In vitro cytotoxicity of a human serum albumin-mediated conjugate of methotrexate with anti-MM46 monoclonal antibody.
In studies on antitumor antibody:drug conjugates as potential antitumor agents, methotrexate (MTX) was conjugated with a murine monoclonal antibody (aMM46) to an antigen on ascitic mouse mammary tumor MM46 cells (MM antigen) with human serum albumin (HSA) as an intermediary. MTX was linked to HSA which had been conditioned to have about 1 mol of thiol group per mol of HSA by dithiothreitol treatment followed by oxidation on standing at 4 degrees C. The MTX linking was performed, without protection of the thiol group of HSA, by using MTX N-succinimidyl ester prepared via MTX intramolecular anhydride. The resulting HSA:MTX was reacted with the immunoglobulin with the maleimide group introduced. The aMM46:HSA:MTX obtained retained both antibody binding and drug activities. The cytotoxicity of aMM46:HSA:MTX against MM antigen-positive MM46 cells was greater than that of control 96.5 (anti-human melanoma-associated antigen, p97):HSA:MTX and was inhibited by unconjugated aMM46. No different cytotoxicity of aMM46:HSA:MTX compared with that of 96.5:HSA:MTX was observed against MM antigen-negative mouse mammary tumor MM48 cells. The presence of ammonium chloride or leupeptin abrogated the selective cytotoxicity against MM46 cells of aMM46 conjugate but did not affect the nonspecific cytotoxicity of 96.5:HSA:MTX. These results support the idea that the selective cytotoxicity of aMM46:HSA:MTX is antibody directed and exhibited through lysosomal degradation of the conjugate.
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal; D000920:Antibody-Dependent Cell Cytotoxicity; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002460:Cell Line; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003602:Cytotoxicity, Immunologic; D007976:Leupeptins / Q000494:pharmacology; D008727:Methotrexate / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D008809:Mice, Inbred C3H; D012709:Serum Albumin / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D007976:Leupeptins; D012709:Serum Albumin; D000643:Ammonium Chloride; C032854:leupeptin; D008727:Methotrexate