Monoclonal antibodies generated in carbonic anhydrase IX-deficient mice recognize different domains of tumour-associated hypoxia-induced carbonic anhydrase IX

www.elsevier.com/locate/jim

Journal of Immunological Methods 282 (2003) 117–134

Monoclonal antibodies generated in carbonic anhydrase

IX-deficient mice recognize different domains of

tumour-associated hypoxia-induced carbonic

anhydrase IX$

Miriam Zat’ovicovaa, Kvetoslava Tarabkovaa, Eliska Svastovaa,Adriana Gibadulinovaa, Vojtech Muchaa, Lydia Jakubıckovaa,

Zuzana Biesovaa, Monika Rafajovaa, Marta Ortova Gutb, Seppo Parkkilac,Anna-Kaisa Parkkilad, Abdul Waheede, Willam S. Slye, Ivan Horakb,

Jaromır Pastoreka, Silvia Pastorekovaa,*

aCentre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, 845 05 Bratislava, Slovak RepublicbDepartment of Molecular Genetics, Institute of Molecular Pharmacology and Medical Centre of Free University of Berlin, Berlin, Germany

c Institute of Medical Technology, University of Tampere and Tampere University Hospital, FinlanddDepartment of Neurology, University of Tampere and Tampere University Hospital, Finland

eEdward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine,

St. Louis, MO 63104, USA

Received 15 May 2003; received in revised form 25 July 2003; accepted 11 August 2003

Abstract

Transmembrane carbonic anhydrase IX (CA IX) is frequently expressed in human tumours in response to hypoxia and

may serve as a tumour marker and therapeutic target. So far, only a single monoclonal antibody (MAb) M75 with an epitope

in the N-terminal proteoglycan (PG)-like region has been available for detection purposes. Attempts to produce MAbs

against other parts of CA IX were unsuccessful due to the immunodominance of the PG region that significantly differs

between human and mouse homologues. To overcome this problem, we used various forms of human CA IX antigen to

immunize CA IX-deficient mice recently produced by targeted disruption of Car9 gene. Here, we describe new MAbs that

react with human, but not mouse CA IX in different immunodetection settings, and show no cross-reactivity with CA I, II

and XII. MAb IV/18 is directed to the PG region, while the other six antibodies bind to the CA domain, as determined by

CA IX deletion variants. IV/18 recognizes a linear epitope, while anti-CA MAbs V/10, V/12, VII/20, VII/28, VII/32 and VII/

38 react with conformational epitopes clustered into three antigenic sites. The new antibodies represent important tools for

improving our knowledge of structure–function relationships in the CA IX molecule and a better understanding of the role

0022-1759/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/j.jim.2003.08.011

Abbreviations: CA, carbonic anhydrase; IHC, immunohistochemistry; MAb, monoclonal antibody; PG, proteoglycan; GST, glutathione-S

transferase.$ According to the carbonic anhydrase nomenclature, human CA isoenzymes are written in capital Roman letters and numbers, while their

genes are written in Italic letters and Arabic numbers.

* Corresponding author. Tel.: +42-1-2-5930-2404; fax: +42-1-2-5477-4284.

E-mail address: [email protected] (S. Pastorekova).

M. Zat’ovicova et al. / Journal of Immunological Methods 282 (2003) 117–134118

of CA IX in cancer development. Moreover, the availability of the MAbs specific for distinct antigenic regions on two

separate extracellular domains offers an opportunity to elaborate a sensitive assay that could be particularly important for CA

IX detection in body fluids of cancer patients.

D 2003 Elsevier B.V. All rights reserved.

Keywords: Monoclonal antibodies; Carbonic anhydrase IX; Cancer detection; CA IX-deficient mice; Antigenic site

1. Introduction clinical research. First, expression of CA IX is very

Carbonic anhydrase IX (CA IX, previously named

MN protein, MN/CA IX) is a highly active member

of the a carbonic anhydrase family of zinc metal-

loenzymes that catalyse the reversible conversion

between carbon dioxide and bicarbonate (Pastorek

et al., 1994; Opavsky et al., 1996; Parkkila, 2000;

Wingo et al., 2001). It is one of 14 isoforms that

exist in mammals and occupy different subcellular

positions, including cytoplasm (CA I, II, III, VII),

mitochondria (CA VA, VB), secretory vesicles (CA

VI) and the plasma membrane (CA IV, IX, XII,

XIV). Some of the isozymes are distributed over a

broad range of tissues (CA I, II, CA IV), others are

more restricted to particular organs (CA VI in sali-

vary and mammary glands), and two isoforms have

been linked to cancer tissues (CA IX, XII) (reviewed

in Parkkila, 2000; Pastorekova and Pastorek, 2003).

Enzyme activity and kinetic properties, as well as

sensitivity to sulfonamide inhibitors range from high

(CA II, CA IX, CA XII, CA IV) to low (CA III)

(Supuran and Scozzafava, 2000). Several isoforms

designated as CA-related proteins (CA-RP VIII, X,

XI) are acatalytic due to an incompletely conserved

active site (Nishimori, 2003). This extraordinary

variability among the genetically related members

of the same family of proteins creates the basis for

their involvement in diverse physiological and path-

ological processes. The catalytic activity is of fun-

damental relevance for the maintenance of acid–base

balance and the exchange of ions and water in

metabolically active tissues. Through this activity,

CAs substantially contribute to respiration, produc-

tion of body fluids (vitreous humor, gastric juice,

cerebrospinal fluid), bone resorption, renal acidifica-

tion, etc. (Parkkila, 2000).

