title: 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 CO(2) to HCO(3)(-) 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 CO(2) 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 0.3μg/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.
authors.affiliation: Department of Internal Medicine-Oncology, University Hospital Zurich, 8091 Zürich, Switzerland.
authors.forename: Margarita T
authors.initials: MT
authors.lastname: Murri-Plesko
authors.forename: Alzbeta
authors.initials: A
authors.lastname: Hulikova
authors.forename: Egbert
authors.initials: E
authors.lastname: Oosterwijk
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Scott
authors.forename: Anna
authors.initials: A
authors.lastname: Zortea
authors.forename: Adrian L
authors.initials: AL
authors.lastname: Harris
authors.forename: Gerd
authors.initials: G
authors.lastname: Ritter
authors.forename: Lloyd
authors.initials: L
authors.lastname: Old
authors.forename: Stefan
authors.initials: S
authors.lastname: Bauer
authors.forename: Pawel
authors.initials: P
authors.lastname: Swietach
authors.forename: Christoph
authors.initials: C
authors.lastname: Renner
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000378:metabolism*; D000071231:Carbonic Anhydrase IX; D002257:Carbonic Anhydrase Inhibitors / Q000276:immunology / Q000494:pharmacology*; D002256:Carbonic Anhydrases / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000201:enzymology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology / Q000494:pharmacology*; D009369:Neoplasms / Q000201:enzymology*; D019151:Peptide Library; D018874:Spheroids, Cellular / Q000187:drug effects / Q000201:enzymology
chemical_list: D000951:Antigens, Neoplasm; D002257:Carbonic Anhydrase Inhibitors; D007128:Immunoglobulin Fragments; D019151:Peptide Library; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases