AntitumorEfﬁcacyofaMonoclonalAntibodyThatInhibitsthe ......Therapeutics, Targets, and Chemical Biology AntitumorEfﬁcacyofaMonoclonalAntibodyThatInhibitsthe Activity of Cancer-Associated

Therapeutics, Targets, and Chemical Biology

Antitumor Efficacy of aMonoclonal Antibody That Inhibits theActivity of Cancer-Associated Carbonic Anhydrase XII

Gabor Gondi1, Josef Mysliwietz2, Alzbeta Hulikova4, Jian Ping Jen4, Pawel Swietach4,Elisabeth Kremmer2, and Reinhard Zeidler1,3

AbstractCarbonic anhydrase XII (CA XII) is a membrane-tethered cell surface enzyme that is highly expressed onmany

human tumor cells. Carbonic anhydrasemembers in this class of exofacial molecules facilitate tumormetabolismby facilitating CO2 venting and intracellular pH regulation. Accordingly, inhibition of exofacial CAs hasbeen proposed as a general therapeutic strategy to target cancer. The recent characterization of 6A10, the firstCAXII-specific inhibitorymonoclonal antibody, offered an opportunity to evaluate this strategywith regard to CAXII-mediated catalysis. Using functional assays, we showed that 6A10 inhibited exofacial CA activity in CA XII-expressing 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 HCO3�supply). These in vitro results argued that the antibody exerted its growth-retarding effect by acting on thecatalytic process, rather than on antigen binding per se. Notably, when administered in a mouse xenograft modelof human cancer, 6A10 exerted a significant delay on tumor outgrowth. These results corroborate the notion thatexofacial CA is critical for cancer cell physiology and they establish the immunotherapeutic efficacy of targetingCA XII using an inhibitory antibody. Cancer Res; 73(21); 6494–503. �2013 AACR.

IntroductionCarbonic anhydrase XII (CA XII) is, alongside a related

isoform IX, a cancer-associated, membrane-tethered exofacialenzyme. Early evidence has suggested that CA XII expression isupregulated under hypoxic conditions (1, 2), akin to CA IX. Incontrast to CA IX, CA XII is upregulated by estrogen andtherefore present in many breast cancers (3). More generally,the enzyme is overexpressed in many forms of human cancer,including renal, pancreatic, gut, oral, brain, lung, and ovarian(2, 4–11), but is also present in some normal tissues, such askidney, gut, and endometrium (4, 6, 12), expressed in combi-nationwith other carbonic anhydrase isoforms. CAXII has alsobeen found to be overexpressed in glaucoma (13) and inadvanced atherosclerotic plaques (14). The enzyme is success-fully used as a serodiagnostic biomarker for lung cancer (15)and as a molecular marker for the detection of breast cancerlymph node metastasis (16).

Despite considerable progress made, the molecular biologyand physiology of CAXII is less well understood than that of CAIX. Like CA IX, CA XII is a catalyst for CO2 hydration (17, 18).Cancer cells produce vast quantities of this metabolic end-product by mitochondrial and pentose shunt decarboxylation,and by titration reactions between acids (e.g., lactic) andbicarbonate (HCO3

�; refs. 19, 20). The catalytic activity ofexofacial carbonic anhydrases has been shown to facilitateCO2 venting from cells (21, 22), optimize extracellular bufferingpower to reduce extracellular pH (pHe) transients during acid/base membrane transport (23), and to supply extracellularHCO3

� (24) for HCO3�-dependent pH-regulating transporter

proteins (e.g., Naþ-HCO3� cotransport). These carbonic anhy-

drase-dependent processes are important for controlling intra-cellular pH (pHi), which must be regulated to within narrowlimits that are permissive for biological functions. The chal-lenge of regulating pHi is particularly apparent in tumors,which are characterized by an elevated metabolic rate andaberrant capillary perfusion (20). It has therefore been hypoth-esized that the cancer-associated carbonic anhydrases (iso-forms IX andXII) play an important role in disease progression.In agreement with the notion that cancer progression selectsfor phenotype, not genotype (25), pHmay play a critical role asa selection pressure driving stable adaptive changes (26, 27).Aside from impairing pHi regulation, inhibition or knockdownof CA IX or CA XII have been shown to suppress tumor growthand migration (17, 28–30). Consequently, the targeting ofcancer-associated exofacial CAs is now being considered asa novel anticancer therapeutic strategy.

The recent characterization of an anti-CA XII monoclonalantibody (6A10) with full inhibitory potency against CAXII (31)

Authors' Affiliations: 1Research Unit Gene Vectors, and 2Institute ofMolecular Immunology, Helmholtz Zentrum M€unchen—German ResearchCenter for Environmental Health; 3Department of Otorhinolaryngology,Ludwig-Maximilians-Universit€at, Munich, Germany; 4Department Of Phys-iology, Anatomy andGenetics, OxfordUniversity, Oxford, UnitedKingdom;Munich, Germany

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Reinhard Zeidler, Helmholtz Zentrum M€unchen,Marchioninistr. 25, D-81377 Munich, Germany. Phone: 498-9318-71239;Fax: 498-9318-74225; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-1110

�2013 American Association for Cancer Research.

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Published OnlineFirst September 12, 2013; DOI: 10.1158/0008-5472.CAN-13-1110

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has made it possible to study the effects of CA XII-specificinhibition on tumor growth. Functional assays have confirmedthat 6A10 inhibits recombinant CA XII at low nanomolarconcentrations and that blocking of CA XII interferes with thegrowth of multicellular tumor spheroids in vitro (32). Thepresent study confirms the inhibitory effect of 6A10 on nativeCAXII expressed in intact cells, and shows that inhibition of CAXII activity slows the growth rate of cancer cells in culture andin xenograft mouse models. Our findings confirm the anti-body's significant antitumor activity and highlight the poten-tial of immunotherapy in the treatment of CA XII-expressingcancers.

Materials and MethodsAntibodies, cell lines, and cell culture methods6A10 is a rat IgG2a monoclonal antibody that has been

described elsewhere (31). The isotype control antibody used inthe in vivo studies is a rat IgG2a antibody specific for Gluta-thione-S-transferase. A549 (ATCC CCL-185), Kato III (ATCCHTB-103), T47D, and HT29 (ATCC HTB-38) are cell linesestablished from a human lung, gastric, ductal breast andcolon cancer, respectively. HT29-shCA9 and HT29-shEV werea kind gift from A. McIntyre (Oxford University, Oxford, UK)and were described in detail elsewhere (28). Cells were grownin Dulbecco's Modified Eagle Medium (DMEM; Sigma) sup-plemented with 10% (v/v) heat-inactivated fetal calf serum(Sigma), Glutamine–Penicillin–Streptomycin mixture (Sigma)and incubated at 37�Cwith 5%CO2 enriched air. Cell lines wereobtained from ATCC and were identified April 2013 by STRDNA testing conducted at Eurofins.To simulate hypoxia before immunofluorescence, cells were

incubated with 1 mmol/L dimethyloxaloylglycine (DMOG), acell permeable, competitive inhibitor of HIF-prolyl hydroxy-lases, for 48 hours. Control cells were treated with the drug-vehicle dimethyl sulfoxide (DMSO) only. Before pHe-imagingexperiments, intact A549 and T47D cells were incubated for 24hours with 20 mg/mL 6A10 to block extracellular-facing CA XIIactivity or with PBS (control conditions); before pHi-imagingexperiments, spheroids were grown for up to 6 days in thepresence of 20 mg/mL 6A10.

ImmunofluorescenceCells were plated on a cover slip in a 35mmPetri dish, grown

to 100% confluency, then fixed using 1 mL of pre-cooledmethanol (�20�C) for 10 minutes at 4�C. Antigen blockingwas conducted with a 1% bovine serum albumin and PBSsolution for 30 minutes. Subsequently, cells were incubatedwith the primary rat monoclonal antibody 6A10 for 1 hour at37�C. The sample was then washed with PBS and 0.2% Tween-20 four times, for 10 minutes each time. Thereafter, cells wereincubated with the secondary goat anti-rat Alexa Fluor 488antibody (green), diluted by 1:1,000 in blocking solution for 1hour at 37�C. To simulate hypoxia cells were treated with 1mmol/L DMOG for 48 hours. Control cells were treated withDMSO for 48 hours to simulate dense normoxic cells. Cellswere imaged with a Zeiss Confocal Laser ScanningMicroscope(LSM) 700 system.

Immunoprecipitation of CA XIIEight micrograms of the anti-CA XII antibody 6A10 was

mixed with 200 mL of protein-L agarose beads in 1 mL PBS andincubated for 1 hour at room temperature. The beads werewashed with PBS thrice, centrifuged for 2 minutes each, thenhalved. The anti-6A10 rat monoclonal antibody was added toeither DMSO or DMOG A549 cell lysates, each 500 mL, and thebead–antibodymixtures were incubated on a shaker overnightat 4�C. Both sampleswere then centrifuged at 15,000 rpm for 20seconds. The lysates were removed, and each sample washedthrice with PBS and centrifuged for 2minutes each time. Forty-five microliters of Laemmli loading buffer containing b-mer-captoethanol were added and samples boiled at 100�C for 5minutes. The samples were then centrifuged and loaded intolanes to conduct polyacrylamide electrophoresis and Westernblot analysis.

Western blottingA549 and T47D cells were lysed at 4�Cwith buffer containing

in (mmol/L): 1% Triton X-100, 0.5% Nonidet P-40 Substitute,NaCl (150), NaF (50), Tris-HCl (50), at pH 7.5, and Completeprotease inhibitors (Roche). Lysed cell fragments were pelletedat 13,000 rpm for 20 minutes at 4�C. Proteins were resolved bySDS-PAGE and transferred to a polyvinylidene difluoridemembrane. Antigen blocking was conducted overnight at 4�Cwith 5% skimmed milk in PBS and 0.2% Nonidet P-40.

The relevant proteins were detected using the followingprimary antibodies: with goat polyclonal anti-CA XII (R&DBiosystems) antibody; monoclonalmouseM75 antibody raisedagainst human CA IX; and polyclonal rabbit antibody raisedagainst CA II (both kind gifts from Prof. S. Pastorekova, SlovakAcademy of Sciences, Bratislava, Slovakia), polyclonal goatantibody raised against actin (Santa Cruz). The primary incu-bation of membranes was followed by incubation with horse-radish peroxidase-conjugated secondary antibodies; both for 1hour on a shaker at room temperature. Enhanced chemilumi-nesence (ECL, Pierce) was used to visualize protein expression,and the membranes developed on X-ray film (Fuji). Acquireddata were normalized to actin.

Preparation of A549/T47D membrane fragmentsCarbonic anhydrase activity was determined from mem-

brane fragments extracted from A549 and T47D cells grownto 90% to 100% confluency. Cells werewashedwith ice-cold PBSand scraped down into ice-cold buffer containing in (mmol/L):NaCl (15), KCl (35), potassiumgluconate (105), HEPES (20),MES(20), Complete protease inhibitors (Roche), pH adjusted to 7.8at room temperature. Cells in suspension were then disruptedby freeze-thaw cycle and centrifuged at 9,000 rpm at 4�C topellet the membrane fraction, which was then resuspended infresh buffer. The centrifugation andbuffer-replacement processwas conducted twice to prevent contamination of cell mem-brane solution with cytosolic carbonic anhydrase isoforms.

Measurement of carbonic anhydrase activity in themembrane fraction of lysed cells

A 0.67 mL aliquot of membrane-fragment suspension wasadded to a 2mL vessel, well stirred, and cooled to 2�C. A narrow

CA XII Inhibiting Antibody

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pH electrode (Biotrode) was inserted tomonitor solution pH at1Hz. To start the carbonic anhydrase-catalyzed reaction, a 0.33mL aliquot of 100%CO2 saturatedwater (2�C)was added to thevessel. CO2 hydration yielded H

þ ions, which could be mea-suredwith the pH electrode. Coolingwas necessary to bring thereaction kinetics within the resolving power of the pH elec-trode. The hydration rate constant was derived from the pHtime-course using afitting algorithmdevelopedpreviously (22).To determine the spontaneous CO2 hydration rate, measure-ments were conducted on membrane-free "blank buffer" sam-ples and also in samples treated with 100 mmol/L acetazol-amide. The increase in CO2 hydration rate above the sponta-neous rate is a measure of carbonic anhydrase catalysis (22).

SolutionsSolutions contained (in mmol/L): CO2/HCO3

� bufferednormal Tyrode (NT): NaCl (130), KCl (4.5), MgCl2 (1), CaCl2 (1),NaHCO3 (12), glucose (11). For ammonium-containing NT,NaCl was substituted with NH4Cl. Solutions were bubbledwith 5% CO2/95% air to attain a pH of 7.2.

Live cell imaging of pHCells were imaged using a Zeiss LSM 700 with a transparent

superfusion chamber (capacity 2mL), the surface of which waspretreated with 0.01% poly-L-lysine to facilitate cell/spheroidadhesion. Solutions were heated to 37�C and delivered at aconstant rate of 2 mL/min. Downstream suction was adjustedto maintain a steady-state solution volume of approximately0.5mL. Cells were exposed for 3minutes tomedium containing50mmol/LWGA-fluorescein (Invitrogen), amembrane-taggingdye that reports pH at the extracellular surface of the cell, nearthe site of CA XII catalysis (21). The dye was excited alternatelyat 405 and 488 nm and fluorescent emission detected at 520�20 nm. The ratio of fluorescence excited at 405 and 488 nmwaslater calibrated using highly buffered solutions (10 mmol/LHEPES, 10mmol/LMES, 120mmol/L NaCl, 4.5mmol/L KCl, 10mmol/L glucose, 1 mmol/L MgCl2, 1 mmol/L CaCl2) at pH 6.8and 7.4.

Statistical analysis of dataSE bars are shown for all data. Data from the measurement

of spontaneous CO2 hydration rates in membrane fractionwere assessed using a two-tailed t test and considered to besignificantly different when P < 0.05. In vitro data were assessedusing paired two-tailed t tests and considered to be significantwhen P < 0.05.

Multicellular spheroidsSpheroids were cultivated as hanging drops. Eight thousand

cells in 20 mL DMEMwith the 6A10 or an isotype antibody at afinal concentration of 20 mg/mL were pipetted onto the innerside of the lid of a cell culture dish. The lid was quickly reversedand placed onto the dish containing PBS to avoid desiccationsand incubated at 37�C. Cell proliferation was calculated withMTT assays.

ImmunocytochemistryExcised xenografts were cryopreserved and cut into 4 mm

sections, stained with a Cy3- or fluorescein isothiocynate-

coupled goat anti-rat secondary antibody and imaged usinga Zeiss AxioImager Z1 microscope.

