title: Human single-chain Fv immunoconjugates targeted to a melanoma-associated chondroitin sulfate proteoglycan mediate specific lysis of human melanoma cells by natural killer cells and complement.
abstract: Two antimelanoma immunoconjugates containing a human single-chain Fv (scFv) targeting domain conjugated to the Fc effector domain of human IgG1 were synthesized as secreted two-chain molecules in Chinese hamster ovary and Drosophila S2 cells, and purified by affinity chromatography on protein A. The scFv targeting domains originally were isolated as melanoma-specific clones from a scFv fusion-phage library, derived from the antibody repertoire of a vaccinated melanoma patient. The purified immunoconjugates showed similar binding specificity as did the fusion-phage clones. Binding occurred to human melanoma cells but not to human melanocytes or to several other types of normal cells and tumor cells. A 250-kDa melanoma protein was immunoprecipitated by the immunoconjugates and analyzed by mass spectrometry, using two independent procedures. A screen of protein sequence databases showed an exact match of several peptide masses between the immunoprecipitated protein and the core protein of a chondroitin sulfate proteoglycan, which is expressed on the surface of most human melanoma cells. The Fc effector domain of the immunoconjugates binds natural killer (NK) cells and also the C1q protein that initiates the complement cascade; both NK cells and complement can activate powerful cytolytic responses against the targeted tumor cells. An in vitro cytolysis assay was used to test for an immunoconjugate-dependent specific cytolytic response against cultured human melanoma cells by NK cells and complement. The melanoma cells, but not the human fibroblast cells used as the control, were efficiently lysed by both NK cells and complement in the presence of the immunoconjugates. The in vitro results suggest that the immunoconjugates also could activate a specific cytolytic immune response against melanoma tumors in vivo.
authors.affiliation: Department of Molecular Biophysics and Biochemistry Yale University, New Haven, CT 06520, USA.
authors.forename: B
authors.initials: B
authors.lastname: Wang
authors.forename: Y B
authors.initials: YB
authors.lastname: Chen
authors.forename: O
authors.initials: O
authors.lastname: Ayalon
authors.forename: J
authors.initials: J
authors.lastname: Bender
authors.forename: A
authors.initials: A
authors.lastname: Garen
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D002478:Cells, Cultured; D011508:Chondroitin Sulfate Proteoglycans / Q000276:immunology*; D002853:Chromatography, Liquid; D003165:Complement System Proteins / Q000276:immunology*; D006224:Cricetinae; D003602:Cytotoxicity, Immunologic; D004331:Drosophila melanogaster; D004730:Endothelium, Vascular / Q000276:immunology / Q000502:physiology; D006801:Humans; D018796:Immunoconjugates / Q000302:isolation & purification / Q000633:toxicity*; D007128:Immunoglobulin Fragments / Q000235:genetics; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007694:Killer Cells, Natural / Q000276:immunology*; D013058:Mass Spectrometry; D008545:Melanoma / Q000276:immunology* / Q000473:pathology; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection; D014407:Tumor Cells, Cultured