lose or reduce CA IX upon conversion to carcinomas (Saarnio et al, 2001; Leppilampi et al, 2003). In the colonic epithelium, CA IX is present normally in the deep crypts and abnormally in the superficial adenomas and carcinomas, with the most intense staining seen in tumors with mucinous component (Saarnio et al, 1998b).

Especially striking is very high proportion of CA IX-positive specimens among the cervical, renal and lung cancers. In the cervical cancer, CA IX immunoreactivity with M75 can be observed in virtually all cervical carcinomas and the majority of cervical intraepithelial neoplasia (Liao et al, 1994). Diffuse CA IX-positive staining signal in normal cervical tissues is found only in concurrent presence of dysplasia or carcinoma and therefore it can be useful as an early diagnostic indicator of cervical neoplasia in Pap smears (Liao and Stanbridge, 1996). In the kidney cancer, CA IX protein expression is selectively linked with the most frequent carcinomas of renal clear cell type (ccRCC). High levels of CA IX are seen in primary, cystic and metastatic ccRCCs, but not in benign lesions (Liao et al, 1997). Simultaneous study using RT PCR has independently proven CA9 mRNA expression in RCC and its absence in benign renal tissue (McKiernan et al, 1997). These findings were further explored into the development of sensitive enhanced RT PCR assay for the detection of RCC cells circulating in the peripheral blood of renal cancer patients (McKiernan et al, 1999). In the lung cancer, CA IX is not found in pre-neoplastic lesions, but is readily present in malignant tumors (Vermylen et al, 1999). Some normally looking bronchial and alveolar epithelia in close vicinity to the tumors contain CA IX positive cells, whereas all other normal lung specimens sampled at a distance from the tumor are negative.

On the basis of the clear-cut division between the tissues with normal and ectopic expression of CA IX as well as on the predominant association of CA IX with different types of tumors, CA IX was proposed as a promising tumor biomarker and further studies strongly supported this view.

VII. Regulation of expression

Unusual distribution of CA IX raised questions about mechanisms that control its differential expression in normal versus tumor cells and contribute to its frequent presence in many tumor types. Experimental evidence that the increased cell density can influence CA IX expression through the promoter activation redirected the attention to a transcriptional regulation of CA9 gene (Lieskovska et al, 1999). CA9 promoter analyzed under conditions of high cell density was shown to possess five regulatory regions containing several cis-acting elements (Kaluz et al, 1999). Two regions adjacent to the transcription initiation site bind AP-1 and SP transcription factors and their synergy is needed for the basic transcriptional activation of CA9 gene (Kaluzova et al, 2001). The most important regulatory element of CA9 promoter is localized on the antisense strand between the SP-1 binding site and the transcription start at position -10/-3 and consists of the nucleotide sequence 5’-TACGTGCA-3’ corresponding to a hypoxia response element (HRE) (Wykoff et al, 2000). HRE element is recognized by HIF-1 transcription factor, which assembles under hypoxic conditions from constitutive b subunit and oxygen-regulated a-subunit. In normoxia, HIF-1a is modified by proline hydroxylases at two conserved proline residues in the central oxygen-dependent degradation domain and subsequently degraded via pVHL tumor suppressor protein (Epstein et al, 2001). Moreover, its transcriptional activity in blocked by factor inhibiting HIF (FIH)-mediated hydroxylation of arginine residue in C-terminal transactivation domain (Mahon et al, 2001). When oxygen level is reduced and the cell is exposed to hypoxic or anoxic conditions, HIF-1a escapes hydroxylation, accumulates and following dimerization with HIF-1b becomes transcriptionally active. Presence of the functional HRE element thus makes CA9 a transcriptional target of HIF-1 and places it among the genes regulated by hypoxia (Semenza, 2003). HRE element is utilized also in a density-induced transcription of CA9, but under these conditions it requires cooperation with juxtaposed SP-1 binding site. Activation of CA9 transcription by increased cell density, which is linked with pericellular hypoxia, involves PI3 kinase pathway and subhypoxic levels of HIF-1a (Kaluz et al, 2002).

Since the level of HIF-1a is controlled by pVHL, it is not surprising that the expression of the wild type VHL transgene can suppress the transcription of CA9 mRNA in the normoxic cells and that VHL deletion or inactivating mutation leads to release of CA9 transcription (Ivanov et al, 1998). Loss of functional pVHL is linked with a majority of clear cell renal cell carcinomas and provides explanation for frequent presence of CA IX in ccRCC. Onset of CA IX expression is an early event occurring in morphologically normal single cells within the renal tubules of patients with VHL disease and therefore provides a robust system for the identification of early foci of VHL inactivation (Mandriota et al, 2002). In addition, transcription of CA9 gene can be modulated by methylation of CpG dinucleotides within the promoter region. In RCC cell lines and in tumors, expression of CA IX is associated with hypomethylation (Cho et al, 2000, 2002). No relationship between CA9 gene expression and promoter methylation was observed in RCC/normal kidney paired specimens by Grabmaier et al, (2002), but it could be missed because the authors did not take into account VHL status and/or presence of hypoxia. Indeed, Ashida et al, (2002) showed that the expression of CA IX in cells with VHL mutation does not occur without hypomethylation of the promoter, particularly CpG sites at positions -74 and -6 with respect to the transcription start, and they proposed that VHL and methylation cooperate in the regulation of CA IX. Methylation of -74 CpG site can also influence the expression of CA IX in the carcinoma cell lines of a different origin than RCC, where it seems to represent adverse factor modifying transcriptional response to cell density (Jakubickova et al, submitted).

It is possible that CA IX expression is regulated also at higher stages of the biosynthetic trail, similarly to some other hypoxia-induced genes. There are several supportive indications, including the presence of consensus phosphorylation sites in the intracytoplasmic tail, which might affect functional performance of CA IX, and the shedding of soluble CA IX, which might control the amount of the plasma-membrane associated protein (Z΅vada et al, 2003). Of course, these assumptions require thorough investigations. So far, it has been clearly shown that the posttranslational stability of CA IX protein is very high (with a half life corresponding to approximately 38 h) and allows for its long persistence in reoxygenated cells (Rafajova et al, 2004). Such extended presence of CA IX on the surface of the post-hypoxic cells may have important implications for its intratumoral distribution.

VIII. Intratumoral expression pattern

The highest level of CA IX expression in vitro is achieved upon mutual aid of high cell density and hypoxia. These conditions are readily found in vivo in solid tumor tissues as consequences of physiological barriers that limit expansive growth of tumor mass. Aberrant formation and functional defects of tumor vasculature cause decreased delivery of oxygen and insufficient removal of metabolic waste exposing distant tumor cell populations to hypoxia and low pH. Depending on severity of these stresses, the affected cells respond by a number of phenotypic changes that may involve necrosis and apoptosis, cessation of the cell cycle, or adaptation enabled by the metabolic shift to anaerobic glycolysis and induction of neoangiogenesis. These changes result from transcriptional activities of HIF, which besides CA IX induces spectrum of functionally relevant genes, including VEGF (vascular endothelial growth factor) and GLUT-1 (glucose transporter) (Semenza, 2003). Hypoxia-triggered architectural and phenotypic rearrangements of tumor tissue finally convert into development of necrotic areas surrounded by the zones of surviving hypoxic cells, which adapted to stress and acquired aggressive behavior. These cells are more inclined to produce metastases and are generally refractive to conventional anticancer treatment modalities. Accordingly, the presence of the hypoxic tumors correlates with poor cancer prognosis (Wouters et al, 2002).

CA IX expression pattern in vivo clearly mirrors a distribution of hypoxic areas. The protein is localized in the perinecrotic regions of various solid tumors, including carcinomas of the breast, skin, ovary, cervix uteri, head and neck, lung, bladder (Wykoff et al, 2000, 2001; Chia et al, 2001, Giatromanolaki et al, 2001; Koukourakis et al, 2001; Loncaster et al, 2001; Olive et al, 2001). According to the measurements in head and neck carcinomas, CA IX expression starts at a distance of 40-140 mm (median 80 mm) from a blood vessel and continues toward necrosis (Beasley et al, 2001). Similar spatial relationship of CA IX to microvessels is found in bladder and lung cancer (Turner et al, 2002, Swinson et al, 2003). When compared to the distribution of HIF-1a and chemical marker of hypoxia EF5 assessed in an independent study (Vukovic et al, 2001), CA IX expression begins more distantly than HIF-1a, but more closely than EF5. This might suggest that CA IX induction requires lower oxygen levels than HIF-1a and that it occurs in a perinecrotic zone, which is larger than the zone labeled by EF5. In support of this assumption, Olive et al, (2001) found that CA IX staining extends beyond the regions binding another chemical marker pimonidazole in cervical carcinomas. Moreover, they demonstrated that CA IX-expressing cells isolated from tumor xenografts are viable, clonogenic and resistant to killing by ionizing radiation. These important findings indicate that at least a fraction of the tumor cells that express CA IX is intermediate in oxygenation and may represent potential source of metastases.