Xenograft studiesNSG mice (NOD/SCID/IL2 receptor gamma chain knock-

out) mice were obtained form Jackson Laboratory and housedat the animal facility of the Helmholtz Center Munich andexperiments were carried out under a license from the State ofBavaria. A549 human lung cancer cells were transduced with avesicular stomatitis virus G protein-pseudotyped lentivirusencoding GFP and Gaussia luciferase (LUC) essentially asdescribed elsewhere (33). Cells expressing the highest GFPlevels were isolated using a BectonDickinsonAria III cell sorterand amplified, yielding a homogenous population with respectto GFP expression (Supplementary Fig. S1). A549/GFP/LUCcells were counted, resuspended in Hank's balanced salt solu-tion (HBSS), and 100,000 cells per mouse in a final volume of200 mL HBSS were injected intraperitoneally. The data shownhere are representative of three independent experiments. Inan earlier experiment, mice were injected with different dosesof antibodies (10–500mg) and 100mg/mL 6A10was determinedof the lowest dose showing an antitumor effect. For theexperiments described herein and starting from day 0 ofinjection, mice were treated with either 100 mg 6A10 or ananti-GST isotype antibody once per week or left untreated (n¼8 for each group). No antibody-related toxicity was observed,that is, body weight, habitus, and behavior were normal andidentical to animals of the untreated group. Also macroscopicpost mortem inspection did not reveal any indication forantibody-related tissue and organ damage. Tumor growthrates were monitored by longitudinal measurements of wholebody bioluminescence signals (photons/second) using an IVISLumina II Imaging System (Caliper Life Sciences) as described(33). Briefly, mice were anesthetized with isofluorane and fixedin the imaging chamber. One hundred micrograms of coelen-terazine (Synchem) was then injected into the tail vein andanimals were immediately imaged for 15 seconds using a fieldof view of 12.5 cmwith open emission filter settings, binning at8 and f/stop at 1. Quantification was conducted with the LivingImage software 4.2 (Caliper). Survival curves were calculatedusing Mantel-Cox tests.

Results6A10 inhibits CA activity at the membrane of CA XIIexpressing A549 and T47D cells

Previous studies have shown an inhibitory effect of 6A10on recombinant CA XII (inhibitory constant IC50 ¼ 5.7nmol/L; 31), the first anti-CA XII monoclonal antibody withbiologic activity on its target enzyme. Confirmation of thisinhibitory effect was tested on CA XII expressed natively byA549 and T47D cell lines. Western blotting showed that bothcell lines express CA XII protein under normoxic incubationand following 48-hour treatment with the hypoxia-induciblefactor (HIF)-stabilizing drug dimethyloxalyl glycine (DMOG; 1mmol/L) to induce hypoxic responses (Fig. 1A). In addition, CAIX expression was also detected in A549 cells (Fig. 1A). Immu-noprecipitation of CA XII protein from A549 whole-cell lysates

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using 6A10 confirmed selective antibody binding to CAXII (Fig.1B). Immunocytochemistry detected CA XII protein at the cellsurface membrane in monolayers of either cell line (Fig. 1C). Asimilar expression patternwas also observed in cells pretreatedwith DMOG (data not shown).

The effect of 6A10 on the catalytic activity of CA XII was firstmeasured in membrane fractions prepared from T47D andA549 cells (Fig. 1D). Samples were prepared by centrifugationand three washing steps to remove soluble intracellular car-bonic anhydrase isoforms. The carbonic anhydrase-catalyzed

Figure 1. Measuring CA XIIexpression, activity, and inhibitionby 6A10. A, Western blot analysisfor CA XII, CA IX, and actin (loadingcontrol) in A549 and T47D celllysates prepared from cellsincubated under control normoxicconditions (DMSO) and in thepresence of 1 mmol/L DMOG toevoke hypoxic signaling.Alternative splice variants ofCA XII underlie the presenceof several bands. B,immunoprecipatation of CA XIIfrom A549 lysates with the 6A10antibody. C, immunofluorescenceof A549 and T47D monolayersstained for CA XII with 6A10 (green)and for nuclei with Hoechst 33342(blue). D, assay for carbonicanhydrase activity. Representativetime courses recorded fromsuspensions containing T47Dmembrane fragments (i).Spontaneous CO2 hydrationkinetics were measured in thepresence of the broad-spectrumcarbonic anhydrase inhibitoracetazolamide (ATZ; 100 mmol/L).Dose–response curve determinedin A549 membrane fragments(n ¼ 5 per dose; ii). Summary ofcarbonic anhydrase activity datafor the soluble and membranefractions obtained from A549lysates (iii). Data for T47D lysates(iv). Doses: acetazolamide (ATZ),100 mmol/L; MSC8, 10 mg/mL;6A10, 10 mg/mL. E, measuringcarbonic anhydrase activity at theexternal surfaceof intact A549 cells(n ¼ 10), tagged with WGAconjugate of fluorescein.Superfusion with 5% CO2/12mmol/L HCO3

� buffered solutions.pHe transients were measuredupon adding and then removing 20mmol/L NH4

þ-containing solution.Exofacial carbonic anhydraseactivity decreases the size of thesepHe transients due to fasterbuffering by CO2/HCO3