CA IX isozyme integrates several properties that

make it an important subject of basic as well as

tightly associated with a broad variety of human

tumours, while it is generally absent from the

corresponding normal tissues (Zavada et al., 1993;

Liao et al., 1994, 1997; Turner et al., 1997; Saarnio et

al., 1998, 2001; Vermylen et al., 1999; Ivanov et al.,

2001; Bartosova et al., 2002). This is principally

related to tumour hypoxia that strongly activates

transcription of the CA9 gene via a hypoxia-inducible

transcription factor binding to a hypoxia-response

element localized just upstream of the transcription

initiation site in the CA9 promoter (Wykoff et al.,

2000). Since tumour hypoxia is an important phe-

nomenon with dramatic implications for cancer de-

velopment and therapy (Hockel and Vaupel, 2001),

CA IX offers significant potential as an intrinsic

hypoxic marker with a prognostic/predictive value

and as a promising therapeutic target (Wykoff et al.,

2000, 2001; Beasley et al., 2001; Giatromanolaki et

al., 2001; Koukourakis et al., 2001). CA IX is an

integral plasma membrane protein with a large extra-

cellular part exposed at the surface of cancer cells and

is thus accessible to many targeting tools, including

specific monoclonal antibodies. Furthermore, in con-

trast to the other CA isozymes, CA IX possesses a

unique proteoglycan (PG)-related region that forms an

N-terminal extension of the extracellular CA domain

and reduces cross-recognition with other isozymes

(Opavsky et al., 1996). Moreover, CA IX has been

functionally implicated in cell adhesion and due to its

high catalytic activity may contribute to acidification

of the extracellular microenvironment (Zavada et al.,

2000; Svastova et al., 2003; Ivanov et al., 1998). In

addition to the potential clinical exploitation of CA

IX, there is an increasing interest in resolving many

basic molecular and functional aspects of the protein

since our knowledge of its precise role in cancer cells,

the contribution of different domains/sequence motifs,

and its regulation remain insufficient.

M. Zat’ovicova et al. / Journal of Immunological Methods 282 (2003) 117–134 119

So far, most of the basic CA IX-related studies have

been performed using a single mouse monoclonal

antibody (MAb) M75 that recognizes the N-terminal

PG region of CA IX (Pastorekova et al., 1992; Zavada

et al., 2000). This antibody proved to be highly

specific and perfectly suitable for certain purposes

including immunohistochemical analyses of cancer

tissue sections (Liao et al., 1994; Saarnio et al.,

1998; Ivanov et al., 2001 and references therein),

targeting hypoxic tumour cells in animal models

(Chrastina et al., 2003), CA IX immunodetection in

vitro and molecular characterization (Pastorek et al.,

1994; Pastorekova et al., 1997; Lieskovska et al.,

1999; Kaluz et al., 1999, 2002; Olive et al., 2001).

On the other hand, CA IX-specific monoclonal anti-

bodies with epitope specificities differing from those

of M75 have become highly desirable for those

approaches that are based on the capture-detection

principle or for studies of mutated variants of CA

IX. However, previous attempts to produce such anti-

bodies were unsuccessful apparently due to the fact

that human CA IX differs from the mouse homologue

predominantly in the N-terminal PG amino acid se-

quence (Ortova Gut et al., 2002). This sequence

appears to be strongly immunogenic possibly because

the immunized mice recognize it as non-self, while

they do not direct a humoral response to the other,

more conserved parts of the human CA IX molecule.

The availability of CA IX-deficient mice (Ortova

Gut et al., 2002) has allowed us to generate hybrid-

oma cell lines producing monoclonal antibodies spe-

cific for different extracellular regions of human CA

IX protein. Here, we provide detailed characterization

of these antibodies and an assessment of their reac-

tivity in various immunodetection procedures, and we

discuss their possible applications.

2. Materials and methods

2.1. Cell culture

Hybridoma cell lines were grown in DMEM me-

dium supplemented with 10% FCS (BioWhittaker,

Verviers, Belgium), 2 mM glutamine and 40 Ag/ml

gentamicin (Lek, Slovenia) at 37 jC in 5% CO2 in air.

The same culture conditions were applied to the

following cell lines that were used either as a source

of CA IX antigen or as negative controls: mouse NIH

3T3 fibroblasts permanently transfected with the full-

length human CA9 cDNA in pSG5C plasmid (NIH

3T3-flCA IX), with the cDNA variants in pSG5C as

described below and corresponding mock transfected

NIH 3T3-neo controls (Pastorek et al., 1994); MDCK

cells transfected with the full-length CA9 cDNA

(MDCK-flCA IX), cDNA variants in pSG5C and

corresponding MDCK-neo controls (Svastova et al.,

2003); human HT-29 colon carcinoma cells as well as

human HeLa cervical carcinoma cells naturally

expressing CA IX; and C33a cervical carcinoma cells

negative for CA IX.

For immunization purposes, the cells grown for 24–

48 h were washed twice in PBS, scraped, collected by

centrifugation and re-suspended in an appropriate

volume of PBS. For the final booster, NIH 3T3-flCA

IX cells were extracted with OCG extraction buffer

composed of 0.5M NaCl, 0.5% octyl-h-D-glucopyra-noside (Sigma, St. Louis, MO), 0.1 mMPMSF (Sigma)

for 30 min at 4 jC, scraped, centrifuged for 5 min at

13000 rpm and dialyzed against PBS for 48 h at 4 jC.

2.2. Cloning and expression of cDNA variants and

recombinant CA IX proteins

Constructs coding for CA IX-PGCA and CA IX-

DCA variants (see Fig. 1) were obtained from the full-

length CA9 cDNA in the pSG5C eukaryotic expres-

sion plasmid (Pastorek et al., 1994) by the removal of

the 3V sequence using EcoRI digestion and removal of

the central CA-coding sequence using double

EcoRV–EcoRI digestion, respectively. The construct

coding for CA IX-DPG was generated from two PCR

products by amplification of the sequences cor-

responding to amino acids 1–41 and 131–459 using

the full-length cDNA and two sets of primers: the

forward primer from the plasmid combined with a

gene-specific DPGS primer (5V-CCATCCCCA-

GAGGTTGCCCAGGGACAAAGAAGGGGATG-

3V) and the reverse primer from the plasmid combined

with DPGA that is complementary to DPGS. The

gene-specific DPGS/DPGA primers were designed so

that their arms were complementary to sequences

flanking the deletion. All the constructs were verified

by sequencing. Recombinant plasmids were co-trans-

fected to NIH 3T3 and MDCK with pSV2neo using

Gene Porter II transfection kit (Gene Therapy System,

Fig. 1. (A) Alignment of amino acid sequences of the human CA IX (HCA IX) and the mouse CA IX (MCA IX) proteins deduced from the

corresponding cDNAs (Pastorek et al., 1994, EMBL Accession No. X66839; Ortova Gut et al., 2002, EMBL Accession No. AJ245857).

Homologous amino acids are written on a grey background. (B) Schematic illustration of CA IX variants used for immunodetection and/or

immunization purposes as described in the body text. Positions of amino acids related to CA IX domain composition and to regions involved in

variants are indicated below. Arrangement of antigenic sites for the antibodies (Y), assumed from the competitive assay shown above the

scheme does not reflect real positions of the epitopes within the protein molecule. Percentage of amino acid homology between two extracellular

domains of human and mouse CA IX proteins is shown inside the linear model of the full-length human CA IX protein, comprising a signal

peptide (SP), proteoglycan-like region (PG), carbonic anhydrase domain (CA), transmembrane anchor (TM) and intra-cytoplasmic tail (IC).



San Diego, CA). The transfected cells were subjected

to 2 weeks of selection in the presence of G418 (Life

Technologies, Gaithersburg, MD), cloned and tested

for expression of CA IX.

The cDNA fragments encoding PG and CA extra-

cellular domains of CA IX (see Fig. 1) were amplified

either individually or together by specific primers using

a full-length CA9 cDNA as a template. The fragment

coding for the PG domain (aa 52–125) was obtained

with PG3 (sense) 5V-TAGAATTCGGCTCTTCTGGG-GAAGAT-3V and PG4 (antisense) 5V-ATACTC-

GAGGGGTTCTTGAGGATCTCC-3V primers, and

the fragment coding for CA domain (aa 121–397)

was obtained with CA5 (sense) 5V-TAGAATTC-GATCCTCAAGAACCCCAG-3Vand CA6 (antisense)5V-AATCTCGAGACTGCTGTCCACTCCAGC-

3Vprimers. The combination of primers PG3 and CA6

was employed to produce cDNA fragment encoding

PG+CA domains (aa 52–397).

The resulting PCR products were cloned into

pGEX 4T-1 bacterial plasmid via EcoRI and XhoI

restriction sites inserted in the primer sequences.

Fusion proteins glutathione-S transferase (GST)–

PG, GST–CA, GST–PGCA were expressed in

Escherichia coli strain DH5a using a standard proce-

dure (Gibadulinova et al., 1998). The full-length

fusion GST–flCA IX protein generated earlier was

produced in parallel. The proteins were purified by

affinity chromatography using Glutathione-S Sephar-

ose (Amersham Pharmacia) and eluted with 10 mM

GSH (reduced glutathione). The proteins obtained

were utilized either as antigens for MAb testing or

as immunogen (GST–CA) for immunization.

2.3. Immunization

For immunization, CA IX-deficient mice generated

and characterized by Ortova Gut et al. (2002) were

used at the age of 8–10 weeks and immunized as

shown in Table 1. The mice were injected intraper-

itoneally (i.p.) with 5� 106 NIH 3T3-flCA IX cells in

0.5 ml PBS. Three weeks later, the mice received

either the same i.p. injection of NIH 3T3-flCA IX

cells or i.p. injection of 5� 106 HT-29 cells in 0.5 ml

PBS. After another 3 weeks, mice were boosted

alternatively as follows: (i) i.p. with 100 Ag of

GST–CA protein bound to Glutathione-S Sepharose

in 0.5 ml PBS, (ii) intravenously (i.v.) with 100 Ag of

eluted GST–CA protein in 200 Al PBS, (iii) i.p. with5� 106 NIH 3T3-flCA IX cells in 0.5 ml PBS and (iv)

i.v. with 200 Al OCG extract of NIH 3T3-CA IX cells.

Splenocytes were harvested 3 days later and fused

with the Sp2/0 myeloma cells.

2.4. Production of MAbs

The fusions of splenocytes from each of the

immunized mice with Sp2/0 cells were carried out

according to Lane et al. (1986). Hybridomas were

selected in DMEM-HAT medium containing hypo-

xanthine, aminopterin and thymidine. The superna-

tants were screened for the specific reactivity towards

CA IX by ELISA as described below. Positive hy-

bridoma cultures were cloned by limiting dilution

using Terasaki microplates. Clonal hybridoma cell

lines were expanded, subjected to freezing–refreezing

and repeatedly tested for reactivity to CA IX. Large

quantities of antibodies were purified from hybridoma

culture medium using affinity chromatography on

Protein A Sepharose CL-4B (Amersham Biosciences,

Uppsala, Sweden).

2.5. ELISA screening

Screening of positive hybridomas was performed

by a sandwich ELISA. Microplate wells were coated

overnight at 37 jC with RIPA extract (specified

below) of NIH 3T3-flCA IX cells, diluted 1/10 in

PBS, and in parallel with an extract of NIH 3T3-neo

cells as a negative control. After blocking with 10%

FCS in PBS, the coated wells were incubated with

undiluted culture media from individual hybridomas.

Peroxidase-labelled pig anti-mouse IgG (Sevapharma,

Prague, Czech Republic) was used as detector. M75

MAb was employed as the positive control.

For the differential ELISA screening, the wells

were coated with the following antigens: 10 ng/well

of GST–flCA IX, 10 ng/well of GST–PGCA, 10 ng/

well of GST–PG, RIPA extract of HeLa cells diluted

1/10 in PBS, RIPA extract of HT-29 cells diluted 1/10

in PBS, and were then assayed as above.

2.6. Determination of isotypes

MAb isotypes were determined by ELISA using

affinity purified rabbit anti-mouse IgG1, IgG2a,

Table 1

Overview of immunization protocols and yields of CA IX-specific hybridomas

Mouse no. Immunization schemea Total no. of clones Non-specificb CA IX-specific hybridomas

Initial no. Subclonedc Stabled

I 1. NIH 3T3-flCA IX i.p. 50 0 1 0 0

2. NIH 3T3-flCA IX i.p.


II, III 1. NIH 3T3-flCA IX i.p. 1700 50 60 6 0


3. HT-29 i.p.

IV 1. NIH 3T3-flCA IX i.p. 800 40 5 5 4


3. OCGexNIH-CA IX i.v.

V, VI 1. NIH 3T3-flCA IX i.p. 1220 17 9 6 2


3. GST–CA i.p.

VII, VIII, IX, X 1. NIH 3T3-flCA i.p. 4400 16 83 12 5

2. HT-29 i.p.

3. GST–CA i.p.

Total 8170 123 158 29 11

Immunogens: NIH 3T3-flCA IX—mouse NIH3T3 fibroblasts stably transfected with the wild-type CA9 cDNA; OCGexNIH-CA IX—extract

from NIH 3T3-flCA IX cells obtained using OCG detergent; HT-29—human colorectal carcinoma cells naturally expressing CA IX; GST–

CA—recombinant bacterial CA domain of CA IX fused to GST.a Brief description of the immunization scheme including order of three consecutive injections given to individual mice that are designated

by Roman numerals, type of immunogen and route of administration (i.p.—intraperitoneal, i.v.—intravenous).b Non-specific hybridomas producing the MAbs against non-CA IX mouse or human cellular proteins of unknown identity.c CA IX-specific hybridomas that were successfully subcloned.d Hybridomas with optimum viability and stable productivity of CA IX-specific MAbs.


IgG2b, IgG3, IgM and IgA antibodies (Mouse Mono-

clonal Antibody Isotyping Reagents, Sigma) accord-

ing to the instructions of the manufacturer.

2.7. Immunofluorescence

Cells grown on glass cover-slips were fixed in ice-

cold methanol at � 20 jC for 5 min. Non-specific

binding was blocked by incubation with PBS con-

taining 1% BSA for 30 min at 37 jC. Then the cells

were sequentially incubated with primary antibodies

diluted in PBS with 0.5% BSA (PBS–BSA) for 1 h at

37 jC, washed three times with PBS–BSA for 10

min, incubated with anti-mouse FITC-conjugated

horse antibody (Vector Laboratories, Burlingame,

CA) diluted 1/300 in PBS–BSA for 1 h at 37 jCand washed as before. Finally, the cells were

mounted onto slides in mounting medium with Citi-

fluor (Agar Scientific, Essex, UK), analysed by a

Nikon E400 epifluorescence microscope equipped

with objectives PlanFluor 20� , 40� and photo-

graphed. Images were acquired using a Nikon Cool-

pix 990 camera.

2.8. Immunoblotting

Cells grown in confluent monolayer were rinsed

twice with cold PBS and solubilised in ice-cold RIPA

buffer (1% Triton X-100 and 1% deoxycholate in

PBS) containing the commercial COMPLETE cock-

tail of protease inhibitors (Roche Diagnostics, Man-

nheim, Germany) for 30 min on ice. The extracts

were collected, cleared by centrifugation at 15000

rpm for 10 min at 4 jC and stored at � 80 jC.Protein concentrations of extracts were quantified

using the BCA protein assay reagent (Pierce, Rock-

ford, IL).

Total cellular extracts (50 Ag of proteins/lane) were

resolved in 10% SDS–PAGE gel under reducing

conditions. The proteins were then transferred to

polyvinylidene difluoride (PVDF) membrane (Amer-

sham Pharmacia Biotech, Little Chalfont Bucking-


hamshire, UK). After blocking in 5% non-fat dry milk

with 0.2% Nonidet P40 in PBS, the membrane was

probed with MAbs (undiluted hybridoma medium),

washed and treated with secondary anti-mouse HRP-

conjugated swine antibody diluted 1/7500 (Seva-

pharma). The protein bands were visualized by en-

hanced chemiluminiscence using the ECL kit

(Amersham Pharmacia Biotech).

2.9. Cell biotinylation

Cells in monolayers of 70%–80% confluence

grown in a 10-cm dish were washed with ice-cold

buffer A (20 mM sodium hydrogen carbonate, 0.15 M

NaCl, pH 8.0). Immediately before use, 1 mg of NHS-

LC-Biotin (Pierce) was dissolved in 50 Al DMSO,

mixed with 4 ml buffer A, added to cells and incu-

bated for 60 min at 4 jC. After biotinylation, the cellswere washed five times with buffer A and extracted as

described above.

2.10. Immunoprecipitation

The test MAb in a volume of 1 ml culture medium

was bound to 25 Al 50% suspension of Protein-A

Sepharose, or Protein-G Sepharose for MAbs of

IgG1 isotype (Pharmacia, Uppsala, Sweden) for 2 h at

RT. Biotinylated cell extract (200 Al) was pre-clearedwith 20 Al of 50% suspension of Protein-A/G Sephar-

ose and then added to the bound MAb. Immunocom-

plexes collected on Protein-A/G Sepharose were

washed according to Williams et al. (1985), boiled 5

min in Laemmli loading buffer and separated by SDS–

PAGE gel (10%) electrophoresis. Afterwards, the pro-

teins were transferred to a PVDF membrane and

revealed with peroxidase-conjugated streptavidin (1/

1000, Pierce) followed by ECL.

2.11. Immunohistochemistry (IHC)

Dissected tumours generated from HeLa cells

xenografted to nude mice (M. Rafajova, unpublished

observations) were fixed in 4% neutral-buffered

formaldehyde, dehydrated and embedded in paraffin.

Dewaxed and rehydrated 5-Am sections were immu-

nostained using the UltraTech HRP Steptavidin–

Biotin Universal Detection System (Immunotech,

Marseilles, France) according to the instructions of

the manufacturer. Briefly, the staining procedure was

performed at room temperature as follows. First,

endogenous peroxidase was blocked by 5 min of

incubation with 3% H2O2 in distilled water. The

sections were treated with the protein blocking agent

for 5 min, incubated for 1 h with MAb in hybridoma

medium diluted 1/10 in PBS containing 1% BSA and

washed with PBS. They were then incubated for 10

min with polyvalent biotinylated secondary antibody,

washed, incubated for 10 min with streptavidin–per-

oxidase reagent, washed again, visualized for 5–15

min in diaminobenzidine solution (Sigma) and, finally,

counterstained with hematoxylin.

2.12. Evaluation of cross-reactivity of anti-CA IX

MAbs to CA I, II and XII

Microplate wells were coated overnight with the

following antigens diluted in PBS: purified CA I (200

ng/well), purified CA II (100 ng/well) and recombi-

nant CA XII (100 ng/well). Then the coated wells

were incubated with undiluted culture media from

individual CA IX-specific hybridomas and with poly-

clonal sera against CA I, CA II and CA XII (all 1/

1000), respectively, as positive controls (Parkkila et

al., 1993; Karhumaa et al., 2000). Peroxidase-labelled

pig anti-mouse IgG and pig anti-rabbit IgG diluted 1/

5000 (Sevapharma) were used as detectors.

Alternatively, the proteins immunoprecipitated

with anti-CA IX MAbs from extracts of HT-29 and

HeLa cells as described above were subjected to

SDS–PAGE and blotting. Non-precipitated extracts

were loaded in parallel to establish the expression of

CA isoforms. The blots were treated with rabbit

polyclonal antisera specific for CA II and CA XII

(1/2000), respectively, followed by HRP-conjugated

pig anti-rabbit antibody diluted 1/5000 (Sevapharma).

Protein bands were visualized by ECL.

2.13. Biotinylation of MAbs

Purified MAbs were labelled with NHS-LC-Biotin

(Pierce) according to the instructions of the manufac-

turer. Briefly, 2 mg of purified IgG dissolved in PBS,

pH 8.0, was incubated with 100 Ag NHS-LC-Biotin

for 2 h on ice. Free biotin was removed using a

microconcentrator (PALL Gelman Lab., Wien, Aus-

tria) or gel filtration.

Table 2

Summary of the isotypes and reactivity of novel CA IX-specific monoclonal antibodies in comparison with M75

Antigen N, HT, G HT, HL, M HT, HL, M M, HT HL tumour

MAb Isotype ELISA Blotting Precipitation Fluorescence IHC

M75 IgG2b + + + + +

IV/6 IgM + + nd + nd

IV/18 IgG2a + + + + +

IV/14 IgM + � nd + nd

IV/11 IgG1 + � � + nd

V/10 IgG2a + � + + �V/12 IgG2a + � + + �VII/13 IgG1 + � � � nd

VII/20 IgG2a + � + + +

VII/28 IgG2a + � + + �VII/32 IgG2a + � + + �VII/38 IgG2a + � + + +

Reactivity of novel MAbs including M75 for comparison was examined using the following antigens: N—extract from transfected mouse NIH

3T3-flCA IX cells expressing human CA IX protein in parallel with corresponding NIH 3T3-neo as negative control; HT—extract from human

HT-29 cells naturally expressing high level of CA IX; HL—extract from human HeLa cells naturally expressing CA IX in dense culture; G—

recombinant GST–flCA IX fusion protein; M—transfected MDCK-flCA IX cells compared to MDCK-neo controls; HL tumour—tissue

sections from HeLa tumour xenografted to nude mice.


2.14. Competitive antibody-binding ELISA

Extract from NIH 3T3-flCA IX cells was adsorbed

on microplate wells at a concentration corresponding

to 50% of maximal binding of labelled MAbs. Coated

plates were washed and saturated with 10% FCS in

PBS. Serial tenfold dilutions of purified MAbs in 30

Al and a constant amount of biotinylated MAb in 30

Al were added and incubated overnight at 4 jC. Theplates were washed and peroxidase-labelled streptavi-

din (Pierce) was used as a detector.

2.15. Capture-detection ELISA

Capture-detection ELISA was performed essential-

ly as described by Pitard et al. (1997). Microplate

wells were coated with 50 Al/well (micro) of individ-

ual purified MAbs diluted in PBS 200 Ag/ml (micro).

After blocking, washing and incubation with the

extract of NIH 3T3-flCA IX cells or control NIH

3T3 (1/100 in PBS) as described above, the same set

Fig. 2. Immunofluorescence detection of CA IX in transfected MDCK

with natural CA IX expression. The cells were grown to confluence,

antibodies followed by FITC-conjugated anti-mouse antibodies. All th

in both cell lines used. Cells with primary antibody omitted and

controls.

of biotinylated antibodies 5 Ag/ml (micro) was added.

Binding of the detector MAbs was determined using

peroxidase-conjugated streptavidin. Results were

expressed as absorbance differences between wells

in which CA IX antigen was present or absent.

3. Results

3.1. Generation and basic characterization of mono-

clonal antibodies to CA IX

To overcome epitope preference caused by the

significant difference in the N-terminal sequences of

the mouse and human CA IX proteins and to produce

monoclonal antibodies directed against other protein

regions (Fig. 1), we have taken the advantage of the

recent availability of mice with a null mutation in the

Car9 gene (Ortova Gut et al., 2002). These mice do

not express their own CA IX protein, and, therefore,

we assumed that they would recognize entire human

polarized epithelial cells and HT-29 colorectal carcinoma cells

fixed by methanol and incubated with representative monoclonal

e antibodies revealed plasma membrane CA IX-specific staining

treated only with the secondary antibody served as negative


Fig. 3. Immunoprecipitation of CA IX from extracts of biotin-

labelled HT-29 human carcinoma cells with the monoclonal

antibodies generated using CA IX-deficient mice. The cells were

grown to confluence, labelled by biotin and extracted with RIPA

buffer. CA IX was immunoprecipitated from the cell extracts as

described in Section 2, separated by SDS–PAGE and blotted. Blots

were incubated with peroxidase-conjugated streptavidin and

developed by ECL.


CA IX molecule as non-self and direct their humoral

response to different portions of the protein.

We examined five immunization protocols based

on different combinations of human cells that natu-

rally express high level of CA IX (HT-29), mouse

cells with ectopic overexpression of CA IX (NIH 3T3-

flCA IX) and, finally, recombinant protein composed

of glutathione-S transferase fused to the CA domain

of CA IX (GST–CA). Three protocols led to the

successful production of CA IX-specific MAbs (Table

1). Altogether, we performed 10 fusions and screened

8170 hybridomas. The 158 hybridomas producing CA

Fig. 4. Detection of CA IX by immunohistochemistry in tumour tissue sect

carcinoma cells were fixed by formaldehyde and embedded in paraffin.

performed with the new monoclonal antibodies directed to CA IX. MAb

membranes of tumour cells. Sections treated with MAb VII/12 did not exh

were also completely negative.

IX reactive antibodies initially selected by ELISA

screening were reduced to 29 hybridomas after sub-

cloning and, finally, to 11 viable and stable clones

after freezing–refreezing. Seven of these antibodies

were of IgG2a isotype, two antibodies were IgG1 and

two were of IgM isotype (Table 2).

The antibodies were first tested in ELISA against

the full-length CA IX antigen expressed in mouse and

human cell lines. All antibodies bound to extracts of

NIH 3T3-flCA IX cells and HT-29 cells, but not to

control NIH 3T3-neo cells and CA IX-negative C33a

human cervical carcinoma cells. They also recognized

GST–CA protein, but not the GST alone, confirming

their specificity for CA IX (Table 2).

Indirect immunofluorescence demonstrated that all

of the antibodies examined bound to an antigen

localized at the cell surface of transfected polarized

MDCK epithelial cells as well as HT-29 human colon

carcinoma cells that naturally express CA IX (Fig. 2).

The distribution of the staining signal was identical to

the distribution of CA IX visualized by M75.

The antibodies of IgG2 isotype were all able to

immunoprecipitate CA IX from the extract of both

HT-29 and HeLa human cells labelled by biotinylation

(Fig. 3). They also immunoprecipitated the CA IX

protein ectopically expressed in MDCK cells. How-

ions. Tumour xenografts generated in nude mice from HeLa cervical

Tissue sections were processed for immunohistochemical staining

VII/20 and M75 displayed CA IX-specific staining of the plasma

ibit any staining. The control sections with omitted primary antibody

Table 3

Target domains of the CA IX-specific monoclonal antibodies based on the reactivity to truncated forms of CA IX

MAb Antigen Target domain

PGCA (G) PG (G) DCA (M) DCA (M) DPG (M)

ELISA Precipitation Fluorescence

M75 + + + + � PG

IV/18 + + + + � PG

V/10 + � � � + CA

V/12 + � � � + CA

VII/20 + � � � + CA

VII/28 + � � � + CA

VII/32 + � � � + CA

VII/38 + � � � + CA

Domain specificity of the monoclonal antibodies was determined with the following antigens: PGCA (G) — N-terminal portion of CA IX

molecule containing both PG and CA domains expressed as GST–PGCA fusion protein; PG (G) — PG domain expressed as GST–PG fusion

protein; DCA (M)—truncated variant of CA IX protein expressed in MDCK cells from transfected cDNAwith deletion in a region encoding CA

domain; DPG (M)—truncated variant of CA IX protein expressed in MDCK cells from transfected cDNAwith deletion in a region encoding PG

domain.


ever, only two of the new MAbs, namely IV/6 and IV/

18, and the old MAb M75 reacted in immunoblotting

with naturally expressed CA IX derived from HT-29

and HeLa cells, respectively, and with CA IX ectop-

ically expressed in MDCK cells.

Examination of the new MAbs for their usefulness

in routine immunohistochemistry revealed that three

MAbs including IV/18, VII/20 and VII/38 were ca-

pable of detecting CA IX in tissue sections from

Fig. 5. Immunofluorescence detection of CA IX and its deletion variants D

grown to confluence, fixed by methanol and incubated with the monoclo

The antibodies revealed plasma membrane CA IX-specific staining only in

Mock-transfected MDCK cells served as negative controls for each of the

paraffin-embedded tumour xenografts generated from

HeLa cells (Table 2). Representative slides are shown

on Fig. 4.

Finally, none of the monoclonal antibodies rec-

ognized the mouse CA IX protein expressed either

in NIH-3T3 cells transfected with the Car9 cDNA

(described in Ortova Gut et al., 2002) or in mouse

L cells with endogenous mouse CA IX (data not

shown).

PG and DCA expressed in transfected MDCK cells. The cells were

nal antibodies followed by FITC-conjugated anti-mouse antibodies.

the cells expressing CA IX variant containing their target domain.

monoclonal antibodies.

Fig. 7. Reactivity of the monoclonal antibodies with reduced CA IX

antigen. An extract of NIH3T3-flCA IX cells was treated by 2-

mercaptoethanol and then coated onto the wells of a microplate and

treated with the MAbs and secondary antibodies. Resulting binding

was compared with the binding of the same MAbs to non-reduced

antigen and expressed as percentage of control.


3.2. Determination of target domains by antibody

binding to recombinant variants of CA IX

The initial tests established the reactivity of the

new monoclonal antibodies towards human CA IX.

To reveal the target protein domains, we selected the

antibodies with the best properties and performed an

additional series of analyses using recombinant var-

iants of CA IX protein produced in bacteria and

variants of CA IX protein expressed from truncated

cDNAs transfected to NIH 3T3 and MDCK cells,

respectively. The results summarized in Table 3 and

shown on Fig. 5 indicate that one of the newly

produced antibodies, namely IV/18, is directed against

the PG domain, similar to M75. The other six anti-

bodies (i.e., V/10, V/12, VII/20, VII/28, VII/32 and

VII/38) reacted with the CA domain, as also con-

firmed by their capacity to immunoprecipitate the full-

length CA IX protein, but not its deletion variant

lacking a large portion of the CA domain (Fig. 6). In

fact, these reagents represent a proof-of-concept that

CA IX-deficient mice are suitable for the generation

of monoclonal antibodies that recognize the regions

outside the immunodominant PG domain.

As described above, the six antibodies directed

against the CA domain reacted with CA IX in

immunoprecipitation but did not recognize blotted

antigen, suggesting that their specificity was towards

flCA IX

∆CA

IV/18 V/10 V/20 VII/38

1 1 12 2 2 1 2

Fig. 6. Immunoprecipitation of CA IX from extracts of MDCK cells

expressing the full-length CA IX protein (fl CA IX, 1) and its DCA

variant (2), respectively. The cells were grown to confluence,

labelled by biotin and extracted with RIPA buffer. CA IX was

immunoprecipitated from the cell extracts as described in Section 2,

separated by SDS–PAGE and blotted. Blots were incubated with

peroxidase-conjugated streptavidin and developed by ECL.

conformational epitopes. The structure of the carbon-

ic anhydrase domain may be stabilized by SUS

bonds between four cysteine residues present in the

extracellular part of CA IX at positions 156, 174,

336 and 409. If so, disruption of these linkages by

reducing agent (e.g., 2-mercaptoethanol) would result

in diminished binding of the antibodies. Based on

this assumption, we added 2-ME to protein extracts

of NIH 3T3-flCA IX cells before coating to micro-

titre plate wells and performing the standard ELISA

procedure. The same procedure without the addition

of 2-ME was done in parallel. As expected, treat-

ment with 2-ME reduced the binding of all CA

domain-specific antibodies, while the reactivity of

the anti-PG antibodies remained almost unchanged

(Fig. 7).

