [6,7]. The indirect conjugate aMM46-HSA-MTX is at least twice as toxic as the direct conjugate.…”

Anti-tumor effects of antibody-alkaline phosphatase conjugates in combination with etoposide phosphate.

“…Such mAbs have been used as carriers of most of the clinically used anticancer agents (5)(6)(7)(8) and also for highly potent toxin molecules such as the A-chain toxins (3). The purpose of much of this work has been to increase the therapeutic effect of the cytotoxic agent by enhancing its localization in the target tissue and, at the same time, to spare the nontarget tissues from its toxic effects.…”

Protein– and Peptide–Drug Conjugates

“…The amino acid-drug metabolites from conjugates with noncleavable linkers are more hydrophilic and much less membrane permeable, which leads to less bystander effects and less nonspecific toxicities compared to conjugates with a cleavable linker (Erickson et al, 2006;Okeley et al, 2010). Early examples for immunoconjugates with noncleavable linkers include immunoconjugates of methotrexate (Endo et al, 1987), daunorubicin (Pimm, Paul, Ogumuyiwa, & Baldwin, 1988), the vinca alkaloids (Spearman, Goodwin, Apelgren, & Bumol, 1987), mitomycin C (Kato, Tsukada, Hara, & Hirai, 1983), idarubicin (Rowland, Pietersz, & McKenzie, 1993), and N-acetyl melphalan (Smyth, Pietersz, & McKenzie, 1987) via amide or succinimide spacers to different murine monoclonal antibodies. At this time, the most commonly used noncleavable linkages in antibody-drug conjugates are succinimide-thioether bonds, which are formed by the reaction of maleimides with thiols.…”