�.20 mg/mL 6A10 inhibited exofacialcarbonic anhydrase activitycompletely (to levels recorded with100 mmol/L acetazolamide).F, experiments repeated onT47D cells (n ¼ 10).






reaction was initiated by adding 100% CO2-saturated water tobuffered suspensions (20 mmol/L HEPES þ 20 mmol/L MES;pH 8.0 at 4�C) containing cell membrane fragments (1:2 v/v).The rate of the Hþ-yielding CO2 hydration reaction (monitoredby measuring medium pH with an electrode) is related tocarbonic anhydrase activity (Fig. 1D, i). The pH dependence ofthe hydration rate constant (kh) was determined by piecewisefitting to the recorded pH time course. Results of the best-fit(Fig. 1D, ii) indicate that the membrane-bound carbonicanhydrase activity is pH-insensitive over the range 6.7–7.7.The addition of 6A10 reduced carbonic anhydrase catalysis in adose-dependent manner (Fig. 1D, iii) towards the spontaneousrate (confirmed by experiments in the presence of the broad-spectrum carbonic anhydrase inhibitor acetazolamide, 100mmol/L). The IC50 of 6A10 measured in A549 membranefragments was 0.96 mg/mL, and therefore 10 mg/mL wasdeemed to inhibit CA XII fully (Fig. 1D, iii). 6A10 (30 mg/mL)did not affect measured carbonic anhydrase activity in thestrongly CA IX-positive breast line MDA-MB-468 (97 � 13% ofcontrol activity; ref. 23), establishing that 6A10 does not cross-react with this other exofacial carbonic anhydrase. The near-full inhibition of carbonic anhydrase activity in A549 mem-brane fragments with 6A10 indicates that endogenous CA IXactivity in this cell line does not contribute significantlytowards total membrane-associated carbonic anhydrase catal-ysis, despite CA IX immunoreactivity (Fig. 1A). This wasconfirmed by the absence of any inhibitory effect of theanti-CA IX antibody MSC8 (which blocks up to 57% of CA IXactivity in CA IX-overexpressing cell lines; Fig. 1D, iv; ref. 34).Results of the assay are summarized in Fig. 1D, iv (A549 cells)and Fig. 1D, v (T47Dcells). Per gramof total protein,membranefragments had higher carbonic anhydrase activity than thesoluble fraction in both cell lines and 10 mg/mL 6A10 potentlyinhibited membrane carbonic anhydrase activity.

Further characterization of 6A10 was conducted on intactcells, loaded with the wheat germ agglutinin (WGA) conjugateoffluorescein, a pH-sensitivefluorescent dye.WGA-fluoresceinreported extracellular pH, close to the site of CA XII activity.Cells were superfused with solutions buffered by 5% CO2/12mmol/LHCO3

� at pH¼ 7.2. Rapid exposure to and subsequentwithdrawal of 20 mmol/L ammonium-containing solution bymeans of solution-switching evoked surface-pHe transients:exposure to ammonium evoked transmembrane NH3 entryand extracellular NH4

þ deprotonation at the extracellularmembrane surface (producing a fall in pHe), whereas ammo-nium removal evoked the reverse reaction (rise in pHe). Theability of extracellular CO2/HCO3

� to buffer these surface-pHetransients depends on exofacial carbonic anhydrase activity.Out-of-equilibrium surface-pHe transients were significantlylarger in the presence of acetazolamide (Fig. 1E and F) in bothT47D and A549 cells, confirming that both cell lines haveconsiderable extracellular-facing carbonic anhydrase activity.Experiments were repeated on cells pretreated with 20 mg/mL6A10 for 24 hours. Surface-pHe transients were larger and nolonger affected by treatment with acetazolamide, indicatingthat 6A10 had inhibited exofacial carbonic anhydrase activity(Fig. 1E and F). Overall, these data show that the 6A10 antibodyis a highly potent, full inhibitor of exofacial CA XII catalytic

activity in A549 and T47D cells natively expressing the protein.In A549 cells, which express both CA IX and CA XII protein, thelatter is the major contributor to total extracellular-facingcarbonic anhydrase activity.

6A10 inhibits cancer cell growthThe effect of CA XII inhibition on the growth of CA XII-

positive cells (A549 and Kato III) was measured using the MTTassay. The starting concentration of HCO3

� was varied, atconstant CO2 partial pressure, to alter the time-course ofmedium [HCO3

�] and pH over a 3-day growth period (wherebyhigher medium [HCO3

�] is associated with more alkaline pH,under the constraints of theHenderson-Hasselbalch equation).Growth of A549 cells was reduced in the presence of 6A10 (Fig.2A). This effect was greater with lower starting [HCO3

�], i.e.,under conditions were HCO3

� availability is reduced through-out the growth period, relative to high-starting [HCO3

�] con-trols. The HCO3

�-dependence of the 6A10 effect was morepronounced in Kato III cells (Fig. 2B). These results indicatethat 6A10 has a significant inhibitory effect on growth in vitro,which is more pronounced when cells are grown inmedia withlower starting [HCO3