3.3. Analysis of the cross-reactivity of CA-domain

specific MAbs with CA I, CA II and CA XII

Because CA IX is the only carbonic anhydrase

isoform containing the PG domain, antibodies

whose epitopes are localized in this domain can

serve as isoform-specific reagents. However, MAbs

against the CA domain of CA IX could recognize

related CA domains in other isozymes. This possi-


bility raised the need to evaluate the cross-reactivity

of the anti-CA MAbs especially to those isoforms

that may be potentially co-expressed with CA IX.

Therefore, we have tested in ELISA the binding of

our monoclonal antibodies to purified CA I, CA II

and CA XII antigens in comparison to polyclonal

sera specific for these antigens. We found that none

of the CA IX-specific MAbs bound to any of the

heterologous CA isoforms. This finding was verified

by an experiment in which protein extracts from

HT-29 carcinoma cells that express several CA iso-

forms, including CA II, CA IX and CA XII (as was

confirmed by immunoblotting), were first immuno-

precipitated by CA IX-specific MAbs, blotted and

immunodetected with anti-CA II and anti CA XII

antisera, respectively. Neither CA II nor CA XII

were coprecipitated with CA IX by CA IX-specific

antibodies, confirming the lack of their cross-reac-

tivity at least with these isoforms (data not shown).

3.4. Mapping of antigenic sites by competitive

binding of MAbs

In order to determine the relative positions of the

antigenic sites recognized by the monoclonal anti-

bodies within their CA IX target domains, we per-

formed a competitive-binding assay. Biotin-labelled

purified antibodies were allowed to bind in the pres-

ence of increasing amounts of non-labelled competi-

tive antibodies. The extent of binding of the labelled

antibody in the presence of the non-labelled compet-

itor was expressed as percentage of binding in the

absence of the competitor. Examination of all different

pairs of the MAbs including the homologous ones and

their classification according to the extent of the

competition allowed the preparation of a basic map

indicating principal relationships between the antibod-

Fig. 8. Chequer-board maps of antigenic sites delineated on the

basis of the competitive binding ELISA (A) and capture-detection

ELISA (B). (A) Antigen-coated plates were incubated with fixed

amount of biotin-labelled antibody and increasing amounts of non-

labelled competitor antibody. Results are expressed as percentage of

absorbance measured in the absence of competitor antibody. (B)

Plates coated with capture antibody were incubated with antigen in

the cellular extract and then with biotinylated detector antibody.

Absorbance resulting from the cooperative binding of capture-

detector antibodies indicates spatial separation between the epitopes

of the paired antibodies.

ies, their clustering and relationships between the

clusters (Fig. 8A). The map shows that the M75 and

IV/18 antibodies recognize an antigenic site in a

separate region, consistent with their proven specific-


ity for the PG domain of CA IX protein. The anti-

bodies targeting the CA domain form three clusters.

Two pairs of MAbs, namely V/12 versus VII/20 and V/

10 versus VII/32, respectively, showed significant

mutual competitions that define two antigenic sites.

However, non-reciprocal competition between V/10

(label) and V/12 (competitor) indicated that the two

sites may partially extend over each other but with

differences in antibody affinity. Alternatively, binding

of the first antibody could cause an allosteric change

that prevented binding of the second one. MAb VII/38

belongs to a distinct site within the CA domain of

CA IX.

3.5. Relationships between the monoclonal antibodies

in a capture-detection assay

Clustering of the MAbs was further explored by a

sandwich ELISA in which each MAb was used

either as a plastic-coated capture or as soluble bio-

tinylated detector. This arrangement permitted spatial

orientation of the antigen bound to the capture

antibody via one antigenic site and exposure of a

distinct non-overlapping antigenic site recognized by

the detector antibody. A map of the antigenic sites

deduced from this analysis principally confirmed the

relationships among the tested antibodies with minor

differences (Fig. 8B). These could be assigned to

differences in the constitution of the assay that

generates a spatial limitation to binding of the

detector antibody to its epitope if it is localized close

to the epitope of the capture MAb. The data obtained

were evaluated on the basis of the absorbance differ-

ences resulting from the cooperation between the

capture and the detector in the presence and absence

of antigen, with the higher absorbance difference

value indicating the bigger mutual distance of the

epitopes on the folded protein molecule. On this

basis, the antigenic sites for the anti-PG antibodies

were clearly separated from the antigenic sites of the

anti-CA antibodies in accord with the competitive

assay. Moreover, the antigenic sites of VII/20 and V/

12 MAbs seemed to localize closer to the PG

domain than those of V/10, VII/28 and VII/32,

although the latter MAb appeared to interfere with

the binding of VII/20 and V/12 when used as a

capture. MAb VII/38 produced inconsistent results

and was withdrawn from this analysis.

4. Discussion

Due to a strong association with cancer tissues and

a proposed functional involvement in tumour progres-

sion, carbonic anhydrase IX has become an interesting

molecule for both basic and clinical research, as

evidenced by the growing number of CA IX-related

papers (summarized in Pastorekova and Pastorek,

2003). Most of these papers have been based on the

utilization of a CA IX-specific monoclonal antibody

M75 (Pastorekova et al., 1992). Although M75 has

facilitated the acquisition of important data, certain

aspects of CA IX could not be evaluated without

additional detection tools, including the monoclonal

antibodies directed to antigenic sites that do not

interfere with that of M75.