Nevertheless, the intratumoral distribution of CA IX suggests that hypoxia is not the only factor driving its expression. Immunohistochemical studies often refer to certain proportion of tumors that do not show signs of hypoxia (such as the presence of necrotic areas, expression of HIF-1a, VEGF, and/or GLUT-1, incorporation of pimonidazole), but still do express CA IX and vice versa, some tumors with apparent hypoxic regions and absence of CA IX (Wykoff et al, 2001, Chia et al, 2001, Swinson et al, 2003). For instance, about 50% of the non-small cell lung carcinoma cases with focal or extensive necrosis do not stain for CA IX and about one fifth of CA IX positive cases do not contain HIF-1a (Giatromanolaki et al, 2001). Among 12 genes induced by hypoxia, CA9 had the greatest magnitude of induction and was induced in the highest number of cell lines (Lal et al, 2001). Coexpression of CA IX with c-ErbB2, EGFR, and MUC-1 indicates possible regulatory involvement of oncogenic pathways (Giatromanolaki et al, 2001; Bartosova et al, 2002), but these observations require further proof.

IX. Relationship to prognosis

There are several studies showing positive correlation between CA IX expression and poor prognosis. In the breast tumors, CA IX is associated with necrosis and high grade of ductal carcinomas in situ (Wykoff et al, 2001), negative estrogen receptor status (Span et al, 2003), higher relapse rate and worse overall survival of patients with invasive carcinomas (Chia et al, 2001). In the head and neck cancer, CA IX expression correlates with necrosis, high microvascular density and advanced stage (Beasley et al, 2001), and with poor complete response rate to chemoradiotherapy and poor local relapse free survival (Koukourakis et al, 2001). In the non-small cell lung cancer, CA IX is a significant factor of poor prognosis independent of angiogenesis (Giatromanolaki et al, 2001) and its stromal expression is associated with advanced tumor stage (Swinson et al, 2003). In bladder cancer patients treated by radical radiotherapy with carbogen and nicotinamide, CA IX expression is significantly linked with worse cause-specific and overall survivals (Hoskin et al, 2003). In the carcinomas of the cervix, extent of CA IX expression correlates with electrode measurements of tumor oxygen and with overall survival and metastasis-free survival after radiation therapy (Loncaster et al, 2001). Additional paper describes lack of these correlations in cervical cancer (Hedley et al, 2003). The authors discuss intratumoral heterogeneity, influence by factors other than hypoxia and technical differences in staining procedures as potential causes of discrepancies and call for further studies.

Quite a different situation with respect to CA IX prognostic relationships is evident in renal cell carcinomas, where overall expression of CA IX decreases with progressing stage, grade and development of metastases, and lower CA IX staining (cutoff 85%) is independently associated with poor survival in advanced RCC (Bui et al, 2003). In addition, CA IX staining is inversely correlated with Ki-67 and combination of these two markers into a single parameter allows for stratification of patients into low, intermediate and high risk groups, significantly predicts survival and can displace histological grade (Bui et al, 2004). It is not clear, whether the expression of CA IX in RCC is predominantly a function of VHL gene mutation or whether it reflects also other factors, nevertheless, it seems to be the most significant molecular marker described in kidney cancer so far (Pantuck et al, 2003).