�]Many cell lines and cancers in vivo coexpress CA XII along

with the exofacial isoform CA IX, raising the possibility that CAXII-specific inhibition could be compensated by intact CA IXactivity. This was tested by comparing the effect of CA XIIinhibition with 6A10 on CA IX-positive and CA IX-negativecells. Experiments were carried out on the HT29 colon cancercell line, which expresses both carbonic anhydrase isozymes.The effects of 6A10 were studied on subclones transduced witheither an expression vector carrying a short-hairpin RNAagainst CA IX (HT29-shCA9) or an empty control vector(HT29-shEV; ref. 30). Cells transduced with shCA9 RNA dis-played no detectable CA IX expression even under hypoxia,whereas the enzyme was evident in HT29-shEV cells (Fig. 2C).Immunoblotting (Fig. 2C) and flow cytometry (Fig. 2D)revealed that knockdown of CA IX had no effect on CA XIIexpression. HT29-shCA9 and HT29-shEV cells were grownunder hypoxia (1% O2) for 3 days in the presence of 6A10 oran isotype control antibody. Inhibition of CA XII activity with6A10 significantly reduced the growth of CA IX-negative HT29-shCA9 in media containing 44 mmol/L bicarbonate and inbicarbonate-free HEPES-buffered medium (Fig. 2E). In con-trast, no inhibitory effects were observed with HT29-shEV cells(Fig. 2F), indicating that the carbonic anhydrase activity con-ferred by CA XII in HT29 cells is adequate to maintain normalgrowth under hypoxia. This observation supports the notion ofa critical role of CA XII catalysis in facilitating growth, which inturn could be targeted efficaciously with 6A10.

6A10 inhibits tumor cell growth in a pH-dependentmanner

The catalytic process mediated by CA XII is strongly inhib-ited by acidic pH substantially lower than 7.0 (35). If thecatalytic process were critical for the effects of 6A10 on growth,the efficacy of the antibody would show pH dependence. Toassess this in vitro, CA XII-expressing A549 cells were grown asspheroids in HEPES-buffered HCO3

�-free medium titrated to

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different starting pH levels. Over the course of growth, thesemedia are expected to acidify further due to metabolism. After3 days of culture, cell growth was assessed by the MTT assay(36). As shown in Fig. 3, overall growth rates of A549 cells werestrongly pH-dependent, with an optimum under conditionswere starting pH was approximately 7.2. Of interest, cells didnot survive pH values higher than 7.3 in nominally HCO3

�-freemedia (data not shown). The inhibitory effect of 6A10 on cellgrowthwas observedwhen cells were grown froma starting pHof 7.1 and 7.3. In contrast, no such effect was observed in cellsgrowth from a more acidic starting pH (between 6.2 and 6.8),that is, under which CA XII catalysis is expected to be inhibitedfrom the start of the experiment. In other words, antitumoralefficacy was not seen under conditions where the enzyme hadbeen inhibited by Hþ ions from the start of incubation. Thesedata show that CA XII enzyme activity is mandatory foroptimal growth of A549 cells, and that the inhibitory effectsof 6A10 correlate directly with CA XII catalytic activity. It isnoteworthy that the relationship between proliferation andambient pH will be acid-shifted relative to the plot of prolif-

eration versus starting pH because of on-going medium acid-ification over 3 days of culture. Overall, the data indicate thatanti-CA XII antibody growth-retarding efficacy relies criticallyon the antibody's ability to block carbonic anhydrase catalyticactivity.

6A10 shows antitumor activity in a xenograft tumormodel

The effects of CAXII inhibitionwith 6A10 on tumor growth invivo were studied in a human xenograft model using immuno-compromised NSGmice. A total of 100,000 A549/GFP/LUC cellswere injected intraperitoneally and xenograft-bearingmiceweretreated with 6A10 (100 mg/mouse), and compared with isotype-treated and untreated controlmice. 6A10waswell tolerated andno side effects were observed. First, CA XII expression and 6A10binding was assessed in established 6A10-treated tumors. Asshown in Fig. 4A, xenografted A549 cells expressed high levels ofmembrane CA XII. Staining of tumors from 6A10-treated ani-mals with a rat-specific secondary antibody alone confirmed6A10 binding to tumor cells. Interestingly, binding was most

Figure 2. 6A10 interferes with thegrowthof tumor cells. A549cells (A)and Kato III (B) cells were grown instandard medium with 44 mmol/Lbicarbonate or in HEPES-bufferedmedium without bicarbonate(0 mmol/L) for 3 days at a constantCO2 partial pressure of 5%. Overthe course of the experiment,medium HCO3

� and pHchanged, but differences betweenexperimental conditions persisted.After 3 days of growth, cellproliferation was measured in anMTT assay at 595 nm. C, CA IXwas induced in HT29-shEV cellsgrowing in hypoxia (1% O2),whereas the gene was efficientlyknocked down in HT29-shCA9cells. CA IX knockdown hadno significant effect on CA XIIexpression levels. GAPDH,loading control. D, CA XII wasexpressed at comparable levels onthe surface of HT29-shCA9 andHT29-shEV cells. Expression wasenhanced modestly under hypoxia(bold line) as compared withnormoxia (dotted line). Tintedhistogram shows the isotypecontrol. HT29-shCA9 cells (E) andHT29-shEV cells (F) were seeded inthe media described in A andproliferation was measured inan MTT assay. �, P < 0.05.






prominent at the tumor margins but was also evident inintratumoral regions (Fig. 4B), Binding of 6A10 to tumors wasalsodetected in vivousing 6A10 labeledwith the infrareddye 800CW (Supplementary Fig. S2). Representative hematoxylin/eosinstains of excised tumors are shown in the Supplementary Fig. S4.

