title: Granzyme B-based cytolytic fusion protein targeting EpCAM specifically kills triple negative breast cancer cells in vitro and inhibits tumor growth in a subcutaneous mouse tumor model.
abstract: Triple-negative breast cancer (TNBC) is associated with poor prognosis and high prevalence among young premenopausal women. Unlike in other breast cancer subtypes, no targeted therapy is currently available. Overexpression of epithelial cell adhesion molecule (EpCAM) in 60% of TNBC tumors correlates with poorer prognosis and is associated with cancer stem cell phenotype. Thus, selective elimination of EpCAM(+) TNBC tumor cells is of clinical importance. Therefore, we constructed a fully human targeted cytolytic fusion protein, designated GbR201K-αEpCAM(scFv), in which an EpCAM-selective single-chain antibody fragment (scFv) is genetically fused to a granzyme B (Gb) mutant with reduced sensitivity to its natural inhibitor serpin B9. In vitro studies confirmed its specific binding, internalization and cytotoxicity toward a panel of EpCAM-expressing TNBC cells. Biodistribution kinetics and tumor-targeting efficacy using MDA-MB-468 cells in a human TNBC xenograft model in mice revealed selective accumulation of GbR201K-αEpCAM(scFv) in the tumors after i.v. injection. Moreover, treatment of tumor-bearing mice demonstrated a prominent inhibition of tumor growth of up to 50 % in this proof-of-concept study. Taken together, our results indicate that GbR201K-αEpCAM(scFv) is a promising novel targeted therapeutic for the treatment of TNBC.
authors.affiliation: Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Pauwelsstr. 20, 52074 Aachen, Germany.
authors.forename: Manal
authors.initials: M
authors.lastname: Amoury
authors.affiliation: Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Pauwelsstr. 20, 52074 Aachen, Germany; Department of Pharmaceutical Product Development, Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstr. 6, 52074 Aachen, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Kolberg
authors.affiliation: Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Pauwelsstr. 20, 52074 Aachen, Germany.
authors.forename: Anh-Tuan
authors.initials: AT
authors.lastname: Pham
authors.affiliation: Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Pauwelsstr. 20, 52074 Aachen, Germany.
authors.forename: Dmitrij
authors.initials: D
authors.lastname: Hristodorov
authors.affiliation: Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Pauwelsstr. 20, 52074 Aachen, Germany.
authors.forename: Radoslav
authors.initials: R
authors.lastname: Mladenov
authors.affiliation: Department of Pharmaceutical Product Development, Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstr. 6, 52074 Aachen, Germany.
authors.forename: Stefano
authors.initials: S
authors.lastname: Di Fiore
authors.affiliation: Department of Surgery, Laboratory for Translational Surgical Oncology, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9700 RB Groningen, Netherlands.
authors.forename: Wijnand
authors.initials: W
authors.lastname: Helfrich
authors.affiliation: Institute for Experimental Molecular Imaging, University Hospital RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany.
authors.forename: Fabian
authors.initials: F
authors.lastname: Kiessling
authors.affiliation: Department of Pharmaceutical Product Development, Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstr. 6, 52074 Aachen, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.affiliation: Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Pauwelsstr. 20, 52074 Aachen, Germany.
authors.forename: Alessa
authors.initials: A
authors.lastname: Pardo
authors.affiliation: Department of Pharmaceutical Product Development, Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstr. 6, 52074 Aachen, Germany.
authors.forename: Theophilus
authors.initials: T
authors.lastname: Thepen
authors.affiliation: Department of Gynecology and Obstetrics, University Hospital RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany.
authors.forename: Ahmad F
authors.initials: AF
authors.lastname: Hussain
authors.affiliation: Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Pauwelsstr. 20, 52074 Aachen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Nachreiner
authors.affiliation: South African Research Chair in Cancer Biotechnology, Institute of Infectious Disease and Molecular Medicine (IDM), Department of Integrative Biomedical Sciences, Faculty of Health Sciences, University of Cape Town, Anzio Road, Observatory, Cape Town 7925, South Africa. Electronic address: stefan.barth@uct.ac.za.
authors.forename: Stefan
authors.initials: S
authors.lastname: Barth
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D015815:Cell Adhesion Molecules / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D000071858:Epithelial Cell Adhesion Molecule; D005260:Female; D053804:Granzymes / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D007167:Immunotherapy / Q000379:methods*; D000922:Immunotoxins / Q000235:genetics / Q000276:immunology / Q000493:pharmacokinetics / Q000494:pharmacology*; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology / Q000493:pharmacokinetics / Q000494:pharmacology*; D014018:Tissue Distribution; D014162:Transfection; D064726:Triple Negative Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D047368:Tumor Burden / Q000187:drug effects*; D023041:Xenograft Model Antitumor Assays
chemical_list: D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D053804:Granzymes
keywords: Granzyme B; Human cytolytic fusion protein; Immunotherapy; Medical biotechnology; Triple negative breast cancer