title: Monoclonal antibody G250 targeting CA Ⅸ: Binding specificity, internalization and therapeutic effects in a non-renal cancer model.
abstract: G250 (Girentuximab) is a chimeric IgG1 monoclonal antibody (MAb) currently being evaluated as an immunotherapy for kidney cancer. It targets carbonic anhydrase protein (CA Ⅸ), a transmembrane carbonic anhydrase (CA) isoform, which is regulated by VHL/HIF pathway and hence expressed in the majority of renal cell carcinomas (RCCs) as well as in hypoxic non‑RCC tumours. CA Ⅸ functions in pH regulation and cell migration/invasion, and supports tumour cell survival in hypoxia and/or acidosis. It contains a highly active extracellular catalytic domain (CA) extended N-terminally with a proteoglycan-like region and C-terminally with short transmembrane and intracellular regions. Here we characterize the binding and internalization properties of G250, as well as its therapeutic effects in animal model, and discuss the impact of G250‑mediated immunotherapy in non‑RCC tumours. We demonstrated that G250 MAb recognizes a conformational epitope in the CA domain, detects the soluble CA Ⅸ ectodomain (ECD), but not the splicing variant, and does not cross-react with CA Ⅰ, Ⅱ, and Ⅻ isoforms. We showed that G250 internalizes via clathrin-coated vesicles, escapes degradation in lysosomes and enters the recycling pathway via the perinuclear compartment. This results in long intracellular persistence and enables consecutive internalization cycles. Moreover, the recycled antibody maintains an intact Fc portion potentially capable of continuous induction of antibody-dependent cell-mediated cytotoxicity (ADCC) response, thus explaining its therapeutic efficacy. Finally, we showed that G250 treatment is effective against HT-29 colorectal carcinoma xenografts that differ from RCC by more heterogeneous, hypoxia-related expression of CA Ⅸ. These results suggest potential therapeutic usefulness of the G250 MAb in non-RCC tumours.
authors.affiliation: Department of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 84505 Bratislava, Slovakia.
authors.forename: Miriam
authors.initials: M
authors.lastname: Zatovicova
authors.affiliation: Department of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 84505 Bratislava, Slovakia.
authors.forename: Lenka
authors.initials: L
authors.lastname: Jelenska
authors.affiliation: Department of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 84505 Bratislava, Slovakia.
authors.forename: Alzbeta
authors.initials: A
authors.lastname: Hulikova
authors.affiliation: Department of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 84505 Bratislava, Slovakia.
authors.forename: Peter
authors.initials: P
authors.lastname: Ditte
authors.affiliation: Department of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 84505 Bratislava, Slovakia.
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Ditte
authors.affiliation: Department of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 84505 Bratislava, Slovakia.
authors.forename: Lucia
authors.initials: L
authors.lastname: Csaderova
authors.affiliation: Department of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 84505 Bratislava, Slovakia.
authors.forename: Eliska
authors.initials: E
authors.lastname: Svastova
authors.affiliation: WILEX AG, 81675 Munich, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Schmalix
authors.affiliation: WILEX AG, 81675 Munich, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Boettger
authors.affiliation: WILEX AG, 81675 Munich, Germany.
authors.forename: Paul
authors.initials: P
authors.lastname: Bevan
authors.affiliation: Department of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 84505 Bratislava, Slovakia.
authors.forename: Jaromir
authors.initials: J
authors.lastname: Pastorek
authors.affiliation: Department of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 84505 Bratislava, Slovakia.
authors.forename: Silvia
authors.initials: S
authors.lastname: Pastorekova
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage* / Q000276:immunology; D000920:Antibody-Dependent Cell Cytotoxicity; D000951:Antigens, Neoplasm / Q000096:biosynthesis* / Q000276:immunology; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000096:biosynthesis* / Q000276:immunology; D002292:Carcinoma, Renal Cell / Q000188:drug therapy / Q000276:immunology / Q000473:pathology; D015179:Colorectal Neoplasms / Q000188:drug therapy* / Q000276:immunology / Q000473:pathology; D019073:HT29 Cells; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007167:Immunotherapy; D051379:Mice; D023041:Xenograft Model Antitumor Assays
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; C106533:G250 monoclonal antibody; D007074:Immunoglobulin G; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases