Carbonic anhydrase IX is a pH-stat that sets an acidic tumour extracellular pH in vivo

Background Tumour Carbonic Anhydrase IX (CAIX), a hypoxia-inducible tumour-associated cell surface enzyme, is thought to acidify the tumour microenvironment by hydrating CO2 to form protons and bicarbonate, but there is no definitive evidence for this in solid tumours in vivo. Methods We used 1H magnetic resonance spectroscopic imaging (MRSI) of the extracellular pH probe imidazolyl succinic acid (ISUCA) to measure and spatially map extracellular pH in HCT116 tumours transfected to express CAIX and empty vector controls in SCID mice. We also measured intracellular pH in situ with 31P MRS and measured lactate in freeze-clamped tumours. Results CAIX expressing tumours had 0.15 pH-unit lower median extracellular pH than control tumours (pH 6.71 tumour vs pH 6.86 control, P = 0.01). Importantly, CAIX expression imposed an upper limit for tumour extracellular pH at 6.93. Despite the increased lactate concentration in CAIX-expressing tumours, 31P MRS showed no difference in intracellular pH, suggesting that CAIX acidifies only the tumour extracellular space. Conclusions CAIX acidifies the tumour microenvironment, and also provides an extracellular pH control mechanism. We propose that CAIX thus acts as an extracellular pH-stat, maintaining an acidic tumour extracellular pH that is tolerated by cancer cells and favours invasion and metastasis.We are grateful for the support of CRUK [grant number C14303/A17197], the Breast Cancer Research Foundation, the Royal Society, Worldwide Cancer Research and the European Research Council [SURVIVE: 723397]. JP-T and SC received support from the Spanish Ministry of Economy and Competitiveness SAF2014-23622

Selection and characterization of antibodies blocking the enzymatic activity of the tumour associated carbonic anhydrase IX

