title: The efficient elimination of solid tumor cells by EGFR-specific and HER2-specific scFv-SNAP fusion proteins conjugated to benzylguanine-modified auristatin F.
abstract: Antibody-drug conjugates (ADCs) combine the potency of cytotoxic drugs with the specificity of monoclonal antibodies (mAbs). Most ADCs are currently generated by the nonspecific conjugation of drug-linker reagents to certain amino acid residues in mAbs, resulting in a heterogeneous product. To overcome this limitation and prepare ADCs with a defined stoichiometry, we use SNAP-tag technology as an alternative conjugation strategy. This allows the site-specific conjugation of O(6)-benzylguanine (BG)-modified small molecules to SNAP-tag fusion proteins. To demonstrate the suitability of this system for the preparation of novel recombinant ADCs, here we conjugated SNAP-tagged single chain antibody fragments (scFvs) to a BG-modified version of auristatin F (AURIF). We used two scFv-SNAP fusion proteins targeting members of the epidermal growth factor receptor (EGFR) family that are frequently overexpressed in breast cancer. The conjugation of BG-AURIF to EGFR-specific 425(scFv)-SNAP and HER2-specific αHER2(scFv)-SNAP resulted in two potent recombinant ADCs that specifically killed breast cancer cell lines by inducing apoptosis when applied at nanomolar concentrations. These data confirm that SNAP-tag technology is a promising tool for the generation of novel recombinant ADCs.
authors: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstr. 6, 52074 Aachen, Germany; Institute of Molecular Biotechnology (Biology VII), RWTH Aachen University, Worringer Weg 1, 52074 Aachen, Germany.
authors: Mira
authors: M
authors: Woitok
authors: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstr. 6, 52074 Aachen, Germany.
authors: Diana
authors: D
authors: Klose
authors: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstr. 6, 52074 Aachen, Germany.
authors: Judith
authors: J
authors: Niesen
authors: TUBE Pharmaceuticals GmbH, Leberstr. 20, 1110 Vienna, Austria.
authors: Wolfgang
authors: W
authors: Richter
authors: TUBE Pharmaceuticals GmbH, Leberstr. 20, 1110 Vienna, Austria.
authors: Muhammad
authors: M
authors: Abbas
authors: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstr. 6, 52074 Aachen, Germany; Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Pauwelsstr. 20, 52074 Aachen, Germany.
authors: Christoph
authors: C
authors: Stein
authors: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstr. 6, 52074 Aachen, Germany.
authors: Rolf
authors: R
authors: Fendel
authors: Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Pauwelsstr. 20, 52074 Aachen, Germany.
authors: Magdalena
authors: M
authors: Bialon
authors: Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Pauwelsstr. 20, 52074 Aachen, Germany.
authors: Christiane
authors: C
authors: Püttmann
authors: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstr. 6, 52074 Aachen, Germany; Institute of Molecular Biotechnology (Biology VII), RWTH Aachen University, Worringer Weg 1, 52074 Aachen, Germany.
authors: Rainer
authors: R
authors: Fischer
authors: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstr. 6, 52074 Aachen, Germany; Faculty of Health Sciences, Department of Integrative Biomedical Sciences, University of Cape Town, Anzio Road Observatory, 7925 Cape Town, South Africa.
authors: Stefan
authors: S
authors: Barth
authors: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstr. 6, 52074 Aachen, Germany; Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Pauwelsstr. 20, 52074 Aachen, Germany. Electronic address: Katharina.Kolberg@ime.fraunhofer.de.
authors: Katharina
authors: K
authors: Kolberg
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004355:Drug Stability; D066246:ErbB Receptors / Q000276:immunology* / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D007167:Immunotherapy / Q000379:methods*; D000922:Immunotoxins / Q000276:immunology / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D051379:Mice; D009842:Oligopeptides / Q000494:pharmacology*; D050682:Q-SNARE Proteins / Q000276:immunology / Q000494:pharmacology*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D057127:Single-Chain Antibodies / Q000276:immunology / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D066246:ErbB Receptors; D006147:Guanine; D000922:Immunotoxins; D009842:Oligopeptides; D050682:Q-SNARE Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C512478:EGFR protein, human; C541485:EGFR protein, mouse; C508053:ERBB2 protein, human; C524056:Erbb2 protein, mouse
keywords: ADC; Auristatin; EGFR; HER2; SNAP-tag technology