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.
Highlights
Carbonic anhydrase (CA) is a ubiquitous enzyme of fundamental physiological importance
Earlier studies showed that G250 monoclonal antibody (MAb) interacts with the native carbonic anhydrase (CA) carbonic anhydrase protein (IX) protein, but its domain specificity has remained unknown [22]
ELISA with these CA IX‐related antigens revealed that G250 MAb bound only to those variants that contained an intact CA domain, including GST‐CA and extracts from MDCK‐human carbonic anhydrase gene (CA9) and MDCK‐ΔPG cells (Fig. 1A)
Summary
Carbonic anhydrase (CA) is a ubiquitous enzyme of fundamental physiological importance. CA IX is often detected in perinecrotic areas and in areas more distant from perfused vasculature and its expression is much more variable (from focal to diffuse) and heterogeneous (from weak to strong) than in RCC This might complicate the immunotherapeutic targeting of such tumours, earlier studies in mouse models indicate that this is not the case [17,36]. This report summarizes the results of such characterization, which indicate that the therapeutic targeting of non‐RCC tumours with cG250 is feasible
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