X. Functions

A. pH regulation

Ectopic expression in tumors, hypoxia-related distribution pattern and correlation with prognosis favor functional involvement of CA IX in cancer development. CA IX is a highly active enzyme with a catalytic performance and proton transfer rate similar to prototype CA II isoform (Wingo et al, 2001). Its activity can be efficiently inhibited by sulfonamides, particularly with certain derivatives that show some selectivity toward CA IX when compared to other isoenzymes (Vullo et al, 2003, Ilies et al, 2003, Abbate et al, 2004, Vullo et al, 2004, Casey et al, 2004). Similarly as other active CA isoforms, CA IX has been proposed to play a role in pH regulation. This proposal seems meaningful especially in relationship to anaerobic tumor metabolism that generates excess of acidic products, such as lactic acid and H⁺, which have to be extruded from the cell interior to maintain the neutral intracellular pH and protect the cells from death. The extrusion of metabolic waste and its poor clearance by inadequate tumor vasculature creates acidic extracellular microenvironment that is more permissive for tumor cell growth and invasion (reviewed in Stubbs et al, 2000). However, lactic acid is not the only source of acidosis and the studies of intratumoral physiological parameters indicate significant contribution of CO₂ (Helmlinger et al, 1997, Helmlinger et al, 2002). A role for CA IX in this process appears to involve catalytic conversion of CO₂ to bicarbonate and proton at the extracellular side of the plasma membrane and facilitation of the bicarbonate transport to the cell cytoplasm. In analogy to another extracellular isoenzyme CA IV, which physically interacts with bicarbonate transporters such as anion exchangers (AE) to form a transport metabolon in differentiated cells (Sterling et al, 2002), CA IX may directly cooperate with AE in tumor cells and help to neutralize their intracellular space. At the same time, the protons produced by CA IX from hydration of CO₂ may remain outside and improve acidosis of microenvironment (Figure 4). Our recent experimental data clearly fit within this concept (Svastova et al, submitted). It is well known, that the acidic extracellular milieu induces production of growth factors, increases genomic instability, perturbs cell-cell adhesion, and facilitates tumor spread and metastasis (Stubbs et al, 2000). Evidence for CA IX as a causal factor of tumor acidosis may thus support its functional involvement in these processes.

Figure 4. Proposed involvement of CA IX in the pH regulation in tumors illustrated on a model that is based on the formation of a transport metabolon composed of anion exchanger (AE) and carbonic anhydrases (in analogy to CA IV-AE-CA II metabolon described by Sterling et al., 2002). CA IX as an extracellular component of the metabolon hydrates carbon dioxide and provides bicarbonate anions to AE, which transports them to the cytoplasm in exchange for chloride anions. At the intracellular side, CA II converts bicarbonate to carbon dioxide, which diffuses out through the plasma membrane. Extracellular CA IX activity also generates protons that contribute to acidification of external pH, whereas cytoplasmic CA II activity allows for consumption of intracellular protons and contributes to neutralization of internal pH.

B. Cell-to-cell adhesion

Besides its enzyme activity, CA IX is also a cell adhesion molecule (CAM), which can mediate attachment of cells to non-adhesive solid support (Z΅vada et al, 2000). This activity resides in the N-terminal end of the molecule, in the proteoglycan-like domain. The adhesion site of CA IX overlaps with the epitope for M75 monoclonal antibody – PGEEDLP, since M75 blocks adhesion of cells to the immobilized CA IX protein (Figure 5). The PG region of CA IX contains three identical repeats of the motif GEEDLP and four modified repetitions. Also an oligopeptide AITFNAQYA identified with the use of a phage display library of random heptapeptides and synthesized with addition of alanine on both ends competed both with binding of CA IX to the M75 antibody and to the cell surface receptor. The amplification of the M75 epitope in the PG region is also reflected by the capacity of CA IX molecules to bind 2-3 times more of M75 IgG molecules than of any of three monoclonal antibodies V10, V12 and VII20 with epitopes in the CA domain (Z΅vada et al, MS in preparation). These CA domain-specific monoclonal antibodies prepared by Zatovicova et al, (2003) represent important tools for study of structure-function relationships in CA IX molecule and allow for sensitive detection of soluble CA IX that is described below.

A remarkable feature of the PG segment of the CA IX molecule is a high content of dicarboxylic aa (24 D + E out of total 59 residues) and at the same time, a low content of basic aa (4 R + K). We observed that the acidic character of the PG is reflected by easy dissociation of CA IX from the complex formed with the M75 antibody or with the cell surface receptor already at slightly acidic pH. This property might facilitate release of cells from the tumors acidified by the products of hypoxic metabolism. The cells might then attach elsewhere in the organism where the pH is neutral or slightly basic, and start a metastatic growth (Z΅vada et al, MS in preparation).