In the next series of experiments, mice were injected withA549/GFP/LUC cells as described above and xenograft-bearinganimals were treated with 6A10 or an isotype control antibodyonce per week (100 mg/mouse) or left untreated. The xenograftgrowth rate was monitored weekly by longitudinal measure-ment of whole body bioluminescence. The bioluminescencedata of day 81 postinoculation are shown exemplarily in Fig. 5Aand the calculated data are presented in Supplementary Fig. S3.Thesemeasurements revealed that 6A10 treatment significant-ly reduced the xenograft growth rate over time as comparedwith the isotype antibody, which had no protective effect (Fig.5B). Consequently, 6A10-treated animals reached a tumor

burden of 2 � 108 light units approximately 20 days later(Fig. 5C) and had a significantly extended overall survival time(Fig. 5D). Post mortem inspections (not shown) showed thattumors had spread mainly to the kidney, the liver, and thespleen. Because immune effector activities are essentiallyabsent in NSG mice (37), the observed antitumor activity of6A10 is solely attributable to the direct inhibition of CA XIIactivity. Taken together, inhibition of CA XII with 6A10 had asignificant antitumor effect in vivo.

DiscussionA characteristic feature of the elevated metabolic activity of

tumor cells is the generation of ATP by constitutive glycolysis,intracellular accumulation of lactic acid, and, consequently,the ensuing challenge of protecting cells from excessive intra-cellular acidification. The aforementioned pH-challenge isexacerbated by typically inadequate blood perfusion delivered

Figure 4. CA XII is expressed onand 6A10 binds to A549xenografts. A, cryosections ofexcised xenografts from a 6A10-treated animal were stained with aCy3-labeled secondary antibody todetect infiltrated 6A10. B, single-cell suspensions of excisedxenografts were stained with 6A10and a Cy5-labeled secondaryantibody and analyzed by flowcytometry.

Figure 3. The inhibitor effect of 6A10 on cell growth depends on the starting pH of culture media. A, A549 cells were seeded in HEPES-buffered, bicarbonate-free cell culture media adjusted to different pH values and incubated in an atmosphere of 5% CO2 over the course of 3 days; media became graduallymore acidic due to metabolism, and medium HCO3

� was given by the Henderson-Hasselbalch equation (higher HCO3� at higher pH). Proliferation

was measured in an MTT assay 3 days later. �, P ¼ 0.026; ��, P ¼ 0.004. B, data replotted from A, showing extent of growth inhibition by 6A10. Proliferationin 6A10 was plotted against proliferation in isotype and matched for starting medium pH (indicated in brackets). Straight line was best-fit through thedata-points at acid pH (6.2–6.8), at which 6A10 did not have a growth-inhibiting effect. Growth inhibition was significant (P < 0.05) for starting pH >7.0.

Gondi et al.





by an aberrant vasculature, which is also responsible for thehypoxic conditions present in rapidly growing solid tumortissues. Cancer cells have evolved a sophisticated and effica-cious homeostatic system to regulate pHi, involving acid/base

transporters in collaboration with buffers and carbonic anhy-drase enzymes. The membrane-bound, exofacial carbonicanhydrase isoforms provide catalysis for facilitating CO2 dif-fusion, extracellular Hþ buffering and supplying extracellular

Figure 5. 6A10 inhibits the growth of xenografts in vivo. A, NSG mice were inoculated with A549/GFP/LUC cells and the tumor growth rate was measured bywhole body bioluminescence once per week as described in Materials and Methods. Shown is the measurement at day 81 after inoculation. Two animalsof the isotype group and one animal of the untreated group did not develop tumors and were therefore excluded from the experiment. B, longitudinalgrowth of xenografts as measured by bioluminescence. C, calculation of the time postinfection when tumor burden reached a light emission of 2 � 108photons/seconds (p/s) as determined by BLI. ��,P < 0.001; ��,P < 0.0001. D, survival curves of tumor-bearing animals.Mantel-Cox tests revealedP¼ 0.02for the 6A10-treated group versus the isotype-treated group and P ¼ 0.037 for the 6A10-treated group versus the untreated group.






