Antitumor Efficacy of a Monoclonal Antibody That Inhibits the Activity of Cancer-Associated Carbonic Anhydrase XII

Abstract

Carbonic anhydrase XII (CA XII) is a membrane-tethered cell surface enzyme that is highly expressed on many human tumor cells. Carbonic anhydrase members in this class of exofacial molecules facilitate tumor metabolism by facilitating CO 2 venting and intracellular pH regulation. Accordingly, inhibition of exofacial CAs has been proposed as a general therapeutic strategy to target cancer. The recent characterization of 6A10, the first CA XII-specific inhibitory monoclonal antibody, offered an opportunity to evaluate this strategy with regard to CA XII-mediated catalysis. Using functional assays, we showed that 6A10 inhibited exofacial CA activity in CA XIIexpressing cancer cells. 6A10 reduced spheroid growth in vitro under culture conditions where CA XII was active (i.e., alkaline pH) and where its catalytic activity was likely rate-limiting (i.e., restricted extracellular HCO 3 À supply). These in vitro results argued that the antibody exerted its growth-retarding effect by acting on the catalytic process, rather than on antigen binding per se. Notably, when administered in a mouse xenograft model of human cancer, 6A10 exerted a significant delay on tumor outgrowth. These results corroborate the notion that exofacial CA is critical for cancer cell physiology and they establish the immunotherapeutic efficacy of targeting CA XII using an inhibitory antibody. Cancer Res; 73(21); 6494-503. Ó2013 AACR.

Esterase activity of carbonic anhydrases serves as surrogate for selecting antibodies blocking hydratase activity

“…MSC8 and 6A10 antibodies showed a dose-dependent inhibition of esterase activity with IC 50 values of 15 mg/ml and 25 mg/ ml for their respective targets, CA9 and CA12. However, the esterase IC 50 values of MSC8 and 6A10 in this study are higher than the published hydratase IC 50 values, 0.32 mg/ml and 0.96 mg/ ml, respectively 20,22 . These differences between the esterase and hydratase IC 50 values could be ascribed to the use of recombinant human CA9 and CA12 for the present esterase activity assays, whereas the published hydratase activity assays were performed with cell derived membrane fractions containing human CA9 and CA12.…”

“…Inhibition with the MSC8 antibody reached a plateau and did not show complete inhibition of the esterase activity even at the highest concentrations tested; in contrast, the 6A10 antibody continued to provide further inhibition with increasing antibody concentration up to the highest concentration tested. A similar tendency was observed for inhibition of hydratase activity 15,20,22 , further supporting the notion that these antibodies have similar mode of inhibition for both esterase and hydratase activities. Complete inhibition of CA12 enzyme activity by 6A10 suggests that the antibody might interact or fit much better with the catalytic pocket leading to the complete inhibition, whereas MSC8 might be lacking some of the crucial interactions required for complete inhibition of the CA9 enzymatic activity.…”

“…Moreover, IC 50 values are generally very dependent on several experimental conditions such as the enzyme and substrate concentrations. Note that variations in the IC 50 values of MSC8 and 6A10 between hydratase and esterase activities need to be interpreted very carefully as we are comparing the IC 50 values of esterase activity from the current data with the IC 50 values of hydratase activity from two independent publications 20,22 . Using the IC 50 values of a series of small molecule compounds on CA1 and CA2 isoforms from literature, we demonstrated that the hydratase and esterase IC 50 values have a correlation.…”

“…Since then, several structural, functional and mutational studies have shown that the hydratase activity and the esterase activity of carbonic anhydrase share similar mechanism, and importantly, the same catalytic pocket, implicating that esterase activity is indicative of hydratase activity [27][28][29][30][31][32] . Using the recently established antibodies that can specifically bind to and inhibit the hydratase enzymatic activity of CA9 (MSC8) and CA12 (6A10), we demonstrate that esterase activity can serve as a rapid, robust and inexpensive surrogate marker for the identification of antibodies blocking carbonic anhydrase enzymatic activity 20,22 .…”

“…So far, little success has been made in identifying carbonic anhydrase-binding antibodies that also block CA enzymatic activity. 6A10 was the first and remains the best-described antibody for the inhibition of CA12 hydratase enzyme activity, with a potential biological role in the reduction of tumor growth 15,20,21 . However, only very few antibodies that can specifically bind and inhibit the hydratase activity of CA9 have appeared in the literature, such as the recently described MSC8 antibody 22,23 .…”