Antibody inhibiting enzymatic activity of tumour-associated carbonic anhydrase isoform IX

Abstract

Carbonic anhydrase IX (CAIX) is a hypoxia-induced, membrane-tethered enzyme that is highly expressed in many cancers. It catalyses the hydration of CO₂ to HCO₃⁻ and H⁺, and the reverse dehydration reaction. Recent studies have shown an important role for CAIX in pH regulation and it has been speculated that CAIX may play a role in supporting cancer progression towards more aggressive forms of the disease. Clinical correlative studies in many tumours have shown that high expression is related to poor outcome. In the present study, we have selected antigen-binding antibody fragments (Fab) against human CAIX by phage-display, and tested these for inhibitory potency on CAIX catalytic activity. Inhibition was assessed from the kinetics of the CAIX-catalysed reaction, using assays performed on intact cells over-expressing CAIX, and their CAIX-containing membrane fragments. Inhibition was also assessed in multi-cellular tissue-models (spheroids) from the kinetics of CO₂ venting. We have identified a Fab antibody, labelled MSC8, and its corresponding full-length IgG that inhibited CAIX by up to 57% and 76%, respectively, with half-maximal inhibition at 0.3 μg/ml. Incubation of CAIX-expressing cells with MSC8 IgG produced a lasting inhibitory effect. The inhibitory effect was prompt and was also observed in isolated membrane-fragments, suggesting that a direct inhibitory interaction takes place between the antibody and CAIX. The inhibitory effects in spheroids argue for a physiological relevance of the antibody. Biologically-active antibodies against CAIX can be used as selective, high-affinity inhibitors in experimental studies to dissect the role of CAIX and, possibly, therapeutically by targeting a catalytically-active cancer-related protein.

Introduction

Cancer progression involves major changes in cellular metabolism and the tumour microenvironment (Fang et al., 2008, Gillies et al., 2002). These adaptations provide a survival advantage to cancer cells (Gatenby and Gillies, 2004), and it has been proposed that inhibitors targeting these adaptive pathways may reduce the survival prospects of cancer cells. A key feature of cancer progression is hypoxia arising from oxygen depletion by elevated metabolism and inadequate oxygen delivery with blood (Kallinowski et al., 1989). Hypoxia feeds back on tumour physiology by regulating gene expression, principally via the hypoxia-inducible factor (HIF) (Semenza, 2003). An important hypoxia-induced protein is carbonic anhydrase IX (CAIX) (Wykoff et al., 2000), a member of a family of enzymes that catalyse the reaction

CO₂ + H₂O ↔ H⁺ + HCO₃⁻.

This reaction involves H⁺-ions, the determinants of pH, and CO₂/HCO₃⁻, a major pH buffering system. CAIX is membrane-tethered and its catalytic site has extracellular-facing topology. The enzyme has been shown to facilitate tumour growth (Chiche et al., 2009) through its role in regulating intracellular pH (Chiche et al., 2009, Swietach et al., 2008) and extracellular pH (Svastova et al., 2004, Swietach et al., 2009). Cancer cells release CO₂ (Holm et al., 1995), a substrate for CAIX, from the intracellular titration reaction between H⁺-ions and HCO₃⁻, from the pentose phosphate shunt or, in aerobic zones, from mitochondrial respiration. CAIX-catalysed extracellular CO₂ hydration helps to maintain a steep efflux gradient for CO₂ venting out of cells. Extracellular HCO₃⁻ that is produced by CAIX can be transported into cells to replenish intracellular HCO₃⁻ supplies for pH buffering (Lee and Tannock, 1998). Extracellular H⁺-ions produced by CAIX can, in some cells, trigger intracellular signalling cascades via surface H⁺-receptors (Huang et al., 2008).

The proposed role for CAIX-catalysis in regulating cancer cell pH and the correlation between CAIX-expression and cancers with poor prognosis (Chia et al., 2001, Generali et al., 2006, Giatromanolaki et al., 2001, Pastorek et al., 1994) has evoked much interest in designing CAIX-inhibitors. Such inhibitors could be used experimentally to investigate the role of CAIX in cancer physiology and provide novel insights into the general importance of extracellular carbonic anhydrase isoforms in cellular physiology. Potentially, inhibitors of CAIX could be used therapeutically to target an important cancer-related protein. Since CAIX is strongly associated with cancer, CAIX-selectivity of such inhibitors would reduce unwanted side-effects on CAIX-negative non-cancerous tissues. A range of membrane-impermeant small-molecule inhibitors are now available for inhibiting extracellular carbonic anhydrase isoforms (Supuran, 2008). Recent studies have shown their ability to inhibit tumour growth in vivo (Ahlskog et al., 2009a). Although some chemical compounds have been designed to bind preferentially to CAIX (Supuran, 2008), it is disputed whether CAIX, among other extracellular isoforms, is being targeted selectively. A more selective approach could be achieved with inhibitory anti-CAIX antibodies. In this study, we have selected a number of CAIX-specific antigen-binding antibody fragments (Fab) by phage display. We have identified an inhibitory antibody, based on tests performed on CAIX-expressing cells and their membrane-fragments, and demonstrated a physiologically-relevant effect of the antibody in CAIX-expressing tissue-growths (spheroids).