Moreover, CA IX appears to play a role in intercellular adhesion. In polarized epithelial MDCK cells transfected with the human CA9 cDNA, CA IX protein co-

tissues, and distribution of the radioactivity was determined. Radiolabeled cG250 showed excellent tumor targeting, with only very low radioactivity in normal tissues. In vivo selectivity of the antibody was among the highest ever reported. Metastases were also strongly radiolabeled. Calculation showed that suitable doses of radioactive cG250 could achieve killing of all tumor cells, without seriously damaging any normal tissue. Scintigraphic images show no accumulation of radioactivity in the stomach, which is normally expressing high levels of the CA IX antigen (a fact not known at the time of this work). The most disappointing observation was a high heterogeneity of intratumoral distribution of ¹³¹I-cG250. This fact proved also later as a main drawback for radioimmunotherapy of RCC (simultaneously with a relatively high radioresistance of RCC). The problem of intratumoral heterogenous distribution of therapeutic antibody could not be solved even by fractionated application (Steffens et al, 1999).

The Wilex company is now advertising that their ¹³¹I-labeled product WX-G250 RIT is intended for the future treatment of biliary cancers (cholangiocarcinoma, gall bladder carcinoma), which are sensitive to radiation.

There are other interesting papers worth mentioning: a mathematical model for antibody clearance from the patients has been produced (Loh et al, 1998); to reduce the radioactive burden to the organism, a two-step treatment comprised the application of bivalent antibody with one arm derived from G250 and the other directed to a chelate binding radioactive indium. The isotope was given after the clearance of unbound antibody (Kranenborg et al, 1998). For the scintigraphic imaging, the cG250 labeled with ^99mTc (using 3 different methods of labeling) was superior to ¹³¹I-labeled antibody (Steffens et al, 1999b).

B. Chimeric and bispecific antibodies

Non-radioactive MAbs were also tested. To activate antibody-dependent effector mechanisms, they were used in combination with cytokines or they were modified so as to amplify their complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). There are two in vitro studies on killing RCC cells with human mononuclear cells from peripheral blood of healthy donors, activated with a combination of cG250 antibody and IL-2 (Surfus et al, 1996; Liu et al, 2002). Both of these groups are suggesting that the combination immunotherapy with cG250 and cytokines such as IL-2 shows promise in the treatment of RCC. Established xenografts of human RCC in nu-nu mice were treated with G250 in combination with IF + TNF. This cocktail proved to be therapeutically more efficient than each of the components alone (Van Dijk et al, 1994).

A chimeric bispecific antibody G250/anti-CD3 was designed to bind simulatenously cytotoxic lymphocytes and RCC cells and induce a T-cell mediated cytolysis of tumor (Luiten et al, 1996a). An alternative way to target cytotoxic cells to the tumor was a chimeric G250 with the Fc fragment of human IgE. Such a construct, upon binding RCC cells, attracted mast cells, which in turn destroyed the tumor (Luiten et al, 1996b; 1997). A bispecific monoclonal antibody directed to CA IX antigen and to cellular membrane-bound complement regulator CD55 enhanced binding of complement C3 and increased the lysis of RCC cells (Blok et al, 1998). However, until now there were no reports on clinical testing of these ingenious engineered antibodies. The Wilex company is advertising the product Rencarex^®(WX-250), which is a chimeric antibody with 25% content of Fab derived from mouse G250 and the rest 75% from human IgG. This product has passed the phase II testing in 36 patients with metastatic RCC, with a partial success. Its mechanism of action is ADCC (antibody dependent cell cytotoxicity).

C. Anti-cancer vaccines

There exist occasional reports on “spontaneous” regression in renal cell carcinoma, sometimes even resulting in disappearence of metastatic cancer. RCC is believed to be a relatively immunogenic tumor (Vissers et al, 1999). Vaccination is one of potential approaches towards immunotherapy of tumors carrying suitable antigens like CA IX. This might afford a life-long active immunity, repeatedly destroying newly emerging tumor recurrences.