Solide Tumoren sind oft schlecht durchblutet. Dadurch haben sie eine verringerte Zufuhr an Sauerstoff und Nährstoffen, wodurch ihr Wachstum limitiert wird. Sauerstoffmangel führt zu Änderungen in der Genexpression – hauptsächlich des Hypoxie-induzierbaren Faktors (HIF) – welche es den Krebszellen erlauben, sich an einen tiefen Sauerstoffgehalt anzupassen. Eines dieser Zielgene ist Carboanhydrase IX (CA IX), ein Transmembranprotein mit extrazellulärer Domäne, welche die reversible Hydratisierung von Kohlenstoffdioxid zu Hydrogencarbonat und Protonen katalysiert. CA IX wird hauptsächlich im Tumorgewebe exprimiert und nur sehr selten in gesundem Gewebe, wie z.B. dem gastrointestinalen Trakt. Die katalytische Aktivität erleichtert das Wachstum von Tumoren durch Senkung des extrazellulären pHs und Neutralisierung des intrazellulären pHs. Dadurch ist CA IX einerseits ein vielversprechender Marker für Tumortargeting und andererseits ein mögliches Ziel für eine die CA IX Aktivität inhibierende Tumortherapie. In der Klinik werden mehrere synthetische Stoffe eingesetzt, welche die CA IX Aktivität inhibieren. Da diese aber auch mit anderen Carboanhydrase Isoformen interagieren, haben sie häufig schwere Nebenwirkungen, und ihr Einsatz in der Klinik ist limitiert. Daneben verwendet man Antikörper, die CA IX sehr spezifisch binden. Diese sind sehr hilfreich im Visualisieren von Tumoren, leider ist ihre therapeutische Wirkung jedoch umstritten. Das Ziel dieser Doktorarbeit war es daher, CA IX spezifische Antikörper mit Enzym-inhibitorischer Aktion zu etablieren, um effizientere Therapien gegen solide Tumoren zu entwickeln. In dieser Arbeit wurden mit Hilfe des Phagendisplays zwölf Fab-Antikörper (MSC 1 – 12) selektioniert, die spezifisch an rekombinantes humanes CA IX binden. Keine unspezifische Bindung an andere Isoformen mit sehr grosser Homologie, namentlich CA II, CA XII oder CA XIV, wurde beobachtet, was die selektive Bindung an CA IX bestätigt. Zusätzlich waren zwei Antikörper, MSC 1 und MSC 3, kreuzreaktiv mit murinem CA IX. MSC 3 IgG färbte murines CA IX spezifisch in Tumorschnitten und kann daher in einem syngenen Mausmodell eingesetzt werden. Fünf Antikörper, MSC 2, MSC 5, MSC 8, MSC 10 und MSC 12, waren in der Lage, die enzymatische Funktion von CA IX in einem biochemischen Assay auf Membranfragmenten teilweise zu inhibieren. Der vielversprechendste der Antikörper, MSC 8, inhibierte die CA IX Aktivität um 57 % als Fab-Antikörper und um 76 % als bivalenter IgG. Zudem inhibierte MSC 8 Fab die CA IX Aktivität auf intakten Einzelzellen um 48 % bzw. MSC 8 IgG um 61 %. Daneben bewirkte MSC 8 IgG die Internalisierung von CA IX, wodurch die CA IX Aktivität auf der Zelloberfläche weiter reduziert werden dürfte. Daher könnte der humane CA IX spezifische IgG MSC 8 einen weitaus grösseren Nutzen haben als zuvor entwickelte anti-CA IX Antikörper, indem Spezifität mit Inhibition der CA IX Enzym- Aktivität gekoppelt wird. Der therapeutische Effekt könnte weiter gesteigert werden, indem biologisch aktive Substanzen mit Hilfe dieser Antikörper spezifisch zu Zielzellen gebracht werden und durch Internalisierung direkt an ihren Wirkungsort gelangen. ABSTRACT The growth of solid tumours is limited by low supply of oxygen and nutrients due to poor vascularisation. Hypoxia leads to changes in gene expression mostly due to the hypoxia inducible factor (HIF), helping cancer cells to adapt to low oxygen levels. One of these target genes is carbonic anhydrase IX (CA IX), a transmembrane protein with an extracellular domain catalysing the reversible hydration of carbon dioxide to hydrogen carbonate and protons. CA IX expression is mostly limited to hypoxic tumour tissues. It is only rarely expressed in normal tissue, e.g. in the gastrointestinal tract. Its catalytic activity facilitates tumour growth by acidification of extracellular pH and neutralisation of intracellular pH. This makes carbonic anhydrase IX not only a promising marker for tumour targeting but also a potential target for tumour therapy by inhibiting CA IX enzymatic activity. Several chemical compounds inhibiting CA IX activity are used in the clinic. However, due to cross-reactivity with other carbonic anhydrase isoforms, they have severe side effects and their clinical use is very much limited. On the other hand, antibodies specifically targeting CA IX have proven to be very useful in tumour imaging, but their therapeutic effect is disputed. The aim of this thesis was therefore to establish CA IX specific antibodies blocking enzymatic activity in order to enhance efficacy of therapies against solid tumours. In this thesis, twelve Fab antibodies, called MSC 1 – 12, specifically recognizing human CA IX were selected by phage display on recombinant human CA IX. No unspecific binding to any of the other highly homologous isoforms CA II, CA XII or CA XIV could be detected, confirming highly selective binding to CA IX. In addition, two of the antibodies, MSC 1 and MSC 3, were cross- reactive to murine CA IX. MSC 3 IgG specifically stained murine CA IX in tumour sections and can thus be used in syngeneic mouse models. Five antibodies, MSC 2, MSC 5, MSC 8, MSC 10 and MSC 12, inhibited CA IX function partially in a biochemical assay on membrane fragments. The most potent of those antibodies, MSC 8, inhibited CA IX activity by up to 57 % as Fab antibody and 76 % as bivalent IgG. In addition, MSC 8 inhibited CA IX activity on intact single cells by 48 % and 61 % for MSC 8 Fab and MSC 8 IgG, respectively. In addition, MSC 8 IgG triggered CA IX internalisation which might further reduce CA IX activity on the cell surface. Therefore, the fully human CA IX-specific IgG antibody MSC 8 may extend beyond the use of previously developed CA IX-antibodies by combining target-specificity with CA IX inhibitory activity. By delivering therapeutic moieties specifically to target cells and triggering internalisation of those compounds the here selected antibodies could further increase therapeutic efficacy

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

Item does not contain fulltextCarbonic 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 0.3mug/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

Application of Monoclonal Antibody G250 Recognizing Carbonic Anhydrase IX in Renal Cell Carcinoma

Contains fulltext : 117856.pdf (publisher's version ) (Open Access)Monoclonal antibody G250 (mAbG250) recognizes a determinant on carbonic anhydrase IX (CAIX). CAIX is expressed by virtually all renal cell carcinomas of the clear cell type (ccRCC), but expression in normal tissues is restricted. The homogeneous CAIX expression in ccRCC and excellent targeting capability of mAbG250 in animal models led to the initiation of the clinical evaluation of mAbG250 in (metastatic) RCC (mRCC) patients. Clinical studies confirmed the outstanding targeting ability of mAbG250 and cG250 PET imaging, as diagnostic modality holds great promise for the future, both in detecting localized and advanced disease. Confirmation of the results obtained in the non-randomized clinical trials with unmodified cG250 is needed to substantiate the value of cG250 treatment in mRCC. cG250-Based radio immuno-therapy (RIT) holds promise for treatment of patients with small-volume disease, and adjuvant treatment with unmodified cG250 may be of value in selected cases. In the upcoming years, ongoing clinical trials should provide evidence for these assumptions. Lastly, whether cG250-based RIT can be combined with tyrosine kinase inhibitors, which constitutes the current standard treatment for mRCC, needs to be established