HCO3� formembrane transporters. Collectively, isoforms such

as CA IX and CA XII play an important role in pH control, andinhibition of these exofacial carbonic anhydrases would dis-turb the pH balance in expressing tumors. As pH is a keyphenotypic variable of the tumor chemical milieu, believed toexert selection pressure and drive disease progression, anymanipulation of pH may change the trajectory of cancerdisease (25). Consequently, targeting cancer metabolism byinterfering with pH regulation is nowadays considered apromising therapeutic approach (38).

Our investigation shows the efficacy of the CA XII-inhibitingmonoclonal antibody, 6A10, in reducing growth of cancer cellsin vitro and in xenograft tumor models in vivo. Our resultsprovide evidence that CA XII optimizes tumor cell growth (Fig.2), but only under conditions where the enzyme is expected tobe catalytically active, that is, when the pH at the start ofincubation was sufficiently alkaline to allow the carbonicanhydrase catalytic process to persist during the growth periodwhenmedia gradually acidify (Fig. 3). As evident particularly inCA XII-positive Kato III cells, the growth-retarding effect of6A10 was particularly strong when starting growth conditionshad low HCO3

� and hence low pH, i.e., characteristic of theacidic tumor milieu (23). The catalytic activity of CA XII seemsto bemore resistant to inhibition at low pH compared with CAIX, as CA XII activity was unaffected over the range 6.7 to 7.7,whereas CAIX is half-inhibited when pH falls to 6.8 (28). Thus,the relative contribution of CA IX and CA XII to total mem-brane-associated carbonic anhydrase catalysis will dependstrongly on ambient pH. Under conditions of limitingHCO3

� supply and low extracellular pH, exofacial carbonicanhydrase activity may be essential for tumor physiology andgrowth. Inhibition of the membrane-tethered carbonic anhy-drases IX and/or XII may result in an antitumor effect arisingfrom inadequate control of intracellular pH caused by insuf-ficientHCO3

�provision and slowpH-buffering byCO2/HCO3�.

Support for this hypothesis comes from our xenograft model oflung cancer grown in NSGmice. In this experiment, 6A10 had asubstantial growth-inhibiting effect in vivo (Fig. 5). The growth-delaying effect of CA XII inhibition is consistent with the rolethat exofacial carbonic anhydrase isoforms play in facilitatingthe venting of cellular acid. Intact CA XII activity normallyallows for facilitated CO2 diffusion, resulting in apparentlysmaller diffusional delays for CO2 removal, allowing tumors toattain larger volumes. NSG mice are heavily immunocompro-mised and show no detectable activities of the adaptive andinnate immune system. Consequently, it is unlikely that effec-tor functions like antibody-dependent cellular cytotoxicity or

complement-dependent cellular cytotoxicity would have con-tributed towards the observed antitumor activity of 6A10.Instead, the effect of the antibody on xenograft growth is mostlikely linked to CA XII inhibition.

Although a significant antitumor effect of 6A10 wasobserved in vivo, our in vitro obtained with HT29 cells, endog-enously expressing both CA IX and CAXII, argue for a degree ofredundancy between exofacial carbonic anhydrase isoforms.Because CA IX and XII coexpression is found in at least sometumor cells (2, 8, 10, 39, 40), and in line with results obtained byChiche and colleagues (17), it is tempting to speculate that theconcurrent and selective inhibition of both enzymes wouldresult in an improved and additive antitumor effect. As hypoxiais already evident in non-vascularized micrometastases, therewould also be mileage in investigating whether CA XII inhibi-tion could also impair metastasis formation.

The results presented herein show that inhibition of CA XIIcatalysis can substantially interfere with tumor growth,highlighting this enzyme as a promising target for novelimmunotherapeutic approaches, also in conjunction with CAIX-targeted therapy.

Disclosure of Potential Conflicts of InterestR. Zeidler has ownership interest (including patents) in patent application. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: G. Gondi, J. Mysliwietz, R. ZeidlerDevelopment of methodology: G. Gondi, J. Mysliwietz, R. ZeidlerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. Gondi, J. Mysliwietz, A. Hulikova, J.P. Jen, P.Swietach, E. KremmerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G. Gondi, A. Hulikova, J.P. Jen, P. Swietach, R. ZeidlerWriting, review, and/or revision of the manuscript: A. Hulikova, J.P. Jen, P.Swietach, E. Kremmer, R. ZeidlerStudy supervision: A. Hulikova, P. Swietach, R. Zeidler

AcknowledgmentsThe authors thank Irmela Jeremias for lentiviral transduction of A549 cells

and Ulrike Buchholz and Axel Walch (Munich, Germany) for excellent help withimmunocytochemistry.

Grant SupportThis study was supported by the Deutsche Forschungsgemeinschaft (Ze 419/

12-1), the Royal Society, Association for International Cancer Research andMedical Research Council, and by institutional grants.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 16, 2013; revised August 6, 2013; accepted August 15, 2013;published OnlineFirst September 12, 2013.

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2013;73:6494-6503. Published OnlineFirst September 12, 2013.Cancer Res Gabor Gondi, Josef Mysliwietz, Alzbeta Hulikova, et al. Activity of Cancer-Associated Carbonic Anhydrase XIIAntitumor Efficacy of a Monoclonal Antibody That Inhibits the

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