Anti-idiotype vaccines represent one possible solution. Their advantages are that a large amount of the immunizing antigen can be produced repeatedly, and that the antigen is in fact not perfectly identical with the original tumor antigen. The patient’s organism is probably tolerant to the antigen like CA IX, which is recognized as “self“, and does not respond to it immunologically. On the other hand, an anti-idiotype vaccine is somewhat different from the original antigen and can conceivably induce formation of antibodies reacting with the immunizing antigen and also cross-reacting with the original tumor. From the mice immunized with G250, six hybridomas were obtained, producing “second“ antibodies (Ab2) reactive with the G250 antibody molecules (Uemura et al. 1994a,b). These “second“ antibodies mimicked the CA IX antigen and they were used for immunization of another group of mice. Their sera after immunization contained polyclonal “third antibodies“ (Ab3), reactive with human RCC cells carrying the CA IX antigen. Indeed, these sera, when injected into nu-nu mice simultaneously with the live RCC cells protected them against development of the tumors. The response was quite spectacular – in certain variants it was a 100% protection. The treatment with Ab3 was highly efficient even in the mice with established human RCC xenografts (Uemura et al. 1995). Let us hope that analogous experiments in the patients will prove as effective as in the mice.

Dendritic cell vaccine is an oligopeptide capable of binding dendritic cells and so to induce a specific cytotoxic lymphocyte response directed towards RCC cells. Vissers et al, (1999) analyzed the sequence of CA IX protein for potential HLA-A2.1 binding peptides using a computer program and found 60 candidate oligopeptides. They immunized with each of them transgenic mice expressing human HLA-A2.1 and found that four of them indeed developed cytotoxic T-lymphocytes (CTLs), capable to lyse transgenic mouse cells expressing human CA IX protein as well as HLA-A2.1. One of these oligopeptides (HLSTAFARV) was able to bind human A2.1 dendritic cells in vitro. After cocultivation with autologous CD8-positive T cells and after their restimulation, peptide-specific human CTL were obtained. They possessed the capacity to destroy target cells expressing the CA IX protein. Hopefully, this is one of the ways towards immunotherapy of RCC. Interestingly, the same CD4 (+) T cells in the context of HLA-DR molecules recognized this sequence also in the naturally processed CA IX protein (Vissers et al, 2002). This finding supports the view that peptide-based vaccines indeed could raise the cytotoxic cell response in the patients.

Other vaccines derived from the sequence of CA IX were tested using mouse RCC cells transfected with human CA9 cDNA. Three nonapeptides were designed so as to be compatible both with murine H-2Kd as well as with human HLA-A24 histocompatibility antigens. One of the peptides was identified as a potential vaccine effective both in mouse and human organisms (Shimizu et al, 2003).

Ringhoffer et al, (2004) have proposed a peptide vaccine combining four antigens which are frequently expressed in renal cell carcinoma: MAGE-1, MAGE-3, CA IX and PRAME. Tumor-specific expression of at least one of these T-cell activating antigens was detected in all of 41 RCC patients examined, 80% of these patients expressed two or more tumor-associated antigens simultaneously.

An alternative method of vaccination is a culture of monocytes, derived from peripheral blood mononuclear cells (PBMC) transduced with the adenovirus-CA9 vector. This system has several advantages: PBMC are easy to obtain from the blood and they express the whole CA IX protein (Mukouyama et al, 2004).

Chimeric protein vaccine was constructed from a part of CA IX protein and a part of GM-CSF (granulocyte/macrophage colony stimulating factor). Tso et al, (2001) produced the fusion gene and inserted it into a baculovirus expression vector system. Fusion protein of M_r66,000 was expressed in insect Sf9 cells and purified by affinity chromatography. The protein retained both antigenic activity of CA IX and immunity-stimulating GM-CSF activities. It proved to be a potent immunostimulant, inducing T-helper cell-supported anti-tumor response.

D. Antifection

One of the key problems of developing new cancer treatment methods is a specific and efficient delivery of therapeutic genes into tumor cells, without transfecting normal tissues. Now a technique termed antifection could provide a solution. In principle it is very simple: therapeutic genes (or vectors) are chemically conjugated with monoclonal antibody directed to a suitable tumor antigen, eg., CA IX. This antibody-vector conjugate can attach to the tumor cell, and is subsequently internalized. Finally, the antifected gene is expressed. This idea indeed works, as shown by Dürrbach et al, (1999), who conjugated CA IX-specific MAb G250 with an IL-2 vector and demonstrated its internalization and prolonged expression in mouse cells previously transfected with a human CA 9 vector. Initially, this technique was only modestly efficient, but several further refinements (Deas et al, 2002) brought about an increased delivery and expression of the reporter gene.

