title: A novel fully-human cytolytic fusion protein based on granzyme B shows in vitro cytotoxicity and ex vivo binding to solid tumors overexpressing the epidermal growth factor receptor.
abstract: Human cytolytic fusion proteins (hCFPs) offer a promising immunotherapeutic approach for the treatment of solid tumors, avoiding the immunogenicity and undesirable side-effects caused by immunotoxins derived from plants or bacteria. The well-characterized human serine protease granzyme B has already been used as a therapeutic pro-apoptotic effector domain. We therefore developed a novel recombinant hCFP (GbR201K-scFv1711) consisting of an epidermal growth factor receptor-specific human antibody fragment and a granzyme B point mutant (R201K) that is insensitive to serpin B9 (PI9), a natural inhibitor of wild-type granzyme B that is often expressed in solid tumors. We found that GbR201K-scFv1711 selectively bound to epidermoid cancer and rhabdomyosarcoma cells and was rapidly internalized by them. Nanomolar concentrations of GbR201K-scFv1711 achieved the specific killing of epidermoid cancer cells by inducing apoptosis, and similar effects were observed in rhabdomyosarcoma cells when GbR201K-scFv1711 was combined with the endosomolytic substance chloroquine. The novel hCFP was stable in serum and bound to human rhabdomyosarcoma tissue ex vivo. These data confirm that GbR201K-scFv1711 is a promising therapeutic candidate suitable for further clinical investigation.
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
authors.forename: Judith
authors.initials: J
authors.lastname: Niesen
authors.affiliation: Pharmedartis GmbH, Aachen, Germany.
authors.forename: Grit
authors.initials: G
authors.lastname: Hehmann-Titt
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany; Institute of Molecular Biotechnology (Biology VII), RWTH Aachen University, Aachen, Germany.
authors.forename: Mira
authors.initials: M
authors.lastname: Woitok
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany; Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Aachen, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Fendel
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany; Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Aachen, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Barth
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany; Institute of Molecular Biotechnology (Biology VII), RWTH Aachen University, Aachen, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany; Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Aachen, Germany. Electronic address: christoph.stein@ime.fraunhofer.de.
authors.forename: Christoph
authors.initials: C
authors.lastname: Stein
mesh_terms: D002294:Carcinoma, Squamous Cell / Q000188:drug therapy* / Q000201:enzymology / Q000276:immunology / Q000473:pathology; D045744:Cell Line, Tumor; D002738:Chloroquine / Q000494:pharmacology; D066246:ErbB Receptors / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D053804:Granzymes / Q000235:genetics / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D000922:Immunotoxins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D008223:Lymphoma / Q000188:drug therapy* / Q000201:enzymology / Q000276:immunology / Q000473:pathology; D008297:Male; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D020298:U937 Cells
chemical_list: D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D002738:Chloroquine; C512478:EGFR protein, human; D066246:ErbB Receptors; D053804:Granzymes
keywords: Chloroquine; EGFR; Granzyme B; Human cytolytic fusion protein; scFv