title: Combinatorial approach to increase efficacy of Cetuximab, Panitumumab and Trastuzumab by dianthin conjugation and co-application of SO1861.
abstract: The therapeutic relevance of immunotoxins is based on the conjugation of monoclonal antibodies to toxins. In cancer therapies, the conjugated antibodies not only direct the binding of immunotoxins to cancer-specific receptors and mediate the elimination of tumor cells through the innate immune system, but also increase target cytotoxicity by the intrinsic toxin activity. In the present study, the therapeutic antibodies Cetuximab (anti-EGFR, Erbitux(®)), Panitumumab (anti-EGFR, Vectibix(®)) and Trastuzumab (anti-HER2, Herceptin(®)) were chemically conjugated to the toxin dianthin. In the first instance, recombinant dianthin was characterized by mass spectrometry and its stability was analyzed by circular dichroism. Dianthin showed increased cytotoxicity on MCF-7 cells when tested in combination with a glycosylated triterpenoid (SO1861) in a real-time impedance-based cytotoxicity assay. In data obtained by live cell imaging, SO1861 specifically mediated the endo/lysosomal escape of dianthin without disrupting the plasma membrane. The purity of immunotoxins was confirmed by SDS-PAGE and Western blot. Their cytotoxicity was evaluated in the presence of SO1861 and dianthin-Cetuximab presented a GI50 (50% growth inhibition) of 5.3pM, dianthin-Panitumumab of 1.5pM, and dianthin-Trastuzumab of 23pM. Finally, the specificity of these immunotoxins was validated in a fluorescence-based real-time assay, where their binding to target cells was prevented by preincubation with an excess of label-free unconjugated antibody. Based on these data, we propose the use of dianthin and SO1861 as a new platform technology to enhance the efficacy of therapeutic antibodies.
authors.affiliation: Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité - Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, D-13353 Berlin, Germany; Department of Medicine, University of Melbourne, Austin Health, Studley Road, VIC 3084 Heidelberg, Australia.
authors.forename: Roger
authors.initials: R
authors.lastname: Gilabert-Oriol
authors.affiliation: Institut für Pharmazie-Pharmazeutische Biologie, Freie Universität Berlin, Königin-Luise-Straße 2+4, D-14195 Berlin, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Weng
authors.affiliation: Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité - Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, D-13353 Berlin, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Trautner
authors.affiliation: Institut für Chemie und Biochemie, Freie Universität Berlin, Thielallee 63, D-14195 Berlin, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Weise
authors.affiliation: Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité - Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, D-13353 Berlin, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Schmid
authors.affiliation: Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité - Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, D-13353 Berlin, Germany.
authors.forename: Cheenu
authors.initials: C
authors.lastname: Bhargava
authors.affiliation: Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité - Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, D-13353 Berlin, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Niesler
authors.affiliation: Department of Medicine, University of Melbourne, Austin Health, Studley Road, VIC 3084 Heidelberg, Australia.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Wookey
authors.affiliation: Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité - Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, D-13353 Berlin, Germany.
authors.forename: Hendrik
authors.initials: H
authors.lastname: Fuchs
authors.affiliation: Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité - Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, D-13353 Berlin, Germany. Electronic address: mayankthakur25@gmail.com.
authors.forename: Mayank
authors.initials: M
authors.lastname: Thakur
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000494:pharmacology*; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D000068818:Cetuximab / Q000008:administration & dosage / Q000494:pharmacology*; D003432:Cross-Linking Reagents / Q000737:chemistry; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism; D005260:Female; D045325:HCT116 Cells; D006801:Humans; D000922:Immunotoxins / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D061986:MCF-7 Cells; D008969:Molecular Sequence Data; D000077544:Panitumumab; D055550:Protein Stability; D011994:Recombinant Proteins; D012503:Saponins / Q000008:administration & dosage / Q000494:pharmacology*; D000068878:Trastuzumab / Q000008:administration & dosage / Q000494:pharmacology*
chemical_list: D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D000922:Immunotoxins; D011994:Recombinant Proteins; C000603609:SO1861; D012503:Saponins; D000077544:Panitumumab; D000068878:Trastuzumab; D000068818:Cetuximab
keywords: Dianthin; Efficacy enhancer; Endosomal escape; SO1861; Therapeutic antibody