E. Genetically engineered cytotoxic cells

Another strategy of the gene therapy is unlimited supply of cytotoxic lymphocytes (CTL) specifically destroying the tumor without damaging normal tissues. Moreover, these CTL should preferably be MHC-independent because (a) tumor cells often do not express MHC; (b) cytotoxic cells should be applicable for all patients carrying the same tumor (or group of tumors) and not tailored for each patient individually with his own combination of MHC antigens. Even this goal appears to be feasible. Weijtens et al, (1996) have engineered CTL armed with a fusion protein scFv/g. The single chain antibody scFv was derived from variable parts of anti-CA IX antibody G250; it has been fused with high affinity Fc(g)RI, which can function in a CD3-independent manner. Such CTLs can specifically lyse target cells via their scFv/g chimeric receptor independent of MHC. They produce cytokines in response to binding with the target cells and like normal CTL, they recycle their scFv/g dictated lytic activity.

In these artificial CTLs, a co-regulatory function of CD2, CD3, CD11a/CD18 and of other molecules was demonstrated (Weijtens et al, 1998). The authors also examined quantitative dependences between the density of T cell chimeric receptor scFv/g and CA IX antigen density on the target cells and their effects on the CTL-mediated cytolysis and production of IL-2 and TNF (Weijtens et al, 2000).

F. Immunomagnetic hyperthermia

Another ingenious and original method of cancer therapy targeted to CA IX-positive tumor cells has been designed (Shinkai et al, 2001). Submicron magnetic particles were enclosed in liposomes with covalently linked Fab fragments of the G250 antibody. Mice with tumors produced by mouse renal carcinoma cells transfected with human CA9 cDNA were injected with these immunomagnetoliposomes (or with control liposomes). After an interval, they were exposed to oscillating magnetic field, which induced vibrations of the magnetic particles. As a consequence, the temperature of the tumors increased to 43°C. This resulted in temporary growth arrest. Control tumors continued growing.

G. Targeted oncolytic viruses

For years, virologists have been thinking of specific oncolysis caused by viruses. Initial experiments were not successful, since the cytopathogenic viruses used (Sindbis, vaccinia) were infectious for both normal and tumor cells. One of possible solutions is retargeting the virus by bispecific scFv with one arm directed against the viral surface antigen (adenovirus knob) and the other against a tumor-associated antigen (CA IX). This virus-antibody complex binds preferably CA IX-expressing tumor cells and eventually destroys them (Jongmans et al, 2003). However, the next virus generation is again a plain adenovirus with no preference for tumor cells.

Another strategy of producing oncolytic viruses was designed by Lim et al, (2004). They constructed a recombinant virus consisting of the CA9 promoter and complete coding sequence of adenovirus. This virus could replicate only in CA IX-expressing cells, which contained relevant transcription factors. At least 100 times higher MOI of such virus was needed for inhibiting CA IX-negative cells than for cells expressing CA IX. Moreover, the viral construct delayed growth of HeLa tumors in nude mice.

XII. Future prospects

There are also several theoretical possibilities to target CA IX function for the anticancer therapeutic purposes and/or utilize the mode of its regulation. These possibilities are based on a solid rationale but certainly require experimental proof of principle.

