title: Potent cytotoxicity of an auristatin-containing antibody-drug conjugate targeting melanoma cells expressing melanotransferrin/p97.
abstract: Identifying factors that determine the sensitivity or resistance of cancer cells to cytotoxicity by antibody-drug conjugates is essential in the development of such conjugates for therapy. Here the monoclonal antibody L49 is used to target melanotransferrin, a glycosylphosphatidylinositol-anchored glycoprotein first identified as p97, a cell-surface marker in melanomas. L49 was conjugated via a proteolytically cleavable valine-citrulline linker to the antimitotic drug, monomethylauristatin F (vcMMAF). Effective drug release from L49-vcMMAF likely requires cellular proteases most commonly located in endosomes and lysosomes. Melanoma cell lines with the highest surface p97 expression (80,000-280,000 sites per cell) were sensitive to L49-vcMMAF whereas most other cancer cell lines with lower p97 expression were resistant, as were normal cells with low copy numbers (< or = 20,000 sites per cell). Cell line sensitivity to L49-vcMMAF was found by immunofluorescence microscopy to correlate with intracellular fate of the conjugate. Specifically, L49-vcMMAF colocalized with the lysosomal marker CD107a within sensitive cell lines such as SK-MEL-5 and A2058. In contrast, in resistant cells expressing lower p97 levels (H3677; 72,000 sites per cell), L49-vcMMAF colocalized with caveolin-1, a protein prominent in caveolae, but not with CD107a. Thus, for antibody-drug conjugates targeting p97, antigen level and trafficking to the lysosomes are important factors for achieving robust in vitro cytotoxicity against cancer cells. Immunohistochemical analysis with L49 revealed that 62% of metastatic melanoma tumors had strong staining for p97. Overexpression of p97 in melanoma as compared with normal tissue, in conjunction with the greater sensitivity of tumor cells to L49-vcMMAF, supports further evaluation of antibody-drug conjugates for targeting p97-overexpressing tumors.
authors.affiliation: Seattle Genetics, Inc., 21823 30th Drive Southeast, Bothell, WA 90021, USA. lsmith@seagen.com
authors.forename: Leia M
authors.initials: LM
authors.lastname: Smith
authors.forename: Albina
authors.initials: A
authors.lastname: Nesterova
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Alley
authors.forename: Michael Y
authors.initials: MY
authors.lastname: Torgov
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Carter
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000627:therapeutic use; D000951:Antigens, Neoplasm / Q000276:immunology*; D014408:Biomarkers, Tumor / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D016503:Drug Delivery Systems; D006801:Humans; D018796:Immunoconjugates / Q000627:therapeutic use*; D008545:Melanoma / Q000188:drug therapy* / Q000276:immunology; D058950:Melanoma-Specific Antigens; D051379:Mice; D009363:Neoplasm Proteins / Q000276:immunology*; D009842:Oligopeptides / Q000627:therapeutic use*; D012878:Skin Neoplasms / Q000188:drug therapy* / Q000276:immunology; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D018796:Immunoconjugates; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins; D009842:Oligopeptides; C495575:monomethyl auristatin E