Preparation and in vitro cytotoxicity of a methotrexate-anti-MM46 monoclonal antibody conjugate via an oligopeptide spacer.
A method was developed by which conjugates of methotrexate (MTX) with antibody were prepared via an oligopeptide spacer which, after internalization of the conjugates into the target cells, would be cleaved by lysosomal enzymes to liberate MTX or its derivative(s) for entry of the drug into the cytoplasm through the lysosomal membrane. The conjugate of MTX with a monoclonal antibody (MAb) (aMM46) to mouse mammary carcinoma MM46 cells prepared by this method via Leu-Ala-Leu-Ala showed potent, antibody-directed cytotoxicity through lysosomal degradation of the conjugate, most likely at the tetrapeptide spacer. This was supported by the following observations: (a) the cytotoxicity of the aMM46 conjugate was more potent than that of the corresponding normal gamma-globulin conjugate, the antibody alone not being cytotoxic; (b) the conjugate retained its potent cytotoxicity to MM46 cells even after removal, by hydroxylamine treatment, of a less stably bound MTX derivative which might have been released extracellularly and have caused non-specific cytotoxicity by entering the cells via the membrane active transport system for MTX; (c) the cytotoxicity was not inhibited by thiamine pyrophosphate, an inhibitor of the MTX transport system; (d) the cytotoxicity was inhibited significantly with ammonium chloride, which inactivates lysosomal enzymes by raising the pH; and (e) the cleavability of the Leu-Ala-Leu-Ala spacer by the lysosomal enzymes was verified by using bio-undegradable poly(D-lysine) and biodegradable poly(L-lysine) as the lysosomotropic macromolecular carriers: unlike the poly(L-lysine) counterpart, the direct MTX-poly(D-lysine) conjugate showed very weak cytotoxicity while the tetrapeptide-mediated conjugate of MTX with poly(D-lysine) exhibited potent cytotoxicity.
Umemoto
N
N
Chemical and Biochemical Research Department, Teijin Institute for Biomedical Research, Tokyo, Japan.
Kato
Y
Y
Endo
N
N
Takeda
Y
Y
Hara
T
T
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification* / Q000633:toxicity; D000951:Antigens, Neoplasm / Q000276:immunology*; D002478:Cells, Cultured; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D004347:Drug Interactions; D004355:Drug Stability; D000922:Immunotoxins / Q000302:isolation & purification* / Q000494:pharmacology / Q000633:toxicity; D007519:Isoantigens / Q000276:immunology*; D008247:Lysosomes / Q000187:drug effects / Q000201:enzymology; D008722:Methods; D008727:Methotrexate / Q000302:isolation & purification* / Q000494:pharmacology / Q000633:toxicity; D051379:Mice; D008809:Mice, Inbred C3H; D008819:Mice, Nude; D009842:Oligopeptides / Q000302:isolation & purification* / Q000494:pharmacology / Q000633:toxicity
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D000922:Immunotoxins; D007519:Isoantigens; D009842:Oligopeptides; D008727:Methotrexate