Unique Biological Properties of Catalytic Domain Directed Human Anti-CAIX Antibodies Discovered through Phage-Display Technology

Abstract

Carbonic anhydrase IX (CAIX, gene G250/MN-encoded transmembrane protein) is highly expressed in various human epithelial tumors such as renal clear cell carcinoma (RCC), but absent from the corresponding normal tissues. Besides the CA signal transduction activity, CAIX may serve as a biomarker in early stages of oncogenesis and also as a reliable marker of hypoxia, which is associated with tumor resistance to chemotherapy and radiotherapy. Although results from preclinical and clinical studies have shown CAIX as a promising target for detection and therapy for RCC, only a limited number of murine monoclonal antibodies (mAbs) and one humanized mAb are available for clinical testing and development. In this study, paramagnetic proteoliposomes of CAIX (CAIX-PMPLs) were constructed and used for anti-CAIX antibody selection from our 27 billion human single-chain antibody (scFv) phage display libraries. A panel of thirteen human scFvs that specifically recognize CAIX expressed on cell surface was identified, epitope mapped primarily to the CA domain, and affinity-binding constants (KD) determined. These human anti-CAIX mAbs are diverse in their functions including induction of surface CAIX internalization into endosomes and inhibition of the carbonic anhydrase activity, the latter being a unique feature that has not been previously reported for anti-CAIX antibodies. These human anti-CAIX antibodies are important reagents for development of new immunotherapies and diagnostic tools for RCC treatment as well as extending our knowledge on the basic structure-function relationships of the CAIX molecule.

Human anti-CAIX antibodies mediate immune cell inhibition of renal cell carcinoma in vitro and in a humanized mouse model in vivo

“…To ascertain the ability of these antibodies to mediate immune cell killing of CAIX-expressing RCC cells, we chose to analyze high affinity scFv-Fc antibodies that either inhibited the CA activity of CAIX, but did not internalize (G37 and G39), or showed minimal to modest inhibition of CA activity and induced internalization of CAIX (G10, G36, and G119) [23]. We utilized RCC cell lines with high CAIX (SKRC-09, Fig.…”

“…Various antibodies targeting the tumor-associated antigen CAIX on RCC cells have been amongst those most extensively studied [8, 21, 23, 24, 34], owing to the antigen’s high expression on RCC. One chimeric anti-CAIX antibody that does not inhibit enzymatic activity, girentuximab, has been evaluated in the clinic [24, 43].…”

New insights into the physiological role of carbonic anhydrase IX in tumour pH regulation

“…These dramatic effects illustrate the importance of extracellular CA activity and may explain the correlation between CAIX/CAXII expression in tumour tissue samples and poor patient prognosis (Saarnio et al, 1998;Liao and Stanbridge, 2000;Chia et al, 2001;Koukourakis et al, 2001;Loncaster et al, 2001;Potter and Harris, 2003;Haapasalo et al, 2006;Haapasalo et al, 2008). These results make a case for clinical trials for studying the responsiveness of tumours in patients to anti-CAIX/anti-CAXII therapies based on small molecule inhibitors (Supuran, 2008;Guler et al, 2010) or selective antibodies with inhibitory effects (Xu et al, 2010). It is noteworthy, however, that some studies on gastric and oesophageal tumours have shown a lack of positive correlation between CAIX expression and cancer progression (Pastorekova et al, 1997;Turner et al, 1997).…”

Esterase activity of carbonic anhydrases serves as surrogate for selecting antibodies blocking hydratase 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 . Lack of effective and efficacious screening methods, which are easy to perform and offer a rapid and high throughput system for identifying CA9 and CA12 blocking antibodies, is believed to be one of the main reasons for this failure.…”