title: Targeting, internalization, and cytotoxicity of methotrexate-monoclonal anti-stage-specific embryonic antigen-1 antibody conjugates in cultured F-9 teratocarcinoma cells.
abstract: Methotrexate (MTX) conjugates of a monoclonal antibody, anti-SSEA-1, containing an average of 45 mol MTX/mol of immunoglobulin M, were prepared by a carbodiimide coupling reaction. Binding experiments indicate that conjugation does not decrease the affinity of the antibody for its antigen. The conjugate strongly inhibits the growth of SSEA-1-bearing F-9 teratocarcinoma cells, with 50% inhibitory dose of 4.5 nM MTX, which makes it more active than free MTX (50% inhibitory dose of 15 nM). The drug-free antibody is not cytotoxic to F-9 cells at the concentrations used. The high efficacy of the conjugated drug may be due in part to the fact that anti-SSEA-1 antibody is an immunoglobulin M. MTX conjugated to nonspecific immunoglobulin M has little inhibitory effect (50% inhibitory dose of 150 nM). When acting on SSEA-1 negative cells, the two conjugates have only a small but identical effect. Thiamine pyrophosphate, an inhibitor of MTX transport, can prevent the cytotoxicity of the free MTX but not that of the anti-SSEA-1 conjugate. Leupeptin, an inhibitor of lysosomal protease, can partially protect F-9 cells against the antibody conjugate but not against free MTX. These results indicate that the MTX antibody conjugate binds specifically to F-9 cells, and is internalized and intracellularly degraded to release a small molecular active drug. Pretreatments of F-9 cells for 1 h with unlabeled antibody inhibits the subsequent uptake of identical concentration of labeled conjugate. The rate of internalization, however, regains almost normal values within 4 h, indicating a rapid reappearance of free antigenic sites at the cell surface.
authors.forename: W C
authors.initials: WC
authors.lastname: Shen
authors.forename: B
authors.initials: B
authors.lastname: Ballou
authors.forename: H J
authors.initials: HJ
authors.lastname: Ryser
authors.forename: T R
authors.initials: TR
authors.lastname: Hakala
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage*; D006017:Glycolipids / Q000276:immunology*; D007074:Immunoglobulin G / Q000008:administration & dosage; D002955:Leucovorin / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D016256:Lewis X Antigen; D008247:Lysosomes / Q000378:metabolism; D008727:Methotrexate / Q000008:administration & dosage* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D013724:Teratoma / Q000276:immunology / Q000473:pathology* / Q000628:therapy; D013835:Thiamine Pyrophosphate / Q000494:pharmacology; D014316:Tritium
chemical_list: D000911:Antibodies, Monoclonal; D006017:Glycolipids; D007074:Immunoglobulin G; D007976:Leupeptins; D016256:Lewis X Antigen; D014316:Tritium; C032854:leupeptin; D002955:Leucovorin; D013835:Thiamine Pyrophosphate; D008727:Methotrexate