CA IX-specific sulfonamides as efficient enzyme inhibitors might potentially reduce CA IX-mediated extracellular acidification of the tumor microenvironment and neutralization of the intracellular pH, thereby decreasing invasion propensity and/or survival of tumor cells. Sulfonamide inhibitors might be also applied together with the conventional chemotherapeutic drugs to improve their uptake and efficiency. This idea is based on the experiences that intracellular accumulation of weakly electrolytic drugs and their therapeutic effects depend on the pH gradient across the plasma membrane (Gerweck, 1998; Raghunand et al, 1999; Stubbs et al, 2000). Indirect arguments in favor of the above assumptions reside in observations that in vitro invasion of tumor cells and in vivo tumor growth in xenotrasplanted animals can be diminished by a non-selective CA inhibitor acetazolamide (Teicher et al, 1993; Parkkila et al, 2000), and that different derivatives of CA inhibitors can retard tumor cell growth in culture even in nanomolar concentrations (Casini et al, 2002). However, the mechanism of sulfonamide action and possible involvement of CA IX in those studies remains unclear. . An important step toward the elucidation of the phenotypic consequences of CA IX inhibition has been recently made by the synthesis of the first CA IX-selective, membrane-impermeable sulfonamides (Casey et al, 2004; Pastorekova et al, 2004).

Function-blocking antibodies might represent alternative CA IX-targeted therapeutic tools, although at this point their potential usefulness is merely a matter of speculation based on the analogy with antibodies against some other cell surface molecules, such as EGFR and c-ErbB2. Nevertheless, series of CA IX-specific monoclonal antibodies is available (Zatovicova et al, 2003) and awaits evaluation of possible anticancer effects.

HRE-driven gene therapy is a strategy designed to hit hypoxic tumor cells and cause their selective destruction (Dachs et al, 1997). This strategy principally utilizes HIF-1 regulated expression of the conditionally cytotoxic gene, which is cloned behind the HRE-element-containing promoter derived from the hypoxia-inducible gene. Obviously, the degree of the selectivity allowing for differential expression between well-oxygenated and hypoxic tumor cells depends on the magnitude of hypoxic induction, which is in turn largely affected by the regulatory context of the HRE element within the corresponding promoter. In different hypoxia-induced genes, position of HRE can range from several dozens to more than thousand nucleotides upstream of the start of transcription. Furthermore, some genes, such as VEGF and GLUT-1, can be significantly induced by the alternative pathways and are also expressed under normoxia, what may partially compromise the therapeutic employment of their promoters. In this respect, CA9 promoter raises a particular hope because its HRE element is localized just in front of the transcription initiation site and its transcriptional control by hypoxia is tighter compared to the other HRE-regulated genes (Lal et al, 2001; Rafajova et al, 2004).

XIII. Where we see the main information gaps?

In spite of the increasing number of scientists and clinicians interested in CA IX, there are some fundamental questions which, when answered, could provide useful leads for drug development. As we see it, these are as follows:

· What are the cell surface receptors/ligands binding CA IX?

· Are the binding partners the same in normal stomach, in the intestines and in various tumors?

· What signals (if any) are transmitted between CA IX and the interacting proteins and what are the downstream targets/effectors?

· Which are additional regulatory pathways upstream of CA IX and what is their biological significance?

· What are the mechanisms underlying intratumoral and intertumoral heterogeneity of CA IX distribution?

· What are the principles determining onset of expression in the course of carcinogenesis, quantity of the protein and percentage of CA IX-positive cells, why CA IX is expressed in tumor stroma?

· What are the phenotypic consequences of CA IX expression in different tumors?

XIV. Conclusion

This review essentially supports the view that CA IX could be useful for targeted cancer therapy. More than sixty papers have reported a partial success, at least in vitro or in animal experiments. What is needed is the amplification of the therapeutic effects. We are showing that several entirely different approaches have been designed. The survey suggests that CA IX study is quite stimulating not only to mount the researchers’ imagination, but also to prompt clinicians’ interests for practical improvements of the management of cancer patients.

Acknowledgements

The research of the authors is supported by Bayer Healthcare, the Academy of Sciences of the Czech Republic (project No. K5011112), the Slovak Scientific Grant Agency (VEGA – 2/3055/23), the Science and Technology Assistance Agency (APVT–51–005802) and by the 6^th Framework Program of the European Commission (EUROXY). The authors wish to thank Dr. Juraj Kopacek (Institute of Virology, Bratislava) for the help with the graphical illustrations and to Dr. Eva Sloncova (Institute of Molecular Genetics, Prague) for the photomicrographs with IHC staining.

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Review Article

Received: 15 July 2004; Accepted: 27 July 2004; electronically published: July 2004

Summary

I. Introduction

II. Classification

A. Radioimmunotherapy

B. Chimeric and bispecific antibodies

C. Anti-cancer vaccines

Acknowledgements

References