In this paper, we describe the generation of such

antibodies using mice deficient for CA IX as donors

of immune splenocytes for fusion and hybridoma

production. This strategy has been chosen in order

to eliminate the epitope preference possibly related to

a high regional homology between the mouse and

human proteins particularly in the sequences outside

the M75 target domain (Ortova Gut et al., 2002). Our

assumption that the mice lacking CA IX would also

recognize these regions as non-self has been con-

firmed by the production of six monoclonal antibodies

directed against the CA domain of CA IX. Interest-

ingly, the immunization protocol that led to the

successful generation of the anti-CA antibodies in-

volved the injection of a recombinant CA IX variant

containing only the CA domain as a key third-dose

component. Instead, injection with the full-length CA

IX antigen extracted from the transfected NIH 3T3

cells resulted in the production of antibodies, among

which MAb IV/18 competes with M75 and two other

antibodies (IV/6 and IV/14) also appear to bind to the

PG region, similar to M75 (not shown). This experi-

ence suggests that even in the absence of endogenous

mouse CA IX protein that would normally compro-

mise the immune recognition of highly homologous

regions of human CA IX (including the CA domain)

by the immunized mice, the M75 MAb-binding PG

domain is strongly immunodominant. The reason for

the apparent immunodominance could reside in both

the N-terminal location and the amino acid sequence

of the PG domain that is highly acidic and composed

of four perfect and two imperfect hexameric repeats


(Opavsky et al., 1996). These repetitions potentially

offer multiple binding sites and the very N-terminal

position of this region may allow for its spatial

protrusion from the presumably globular CA domain

in the extracellular portion of CA IX (Pastorek et al.,

1994).

These attributes of the PG domain may also

explain the finding that anti-PG MAbs recognize

denatured CA IX protein in immunoblotting, while

anti-CA MAbs fail to do so. One may speculate that a

relatively short PG domain with repetitive motifs

constitutes a linear antigenic region that is not sus-

ceptible to denaturation, while a presumably globular

structure of the large CA domain carrying putative

conformational epitopes can be completely disrupted.

Indeed, reduction of SUS bond between cysteine

residues in the extracellular portion of CA IX perturbs

recognition of these CA domain-related epitopes

thereby supporting this idea. It is also consistent with

the finding that both anti-PG and anti-CA are capable

of immunoprecipitating native CA IX antigen

extracted with mild detergents as well as the antigen

on the surface of transfected MDCK cells that were

fixed by methanol. Such antigen apparently possesses

intact epitopes irrespective of whether they are linear

or conformational.

As delineated by the competitive immunoassay

performed with the repertoire of new MAbs, the CA

domain contains three distinct antigenic sites, two of

which partially overlap. While it is possible to pro-

pose relative mutual positions for these sites, our data

do not permit prediction of their localization on the

CA domain. Based on the extent of the deletion

associated with the loss of binding of these MAbs,

we can at least conclude that the CA antigenic region

spans the amino acids 166–397 of CA IX. However,

it cannot be excluded that the deletion led to disloca-

tion of the antigenic site(s) partly located also on the

amino acids 135–165 that remained preserved in the

DCA deletion variant.

Furthermore, the data from capture-detection anal-

ysis indicate that the antigenic area reactive with

MAbs VII/32, V/10 and VII/28 is localized more

distantly with respect to the PG domain, when com-

pared to the antigenic area reacting with V/12 and VII/

20 MAbs. However, it is difficult to reach any

definitive conclusion regarding the mutual distance

and relationship between the antigenic sites of the CA

domain because of their conformational character.

Nevertheless, based on our data, a combination of

anti-PG MAbs M75 and IV/18 with the anti-CA

MAbs V/10 and VII/32 appears most promising for

CA IX capture-detection purposes.

The characteristics of the new CA IX-specific

monoclonal antibodies suggest their possible appli-

cations. In basic research, these antibodies represent

important reagents for the study of functional con-

tributions of the PG and CA domains, respectively.

Each of these two extracellular domains of CA IX

has been associated with different aspects of tumour

progression. The catalytically active CA domain is

thought to contribute to acidification of the tumour

microenvironment whereas the PG domain (absent

from other CA isoforms) is believed to determine

participation in adhesion-related processes. Using

the present set of MAbs, it will be possible to

differentiate between biological effects exerted by

these domains expressed separately in the form of

deletion variants or mutants of CA IX. The exclu-

sive reactivity of these MAbs with CA IX but not

with the other CAs tested here further increases

their research and clinical value.

The main potential of the new MAbs is in their

clinical application. Many clinical studies of different

tumour tissues have recently demonstrated the predic-

tive and prognostic value of CA IX, especially in

relation to tumour hypoxia (Loncaster et al., 2001;

Chia et al., 2001; Giatromanolaki et al., 2001; Kou-

kourakis et al., 2001; Hui et al., 2002). In this respect,

it is obvious that the new MAbs can be utilized for

detection and/or targeting of CA IX-expressing cancer

cells in different settings (as reviewed in von Mehren

et al., 2003). The latter option seems especially prom-

ising given the capacity of all the MAbs to recognize

CA IX antigen in its native form as confirmed in this

paper. In addition, three of the new MAbs, namely the

PG domain-specific IV/18 and the CA domain-specific

VII/20 and VII/38 antibodies can be used in immuno-

histochemical studies of cancer tissue sections. More-

over, future experiments designed to examine the

biological activity of these MAbs will show whether

they could be of any significance for antibody-medi-

ated anti-cancer therapy, analogous to the use of well-

known MAbs directed against the oncoproteins ErbB2

and EGFR (epidermal growth factor receptor). Last but

not least, the availability of non-competing antibodies


specific for distinct antigenic sites on two separate

extracellular domains offers an opportunity to elabo-

rate a sensitive assay that could be particularly impor-

tant for the detection of CA IX in the body fluids of

cancer patients. It has been shown recently that CA IX

is shed from the tumour cell surface and can be

detected in the blood and urine of cancer patients by

immunoprecipitation (Zavada et al., 2003; Chrastina et

al., 2003). Development of a fast and reliable micro-

assay based on a combination of the antibodies de-

scribed here could potentially permit non-invasive

monitoring of cancer patients. In summary, the anti-

human CA IX monoclonal antibodies generated using

CA IX-deficient mice and characterized in this paper

represent important tools for improving our knowledge

of structure–function relationships in the CA IX

molecule. They should provide a better understanding

of the role of CA IX in cancer development and

improve clinically relevant detection of CA IX in

biological materials.

Acknowledgements

This work was supported by grants from Bayer,

from the Slovak Scientific Grant Agency (VEGA-2/

3055/23 and 2025/22) and from the Science and

Technology Assistance Agency (contract APVT-51-

005802). SPc is a recipient of a grant from Sigrid

Juselius Foundation.

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